THE PHOTOPROTECTIVE ROLE OF ANTHOCYANIN PIGMENTS

IN LEAF TISSUES

By

NICOLE M. HUGHES

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY

GRADUATE SCHOOL OF ARTS AND SCIENCES

In Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

in the Department of Biology

May 2009

Winston-Salem, NC

Approved by:

William K. Smith, Ph.D., Advisor

Examining Committee:

Gloria K. Muday, Ph.D.

Peter D. Weigl, Ph.D.

Kathleen A. Kron, Ph.D.

Taylor S. Feild, Ph.D.

 ACKNOWLEDGEMENTS

I would first like to thank my parents, Randy and Theresa, for their love and support in

everything I’ve ever done in my life. At the interface between family and science, I

thank my science fathers—Bill Smith and Howie Neufeld, for their guidance, advice, and

support. I also thank my fellow lab mate, good friend, and mentor, Dan Johnson. I don’t

thank Keith Reinhardt for anything, but I acknowledge that he has served some purpose

in my life here as my scientific and personal antagonist for three years. Thanks also to

my dearest friends—Alison Northup, Ali Farr, Adriana Sanchez, Ellen and Adam

Koehler, Bruce Moser, Catherine Reinhardt, Sara Venables, Lauren Eiter, and Sarah

Higgins.

Chapter-specific acknowledgements

Chapter 1- I thank Drs. L. Donovan and H. Neufeld for use of the spectroradiometric

equipment, Dr. A. McCauley and the microscopy facility at WFU, C. Machalaba and S.

Bissett for technical assistance, and the anonymous reviewers of this work, whose helpful

suggestions greatly improved the quality of this manuscript. This work was supported

financially by the graduate school at Wake Forest University.

Chapter 2- Craig Brodersen and Nate Poirier (University of Vermont) provided technical

assistance with fluorescence profiling, Dr. Howard Neufeld (Appalachian State

ii
University) provided use of optical filters and the Li-1800, and Tim Anderson

(PalmHammock Orchid Estate) graciously donated plant materials.

Chapter 3- I would like to thank Spencer Bissett for technical assistance and Dr. Anita

McCauley of the microscopy facility at WFU for help with microscopic imaging. This

study was supported financially by Wake Forest University and a Research Experience

for K-12 Teachers (RET) grant to CBM (National Science Foundation).

Chapter 4- Spencer Bissett and Kelsey McDowell were instrumental in their help in the

field and lab, respectively. Funding for this project was provided by the Vecellio Fund at

Wake Forest University.

iii
 TABLE OF CONTENTS

ACKNOWLEDGEMENTS……………………………………………………………….ii

LIST OF TABLES……………………………………………………………………….vii

LIST OF FIGURES……………………………………………………………………..viii

ABSTRACT……………………………………………………………………………….x

CHAPTER I

Anthocyanin pigments: An introduction………………...………………………..1

CHAPTER II

Attenuation of incident light in Galax urceolata (Diapensiaceae): Concerted

influence of adaxial and abaxial anthocyanic layers on photoprotection.

Published in American Journal of Botany (2007)

ABSTRACT……………………………………………………………...25

INTRODUCTION…………….…………………………………………26

MATERIALS AND METHODS………………………………………...29

RESULTS……………………………………………………..…………33

DISCUSSION……………………………………………………………35

LITERATURE CITED………………………………………………..…39

CHAPTER III

Optical effects of abaxial anthocyanin on absorption of red wavelengths by

understory species: revisiting the back-scatter hypothesis.

Published in Journal of Experimental Botany (2008)

iv
 ABSTRACT……………………………………………………………...51

INTRODUCTION……………………………………………………….52

MATERIALS AND METHODS………………………………………...55

RESULTS & DISCUSSION…………………………………………….59

LITERATURE CITED…………………………………………………..65

CHAPTER IV
Blackwell Publishing Ltd

Coordination of anthocyanin decline and photosynthetic maturation in juvenile

leaves of three deciduous tree species.

Published in New Phytologist (2007)

ABSTRACT…………………………………………………………...…82

INTRODUCTION…………………………………………………….…83

MATERIALS AND METHODS……………………………….……..…86

RESULTS…………………………………………………………….….91

DISCUSSION……………………………………………………………96

LITERATURE CITED…………………………………………………103

CHAPTER V

Seasonal photosynthesis and anthocyanin production in ten broadleaf evergreen

species.

Published in Functional Plant Biology (2008)

ABSTRACT…………………………………………………………….118

INTRODUCTION…………………………………………………...…119

MATERIALS AND METHODS……………………………………….122

RESULTS………………………………………………………………127

v
 DISCUSSION………………………………………………………..…130

LITERATURE CITED…………………………………………………133

CHAPTER VI

Conclusions and Future Directions……………………………………………..149

CURRICULUM VITAE……………………………………………………………..…153

vi
 LIST OF TABLES

Table 2.1. Pigment content and leaf thickness values for the four leaf types of Galax

urceolata used in abaxial/adaxial optical experiments…………………….…...43

Table 3.1. Chlorophyll data for abaxially anthocyanic (red) and acyanic (green) portions

of Begonia heracleifolia leaves…………………………………………………80

Table 5.1. Detailed descriptions of five red and five green broadleaf evergreen

species ……………………………………………………………...…………..137

Table 5.2. Winter photosynthetic and non-photosynthetic pigment content of red and

green broadleaf evergreen species………………………………………..…….139

vii
 LIST OF FIGURES

Figure 1.1. Anthocyanins depicted during all life stages and seasons…………..………17

Figure 1.2. The biosynthetic pathway for anthocyanin………………………….………19

Figure 1.3. Leaves exhibiting light-dependence of anthocyanin synthesis…………..…21

Figure 1.4. Anatomical location of anthocyanins………………………………….……23

Figure 2.1. Cross sections of Galax urceolata leaves exhibiting the four combinations of

pigmented surfaces seen in winter leaves………………..………………………45

Figure 2.2. Spectral scans of Galax urceolata leaves exhibiting four combinations of

pigmented surfaces seen in winter leaves………………………………………..47

Figure 2.3. Recovery of maximum PSII efficiency (Fv/Fm) for abaxial and adaxial

surfaces of red and green Galax urceolata leaves……………...………………..49

Figure 3.1. Adaxial view of Begonia heracleifolia leaf, and chlorophyll fluorescence

profiles in red and green variegated leaf sections under red, green, and

blue light……………………………………………………………………...….70

Figure 3.2. Spectral distribution of ambientlight, the red optical filter used in

experiments, and light reflected by red abaxial surfaces B. heracleifolia ………72

Figure 3.3. Optical properties (absorptance, transmittance, and reflectance) of adaxial

and abaxial surfaces of anthocyanic and acyanic tissues of B. heracleifolia…....74

Figure 3.4. Quantification of chlorophyll fluorescence profiles according to leaf depth

for abaxially anthocyanic and acyanic B. heracleifolia tissues under red, blue, and

green light………………………………………………………………………..76

viii
Figure 3.5. Photosynthetic light response curves of abaxially acyanic/green and

anthocyanic/red leaf tissues under red light……………………………...………78

Figure 4.1. Whole branches and microscopic sections of leaves from nodes 1-5 of

L. styraciflua, A. rubrum, and C. canadensis …………………………...……...108

Figure 4.2. Pigment concentrations and leaf thicknesses expressed as a percent of mature

values for L. styraciflua, A. rubrum, and C. canadensis…………………….….110

Figure 4.3. Percent maximum photosynthesis (Asat) and leaf conductance of water vapor

(g) values by leaf node, expressed as a percent of mature-leaf values, for L.

styraciflua, A. rubrum, and C. canadensis…………...…………………………112

Figure 4.4. Tip-down (nodes 1-5) chlorophyll a/b ratios for species with green

developing leaves and red developing leaves……..............................................114

Figure 4.5. The relationships between photosynthesis maturation and corresponding

changes in chlorophyll per unit area, leaf thickness, and anthocyanin for L.

styraciflua, A. rubrum, and C. canadensis………………………………...……116

Figure 5.1. Winter maximum light capture efficiency of PSII (Fv/Fm), light-saturated

photosynthesis (Asat), and leaf conductance (g) for red and green broadleaf

evergreen species measured across a range of ambient temperatures…………141

Figure 5.2. Cold, mild, and warm winter day measurements of Asat, g, and Fv/Fm for

green-leaf and red-leaf species…………………………………………………143

Figure 5.3. Seasonal light response curves for red and green broadleaf evergreen

species…………………………………………………………………………..145

Figure 5.4. Percent decrease in maximum observed winter Asat relative to summer values

for red and green broadleaf evergreen species…………………………………147

ix
 ABSTRACT

Hughes, Nicole M.

THE PHOTOPROTECTIVE ROLE OF ANTHOCYANIN PIGMENTS

IN LEAF TISSUES

Dissertation under the direction of William K. Smith, Ph.D.,

Charles H. Babcock Professor of Botany

Anthocyanins are vacuolar pigments most commonly responsible for red to purple

coloration in plant tissues. Because they absorb strongly in the blue-green waveband,

anthocyanins effectively reduce internal light within the leaf, which may be beneficial

under high-light stress conditions. However, pigment patterns in natural systems often

appear inconsistent with a photoprotective function (e.g. anthocyanins in abaxial leaf

surfaces), and their absence in many species exposed to high-light stress suggests that

anthocyanins may not be necessary for photoprotection. Furthermore, the presumed

dynamic interaction between anthocyanin synthesis and relative need for photoprotection

has yet to be quantitatively described. These issues, among others, have stalled acceptance

of a photoprotective function of anthocyanin pigments, and are addressed in the studies

presented here.

Measurements of leaf optics and chlorophyll fluorescence were used to compare

optical and photosynthetic effects of abaxial anthocyanins in vivo. In a species where

abaxial leaf surfaces are naturally exposed to high light stress in the field (Galax urceolata),

anthocyanins appeared to function similarly to anthocyanins in adaxial surfaces—absorbing

x
strongly in the blue-green wavelengths, and reducing high-light stress (i.e. photoinhibition

of photosynthesis). In a shade-adapted, abaxially-red understory species (Begonia

heracleifolia), a photoprotective function was also supported, but through attenuation of

internally-scattered green light transmitted through the upper leaf surface. This function

may be adaptive during periodic exposure to high intensity sunflecks or sun-patches, which

are potentially damaging to shade-adapted plants.

In developing leaves of three deciduous-tree species, anthocyanin disappearance

corresponded with development of ~50% mature photopigment concentrations, ~80%

lamina thickness, and differentiation of the mesophyll into palisade and spongy layers.

This conserved pattern in anthocyanin loss during development between species is

consistent with a controlled coupling of anthocyanin concentration and relative need for

photoprotection.

Relative photosynthetic capacity was compared for five species exhibiting

anthocyanin production in winter leaves and five species lacking anthocyanin. It was

expected that species with red winter leaves would correspond with those exhibiting

diminished photosynthetic capacity, rendering them in greater need for photoprotection.

This hypothesis was not supported, however, and the reason why some species require

anthocyanin pigments while others do not during winter remains unknown.

In conclusion, these studies are generally consistent with a photoprotective function

of anthocyanins in leaf tissues.

xi
 CHAPTER I

ANTHOCYANIN PIGMENTS: AN INTRODUCTION

1
 Anthocyanins are vacuolar, flavonoid pigments most commonly responsible for red,

purple, and blue coloration in plant tissues. They are most often associated with coloration

in flowers and fruits, though anthocyanins may be synthesized in virtually all plant tissues,

and observed in leaves during all ontogenetic stages and seasons (Figure 1). Though the

evolutionary history of anthocyanin has yet to be fully characterized, it is known that the

pigments are absent in algae, present in some bryophytes, ferns, and conifers, and are

virtually ubiquitous in angiosperms (Lee and Gould 2002). Despite their widespread

distribution, however, the functional significance of anthocyanin pigments remains

unresolved (see reviews by Chalker-Scott 1999, Manetas 2006, Ougham et al. 2008).

Furthermore, factors which dictate their presence in certain ontogenetic stages, tissues, and

species, but not others, are not yet fully understood (Hughes and Smith 2007). The

objective of this dissertation was to address these unresolved issues in anthocyanin research

using naturally anthocyanic and acyanic plant systems and current ecophysiological

techniques.

Biochemistry of anthocyanin synthesis

Anthocyanins are secondary metabolites derived from the flavonoid branch of the

phenylpropanoid pathway. Flavonoids are derived from the precursors malonyl-CoA and

p-coumaroyl-CoA, and the biochemical pathway of anthocyanin synthesis from these

precursors is illustrated in Figure 2 (derived from Holton and Cornish 1995). Regulatory

mechanisms for flavonoid synthesis are primarily at the level of transcriptional control for

the structural genes involved in each step of the pathway (Davies and Schwinn 2003).

2
 Anthocyanin gene expression is almost always dependent on light (Mancinelli

1993; Figure 3A), the intensity of which is also directly proportional to anthocyanin

concentration (Figure 3B; Mancinelli 1993; Hughes et al. 2005). Genetic expression

occurs under two distinct light conditions, involving two different processes and groups of

photoreceptors. Under low-fluence conditions (i.e. light intensities typical of the

understory), anthocyanin production is significant, but generally low in quantity, and shows

the general characteristics of a low fluence response (LFR) of phytochrome B (e.g. an

action spectrum with only one peak in the red wavelengths, activation is reversible under

far-red light and follows the Bunsen-Roscoe law of reciprocity) (Mancinelli 1993).

Anthocyanins may be induced in greater quantities under prolonged exposure to high

irradiance as a high-irradiance response (HIR). This process shows peaks in the action

spectra not only in the red, but also UV-A, UV-B and blue wavelengths, indicating

involvement of phytochrome A (activated by red light), cryptochrome (blue and UV-A),

and another, currently unknown, UV-B receptor (Mancinelli, 1993). Under high light,

phytochrome and cryptochrome are synergistic, and anthocyanin synthesis under exposure

to red + blue/UV-A is greater than the sum of anthocyanin production under red light alone

and blue/UV-A light alone. Once these sensory pigments are activated, they themselves

activate (i.e. phosphorylate or reduce) transcription factors involved in expression of the

anthocyanin structural genes.

However, high light alone is not sufficient to induce anthocyanin synthesis. Clearly,

not every plant growing in full sunlight is red, and many leaves growing under high light

will turn red only under certain growth conditions, usually those associated with abiotic

stress. More specifically, heat/cold, drought, osmotic, and nutrient stress are known to

3
induce anthocyanin synthesis in many species under high light (see Chalker-Scott 1999 for

review), as will the onset of certain ontogenetic stages or seasons which render plants

vulnerable to these stresses. The general requisite of stress in addition to light suggests that

additional regulatory mechanisms are in place to control anthocyanin synthesis in addition

to those which are light-mediated.

Hydrogen peroxide (H2O2) is known to function as a regulatory molecule in stress

perception and signal transduction, including anthocyanin synthesis (Vanderauwera et al.

2005). Indeed, many, if not all, of the conditions described above also correspond with

elevated production of radical oxygen species (ROS) and hydrogen peroxide. As expected,

the H2O2-inducible transcriptional cluster involved in anthocyanin synthesis is distinct from

the cluster inducible by light (Vanderauwera et al. 2005), though the interplay between

these two systems is not yet clear. In addition to hydrogen peroxide, sugars also play a role

in stress signaling in plant systems (Smeekens 2000; Rolland et al. 2002; Rook and Bevan

2003), and elevated levels of reducing sugars are known to induce anthocyanin synthesis

(e.g. Weiss 2000, Teng et al. 2005; Hughes et al. 2005; Murakami et al. 2008); however,

the protein(s) involved in sugar sensing, and the downstream effects on anthocyanin

synthesis, have not yet been fully characterized, though several signaling intermediates

have been identified (Vitrac et al. 2000; Teng et al. 2005). In a general ecophysiological

context, it is possible that stressful conditions which require anthocyanin may also

correspond with those that have high sugar concentrations and/or elevated production of

H2O2, which may act as a natural signal inducing anthocyanin gene expression (Teng et al.

2005; Hughes et al. 2005).

4
 Synthesis of anthocyanins occurs in the cytoplasm until the precursor anthocyanidin

(last row of molecules in Figure 2). Up to 19 different naturally-occurring anthocyanidins

have been reported in plant tissues (Iwashina 2000), though the bronze-colored cyanidin-3-

glucoside is the most common form encountered in leaves (Lee and Gould, 2002). In the

cytoplasm, anthocyanidin is tagged with a glutathione by glutathione-S transferase, and is

transported via glutathione pump into the vacuole (Marrs et al. 1995). It is within the

vacuole (Figure 4) that anthocyanins take on their species-specific coloration, which results

from hydroxylation, methylation, sugar addition, or acylation.

Physiological function of anthocyanins in plant tissues

From within the vacuole, anthocyanins may function physiologically in many ways.

Because they absorb strongly in the blue-green waveband, anthocyanins effectively reduce

internal light within the leaf. It is important to note, however, that because these pigments

are spatially separated from the photosynthetic pigments, light-energy absorbed by

anthocyanins is not used for photosynthesis, but is rather dissipated as heat. The remaining,

un-absorbed wavelengths (i.e. those reflected or transmitted) will continue to be transmitted

through leaf cells, where they may or may not be absorbed by photosynthetic pigments

(Gould 2004). The optical properties of anthocyanins are therefore of functional

importance, as they directly influence the wavelengths and intensity of light available to be

used for photosynthesis within a leaf. Pietrini et al. (2002) estimated that anthocyanins in

Zea mays could intercept up to 43% incoming photosynthetically active radiation (PAR),

primarily between 400-600 nm, which reduced apparent quantum yield of CO2 fixation and

non-cyclic electron transport of PSII proportionately. Considering that blue-green light

5
contains the most highly-abundant, high-energy wavelengths of the solar spectrum, that

blue-green light deeply penetrates leaf tissues (Cui et al. 1991), and that wavelengths

responsible for photoinhibition and photo-bleaching are primarily between 520 and 680 nm

(Merzlyak and Chivkunova 2000), the value of blue-green light absorbing pigments during

high sunlight exposure becomes increasingly apparent. The down-regulation of internal

light for photoprotection has thus become a popular explanation for the widespread

occurrence of anthocyanin pigments in leaves and other photosynthetic tissues, especially

in high-light habitats. In vivo, red tissues exhibit less photoinhibition of photosynthesis

under blue and green light than green tissues of the same species, but equal photoinhibition

under red light, which is poorly absorbed by red pigments (Smillie and Hetherington 1999;

Feild et al. 2001; Hughes et al. 2005). Red leaves also appear to exhibit shade

characteristics relative to green conspecifics (Gould et al. 2002; Manetas et al. 2003;

Hughes and Smith 2007; Kyparissis et al. 2007). However, other studies have found no

evidence for a photoprotective function (e.g. Burger and Edwards 1996; Dodd et al. 1998;

Lee et al. 2003; Kyparissis et al. 2007).

Because anthocyanins increase energy absorption by the leaf, and anthocyanins

convert light energy into heat, it has also been proposed that anthocyanins may function to

warm leaves and increase rates of transpiration and metabolism, or protect against cold

temperatures (Smith 1909). This would be most effective in colder climates, as leaf energy

balance models dictate that leaves may be more easily uncoupled from air temperature

when air temperature is low (Campbell and Norman 1998). Indeed, some evidence exists

for a warming function of anthocyanin in other plant tissues (i.e. cones) at high altitudes

(Sturgeon and Mitton, 1980). In the case of leaves, however, there is much evidence

6
against a warming function of anthocyanins in tropical and temperate climates (e.g. Lee et

al. 1979; Lee et al. 1987; Gould et al. 1995; Lee et al. 2003), though leaf temperatures have

not been compared under very cold temperatures. Also inconsistent with a warming

function of anthocyanin is the tendency for leaves on the outer (most sunlit) portions of the

crown to synthesize anthocyanins, as these are the leaves that are exposed to the greatest

windflow, and thus, the most coupled with air temperature within the canopy (Campbell

and Norman 1998).

In addition to optical functions of anthocyanins, the pigment has also been

implicated as functioning biochemically as an in vivo antioxidant. There has been some

debate, however, as to how effective a vacuolar antioxidant could be in protecting tissues

against radical oxygen species (ROS) generated in the chloroplast (Manetas 2006). It has

recently been shown, however, that H2O2 is capable of penetrating both chloroplast and

vacuolar membranes, and that anthocyanins can effectively neutralize these ROS in cells

proximate to high light stress (Yamasaki et al. 1996; Gould et al. 2002; Nagata et al. 2003;

Kytridis and Manetas 2006).

There also exists some evidence that anthocyanins function as a solute to decrease

leaf osmotic potential, thereby contributing to osmotic adjustment and maintenance of

turgor pressure in the presence of drought stress during senescence (Chalker-Scott 1999).

In general, anthocyanin synthesis is known to be inducible under conditions which promote

osmotic stress, including high salinity (Dutt et al. 1991 Ramanjulu et al. 1993;

Kaliamoorthy and Rao 1994), drought (Zhi-min et al. 2000; Allen et al. 1999; Spyropoulos

and Mavormmatis 1978), and high sugar treatment (Tholakalabavi et al. 1997, Sakamoto et

al. 1994; Suzuki, 1995 and others). Furthermore, species with high anthocyanin content

7
seem to be common in environments characterized by low soil moisture (Spyropoulos and

Mavormmatis 1978; Schemske and Bierzychudek 2001), and appear to be more tolerant of

drought conditions (Diamantoglou et al. 1989; Knox 1989; Beeson 1992; Paine et al. 1992).

The precise role of anthocyanins within the context of water stress, however, remains

unclear. It has been suggested that anthocyanins may be directly involved in osmotic

regulation, preventing transpirational water loss while helping maintain turgor pressure

(Chalker-Scott 1999, 2002). However, Manetas (2006) reports that, even at very high

concentrations, anthocyanin content in leaf cells is generally much too low to sufficiently

support an osmoregulatory function, and may contribute less than 1% to the osmotic

potential of a leaf. Furthermore, soluble sugars and K+ are ubiquitous among plants while

anthocyanins are not, are less metabolically costly than anthocyanin, and are known to be

the primary contributors to osmotic adjustment in several species (Handa et al. 1983;

Ranney et al. 1991). Another explanation for the association between anthocyanins and

water stress is that anthocyanins are synthesized to alleviate oxidative stress resulting from

low water potentials, either through acting as an antioxidant, or in photoprotection (as

previously described). Under this latter scenario, anthocyanins would be inducible by

water stress, but may not function directly in its alleviation.

Seasonal and ontogenetic occurrence of anthocyanins in photosynthetic tissues

Anthocyanins can be observed in leaves across all ontogenetic stages and seasons

(Figure 1). In most cases, their synthesis corresponds with transient high-light stress, either

due to environmental or ontogenetic limitations to photosynthesis. A decreased capacity

for photosynthesis is known to render plants intrinsically more vulnerable to

8
photoinhibition, due to a reduction in energy sinks available for energy dissipation (i.e.

photochemical quenching, thus increasing their need for photoprotection correspondingly

(Osmond 1981; Powles 1984). For example, immature leaves in the spring and summer

tend to be especially vulnerable to high-light stress due to such factors as: immature

chloroplast structure (Pettigrew and Vaughn 1998; Choinski et al. 2003), reduced capacity

and quantity of photosynthetic enzymes (Miranda et al. 1981a; Pettigrew and Vaughn

1998), and limited stomatal and cellular conductance of CO2 (Miranda et al. 1981b;

Choinski et al. 2003). For this reason, juvenile leaves frequently exhibit biochemical and

structural adaptations that reduce light absorption, including (but not limited to)

anthocyanins (Figure 1A). Seasonal changes may also introduce limitations on

photosynthesis that could render the synthesis of anthocyanin beneficial. Low

temperatures inhibit the biochemical reactions of the Calvin cycle, but do not significantly

affect light capture, which may result in excessive energy captured relative to that used in

carbon fixation. This imbalance leads to an increase in energy and electron transfer to

molecular oxygen, production of radical oxygen species, and increased vulnerability to

photo-oxidative damage (Mittler 2002). This vulnerability to high light under cold

temperatures may have been one of the factors behind evolution of the needle-leaf shape in

conifers during cold, bright glacial periods (Feild et al. 2003), as a cylindrical shape

reduces direct light incidence by the cosine law (Johnson et al. 2005). Similarly,

anthocyanins may represent a more transient photoprotective strategy employed by broad-

leaved evergreen species, employed only when seasonal changes (e.g. winter) render their

synthesis necessary.

9
Objectives

In the studies presented here, unresolved conflicts or questions associated with a

photoprotective function of anthocyanin pigments are addressed. First, the optical and

photosynthetic effects of anthocyanins in abaxial surfaces of leaves are assessed in two

systems—1) plants which encounter high abaxial irradiance, and 2) plants which grow in

deeply shaded environments characterized by low light—in order to determine whether a

photoprotective function is relevant in either (or both) these cases. Secondly, the dynamic

interaction between anthocyanin synthesis and relative need for photoprotection is

quantified using developing leaves of three deciduous tree species as they transition from

red to green. Finally, the first functional hypothesis behind why some broad-leaf evergreen

species synthesize anthocyanin in winter leaves while others do not, is proposed and tested.

10
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15
Figure 1.1

Anthocyanins may be observed in leaves during all life stages and seasons. Examples: (A)

Quercus alba synthesizes anthocyanin in juvenile leaves during spring; (B) Saxifraga

michauxii exhibits abaxial reddening during summer months in mature leaves; (C) Acer

saccharum synthesizes anthocyanin in senescing leaves during autumn; (D) the evergreen

herb Galax urceolata exhibits reddening due to anthocyanin accumulation in mature leaves

during winter.

16
Figure 1.1

17
Figure1.2

The biosynthetic pathway for anthocyanin, beginning at malonyl-CoA and p-coumaroyl-

CoA (the precursors of all flavonoids), showing how the three general colors of

anthocyanin are synthesized (from Holton and Cornish, 1995).

18
Figure 1.2

19
Figure 1.3. (A) Photoinduction of anthocyanin synthesis in Lonicera japonica, (B) the

direct relationship between light intensity and anthocyanin accumulation, illustrated in

Galax urceolata.

20
Figure 1.3

21
Figure 1.4. (A) Vacuolar sequestration of anthocyanin in palisade mesophyll cells of

Rhododendron spp. (B) Adaxial anthocyanin in palisade mesophyll cells of Leucothoe

fontanesiana.

22
Figure 1.4

23
 CHAPTER II

ATTENUATION OF INCIDENT LIGHT IN GALAX URCEOLATA (DIAPENSIACEAE):

CONCERTED INFLUENCE OF ADAXIAL AND ABAXIAL ANTHOCYANIC

LAYERS ON PHOTOPROTECTION

Nicole M. Hughes and William K. Smith

The following manuscript was published in American Journal of Botany, volume 94, pages

784-790, 2007, and is reprinted with permission. Stylistic variations are due to the

requirements of the journal. N.M. Hughes performed the field work, data analysis, and

prepared the manuscript. W.K. Smith provided logistical support, advice and ideas, and

editorial assistance.

24
Abstract

Although anthocyanin coloration in lower (abaxial) leaf cells has been documented

for numerous species, the functional significance of this character has not been

comprehensively investigated according to habitat or leaf orientation. In the current study,

we demonstrate that abaxial anthocyanin may function as a photoprotectant, similarly to its

purported role in upper (adaxial) cells, in leaves vulnerable to high irradiance incident on

abaxial surfaces. Spectral scans were derived for Galax urceolata leaves exhibiting the

following phenotypes: abaxial or adaxial anthocyanin only, abaxial and adaxial

anthocyanin, and no anthocyanin. To determine whether anthocyanins conferred protection

from photoinhibition, maximum photosystem II efficiencies of red (anthocyanic) and green

(acyanic) surfaces were compared during and after exposure to photoinhibitory conditions.

Leaves were either positioned with their adaxial surfaces facing the light source or inverted

to expose abaxial surfaces. Spectral scans showed increased absorptance of 500-600 nm

wavelengths by red surfaces (consistent with the absorbance spectrum of anthocyanin),

regardless of whether that surface was abaxial or adaxial. Leaves with anthocyanin in

either illuminated surface were also photoinhibited less than leaves lacking anthocyanin in

that surface. These results suggest that anthocyanic layers reduce absorbed sunlight in the

mesophyll not only for adaxial surfaces, but abaxial as well. Adaxial/abaxial anthocyanin

plasticity may therefore be adaptive in high-light environments or during light-sensitive

developmental stages where leaf orientation and/or substrate albedo are variable.

25
Introduction

Anthocyanins have received considerable attention in the recent literature due to

their proposed role as light-attenuators in leaves vulnerable to high light stress (Drumm-

Herrel and Mohr, 1985; Landry et al., 1995; Smillie and Hetherington, 1999; Gould et al.,

2000; Feild et al., 2001; Gould et al., 2002; Pietrini et al., 2002; Hoch et al., 2003; Neill

and Gould, 2003; Manetas et al., 2003, Hughes et al., 2005; Karageorgou and Manetas,

2006). By absorbing blue-green light that may otherwise be absorbed by chlorophyll b in

subjacent mesophyll cells, anthocyanins are thought to relieve the potential damage to the

photosystems that occurs when leaves are exposed to more light energy than can be

effectively assimilated for photosynthesis or dissipated as heat. The physiological

advantage that this confers has been evidenced in studies comparing light capture

efficiency of anthocyanic and cyanic leaves under photoinhibitory conditions. When red

and green leaf surfaces (typically adaxial) are exposed to high light stress, red leaves tend

to be less photoinhibited than green leaves when other photoprotective mechanisms are

equal (Feild et al., 2001; Hoch et al., 2003; Hughes et al., 2005). In the current study, we

test whether abaxial anthocyanin may perform the same function in leaves where the

abaxial surface is incident to high light rather than (or in addition to) the adaxial surface.

Exposure to high abaxial irradiance occurs under a broad range of conditions: in

environments with high substrate albedos (e.g. snow and sand), among plants with inclined

leaf angles that expose abaxial surfaces to ambient sunlight, and in species where

developing juvenile leaves emerge with abaxial surfaces facing outwards (Drumm-Herrel

and Mohr, 1985; Sherwin and Farrant, 1998; personal observation). However, little is

known about adaptations of abaxial cells to withstand high irradiance, which is surprising

26
given the increased sensitivity of abaxial surfaces relative to adaxial in dorsiventrally

asymmetric leaves (Sun et al., 1996; Sun and Nishio, 2001). Although the synthesis of

anthocyanin in abaxial cells in response to high light stress has been reported (Barker et al.,

1997; Sherwin and Farrant, 1998; Pietrini et al., 2002; Hughes et al., 2005), and

development of photostability has been shown to occur more quickly in seedlings with

abaxial anthocyanin (Drumm-Herrel and Mohr, 1985), no studies have directly tested the

prospect that these pigments decrease susceptibility to photoinhibition through light

attenuation in abaxial cells.

Previous studies on abaxial anthocyanin have focused almost exclusively on

understory species adapted to low light environments, where red and purple abaxial

surfaces are also a common morphological character (Richards 1952; Lee et al., 1979; Lee

and Graham, 1986; Lee and Collins, 2001). Under these conditions it has been suggested

that abaxial anthocyanin functions to back-scatter red wavelengths that enter the leaf from

the adaxial surface, thereby increasing light capture efficiency in low-intensity and poor-

light quality environments (Lee et al., 1979). However, this interpretation has not been

well-supported, and seems insufficient to explain the appearance of anthocyanin in abaxial

cells of leaves experiencing high light stress (where an increase in light capture would

likely exacerbate photoinhibition). Under such conditions, it is more likely that

anthocyanins are functioning as light attenuators to reduce photoinhibition of abaxial cells.

To determine whether abaxial anthocyanin functions to decrease susceptibility to

photoinhibition similarly to adaxial anthocyanin, we take advantage of the phenotypic

plasticity found in the evergreen understory herb Galax urceolata (Poir.) Brummitt for

synthesizing anthocyanin on either or both leaf surfaces receiving moderately high levels of

27
irradiance (i.e. > 750 µmol · m-2 · s-1) during the winter. In its natural environment, G.

urceolata leaves are typically oriented with adaxial surfaces facing canopy gaps, with leaf

angles ranging from 0° to 90° (most commonly between 40° and 60°). Although highest

irradiances are typically incident on adaxial surfaces (resulting in adaxial anthocyanin),

abaxial surfaces of leaves with more vertical angles often experience relatively high

irradiance from snow albedo and direct and diffuse light, resulting in anthocyanin synthesis

in abaxial surfaces as well. Less frequently, leaf litter or debris will cause leaves to be

entirely inverted, and only abaxial surfaces will appear red. By comparing the optical

properties and ecophysiology of leaves with and without adaxial and abaxial anthocyanin,

we demonstrate that anthocyanin located within abaxial tissues attenuates light in the same

manner as anthocyanin located within adaxial cells, effectively reducing photoinhibition of

abaxial leaf tissues vulnerable to high light stress.

28
Materials and Methods

To compare optical profiles of abaxially- and adaxially-anthocyanic leaves, spectral

scans were derived for leaves with the following pigment patterns: entirely green (green

adaxial and green abaxial), bicolored (either green adaxial with red abaxial, or red adaxial

with green abaxial), or entirely red (red adaxial, red abaxial) (Fig. 1). Three leaves of each

type were removed from the field and stored under cold, moist conditions until analysis

later that day. Spectral scans for reflectance and transmittance of photosynthetically active

radiation (PAR), representing 400-700 nm, of both adaxial and abaxial surfaces were

derived using a Li-Cor 1800 spectroradiometer with external integrating sphere (Li-Cor,

Inc., Lincoln, Nebraska, USA). Measurements were taken at 1-nm intervals. Absorptance

was calculated as (1 – reflectance – transmittance). The area tested was marked, and leaf

thickness and pigment concentrations were subsequently quantified. Leaves were all first

year leaves obtained from a wooded area in Jonas Ridge, North Carolina, USA (35°57’20”

N, 81°53’55” W) in February 2006.

Leaf thickness was measured using digital calipers with 0.01 mm precision.

Chlorophylls were extracted by placing three 0.33 cm2 leaf discs in 3 mL N,N’ -

dimethylformamide in the dark for 24 h. Concentrations were determined using the

equations described by Porra (2002). Anthocyanins were extracted by placing three discs

in 3 mL 6M HCl:H2O:MeOH (7:23:70) in the dark at 4oC for 24 h. Following extraction, 1

mL of the solution was removed, and to it 1 mL water and 1mL of chloroform were added

to separate anthocyanins (insoluble in chloroform) from chlorophylls. The solutions were

centrifuged for 15 minutes at 2630 g and 1 mL of the top layer (containing anthocyanins)

was used for analysis. Anthocyanin concentration was determined as A530, using an

29
extinction coefficient of 30 000 l mol-1 cm-1 (Murray and Hackett, 1991). Prior to

extraction, all leaf discs were submerged in liquid nitrogen to infiltrate cuticular waxes and

disrupt cell membranes.

Chlorophyll a and b concentrations, ratios, total chlorophyll content, and leaf

thickness of the four leaf groups were compared using a single factor ANOVA with Tukey-

Kramer test for means difference, using JMP statistical software (SAS Institute, Cary, NC)

(Zar, 1999). Anthocyanin concentrations were compared using a single factor ANOVA

with planned contrast. Completely green leaves were predicted a priori to exhibit

significantly less anthocyanin than all other groups, and completely red leaves were

predicted to exhibit significantly more.

For microscopic imaging, leaves were sectioned into 50-100 µm sections using a

vibratome. Sections were mounted on a Zeiss Axioplan upright microscope (Carl Zeiss

Inc., Thornwood, NY), viewed using under differential interference contrast (DIC)

microscopy, and images were captured using a Hamamatsu C5810 three-chip cooled color

CCD camera (Hamamatsu Photonics; Hamamatsu City, Japan). Photographs of leaf

sections were rotated and adjusted for brightness and image sharpness using Adobe

Photoshop CS (Adobe Systems, San Jose, California).

Another set of G. urceolata leaves was used to compare maximum light capture

efficiency (Fv/Fm) of the leaf types during and following exposure to photoinhibitory

conditions. Leaves were derived from the same plots described above, also in February.

Fluorescence measurements were taken using a Handy-PEA 1000 fluorescence analyzer

(Hansatech Institute, Cambridge, UK), emitting a two second, 3 mmol · m-2 · s-1 saturating

pulse. Prior to measurement, plants were dark-adapted for 30 min using Handy-PEA leaf

30
clips. Five leaves with red adaxial surfaces and five leaves with green adaxial surfaces

(both having green abaxial surfaces) were used to test adaxial response to high light stress.

Abaxial surfaces were tested in a separate experiment using five leaves for each of three

phenotypes: green adaxial, green abaxial; red adaxial, red abaxial; red adaxial, green

abaxial. Experiments differed only in the surface of the leaf exposed to the light source.

Because results from abaxial experiments showed no effect of anthocyanin in the non-

illuminated surface on photoprotection, it did not seem necessary to test all four

combinations of anthocyanic layers in this study.

Leaves were collected from the field, and petioles were re-cut underwater and

remained submerged until experimentation. To equalize starting Fv/Fm values, all leaves

were placed in low stress environments for four days until no significant difference

between Fv/Fm values of leaf groups was observed (for detailed description of why field

Fv/Fm values of red and green leaves differ and why standardization of Fv/Fm is necessary,

see Hughes et al., 2005). During this time, leaves with anthocyanic surfaces were kept at

constant temperature (18°C) and exposed to 10 h of low light (175 μmol · m-2 · s-1),

provided by a 1000 W metal halide bulb equipped with water bath and neutral density

shade cloth to obtain desired PPFDs, to allow Fv/Fm to recover to values similar to those of

green leaves. Green leaves were placed under similar light conditions, but exposed to field

temperatures (−10 to 15°C) to prevent Fv/Fm values from exceeding 0.6-0.7 (which red

leaves could not recover to during the winter). Once values of Fv/Fm were no longer

significantly different between the leaf groups, all leaves were placed in the outdoor,

protected enclosure overnight. At 7 am the following morning, leaves were exposed to a

high stress treatment, consisting of high light (1150 µmol · m-2 · s-1 provided by a 1000 W

31
metal halide bulb) and low temperatures (circulating outside air, with daily temperatures

ranging from 0 to 10oC during the day, and −15 to 0oC during the night). High light stress

was applied for 10 h per day over 3 consecutive days. On the fourth day, leaves were

transferred into a low stress environment (same conditions as those described above to pre-

treat red leaves) until Fv/Fm recovered to near starting values. Fluorescence readings were

taken at 5 pm each day throughout the experiment. A repeated measures MANOVA was

used to compare fluorescence between leaf types during the high light treatments.

32
Results

Levels of chlorophyll a and b, total chlorophyll content, chlorophyll a:b, and leaf

thickness did not significantly differ between the four leaf phenotypes in G. urceolata (p=

0.96, 0.85, 0.85, 0.27, and 0.26 respectively) (Table 1). Leaves with anthocyanin occurring

on both adaxial and abaxial surfaces had significantly higher anthocyanin content than the

other 3 leaf types (p< 0.01), exhibiting 2-fold higher anthocyanin concentration than

bicolored leaves, and >7-fold higher concentration than entirely green leaves (which

contained virtually no anthocyanin). Adaxially and abaxially-bicolored leaves did not

significantly differ in anthocyanin concentration (p= 0.86), but did contain significantly

more anthocyanin than green leaves (p= 0.037) (Table 1).

Leaves with anthocyanin in both surfaces absorbed the most blue-green to yellow

(500-600 nm) light of the four leaf types, absorbing up to 20% more of the green

wavelengths than entirely green leaves, and 2-3% more than bicolored leaves (Figs. 2A and

B). Absorption spectra of red and green bicolored leaves were similar to those of entirely

red leaves when light was shone on the anthocyanic surface, though absorption was

reduced by roughly 10% when the acyanic surface was illuminated. However, absorptance

of green wavelengths was still much higher (by up to 10%) than entirely green leaves. The

increased absorptance of green light by anthocyanic tissues resulted in a dramatic decrease

in transmittance of green wavelengths for anthocyanic leaves relative to entirely green

leaves (Figs. 2C and D), with entirely red leaves reducing transmittance of blue-green light

(500-550 nm) to 0%, and bicolored leaves to 2-3% (10% lower than green leaves). The

increased absorptance of green light by anthocyanin also corresponded to a decrease in

reflectance of these wavelengths when the red surface was illuminated (by up to 10%),

33
though reflectance was not affected when light was incident on the green surface (Figs. 2E

and F). In all leaves containing anthocyanin, roughly 1% more incident red light (600-675

nm) was reflected from the leaf surface compared to entirely green leaves, regardless of

which surface was illuminated (acyanic or anthocyanic).

A comparison of spectral scans of abaxial and adaxial surfaces showed that leaves

absorbed more light (5-7% between 450 and 600 nm wavelengths) when light was incident

upon the adaxial surface, irregardless of pigmentation (Figs. 2A and B). Furthermore,

leaves reflected more light of all wavelengths when the light was incident on the abaxial

surface (Figs. 2D and E). However, transmittance across the leaf was not affected by the

directionality of the light source (Figs. 2C and D).

Green leaf Fv/Fm declined by 86% compared to only 55% for red leaves following

exposure to photoinhibitory conditions (Fig. 3A), a difference which was highly significant

(p<0.0001). This trend was also observed for abaxial surfaces (Fig. 3B, p<0.001), where

red undersides exhibited significantly less of a decline in Fv/Fm than green undersides

(regardless of whether the opposite surface was red or green). Photoinhibition of red

abaxial cells was approximately 10% higher than that of red adaxial cells, though patterns

of photoinhibition between all green surfaces (adaxial and abaxial) were similar. Recovery

from photoinhibition also appeared to be affected by anthocyanin, as Fv/Fm values of red

adaxial surfaces recovered to near-starting values after 1 d, while green adaxial surfaces

required 5 d. This trend was also observed with red abaxial surfaces compared to green.

34
Discussion

Analyses of leaf pigments and thicknesses (Table 1) showed that the leaf

phenotypes measured here did not significantly differ in chlorophyll content or leaf

thickness, only anthocyanin concentration. Therefore, any deviations in optical properties

between leaf types were most likely due to the effect of anthocyanin.

Consistent with known spectral characteristics of anthocyanin, spectral scans

indicated that anthocyanins affected leaf optical properties most apparently through their

attenuation of blue-green to yellow (500-600 nm) light (Fig. 2), with peak absorptance

occurring between 530-550 nm. When light was incident on red surfaces, absorptance of

these wavelengths increased by up to 20%, regardless of whether that surface was abaxial

or adaxial, or whether the opposite surface contained anthocyanin or not. This suggests

that abaxial and adaxial anthocyanic layers may function independently to protect adjacent

mesophyll cells, or in concert to protect the leaf entire. Increased absorptance of blue-

green to yellow light also corresponded with decreased transmittance (Figs. 2C and D) and

reflectance (Figs. 2E and F) of these wavelengths. Because absorptance of blue-green light

by anthocyanin has been shown to protect leaves from photoinhibition (Feild et al., 2001;

Hughes et al., 2005), these data support the idea that anthocyanins on either leaf surface

confer photoprotection.

Because leaves with high anthocyanin concentrations appear red, the

photoprotective effect of anthocyanin may be misattributed to an increase in reflectance of

red light. Because red light is preferentially absorbed by chlorophyll, increasing

reflectance of these wavelengths would indeed be effective in curtailing photoinhibition.

However, as show in Figures 2E and F, the presence of anthocyanin does not necessarily

35
increase the percentage of red light reflected by the leaf. Instead, leaves appear red due to

the diminished reflectance of other wavelengths (primarily green and blue). As a result, the

reflectance peak shifts from green to red as leaves accumulate anthocyanin, though the

magnitude of reflectance of red wavelengths does not markedly change (i.e. greater than

1%). In addition, previous work found no difference in photoinhibition between red and

green G. urceolata leaves illuminated with red light (Hughes et al., 2005). Therefore, any

apparent increase in reflectance of red wavelengths afforded by anthocyanin does not

convey a photoprotective effect in this species.

When the green surfaces of bicolored leaves were illuminated, it was found that

anthocyanins were still able to intercept up to 10% of green wavelengths that would

otherwise have been transmitted through the leaf (Fig. 2A and B). Since an increase in

sunlight absorptance often translates into an increase in leaf temperature, it has been

proposed in previous studies that abaxial pigment layers may function to elevate leaf

temperature (Smith, 1909; Lee et al., 1979; Lee et al., 1987). Although this function would

seem adaptive in plants susceptible to low temperature photoinhibition, several studies of

abaxially anthocyanic plants (including G. urceolata) have found no difference in leaf

temperature between anthocyanic and acyanic leaves (Lee et al., 1979; Lee et al., 1987;

Hughes, 2004).

The effects of anthocyanin on maximum photosystem II efficiency (Fv/Fm) during

photoinhibitory conditions are shown in Figure 3. Leaves with red surfaces appeared less

susceptible to low temperature photoinhibition (as evidenced by higher Fv/Fm) during the

high stress treatment than leaves with green surfaces, regardless of whether that surface

was adaxial (3A) or abaxial (3B). Furthermore, anthocyanic surfaces recovered to starting

36
Fv/Fm values more quickly than acyanic surfaces, suggesting a lesser degree of

photodamage had been incurred. In a previous study on G. urceolata by the authors

(Hughes et al., 2005), it was further shown that photoinhibition of red and green adaxial

surfaces were similar under red light (which anthocyanins do not absorb), but under green

light (which anthocyanins absorb strongly) red surfaces were significantly less

photoinhibited than green. Therefore, we can presume in the case of abaxial anthocyanins

as well, that any difference in Fv/Fm is due to the presence of anthocyanin, and not, for

example, a difference in the pre-treatments (which would have been evident under both red

and green light conditions). Finally, it was also observed that the presence of anthocyanin

in the non-illuminated surface did not appear to have an effect on Fv/Fm (3B), again

suggesting an independent function of these layers on photoprotection of their respective

surface.

An additional trend that is apparent from these data is the increased susceptibility of

red abaxial cells to photoinhibition compared to red adaxial cells. This is most likely due to

lower anthocyanin content in abaxial relative to adaxial cells of leaves tested in this

experiment (data not shown), as natural leaf angles of this species generally cause adaxial

surfaces to receive the greatest incoming radiation. This is most likely due to lower

anthocyanin content in abaxial relative to adaxial cells (data not shown) due to less intense

incident PAR (natural leaf angles of this species cause adaxial surfaces receive the greatest

incoming radiation). Another trend worth noting is that the protection afforded by

anthocyanin appears to be independent of the presence of anthocyanin on the opposite

surface (3B), again suggesting that leaf surfaces function independently with regards to

protecting tissues from light incident on that surface.

37
 Given the above findings, which support the idea that abaxial anthocyanins function

in light attenuation, what might be the function of abaxial anthocyanins in leaves of deeply

shaded understory plants (where abaxial red coloration is most common)? As mentioned

previously, although no studies have been able to provide a well-supported explanation, it

is possible that abaxial anthocyanins are acting as light-attenuators in shade plants as well.

For example, it is possible that abaxial anthocyanin protects the mesophyll from high

ambient light (either diffuse or caused by sunflecks) striking the extremely-dark adapted

abaxial surfaces. Another possibility, based on current research by the authors on Begonia

spp., is that abaxial anthocyanins attenuate internally-scattered green light (entering the leaf

from the adaxial surface), thereby buffering internal light levels during high-intensity

sunflecks. This function is consistent with the findings of Gould et al. (1995), where

abaxially-red tropical understory plants had significantly higher Fv/Fm than abaxially-green

conspecifics.

In conclusion, the results reported here for Galax urceolata support the idea that

anthocyanins located in either adaxial or abaxial surfaces may confer a photoprotective

advantage in tissues exposed to photoinhibitory conditions by effectively intercepting PAR

incident on the respective surface. Moreover, the abaxial/adaxial anthocyanic plasticity

observed in G. urceolata and numerous other species may be an important adaptation in

photoinhibitory environments and/or during light-sensitive developmental stages where

leaf orientation and/or substrate albedo are variable.

38
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LEE D. W., AND T. M. COLLINS. 2001. Phylogenetic and ontogenetic influences on the
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MANETAS Y., Y. PETROPOULOU, G. K. PSARAS, AND A. DRINIA. 2003. Exposed

red (anthocyanic) leaves of Quercus coccifera display shade characteristics. Functional
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MURRAY J. R., AND W. P. HACKETT. 1991. Dihydroflavonol reductase activity in

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antioxidants? Functional Plant Biology 30: 865–873.

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in the illuminated surface of maize leaves enhances protection from photo-inhibitory risks
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SHERWIN H. W., AND J. M. FARRANT. 1998. Protection mechanisms against excess

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41
Table 2.1

Pigment content and leaf thickness in the four leaf types of Galax urceolata tested in this

study. Values represent means of 3 samples (±SE) for chlorophyll a (Chl a), chlorophyll b

(Chl b), total chlorophyll (Total Chl), ratio of chlorophyll a to chlorophyll b (Chl a/b),

anthocyanin, and leaf thickness. Row headings describe the coloration of the

adaxial/abaxial surfaces. Green surfaces contain no visible anthocyanin, red surfaces

contain visible anthocyanin.

42
Table 2.1

Chl a Chl b Total Chl Chl a/b Anthocyanin Leaf thickness

(µg/cm ) (µg/cm ) (µg/cm2)
2 2
(µmol/cm2) (mm)
Green/Green 35.9 a 13.5 a 49.4 a 2.64 a 0.0622 a 0.223 a
(3.8) (0.90) (4.2) (0.074) (0.062) (0.007)
Green/Red 35.4 a 15.0 a 50.4 a 2.36 a 11.2 b 0.273 a
(3.3) (0.93) (4.2) (0.099) (5.9) (0.022)
a a a a b
Red/Green 36.5 14.9 51.4 2.48 12.2 0.257 a
(4.2) (2.3) (6.4) (0.12) (2.8) (0.0033)
a a a a c
Red/Red 34.2 14.5 48.6 2.36 23.7 0.273 a
(0.88) (0.21) (0.68) (0.16) (3.8) (0.014)
Note: Means within a column followed by different letters are different at p< 0.05.

43
Figure 2.1

Cross sections of leaves exhibiting the range of spatial distribution of anthocyanin seen in

winter leaves of the evergreen herb, Galax urceolata. Depicted here are leaves with: (A)

Acyanic adaxial and abaxial, (B) Anthocyanic adaxial and acyanic abaxial, (C) Acyanic

adaxial and anthocyanic abaxial, and (D) Anthocyanic adaxial and abaxial surfaces. In all

pictures the adaxial surface is facing upwards.

44
Figure 2.1

45
Figure 2.2

Spectral scans of Galax urceolata leaves exhibiting four combinations of pigmented

surfaces: green adaxial and green abaxial (solid line), green adaxial and red abaxial (dotted

line), red adaxial and green abaxial (dashed line), and red adaxial and red abaxial (dashed

and dotted line). Graphs in left column represent (A) Absorptance, (C) Transmittance, and

(E) Reflectance curves of the leaves when light was shone on the lower surface; graphs in

the right column represent (B) Absorptance, (D) Transmittance, and (F) Reflectance

curves of the leaves when light was shone on the upper surface. Lines are means of three

leaves, and the color (black or gray) indicates the color of the surface that received

experimental illumination; black represents green surfaces, gray represents red surfaces.

46
Figure 2.2

47
Figure 2.3

Recovery of maximum PSII efficiency (Fv/Fm) for red and green Galax urceolata leaves.

In A, light was shone on adaxial surfaces only; in B, only abaxial surfaces were illuminated.

White areas represent high stress treatments and gray areas represent recovery periods.

Closed circles represent leaves with green adaxial and abaxial surfaces; open circles, leaves

with red adaxial and green abaxial surfaces; open triangles, leaves with red adaxial and

abaxial surfaces. Individual values are means ± SE of five replicates. Graph A and some

data from Graph B are from Hughes et al., (2005).

48
Figure 2.3

49
 CHAPTER III

OPTICAL EFFECTS OF ABAXIAL ANTHOCYANIN ON ABSORPTION OF RED

WAVELENGTHS BY UNDERSTORY SPECIES: REVISITING THE BACK-SCATTER

HYPOTHESIS.

Nicole M. Hughes, Thomas C. Vogelmann, and William K. Smith

The following manuscript was published in Journal of Experimental Botany, volume 59,

pages 3435-3442, 2008, and is reprinted with permission. Stylistic variations are due to the

requirements of the journal. N.M. Hughes performed the laboratory and field work, data

analysis, and prepared the manuscript. T.C. Vogelmann provided ideas, equipment, and

technical assistance, W.K. Smith provided equipment, logistical support, and editorial

assistance.

50
Abstract

Red/purple coloration of lower (abaxial) leaf surfaces is commonly observed in

deeply-shaded understory plants, especially in the tropics. However, the functional

significance of red abaxial coloration, including its role in photosynthetic adaptation,

remains unclear. The objective of this study was to test the back-scatter hypothesis for

abaxial leaf coloration, which posits that red pigments internally reflect/scatter red light

transmitted through the upper leaf surface, thereby enhancing photon capture by the

mesophyll in light-limited environments. Abaxially red/non-red variegated leaves of

Begonia heracleifolia (L.) were used to compare reflectance spectra and chlorophyll

fluorescence profiles of abaxially anthocyanic (red) and acyanic (non-red) tissues under red

light. Photosynthetic gas exchange in response to red light was also compared for abaxially

red/non-red leaf sections. Results did not support a back-scattering function, as

anthocyanic leaf surfaces were not more reflective of red light than acyanic surfaces.

Anthocyanic tissues also did not exhibit increases in mesophyll absorptance of red light, or

increased photosynthetic gas exchange under red light at any intensity, relative to acyanic

tissues. These results suggest that abaxial anthocyanins do not significantly enhance

absorption of red light in the species tested, and alternative functions are discussed.

51
Introduction

Anthocyanins are vacuolar pigments commonly responsible for red, purple, and

blue coloration of plant tissues. They can be found in plant species across a broad range of

habitats, and are especially common in understory plants of the tropics (for species surveys

see Forsyth and Simmonds, 1954; Lee and Collins, 2001; Dominy et al., 2002). Among

tropical species, leaf coloration may be transient—occurring in upper (adaxial) and/or

lower (abaxial) cell layers during development and senescence or, alternatively, coloration

may be permanent (Lee and Collins, 2001). In permanently pigmented leaves of the tropics,

anthocyanins are most commonly abaxial (Lee et al., 1979; Lee and Collins, 2001).

Despite the widespread distribution of abaxial coloration among tropical taxa, very little is

known regarding the functional significance of this trait. Previous studies on the

ecophysiology of foliar anthocyanin have focused almost exclusively on plants with

pigmentation in the upper (adaxial) surface of leaves. In high-light environments, for

example, it has been demonstrated that adaxial pigmentation may function in light-

attenuation, protecting underlying cells from photoinhibition through absorption of high

energy blue-green wavelengths (Gould et al., 2002; for reviews see Chalker-Scott, 1999,

Close and Beadle, 2003, and Gould, 2004). Abaxial anthocyanins have also been

implicated to function in photoprotection in plants where exposed abaxial leaf surfaces are

vulnerable to high-incident light (Drumm-Herrel and Mohr, 1985; Sherwin and Farrant,

1998; Hughes and Smith, 2007). However, several studies have also yielded results that do

not support a photoprotective function (e.g. Burger and Edwards, 1996; Dodd et al., 1998;

Lee et al., 2003; and Kyparissis et al., 2007). In addition to functioning in light attenuation,

anthocyanins in upper cell layers have also been suggested to function in insect avoidance

52
and/or deterrence (Stone, 1966; Hamilton and Brown, 2001; Archetti and Brown, 2004;

Lev-Yadun et al., 2004; Karageorgou and Manetas, 2006), act as a fungicide (Coley and

Aide, 1989), function as a carbon sink (Arnold et al., 2004), or as antioxidants during

periods of high photo-oxidative stress (Neill et al., 2002; Neill and Gould, 2003). However,

none of these hypotheses seem appropriate for explaining the occurrence of anthocyanin in

the abaxial cells of understory species.

Smith (1909) proposed the first functional explanation for abaxial coloration in

understory plants, suggesting that anthocyanin pigments function to elevate leaf

temperatures by increasing sunlight absorption, possibly increasing transpiration, nutrient

uptake, metabolism, and growth rates in understory plants. The idea that anthocyanins

increase leaf temperature has not received experimental support, as field measurements

have shown no differences in leaf temperature between abaxially anthocyanic and acyanic

leaves (Lee et al., 1979; Lee et al., 1987; Gould et al. 1995). Lee et al. (1979) observed

that the abaxial leaf surfaces of some abaxially anthocyanic species appeared to reflect

more red light than green surfaces. This observation led to the idea that anthocyanin

pigments near the lower epidermis may function to reflect adaxially-transmitted red light

back into the mesophyll, possibly maximizing capture of red photons in environments

where light is limiting. This “back-scatter” hypothesis has yet to receive experimental

validation beyond these initial observations, though it is still frequently invoked to explain

abaxial coloration in understory plants. In the current study, we employed a cultivated

variety of Begonia heracleifolia (L.) with abaxial anthocyanic/acyanic variegation (Fig. 1),

along with recently-developed, non-destructive optical techniques for measuring light

absorbance within leaf tissues, to test the back-scatter hypothesis. We also compared the

53
light response of photosynthesis under red light for abaxially-anthocyanic and -acyanic leaf

sections to evaluate whether abaxial anthocyanins confer a significant photosynthetic

advantage in leaf tissues.

54
Materials and methods

Plant material

Begonia heracleifolia plants were grown in shaded greenhouse conditions with

periodic exposure to lower intensity, short-lived sunflecks to longer lasting, more intense

sun-patches (Smith et al., 1989). Photosynthetic photon flux densities (PPFDs) in the

shade averaged ca.50 μmol m-2 s-1, while irradiance during sunflecks and sun-patches

ranged from ca. 150 - 1500 μmol m-2 s-1. In all measurements comparing abaxially red and

non-red tissues, adjacent variegated sections of individual leaves were used.

Leaf sections for light microscopy were cut from fresh leaves using a razor blade,

and mounted on a Zeiss Axioplan upright microscope (Carl Zeiss Inc., Thornwood, NY,

USA). Sections were viewed using differential interference contrast (DIC) microscopy,

and images were captured using a Hamamatsu C5810 three-chip cooled color CCD camera

(Hamamatsu Photonics, Hamamatsu City, Japan). Photographs of leaf sections were

rotated and adjusted for brightness and image sharpness using Adobe Photoshop CS

(Adobe Systems, San Jose, CA, USA).

Spectral scans

Five leaves were removed from individual plants, kept under cool, moist conditions,

and analyzed within 8 hrs using a Li-Cor 1800 spectroradiometer with external integrating

sphere (Li-Cor, Inc., Lincoln, Nebraska, USA). Measurements were taken at 2 nm intervals,

and absorbance was calculated as (1 – reflectance – transmittance). Spectral data were

compared by calculating 95% confidence intervals for five replicates; areas of error-bar

overlap were considered not considered to be significantly different. For ambient sunlight

55
measurements, the spectroradiometer was positioned horizontally on a flat surface on a

clear, sunny day in Winston-Salem, NC, USA. For transmission scans of the red optical

filter used in photosynthesis experiments (transmittance spectrum shown in Figure 2)

(Schott Glass, Grünenplan, Germany), the filter was placed in the water bath beneath a

1000W metal halide bulb, and the spectroradiometer was placed in at the same height

below the bulb as the cuvette used in experimentation.

Pigments

Chlorophyll was extracted from the same leaves for which spectral curves had been

measured. Chlorophylls were extracted by removing three 0.33 cm2 leaf discs from either

abaxially anthocyanic or acyanic leaf sections, and extracting the tissues overnight in 3 mL

N, N’-dimethylformamide. Chlorophyll a and b concentrations were determined

spectrophotometrically using the equations described by Porra (2002). Chlorophyll a/b

ratios were calculated to assess relative emphasis on light capture versus processing for red

and green sections, as higher chlorophyll a/b ratios reflect both an increase in core

(exclusively chl a) relative to light-harvesting (both chl a and b) complexes, and/or higher

ratios of Photosystem I (4 / 1 ratio of chl a/b) to Photosystem II (1.2 / 1 ratio) (Cui et al.,

1991; Demmig-Adams, 1998; Hopkins and Hüner, 2004). Pigment concentrations of

adjacent red and green sections were compared using a one-tailed, paired t-test, with p <

0.05 used to determine significance.

56
Chlorophyll fluorescence profiles

Spatial distribution of light absorption within leaf tissues was determined as

described in Vogelmann and Han (2000). Briefly, cm2 sections were cut from six

different variegated leaves, such that one half of each section contained abaxial

anthocyanin and the other half did not (Fig. 1D). The sections were then mounted

on an inverted microscope and upper (adaxial) surfaces were irradiated using filtered blue,

green, and red light (intensity of the beam was ca. 500 μmol m-2 s-1 for blue, 300 for green,

and 200 for red light). Resulting leaf chlorophyll fluorescence was filtered through a

narrow band-pass filter (680 nm, half band width = 16nm, Corion Corp., Holliston, MA),

and captured using a cryogenically cooled charged-coupled device (CCD) camera (CH270

camera head, CF200A 16/40 camera electronics unit, AT200 controller board, 35-mm

shutter; all from Photometrics, Tuscon, AZ). Intensity of chlorophyll fluorescence was

used to quantify light absorbance by chlorophylls (with highest intensities corresponding

with greatest absorbance). Chlorophyll fluorescence according to leaf depth for each image

was quantified using Image Pro Plus software (version 4.1, Media Cybernetics, Silver

Springs, MD). It is important to note that chlorophyll fluorescence in all cases was emitted

exclusively by cells containing chlorophyll (i.e. the mesophyll), though most of this light

appeared to scatter in the adjacent clear epidermal lens cells (as described in Brodersen and

Vogelmann, 2007). Mean percent chlorophyll fluorescence for abaxially anthocyanic and

acyanic tissues was plotted versus leaf depth (from adaxial surface) and 95% confidence

intervals were calculated. Areas of confidence interval overlap were not significantly

different.

57
Light response of photosynthesis

To further test the idea that abaxial anthocyanins enhance capture of red photons for

use in photosynthesis, photosynthetic light response curves were measured for

abaxially anthocyanic/acyanic leaf sections under red light using a Li-Cor 6400

infra-red gas analyzer with clear-top chamber (Licor, Lincoln, Nebraska, USA).

Light was supplemented by a 1000-W metal halide lamp equipped with a UV filter and

water bath, and irradiance was filtered through a red optical filter (transmittance spectrum

shown in Figure 2) (Schott Glass, Grünenplan, Germany). Leaves were positioned

horizontally, with upper surfaces facing the light source. Because variegated leaf sections

were smaller in area than the leaf chamber, an opaque tape with a 2 cm2 square opening

was used to cover the areas of leaves not being measured on both adaxial and abaxial

surfaces. The tape effectively reduced stomatal gas exchange to negligible amounts (i.e.

<1-2%), as determined by experiments on leaf surfaces entirely covered in tape. Neutral

density shade cloths were used to obtain desired PPFDs, which ranged from 0 – 600 µmol

m-2s-1 (the latter representing roughly the maximum intensity of red light in full sunlight;

Lee and Downum, 1991). Quantum yield of photosynthesis was determined as the slope of

the linear portion of the curve, and compared using a paired t-test for adjacent red and

green sections on the same leaf. Sample size for gas exchange experiments was five leaves.

Light response curves for red and green leaf sections were compared using repeated

measures ANOVA.

58
Results and Discussion

Optical effects of abaxial anthocyanin

In contrast to the findings of Lee et al. (1979), red (anthocyanic) abaxial surfaces

of B. heracleifolia were not more reflective of red wavelengths than green (acyanic)

abaxial surfaces, but rather, acyanic leaf surfaces were significantly more reflective of all

wavelengths than anthocyanic (Figs. 3A, B). This was likely due both to the presence of

anthocyanin and higher chlorophyll concentrations in anthocyanic tissues (both of which

increase leaf absorptance, decreasing reflectance) (Table 1, Fig. 3). Increased chlorophyll

content has been observed in several anthocyanic species relative to acyanic phenotypes

(e.g. Gould et al., 1995; Manetas et al., 2003; Hughes et al., 2005), and has been attributed

to shading effects of anthocyanin (discussed in further detail below). Therefore, although

not ideal for quantifying the optical effects of anthocyanin alone (due to confounding

effects of chlorophyll), these scans are likely representative of the realistic effects of

anthocyanin on leaf optics in vivo. Furthermore, important information relevant to the

back-scatter question can still be garnered from these scans. For example, absorbance

spectra showed high absorbance efficiency (~90%) of red light (650-700 nm) in both tissue

types, regardless of the presence of anthocyanin, with less than 5% being lost via

transmittance through the leaf lamina (Fig. 3). This degree of efficiency in photon

absorption is not unique to Begonia, as shade plants are generally known to increase

allocation of resources towards light harvesting capacity, resulting in relatively high

quantum yield of photosynthesis under low irradiances (Larcher, 2003). If abaxial surfaces

of understory plants have evolved to maximize light capture via red-light back-scattering,

they seem to have done so for a narrow waveband that is already being (relatively)

59
efficiently captured. Instead, it would seem that a white reflective layer (which is fairly

common in the plant kingdom) would be of greater benefit than one that reflects red alone,

as this would enhance capture of wavelengths more readily lost through transmittance, such

as green and yellow (Woolley, 1971; Lin and Ehleringer, 1983; DeLucia et al., 1986).

Comparing these scans to those published for other species, the lack of increased

reflectance of red wavelengths in anthocyanic versus acyanic tissues has also been reported

in other studies (Gould et al., 1995; Hughes and Smith, 2007). Slight (1-2%) increases in

reflectance of red wavelengths have also been reported for anthocyanic tissues relative to

acyanic (Burger and Edwards, 1996; Liakopoulos et al., 2006), though differences on the

order of the magnitude of those reported in Lee et al. (1979) (i.e. 20% higher reflectance of

red wavelengths by red tissues) have not been reported. It is possible that repeated

analyses of the species tested by Lee et al. (1979) using newer equipment may yield

different results (D.W. Lee, personal communication).

Effects of abaxial anthocyanin on light capture and photosynthesis

Chlorophyll fluorescence profiles also did not support a back-scattering function of

the abaxial anthocyanin layer. If back-scattering was occurring, increased fluorescence

should have been observed in anthocyanic tissues relative to acyanic under red light,

especially in the most abaxial mesophyll cells. Instead, chlorophyll fluorescence was

similar at all depths in anthocyanic and acyanic leaf sections under red light, with very little

fluorescence observed in abaxial cells layers in either case (Figs. 1A and 4A). This effect

was even more pronounced under blue light (Fig. 1B), where virtually no fluorescence was

observed to be emitted from abaxial mesophyll cells. This indicates that most of the

60
incident red and blue light was absorbed by chloroplasts in the adaxial mesophyll, and that

very little was transmitted to abaxial cells (consistent with our interpretation of spectral

scans). Because the light levels used here (i.e. 200 μmol m-2s-1 for red light) were

substantially more intense than PPFDs reported for the shade of tropical forest understories

(total PPFDs ranging from ca. 2-45 µmol m 2s-1, Lee, 1987), it is unlikely that ambient red

wavelengths in the field would be sufficient to penetrate mesophyll cells unabsorbed, much

less penetrate them, then strike the anthocyanin layer and be back-scattered. Furthermore,

because understory species photosynthetically saturate at relatively low PPFDs (i.e. 200-

300 μmol m-2s-1; Björkman and Demmig, 1987; Larcher, 2003), irradiance greater than 200

μmol m-2s-1, such as that experienced during longer-lasting, more intense sunflecks or sun-

patches, would likely be sufficient to saturate photosynthesis without the assistance of

back-scattering. Consistent with this interpretation, no significant difference in

photosynthesis between abaxially-red and green leaf sections was observed either under

low intensities of red light (0 – 200 μmol m-2s-1; Fig. 5A), or when higher irradiances were

included (0 – 600 μmol m-2s-1; Fig. 5B) (p = 0.73 and 0.68 respectively), even despite

higher chlorophyll content in red tissues. At higher light intensities, some variation in

photosynthetic gas exchange was observed between leaves (most likely due to differences

in stomatal conductance, not shown), though within-leaf comparisons of adjacent red and

green sections consistently showed nearly identical rates of photosynthesis at all intensities

(e.g. Fig. 5C).

61
Alternative functions of the anthocyanic layer

Although no difference in mesophyll absorptance of red light between abaxially red

and green tissues was observed, significant differences in green-light absorption were

apparent in our study. Spectral scans showed that anthocyanic tissues absorbed

significantly more blue-green light (500-600 nm) than acyanic tissues (Fig. 3E),

corresponding with anthocyanin’s absorbance peak. Perhaps more interestingly,

chlorophyll fluorescence profiles of abaxially-red tissues showed significantly reduced

absorption of green light by mesophyll cells (i.e. reduced chlorophyll fluorescence)

compared to acyanic tissues, with effects being greatest in cells most proximate to the

anthocyanic layer (Figs. 1C and 4C). These results are consistent with an alternative

explanation for abaxial coloration proposed by Gould et al. (1995), which purports that

abaxial anthocyanins in understory plants function in light attenuation (similarly to their

function in adaxial cells) during intermittent exposure to high-intensity sunlight (i.e. sun-

patches). Gould et al. based this hypothesis on the observation that abaxially-anthocyanic

leaves had significantly higher maximum quantum yield efficiency of photosystem II

(Fv/Fm) than acyanic leaves, as well has higher chlorophyll content and higher maximum

photosynthesis. However, no details were presented to explain how abaxial pigments

might protect leaves in this way.

From the chlorophyll fluorescence profiles presented here, we suspect that

attenuation of green wavelengths by anthocyanins may be occurring (1) as adaxially-

incident light passes through the anthocyanic spongy mesophyll (reducing absorbance by

subjacent chloroplasts), and/or (2) when remaining green light reaches anthocyanic-abaxial

lens cells, being absorbed before internal scattering may occur (i.e. the opposite of back-

62
scattering). This photoprotective function may be especially adaptive in shade-adapted

understory plants, which photosynthetically saturate (and thus photoinhibit) at relatively

low irradiances, and yet frequently encounter potentially damaging irradiance via high-

intensity sunflecks and sun-patches. The occurrence of the pigment on the abaxial surface

rather than the adaxial might prevent interference of light absorption by anthocyanin at low

light intensities, providing photoprotection only when intensities are high enough to

penetrate the mesophyll (which would likely correspond with damaging levels of photons).

This photoprotective function is further supported by the chlorophyll data (Table 1). As

shown in previous studies on anthocyanic versus acyanic phenotypes (e.g. Gould et al.,

1995, Manetas et al., 2003; Hughes et al., 2005), abaxially-red tissues measured here had

significantly higher total chlorophyll content, and lower chlorophyll a/b ratios than non-red

tissues (p < 0.01 for both). High chlorophyll content and low a/b ratios generally reflect

increased emphasis on light capture relative to processing—a characteristic associated with

more shade-adapted tissues (Demmig-Adams, 1998). These shade-like qualities make

sense for adaxially-anthocyanic tissues (which are directly shaded by the overlying

anthocyanic layer), and also for abaxially-anthocyanic tissues when abaxial attenuation is

considered. However, it is also possible that this effect may be due to increased shading

due to higher chlorophyll content, though this too might be an indirect result of shading by

anthocyanin (as mentioned above).

Conclusion

In summary, the results reported here clearly do not support a back-scattering

function of abaxial red coloration. Red abaxial surfaces were not more reflective of red

63
light than non-red abaxial surfaces, and the presence of a red abaxial layer did not result in

increased mesophyll absorption of red light, or an increase in photosynthetic gas exchange.

However, these results do appear consistent with a photoprotective function of abaxial

anthocyanin, though a more detailed comparison of photoinhibition of photosynthesis for

abaxially anthocyanic versus acyanic tissues is needed to further substantiate this

explanation.

64
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68
Figure 3.1

Top: adaxial view of Begonia heracleifolia leaf. Bottom: chlorophyll fluorescence in

partially red and green variegated leaf sections with upper (adaxial) surface illuminated. In

all pictures, adaxial surface is facing upwards and anthocyanins are present only in the left

half of each photograph, as shown in (D). Images represent chlorophyll fluorescence under

red (A), blue (B), and green (C) light. Note that, while fluorescence is only emitted by

chlorophyll-containing cells, it may also be apparent via scatter within adjacent clear,

epidermal lens cells (see text). Scale bar represents 0.5 mm.

69
Figure 3.1

70
Figure 3.2

Spectral scans. Solid line represents percent mean leaf reflectance for the red abaxial

surface of Begonia heracleifolia leaves. Dotted line represents the transmission spectra for

the red filter used in photosynthesis measurements. The ambient solar spectrum is

represented by the dashed line.

71
Figure 3.2

72
Figure 3.3.

Optical properties (absorptance, transmittance, and reflectance) of the

adaxial and abaxial surfaces of anthocyanic (black lines) and acyanic (grey lines)

tissues of Begonia heracleifolia. Lines represent means of five leaves;

error bars are 95% confidence intervals. The waveband depicted represents the visible

spectrum, with violet-blue wavelengths depicted between 400-500 nm, green 500-580,

yellow-orange 580-630, and red 630-700 nm.

73
Figure 3.3

74
Figure 3.4

Percent chlorophyll fluorescence versus leaf depth for abaxially

anthocyanic (black lines) and acyanic (grey lines) tissues of Begonia heracleifolia

under red, blue, and green light. Error bars represent 95% confidence intervals for six

replicates.

75
Figure 3.4

76
Figure 3.5

Photosynthetic light response curves of abaxially acyanic/green (open circles, dotted lines)

and anthocyanic/red (closed circles, solid lines) leaf tissues under red light. (A) Linear

portion of the light response curves shown in (B); (B) light response curves from 5 leaves

(with each having a single red and green section tested), and (C), a representative pair of

light response curves derived from the same leaf. Error bars represent SE; n = 5.

77
Figure 3.5

78
Table 3.1

Chlorophyll data for abaxially anthocyanic (red) and acyanic (green) portions of Begonia

heracleifolia leaves. Values are means for 5 replicates (± SD). Means within columns not

followed by the same letter are different at p < 0.05.

79
Table 3.1

Abaxial Chl a Chl b Total Chl Chl a/b

coloration (μg/cm2) (μg/cm2) (μg/cm2)
Red 17.9 a 7.97 a 25.8 a 2.25 a
(0.33) (0.30) (0.39) (0.040)

Green 13.7 b 5.44 b 19.1 b 2.51 b

(1.0) (0.21) (1.5) (0.096)

80
 CHAPTER IV

THE COORDINATION OF ANTHOCYANIN DECLINE AND PHOTOSYNTHETIC

MATURATION IN DEVELOPING LEAVES OF THREE DECIDUOUS TREE SPECIES

Nicole M. Hughes, Christianna B. Morley, and William K. Smith

The following manuscript was published in New Phytologist, volume 175, 675-685, and is

reprinted with permission. Stylistic variations are due to the requirements of the journal.

N.M. Hughes performed the laboratory and field work, data analysis, and prepared the

manuscript, C.B. Morley was involved in early organization of the project and ideas, and

W.K. Smith provided equipment, logistical support, and editorial assistance.

81
Abstract

Juvenile leaves in high-light environments commonly appear red due to

anthocyanin pigments, which play a photoprotective role during light-sensitive ontogenetic

stages. The loss of anthocyanin during leaf development presumably corresponds to a

decreased need for photoprotection, as photosynthetic maturation allows leaves to utilize

higher light levels. However, the relationship between photosynthetic development and

anthocyanin decline has yet to be quantitatively described. In the current study,

anthocyanin concentration was measured against photopigment content, lamina thickness,

anatomical development, and photosynthetic CO2 exchange in developing leaves of three

deciduous-tree species. In all species, anthocyanin disappearance corresponded with

development of ~50% mature photopigment concentrations, ~80% lamina thickness, and

differentiation of the mesophyll into palisade and spongy layers. Photosynthetic gas

exchange correlated positively with leaf thickness and chlorophyll content, and negatively

with anthocyanin concentration. Species with more rapid photosynthetic maturation lost

anthocyanin earliest in development. Chlorophyll a/b ratios increased with leaf age, and

were lower than those of acyanic species, consistent with a shading effect of anthocyanin.

These results suggest that anthocyanin re-assimilation is linked closely with chloroplast and

whole-leaf developmental processes, supporting the idea that anthocyanins protect tissues

until light-processing and carbon-fixation have matured to balance energy capture with

utilization.

82
Introduction

One of the most conspicuous developmental changes observed in juvenile leaves as

they mature is color change, with young leaves on new growth tips of many species first

appearing red, purple, pink, or less commonly blue or white, and becoming greener with

leaf age. The red-to-blue coloration of young leaves is most commonly due to the pigment

anthocyanin, appearing within vacuoles of epidermal and/or mesophyll cells within hours

to days of seedling germination, and then decreasing concomitantly with leaf expansion

and maturation (Choinski et al., 2003; Cai et al., 2005). Anthocyanic coloration of juvenile

leaves is nearly ubiquitous in tree species at equatorial latitudes (for species surveys see

Lee & Collins, 2001 and Dominy et al., 2002), where coloration has been observed to

precede and accompany chlorophyll development in species with delayed greening (Kursar

& Coley, 1992; Cai et al., 2005). Expanding leaves of both woody perennials (for species

survey see Price & Sturgess, 1938) and herbaceous species of the temperate zone (see

studies listed in Chalker-Scott, 1999), also commonly exhibit anthocyanin, but during a

more gradual greening process.

Despite its widespread phylogenetic distribution, the functional significance of

anthocyanin in juvenile leaves remains unresolved. Some hypotheses, such as the elevation

of leaf temperature (Smith, 1909), have been clearly dismissed (Lee et al., 1979; Lee et al.,

1987; Hughes, 2004), while others, including camouflage against herbivory (Stone, 1979),

fungicide (Coley & Aide, 1989) or antioxidant activity (Rice-Evans et al., 1997; Wang et

al., 1997), or a signal indicating the presence of unpalatable phenolics (Hamilton & Brown,

2001) have received mixed support (Coley, 1981, 1983; Kursar & Coley, 1992; Dominy et

al., 2002; Neill et al., 2002; Numata et al., 2004; Karageorgou & Manetas, 2006; Manetas,

83
2006). Perhaps the most compelling current explanation for anthocyanin in developing

tissues is that the pigments act as light-attenuators, protecting underlying cells from high

irradiance through absorption of high energy blue-green (and possibly UV) wavelengths of

the solar spectrum (for reviews see Chalker-Scott, 1999 and Gould, 2004). While this latter

function may not be beneficial to species in deeply-shaded habitats, light-attenuation

should be particularly advantageous to species with leaves that initiate maturation at more

sun-exposed, apical tips.

Immature leaves tend to be especially vulnerable to high-light stress due to a

combination of several factors, including immature chloroplast structure (Pettigrew &

Vaughn, 1998; Choinski et al., 2003), reduced capacity and quantity of photosynthetic

enzymes (Miranda et al., 1981a; Pettigrew & Vaughn, 1998), and limited stomatal and

cellular conductance of CO2 (Miranda et al., 1981b; Choinski et al., 2003). As a result,

young leaves growing under high irradiances tend to photosynthetically saturate, as well as

photoinhibit, under substantially lower sunlight levels than mature leaves (Hoflacher &

Bauer, 1982). It is therefore generally beneficial for light capture to be down-regulated

early in leaf development. Observed strategies to decrease light capture in juvenile leaves

include increased xanthophyll pigments (Krause et al., 1995; Barker et al., 1997), inclined

leaf orientation (Jiang et al., 2006), pubescence (Liakopoulos et al., 2006), decreased

chlorophyll content (Choinski et al., 2003; Cai et al., 2005), and light-attenuating layers

such as anthocyanin (Manetas et al., 2002; Karageorgou & Manetas, 2006; Liakopoulos et

al., 2006). Although the physiological benefits of these processes have been addressed and

supported in several studies, little information exists which quantitatively describes the

coordination of photoprotective downregulation with the maturation of the photosynthetic

84
processes which they are assumed to protect. The objective of the present study was to

quantitatively describe the relationship between anthocyanin diminution and photosynthetic

maturation, including development of leaf structure, photopigment accumulation, and

photosynthetic CO2 exchange in three deciduous tree species common in the temperate

zone.

85
Materials and Methods

Pigment composition, lamina developmental characteristics (i.e. thickness and

anatomy), and photosynthetic CO2 exchange were measured for developing leaves of three

deciduous tree species under natural field conditions. These parameters were related to the

disappearance of visible anthocyanin in order to identify possible functional interactions.

Measurements were made at consecutive apical nodes representing increasing stages of

maturation.

Plant material

Species observed in this study were sweetgum (Liquidambar styraciflua L.,

Hamamelidaceae), red maple (Acer rubrum L., Aceraceae), and Eastern redbud (Cercis

canadensis L., Fabaceae) growing along disturbed high sunlight-exposed roadsides in

Winston-Salem, NC, USA (36° 8' N 80° 13' W). For acyanic juvenile leaf comparison of

chlorophyll a/b values, juvenile leaves of English ivy (Hedera helix L., Araliaceae),

forsythia (Forsythia suspensa Thunb., Oleaceae), and Japanese honeysuckle (Lonicera

japonica Thunb., Caprifoliaceae) growing in the same areas were also included. All of

these species continuously produce new growth throughout the summer, and measurements

were taken between July and early September. Since some variation exists in the number

of apical red leaves per branch of a given species, we used branches exhibiting the most

commonly observed number of red leaves per branch in each study: sweetgum branches

most commonly had two apical red leaves; red maple, three; redbud, four (Figure 1).

Leaves were determined to be without anthocyanin when anthocyanin levels were less than

60 μmol m-2, corresponding to values where anthocyanins were no longer visible with the

86
unaided eye. In all experiments, at least five different trees growing in fully-exposed (i.e.

at least 7 h of full sunlight per day) habitats were sampled, using one branch on the east

side of each tree. All trees were saplings < 3 m in height. The most basal leaves of the

same branch on which juvenile leaves were sampled were used to represent “mature”

leaves. Maturity was assumed only if leaf thickness, photosynthesis, and chlorophyll

contents remained constant compared to younger leaves on the branch. All mature leaves

were also exposed to full irradiance (> 1350 µmol m-2 s-1) during much of the day,

including the time of sampling.

Microscopic sectioning

Branches of each species were removed from plants, re-cut underwater, and kept

hydrated until sectioned into 50-100 µm sections using a Vibratome sectioner. Sections

were mounted on a Zeiss Axioplan upright microscope (Carl Zeiss Inc., Thornwood, NY),

viewed using differential interference contrast (DIC) microscopy, and images were

captured using a Hamamatsu C5810 three-chip cooled color CCD camera (Hamamatsu

Photonics; Hamamatsu City, Japan). Photographs of leaf sections were rotated and

adjusted for brightness and image sharpness using Adobe Photoshop CS (Adobe Systems,

San Jose, CA). The backgrounds of all images were also converted to white using Adobe

Photoshop CS.

Leaf thickness

Six whole branches of each species containing the leaf nodes of interest were cut

from trees between 10 : 00 and 11 : 00 h, re-cut underwater, and remained submerged until

87
analysis within 2 h. During this time, no visible wilting was observed to occur, and leaf

thicknesses were not found to significantly differ from non-excised branches (data not

shown). Three to six pieces (depending on variance) of leaf tissue visibly free of

protruding veins were cut out from along the mid-rib of each leaf and immediately

measured using IP54 electronic micrometer calipers with 1µm precision (Fred V. Fowler

Co., Inc, Newton, MA). Average values were compared to those of microscopic sections

measured using a reticle to assure accuracy. No significant difference was observed, as

microscopic values fell within ranges derived using calipers (data not shown). Mean

thickness of each leaf was divided by the average thickness of fully expanded leaves of that

species to estimate percent-mature leaf thickness for each node.

Pigment quantification

Branches were collected between 10 : 00 and 11 : 00 h, during which time all plants

had been exposed to full sunlight for at least 3 h. A standard hole puncher was used to

excise three 0.33 cm2 leaf discs, which were then placed in 3 mL N,N’ -dimethylformamide

to extract in the dark for 24 h. Chlorophyll concentrations were determined

spectrophotometrically using the equations described by Porra (2002), and total carotenoid

concentrations were calculated using equations described by Wellburn (1994). Chlorophyll

concentrations were reported as a function of both volume (using mean leaf thicknesses

calculated for each node, derived from other branches previously measured) and leaf area.

This was done to determine whether increases in chlorophyll content corresponded to

increased leaf thickness, as opposed to increased intracellular chlorophyll (the former being

evidenced by changes in chlorophyll per unit area, but not volume). From the same leaves,

88
three additional leaf discs were excised and placed in 3 mL 6M HCl:H2O:MeOH (7:23:70)

for 24 h at 4°C to extract anthocyanins. Anthocyanin concentration was measured

spectrophotometrically as A530 – 0.24 A653 using an extinction coefficient of 30 000 l mol-1

cm-1 (Murray & Hackett, 1991). All extracts were analyzed using a Hewlett Packard 8453

UV-VIS spectrophotometer (Hewlett Packard, Palo Alto, CA). Concentrations of

chlorophyll and carotenoid pigments for leaves of each node were divided by average

values of fully expanded leaves to estimate the percentage of leaf pigments present relative

to mature leaves.

Chlorophyll a/b ratios were calculated to assess relative emphasis on light capture

versus processing during maturation, as higher chlorophyll a/b ratios reflect both an

increase in core (exclusively chl a) relative to light-harvesting (both chl a and b) complexes,

and/or higher ratios of Photosystem I (4/1 ratio of chl a/b) to Photosystem II (1.2/1 ratio)

(Cui et al., 1991; Demmig-Adams 1998; Hopkins and Hüner, 2004a).

Carotenoid/chlorophyll ratios were also calculated to compare relative photoprotection by

the xanthophylls through leaf development to photosynthetic maturity.

Photosynthetic gas exchange

A portable photosynthesis system (PP Systems Inc. model TPS-1, Amesbury, MA)

was used to measure leaf conductance and photosynthesis for plants in the field.

Measurements were taken between 9 : 00 and 12 : 00 h on plants under clear-sky ambient

sunlight (>1350 µmol m-2 s-1) after the leaf had been enclosed in the cuvette for at least one

minute and oriented directly towards the sun, to allow for light-saturated photosynthesis.

Measured light response curves of all age-class leaves were light-saturated (85% of mean

89
maximum values) at well below (<48%) full sunlight (data not shown), consistent with the

findings of Hoflacher and Bauer (1982) and Wallace and Dunn (1980). Thus, measured

photosynthesis under full sunlight was assumed to represent light-saturated photosynthesis

(Asat) under ambient CO2. The most basal (non-senescent) leaves on the branch were

measured to represent fully mature leaves. Leaf area was measured using a Delta-T leaf

area measurement system (Delta-T Devices Ltd, Cambridge, UK) and photosynthesis was

expressed on a per unit leaf area (projected) and volume basis. Leaf mass was not used to

express photosynthesis due to potential problems associated with variations in mass per

unit volume (e.g. cell wall development).

Statistics

To compare levels of photosynthetic pigments, leaf thickness, and gas exchange of

youngest nodes lacking visible anthocyanin, measurements for the first non-red leaf node

(node 3 for sweetgum, 4 for red maple, and 5 for redbud) were compared using a single-

factor ANOVA with Tukey-Kramer test for means difference. ANOVA with Tukey-

Kramer test was also used to compare these parameters for fully mature leaves. A Pearson

Product Moment Correlation test was used to examine the relationship between leaf node

and pigment concentrations, pigment ratios, leaf thicknesses, and photosynthesis for

individual leaves (as opposed to means). Sample size (N) was five or greater for all

comparisons, with N-1 degrees of freedom. Significance for all tests was determined at p <

0.05.

90
Results

Leaf anatomy

Anthocyanins were either contained within the epidermis (redbud), or mesophyll

cells (red maple and sweetgum) (Figure 1). Red maple and sweetgum contained

significantly more anthocyanin per unit area than redbud in the youngest two leaf nodes (p

< 0.0001) (Figure 2), likely reflecting differences in mesophyll versus epidermal

pigmentation. In red maple and sweetgum, all mesophyll cells initially exhibited reddening,

although anthocyanins eventually became restricted to palisade cells in both species prior

to their disappearance. While reddening was observed to occur in upper (adaxial)

mesophyll cells of all species, the youngest leaves of redbud exhibited intense reddening in

abaxial epidermal cells, which dissipated as leaves matured. Anthocyanins were also

observed in abaxial epidermal cells surrounding protruding leaf veins in redbud and red

maple, even when other abaxial epidermal and/or mesophyll cells contained little or no

anthocyanin.

In all species, anthocyanin did not disappear until after palisade layer differentiation

had begun or was complete (Figure 1). In sweetgum, double palisade layers occurred in the

youngest, expanded leaves observed (apical node 1), and anthocyanin appeared to be

present both during and after palisade formation. In red maple and redbud, anthocyanins

dissipated concomitantly with differentiation and elongation of the palisade layer at nodes

3 and 4, respectively.

Sweetgum leaves were significantly thicker than the other species during early

development (p = 0.022 for node 1, p = 0.0036 node 2, p < 0.0001 node 3), but only

significantly thicker than redbud in later development through maturity, (p < 0.0001 node 4,

91
p < 0.0001 for mature leaves) (Table 1). Thickness of red maple leaves did not

significantly differ from redbud leaves until node 4, after which they became increasingly

thicker (p < 0.0001 for node 4, p < 0.0001 node 5, p = 0.0002 for mature leaves). At the

time of anthocyanin disappearance, lamina thicknesses relative to mature leaves upon

anthocyanin loss did not differ significantly (p = 0.596) between species, and complete

disappearance had occurred by the time leaves were ~80% mature leaf thickness (Figure 2).

Pigment accumulation

In all three species, leaf chlorophyll concentration significantly increased with node

position when expressed on a leaf area basis (r2 = 0.77, 0.68, 0.48 for sweetgum, red maple,

and redbud, respectively; p < 0.0001 in all cases; Table 1 and Figure 2). In contrast,

chlorophyll concentrations did not significantly correlate with leaf node when expressed on

a leaf volume basis (r2 = 0.00, 0.15, 0.14 and p = 0.91, 0.060, 0.061 for sweetgum, red

maple, and redbud, respectively) (Table 1 and Figure 2). All three species had

microscopically visible anthocyanins until mean values of 44% of total chlorophyll per unit

area (relative to mature leaves) had developed. This percentage did not vary significantly

between species (p = 0.262). For chlorophyll expressed on a leaf volume basis, this

percentage was 58% and also did not differ among species (p = 0.182). Anthocyanins were

retained until roughly 49% of mature-leaf carotenoids (per unit area) had developed in all

three species (p = 0.058).

When absolute pigment concentrations were compared (as illustrated in Table 1), as

opposed to percents of maturation, anthocyanin in sweetgum leaves disappeared

significantly earlier (p < 0.01) with regards to chlorophyll content on a unit volume basis

92
compared to red maple and redbud (which did not differ). This translated into 0.797 kg m-3

for sweetgum, and 1.22 and 1.27 kg m-3 for red maple and redbud. On a unit area basis,

anthocyanin loss also occurred significantly earlier in sweetgum compared to red maple (p

= 0.048), but did not significantly differ from redud. On average, sweetgum leaves lost

anthocyanin by the time 138 mg m-2 chlorophyll had developed, while red maple and red

bud retained anthocyanin until 298 and 159 mg m-2 chlorophyll had developed

(respectively). Carotenoids concentration did not significantly vary between species in this

respect (p = 0.19), as anthocyanins had disappeared by the time mean carotenoid levels

reached 63 mg m-2 in all species.

While leaf chlorophyll a/b ratios decreased from the tip-down in acyanic species

(honeysuckle r2 = 0.416, ivy r2 = 0.330, forsythia r2 = 0.561; p < 0.005 for all species), chl

a/b increased in all three anthocyanic species (r2 = 0.76, 0.56, 0.31 for sweetgum, red

maple, and redbud respectively; p < 0.01 for all species) (Figure 2, Table 1). However, the

range of chlorophyll a/b values varied substantially among the three species. Sweetgum

leaves had the broadest range and lowest chlorophyll a/b ratios of all species, ranging

between 1.5 and 3.0 for the nodes examined. Chlorophyll a/b of red maple ranged from 2.4

to 3.3, and from 3.4 to 3.7 in redbud. Carotenoid/chlorophyll ratios significantly decreased

from the tip-down in all anthocyanic species (r2 = 0.79, 0.62, 0.63 for sweetgum, red maple,

and redbud, respectively; p < 0.001 for all species) (Figure 2, Table 1), but of the acyanic

species, only ivy showed significant declines (r2 = 0.56; p < 0.0001; p = 0.18 and 0.12 for

honeysuckle and forsythia respectively; data not shown). Carotenoid/chlorophyll ratios of

the youngest non-red leaf were similar among the anthocyanic species (~108% values of

mature leaves), though a Tukey-Kramer test indicated car/chl ratios of sweetgum to be

93
significantly higher than those of red maple (p = 0.0031), while other species combinations

did not significantly differ. In all anthocyanic species, ranges of carotenoid chlorophyll

ratios of leaf nodes measured were similar (mean of 140% for the youngest leaf node,

100% for the 2nd non-red leaf node).

Gas exchange

The youngest non-red juvenile leaves of sweetgum (third node) had significantly

higher photosynthesis under saturating light conditions (Asat) for both absolute and percent-

mature values compared to the corresponding values for juvenile leaves of red maple and

redbud (p = 0.0003 for absolute values and 0.0029 for percent mature values per unit area;

p = 0.0018 and 0.002 per unit volume, respectively) (Table 1, Figure 3). Mean Asat (per

unit area) observed for the youngest non-red leaves of sweetgum was 11.5 µmol m-2 s-1

(corresponding to 99% full mature maximum photosynthetic values), while red bud and red

maple averaged 6.5 µmol m-2 s-1 (corresponding to ~60% of mature values). On a per unit

volume basis, Asat for the first non-red leaf of sweetgum was 66.4 mmol m-3 s-1 (130%

mature photosynthesis), 55.5 mmol m-3 s-1 for redbud (77.6% of mature), and 38.1 mmol m-
3 -1
s for red maple (81.6% of mature). In fully mature leaves, the three species did not

differ significantly with respect to photosynthesis per unit leaf area, which averaged 11

µmol m-2 s-1 (p = 0.275). However, on a per unit volume basis, mature leaves of redbud

had significantly (~33%) higher photosynthesis compared to red maple and sweetgum (p =

0.005). Mean values for mature leaf Asat on a unit volume basis were 72.4 (redbud), 46.7

(red maple), and 50.7 mmol m-3 s-1 (sweetgum). Percent mature photosynthesis in

sweetgum, red maple, and redbud correlated positively to the percent of mature chlorophyll

94
content (area basis) (r2 = 0.53, 0.98, 0.91, and p = 0.001, < 0.0001, < 0.0001 respectively)

and leaf thickness (r2 = 0.86, 0.97, and 0.93; p < 0.0001 for all species), and negatively

correlated with anthocyanin (r2 = 0.90, 0.80, 0.60; p < 0.0001 for all species) (Figure 5).

Though ANOVA tests showed a significant difference between leaf conductance rates of

the three species at the first non-red leaf node (p = 0.0369), Tukey-Kramer test for means

difference did not indicate a significant difference between any two pairs. Mean leaf

conductance values for the youngest non-red leaves of the three species corresponded to

143 (sweetgum), 104 (red maple), and 130 mmol m-2 s-1 (redbud). Similarly, there were no

differences among the three species in the percent of mean maximum leaf conductance

relative to mature leaves at the time of anthocyanin disappearance (p = 0.1850), and the

youngest, fully green leaves of all species averaged 68% of the mean maximum leaf

conductance measured for mature leaves.

95
Discussion

Photopigment accumulation

In all species examined, anthocyanins were visible until approximately 44%

chlorophyll (per unit area), 58% chlorophyll (per unit volume) and 49% of total carotenoid

content (per unit area) had developed (Figure 2). These values did not vary significantly

between species. Similar chlorophyll contents have also been reported for developing

leaves of Rosa sp. and Ricinus communis, in which anthocyanic leaves had up to ~50%

chlorophyll and carotenoid contents of mature leaves (per unit area) (Manetas et al., 2002).

This interspecific similarity in photopigment concentrations observed at the time of

anthocyanin disappearance suggests a level of photosynthetic maturity that no longer

requires photoprotection by anthocyanin. Perhaps photopigment concentrations near 50%

are low enough to avoid excessive light capture (i.e. beyond that which can be efficiently

processed by the immature chloroplast and/or anatomical structure), and/or concentrations

are high enough to contribute significantly to self-shading and (in the case of the

xanthophylls) non-photochemical quenching. Interestingly, these values also correspond

closely with those reported by Lee et al. (2003) during anthocyanin formation in a variety

of temperate deciduous tree species during autumn leaf senescence. In that study,

anthocyanin synthesis was found to be initiated when <50% chlorophyll still remained,

corresponding to <200 mg chl m-2. Here, we report a similar pattern for a different life and

phenological stage, i.e. anthocyanins were present only until ~50% mature chlorophyll

content was reached in maturing leaves, corresponding also to <200 mg m-2.

It is notable that, compared to the more constant increase in chlorophyll per unit

area during leaf development, chlorophyll per unit volume remained relatively unchanged

96
at ~58% of mature leaf values at all measured nodes for all three species (Figure 2; Table

1). This suggests that much of the increase in chlorophyll content per unit area observed

during maturation may be attributable to mesophyll thickening, rather than increased

intracellular chlorophyll content. This is supported by the observations that increases in

chlorophyll per unit area paralleled increases in leaf thickness when plotted against

photosynthetic capacity (Figure 5), and also by the visible elongation of palisade cells

during mesophyll thickening (Figure 1). The maintenance of relatively low chlorophyll

content per unit volume (~58% in young leaves) further supports a decreased emphasis on

light capture in younger leaves, with additional chlorophyll likely being added later in

development following completion of additional leaf-structural and chloroplast maturation

(Smith et al., 1997; Pettigrew & Vaughn, 1998; Niinemets et al., 2004). Although not

significant, it should be noted that chlorophyll per unit volume did increase somewhat

during development in all species (Figure 2), indicating the addition of some intracellular

chlorophyll.

In addition to decreased anthocyanin concentrations during leaf development,

carotenoid/chlorophyll ratios also significantly declined (Figure 2), probably reflecting a

decreased need for photoprotection through non-photochemical quenching (NPQ) as leaves

matured. These results are consistent with findings reported for both acyanic and

anthocyanic juvenile-leafed species (Krause et al., 1995; Barker et al., 1997 respectively),

but contrast results of others reporting no significant change in carotenoid pools during

development, even when juvenile leaves were anthocyanic (Manetas et al., 2002). It has

been suggested that species with elevated carotenoids during juvenile phases may develop

other photoprotective structures when leaves mature (such as highly reflective layers),

97
which effectively replace photoprotection by anthocyanins and/or elevated carotenoid pools

(Manetas et al., 2002). In all species examined here, leaf maturation was accompanied by

gradual whitening (presumably due to the thickening of highly-reflective epicuticular

waxes) on abaxial surfaces (data not shown). These layers likely function to increase

reflectance of incident sunlight on abaxial surfaces of these dorsiventrally asymmetric

leaves, which tend to be more light-sensitive than adaxial surfaces (Sun et al., 1996; Smith

et al. 1997; Sun & Nishio, 2001). Indeed, in redbud and Ailanthus altissima (data not

shown), anthocyanin in the abaxial surface remained visible longer than adaxial

anthocyanin, supporting the importance of abaxial-specific photoprotection in both juvenile

and mature leaves (Hughes & Smith, 2007). The concomitant decline of high

carotenoid/chlorophyll ratios and anthocyanins observed here may therefore represent

another level of coordination leaves exhibit during development— the concerted decline of

juvenile-specific photoprotective strategies with the maturation of others more

characteristic of fully expanded leaves.

Previous studies have demonstrated that developing chloroplasts generally exhibit

higher proportions of chl b relative to chl a in developing leaves, corresponding with higher

proportions of PSII relative to PSI (Pettigrew & Vaughn, 1998). The lag in PSI

development during chloroplast maturation corresponds with decreased unappressed

thylakoid membrane surface area availability early in chloroplast development, which is

where PSI complexes primarily occur (Pettigrew & Vaughn, 1998; Hopkins & Hüner,

2004b). Chl a levels increase as unappressed membranes increase in number. Consistent

with this trend, the anthocyanic species evaluated here had significant increases in

chlorophyll a/b during development (Figures 2 and 4; Table 1). However, these values

98
were much lower than those observed for species with acyanic juvenile leaves, with chl a/b

ratios for two acyanic species actually remaining relatively unchanged during development

(Figure 4). The reduced chlorophyll a/b ratios in red leaves relative to green are likely

attributable to the shading effect of anthocyanin. Previous studies have shown that shade

both retards growth and developmental rates (thus reducing the rate of chl a increase), and

also decreases the ratio of core (exclusively chl a) relative to light-harvesting (both chl a

and b) complexes (Cui et al., 1991; Jones, 1995; Demmig-Adams 1998; Hopkins & Hüner,

2004a). This explanation is supported by the observation that redbud, containing

significantly less anthocyanin in young nodes than the other two species, had the highest

chlorophyll a/b ratios of the three species, presumably due to less shading (Figure 2, Table

1). The shading effect of anthocyanin in juvenile leaves has been investigated in greater

detail by Manetas et al. (2003) using red and non-red variants of Quercus coccifera. In

addition to lower chlorophyll a/b ratios, red juvenile leaves of Q. coccifera had a suite of

other shade characteristics, including thinner leaves and lower levels of xanthophyll cycle

pigments relative to acyanic variants. Presumably, these differences were due to shading

by anthocyanic layers which, as previously mentioned, reduce incoming sunlight via strong

absorption of blue-green light.

Leaf structure and gas exchange

Other factors in addition to photopigment content must also be considered when

evaluating photon absorption and processing, including leaf (mesophyll) thickness,

structural maturation (e.g. spongy and palisade mesophyll differentiation and chloroplast

99
development), leaf conductance of CO2, and photosynthetic CO2 fixation (see Smith et al.,

1997; Evans et al., 2004 for reviews).

A strong relationship was found between leaf thickness and the disappearance of

anthocyanin during leaf maturation. On average, anthocyanin loss occurred only when

~80% of mature leaf thickness had been attained (Figure 2). These values did not

significantly vary between species. Photosynthesis (computed on a leaf area basis) also

showed a strong, positive correlation with leaf thickness (Figure 5), most likely due to

increases in chlorophyll per unit area and chloroplast development (as previously

described), as well as leaf structural maturation.

To infer structural maturation, chlorophyll content expressed per unit volume may

be compared to photosynthesis per unit volume. In red maple and redbud, photosynthesis

expressed per unit leaf volume continued to increase through node five (Figure 3), even

though chlorophyll content per unit volume did not change significantly (Figure 2). This

suggests that either (or both) chloroplast development was still ongoing (in terms of

thylakoid number per granum, membrane complexity, stroma lamellae development, etc.),

or that leaf anatomical structure facilitating light distribution and/or CO2 diffusion was still

in the process of maturation. Although we did not observe chloroplast development in this

study, we were able to observe differentiation of mesophyll into palisade and spongy layers

(Figure 1). These structures can be important for enhancing light propagation and

processing by distributing absorbed light more uniformly throughout the mesophyll (in the

case of palisade cells) and facilitating CO2 diffusion (spongy) (Vogelmann et al., 1996;

Smith et al., 1997). Moreover, these structures were not fully differentiated until the third

leaf node in red maple and redbud (Figure 1). In contrast, sweetgum had more constant

100
(~100% mature values) photosynthesis (expressed per unit volume) between nodes two and

five (Figure 3), with fully differentiated, double palisade layers appearing in the youngest

leaf node with expanded leaves (Figure 1).

Paralleling increases in photosynthesis, leaf conductance of CO2 also increased

significantly during development for all species (Figure 3), with the most rapid increase

between leaf nodes occurring in sweetgum. Because mesophyll conductance is a

significant factor affecting photosynthetic potential, it is to be expected that stomata would

not open fully until leaves are structurally and biochemically mature enough to process

increased internal CO2. This may explain why conductance values in sweetgum

approached mature values earlier than red maple and redbud, corresponding to its more

rapid structural maturity (Figure 1).

The increase in photosynthetic carbon fixation facilitated by a mature internal leaf

anatomy may also provide a greater sink for dissipating excessive sunlight energy,

potentially decreasing the requirement of photoprotective mechanisms such as anthocyanin.

In support of this idea, sweetgum (having the highest photosynthetic rates in young leaves)

lost anthocyanin more quickly during development (i.e. at significantly lower chlorophyll

content) than the other two species examined. By the third leaf node, where leaf

anthocyanin was no longer visible in sweetgum, photosynthesis per unit area had reached

~99% full capacity, although only ~138 mg m-2 (44% of mature) chlorophyll had

developed (Figure 3, Table 1). In contrast, anthocyanin remained visible until the fourth

leaf node in red maple (which had only reached ~60% of full photosynthetic capacity, with

~298 mg m-2 chlorophyll), and the fifth node in redbud (~40% full capacity, and ~159 mg

m-2). This trend of prolonged anthocyanin presence in species with slower photosynthetic

101
maturation is supported further by values reported for C. gummifera (Choinski et al., 2003).

C. gummifera had substantially lower photosynthesis during development relative to the

species here, but also retained anthocyanin until much later in development (i.e. until

~100% full leaf thickness had been obtained and ~400 mg chl m-2 had formed; note,

however, that C. gummifera leaves were substantially thicker than leaves measured here).

The association of rapid structural and photosynthetic maturation with rapid anthocyanin

decline provides further evidence for a strong functional coupling between leaf structure,

anthocyanin presence, and photosynthetic maturation.

102
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106
Figure 4.1

Whole branches and microscopic sections of leaves from nodes 1-5 of Liquidambar

styraciflua (sweetgum), Acer rubrum (red maple), and Cercis canadensis (redbud). All

scale bars represent 100µm. In sweetgum, leaf sections were magnified at 100X; red maple,

200X for node 1, 100X for nodes 4-5; redbud, 200X.

107
Figure 4.1

108
Figure 4.2

Pigment concentrations and leaf thicknesses expressed as a percent of mature values.

Shaded areas represent visible presence of anthocyanin. Illustrated are: anthocyanin

content, % chlorophyll content per unit area (closed circles) and per unit volume (open

circles), % chlorophyll a/b, % total carotenoids (per unit area), and % leaf thickness for

Liquidambar styraciflua (sweetgum); Acer rubrum (red maple); and Cercis canadensis

(redbud). Points represent means of five replicates ± SD for pigments and ± SE for lamina

thickness.

109
Figure 4.2

110
Figure 4.3

Percent maximum photosynthesis (Asat) and leaf conductance of water vapor (g) values by

leaf node, expressed as a percent of mature-leaf values. Shaded areas represent the visible

presence of anthocyanin. Illustrated are: percent photosynthesis expressed as per unit area

(closed circles; µmol m-2 s-1) and per unit volume (open circles; mmol m-3s-1) and %

conductance for Liquidambar styraciflua (sweetgum); Acer rubrum (red maple); and

Cercis canadensis (redbud). Points represent means of 5-10 replicates ± SD.

111
Figure 4.3

112
Figure 4.4

Tip-down (nodes 1-5) chlorophyll a/b ratios for species with green developing leaves

(closed symbols) and red (open), compared to mature leaf values. Green (acyanic) species

include Hedera helix (circle), Forsythia suspensa (triangle), and Lonicera japonica

(square). Red species include Acer rubrum (circle), Liquidambar styraciflua (triangle), and

Cercis canadensis (square). Points represent means of 5 replicates ± SD.

113
Figure 4.4

114
Figure 4.5

The relationships between photosynthesis maturation and corresponding changes in

chlorophyll per unit area (thick black), leaf thickness (thick gray), and anthocyanin (thin

black). Sweetgum is represented by the solid lines, red maple by long dashes, and redbud

by dotted lines. Percent of mature photosynthesis in sweetgum, red maple, and redbud

correlated positively to percent of mature chlorophyll content (r2 = 0.53, 0.98, 0.91

respectively) and leaf thickness (r2 = 0.86, 0.97, and 0.93), and correlated negatively to

anthocyanin (r2 = 0.90, 0.80, 0.60). Slopes of these lines were also statistically similar

between species. For chlorophyll content, slopes were 0.179, 0.375, 0.340 for sweetgum,

red maple, and redbud, respectively; for thickness, 0.304, 0.500, 0.393, and for anthocyanin,

-0.750, -0.985, and -0.775.

115
Figure 4.5

116
 CHAPTER V

SEASONAL PHOTOSYNTHESIS AND ANTHOCYANIN PRODUCTION IN TEN

BROADLEAF EVERGREEN SPECIES

Nicole M. Hughes and William K. Smith

The following manuscript was published in Functional Plant Biology, volume 34, 1072-

1079, and is reprinted with permission. Stylistic variations are due to the requirements of

the journal. N.M. Hughes performed the laboratory and field work, data analysis, and

prepared the manuscript, W.K. Smith provided equipment, logistical support, and editorial

assistance.

117
Abstract

Leaves of many evergreen species turn red when exposed to high sunlight during

winter due to production of photoprotective anthocyanin pigments, while leaves of other

species, lacking anthocyanin, remain green. Why some species synthesize anthocyanin

pigments while others do not is currently unknown. Furthermore, the relative

photosynthetic performance of anthocyanic (red) and acyanic (green) evergreens has yet to

be described. Here we present seasonal ecophysiological data for five red and green

broadleaf evergreen species. We hypothesize that species which synthesize anthocyanins

in winter leaves correspond to those with the most drastic seasonal photosynthetic declines,

as reduced energy sinks increase vulnerability to photoinhibition and need for

photoprotection. Our results did not support this hypothesis, as gas exchange

measurements showed no difference in mean seasonal photosynthetic capacity between red

and green-leaf species. Consistent with anthocyanin’s shading effect, red-leaf species had

significantly higher chlorophyll content, lower chlorophyll a/b ratios, and higher maximum

light capture efficiency of PSII (Fv/Fm) than green-leaf species during the winter, but not

during the summer (when all leaves were green). We conclude that anthocyanin

production during winter is likely not associated with diminished photosynthetic capacity,

and may simply represent an alternative photoprotective strategy utilized by some species

during winter.

118
Introduction

Evergreen plants exhibit a broad range of adaptations enabling extended

photosynthetic carbon gain during winter months, despite a variety of abiotic stresses (for

reviews see Tranquillini 1964; Nilsen 1992; Uemera and Steponkus 1999; Bigras and

Colombo 2001; Öquist and Huner 2003; Adams et al. 2004). Of particular importance are

adaptations which allow photosynthetic tissues to avoid and/or dissipate excess light energy

(Krause 1994). Because low temperatures inhibit the biochemical reactions of the Calvin

cycle, but do not significantly affect light capture, high-sunlight exposure in combination

with low temperatures may lead to excessive energy captured relative to that used in carbon

fixation (Hüner et al. 1998). This imbalance leads to an increase in energy and electron

transfer to molecular oxygen, production of radical oxygen species, and increased

vulnerability to photo-oxidative damage (Mittler 2002). Adaptations which curtail these

processes generally do so by either reducing photosynthetically active radiation (PAR)

incident on the leaf or chloroplasts (i.e. avoidance strategies) or quenching/dissipating

absorbed energy before transfer to the chlorophyll reaction centers (non-photochemical

quenching). Anthocyanin pigments are an example of an avoidance strategy, as they are

thought to intercept incident blue-green light and dissipate the absorbed quantum energy as

heat, thereby protecting underlying photosynthetic cells from photoinhibition (Lee and

Gould 2002). This shade function has been supported by studies showing reduced

photosynthesis in red-leaf morphotypes of several species relative to green (e.g. Bahler et

al. 1991; Burger and Edwards 1996; Gould et al. 2002) as well as reduced photoinhibition

(e.g. Feild et al. 2001; Hughes et al. 2005; Liakopoulos et al. 2006), often resulting in

119
elevated photosynthesis in anthocyanic leaves relative to acyanic leaves under

photoinhibitory conditions (e.g. Gould et al. 1995; Liakopoulos et al. 2006).

Although the capability for synthesizing anthocyanins is nearly ubiquitous among

angiosperms, not all evergreen angiosperms synthesize anthocyanins in leaves during

winter, when photoprotective mechanisms are generally highly engaged (Adams et al.

2004). Interestingly, many of the evergreen species which do not synthesize anthocyanin

in winter leaves do so in other tissues or during different ontogenetic stages, such as in

juvenile leaves, flowers, stems, roots, senescing leaves, and/or in response to pathogen

infection (see Table 1). Their failure to produce anthocyanins in winter leaves therefore

suggests that anthocyanins are not necessary or beneficial for these species during the

winter season. However, this assumption has not been tested, and no explanation currently

exists for why some broadleaf evergreen species synthesize anthocyanins in winter leaves

while others do not.

One possibility is that anthocyanin synthesis only occurs in species with drastic

seasonal decreases in photosynthetic capacity during the winter, corresponding with an

increased need for photoprotection. A decreased capacity for photosynthesis is known to

render plants intrinsically more vulnerable to photoinhibition, due to a reduction in energy

sinks available for energy dissipation (i.e. photochemical quenching; Osmond 1981;

Powles 1984). This idea has been supported by previous research on anthocyanins in

deciduous tree species, where it has been shown that shade-intolerant species

(characterized by high maximum photosynthesis) tend to not produce anthocyanins during

the autumn, while shade-adapted species (lower maximum photosynthesis) do (Koike

1990; Hoch et al. 2001). Indeed, evergreen species are known to vary greatly in seasonal

120
photosynthetic capacity due to either differences in seasonal growth, and/or general

differences in intrinsic limitations to photosynthesis (e.g. vulnerability to cavitation, water-

stress tolerance, freezing damage) (Davis et al. 1999; Uemera and Steponkus 1999;

Verhoeven et al. 1999; Adams et al. 2002; Taneda and Tateno 2005). However, it is

unknown whether such differences correspond with red/green leaf coloration in winter.

To determine whether color change corresponds with any observable patterns in seasonal

photosynthesis, we measured photosynthetic gas exchange, photosynthetic light response

curves, and maximum light capture efficiency of photosystem II (Fv/Fm) for ten broadleaf

evergreen species (representing five different plant families) differing in their production of

anthocyanin during winter.

121
Materials and methods

Sites and species

Species selected for study were mature field plants growing along sun-exposed

road-sides in Jonas Ridge, NC, USA (35° 57’ 20” N, 81° 53’ 55”W; altitude: ca. 1200 m)

on south or southeast-facing sites receiving >6 h full sunlight (i.e. >1350 µmol m-2 s-1 on a

horizontal surface) per day during both summer and winter months. Measurements were

taken on clear sunny days, with little or no cloud cover. Detailed descriptions of the

individual species examined are in Table 1. All hourly time designations are expressed as

Solar Time (List 1971).

Photosynthetic gas exchange

Winter photosynthesis measurements were taken between December 4, 2005 and

March 4, 2006, and December 15 – 17, 2007, and summer measurements on August 2-4,

2007. All measurements were made on first year leaves under full ambient sunlight (>1350

µmol m-2 s-1), and plants were sampled via a standard random walk procedure (Codling and

Hill 2005). Measurements were replicated at times of the day that corresponded to periods

with the highest stomatal conductance. During the summer, these higher conductance

periods occurred in early morning (between 800 and 1100 h) and late afternoon (1600 and

1900 h) when air temperatures were coolest. During the winter, measurements were taken

in mid-afternoon (1100 until 1500 h), when air temperatures were the warmest. A portable

photosynthesis system (PP Systems Inc., model TPS-1, Amesbury, MA, USA) was used to

measure leaf conductance, photosynthetic CO2 assimilation, leaf temperatures, and air

temperature for gas exchange measurements. This instrument was chosen because its small

122
size facilitated the substantial climbing involved in sampling. Measured values were

compared on three different occasions and compared to simultaneous measurements using

a LICOR model Li-6400 (Li-Cor, Inc., Lincoln, NE), and were found to be within ±3%.

Photosynthetic response to light was measured in the field on warm winter days (daytime

maximum air temperatures >17°C) following at least three consecutive days with night

time temperatures >2°C. Measurements were made between 1100 and 1500 h using a Li-

Cor 6400 infrared gas analyzer with red and blue LED light sources (Li-Cor, Inc., Lincoln,

NE). This device was used for light-response curves because the LED transmittance curve

avoids the absorbance spectrum of anthocyanin, allowing photosynthesis to be measured

without the pigment’s shading effect (LED spectrum illustrated in Figure 8-3 of Li-6400

Instruction Manual); this allowed light exposure to be standardized for red and green leaves.

The temperature of the leaf cuvette was set to 20°C during the winter to normalize

measurements between samples, while summer measurements were taken at ambient

temperatures (29 – 35° C). Measurements were initiated at ambient photosynthetic photon

flux densities (PPFD), then increased up to 2000 μmol m−2 s−1, and then decreased

incrementally until irradiance was zero. All measurements were taken on plants that had

been exposed to full sunlight for at least 2 hrs to ensure maximal stomatal opening

(checked by measuring leaf conductance values). The order of individual plant

measurements was randomized each day and sample size was at least n = 3, with different

individuals tested for each species each day.

123
Chlorophyll fluorescence

Maximum light capture efficiency of photosystem II (Fv/Fm) was measured in the

field on mature, current-year leaves between December 4, 2005 and March 4, 2006, and

December 15, 2006 and March 15, 2007, and in summer August 2-4, 2007. Measurements

were randomized according to plant and leaf, and taken between 1100 and 1400h. A PAM

Fluorescence System (Hansatech Institute, model FMS-2, Cambridge, UK) emitting a two

second long, 3 mmol m-2 s-1, 594 nm saturating pulse was used to derive Fv/Fm for all

sampled leaves. Prior to each measurement, plants were dark-adapted for 30 min using

model FMS-2 leaf clips.

Pigment analyses

Chlorophyll concentrations were measured and a/b ratios were calculated to assess

the relative emphasis of light capture versus processing in winter leaves. Increased

chlorophyll content is associated with an emphasis on light capture, as well as increased

chlorophyll a/b ratios, which reflect an increase in core (exclusively chl a) relative to light-

harvesting (both chl a and b) complexes, and/or higher ratios of Photosystem I (4/1 ratio of

chl a/b) to Photosystem II (1.2/1 ratio) (Cui et al. 1991; Demmig-Adams 1998; Hopkins

and Hüner 2004).

On February 16, 2007, one mature (fully expanded) and healthy-appearing leaf was

removed from five separate individuals in the field between 1100 and 1300 h, after all

plants had been exposed to full sunlight for at least 3 hrs. A standard hole puncher was

used to excise three 0.33 cm2 leaf discs, which were immediately placed in 3 mL N,N’ -

dimethylformamide to extract in the dark for 24 hrs. Chlorophyll concentrations were

124
determined spectrophotometrically using the equations described by Porra (2002). From

the same leaves, three additional leaf discs were excised and placed in 3 mL 6M

HCl:H2O:MeOH (7:23:70) for 24 hrs at 4°C to extract anthocyanins. Anthocyanin

concentration was measured spectrophotometrically as A530 – 0.24 A653 using an extinction

coefficient of 30 000 l mol-1 cm-1 (Murray and Hackett 1991). All extracts were analyzed

using a Hewlett Packard 8453 UV-VIS spectrophotometer (Hewlett Packard, Palo Alto,

CA).

Statistics

Linear regression analysis was used to evaluate correlations between temperature

and photosynthesis, leaf conductance, and Fv/Fm, for each species. Slopes and Y-intercepts

of best fit lines for these values were grouped according to the presence or absence of

anthocyanin (i.e. red or green winter leaf color, respectively) and compared using a two-

sample, two-tailed Student’s t-test (Zar 1999). The effects of leaf color on Fv/Fm,

photosynthesis, and leaf conductance values were analyzed using a nested MANOVA (with

species nested within color), on three winter dates differing in air temperature (Cold: low

temp -9°C, high -3°C; Mild: low -3°C, high 10°C; and Warm: low 8°C, high 20°C; dates

are given in Fig. 2 legend); measurements were also made on one summer day for

comparison (low 31°C, high 18°C). Measurements on different days used random leaves

sampled from multiple (i.e. > 5) plants throughout the plot, invalidating use of the repeated

measures technique (Zar 1999).

For light saturation curves, nested MANOVAs were also used to compare dark

respiration, light-saturated photosynthesis at ambient CO2 (Asat), and photosynthesis (A) at

125
individual irradiance levels for species differing in color. A one-tailed t-test was used to

compare linear slopes of photosynthesis below light saturation for red and green species

during summer and winter. To compare seasonal photosynthetic capacity, summer Asat (i.e.

maximum values observed during the summer) were compared to Asat observed on a warm

winter day (low 8°C, high 20°C, representing the maximum values observed during the

winter) for each species using a one-tailed t-test. A one-tailed Student’s t-test was also

used to compare mean seasonal percent declines in Asat for red and green species.

Photopigment concentrations and chlorophyll a/b ratios of red and green species were

compared using a two-way nested MANOVA, with species nested within leaf color. All

tests employed met distributional and sample size requirements described in Zar (1999),

and significance was accepted at p < 0.05.

126
Results

Winter photosynthesis

Photosynthesis significantly (p < 0.0001) increased with air temperature for all

species during the winter (r2 values for green species: K. latifolia 0.583, R. maximum 0.475,

R. catawbiense 0.711, V. minor 0.627, Rhododendron azalea spp. 0.766; r2 values for red

species: G. urceolata 0.428, L. fontanesiana 0.627, L. japonica 0.772, H. shuttleworthii

0.779, R. azalea 0.857) (Figs 1 & 2). The slope of this increase (m = 0.219) did not

statistically differ between red and green species as a group (p = 0.827).

Leaf conductance was more variable during the winter than photosynthesis, but did

increase significantly with temperature for all species except the green-leafed R. maximum

and V. minor during the winter (p = 0.99 and 0.383 R. maximum and V. minor respectively,

p < 0.05 for all remaining species; Fig. 1). The slope of the increase in conductance

according to temperature during the winter (m = 1.87) did not significantly differ for red

and green species (p = 0.136).

On cold winter sample days, photosynthesis for all plants was at or near zero,

corresponding with near zero leaf conductance values (Fig. 2). There was no significant

difference in Asat or leaf conductance between species differing in leaf color on a cold

winter day (p = 0.303 and 0.754, respectively) or a mild winter day (p = 0.101 and 0.607).

However, on warm days, red-leafed species photosynthesized at significantly higher rates

than green-leafed species (p < 0.0001), and also had significantly higher leaf conductance

(p < 0.0001). However, there was also a significant species effect on warm days (p <

0.0001), with very high Asat and g values for the red-leaf L. japonica, and very low values

127
for the green-leaf V. minor driving statistical differences between groups; values for other

species generally overlapped (Fig. 2).

For light-response curves (Fig. 3), red species had significantly higher Asat than

green species during the winter (p = 0.01), although, again, there were highly significant

interspecific differences (p < 0.0001), and with the exception of L. japonica and red R.

azalea spp., Asat for red and green-leaf species was comparable. Similarly, there was no

difference in Asat between red and green-leaf species during the summer when all leaves

were green (p = 0.75). During winter, red-leaf species also showed significantly higher A

than green species at low PAR values (i.e. < 500 µmol m-2 s-1) (p < 0.01 at 200 and 100

µmol m-2 s-1), and had significantly greater quantum yield of photosynthesis at low PAR

(as evidenced by steeper slopes below the light saturation point) (p = 0.02, where mean m

for red species = 0.016, and green species = 0.0098); the groups did not differ in any of

these parameters during the summer (p > 0.50). During both summer and winter seasons,

dark respiration rates of red and green-leaf species did not differ (p > 0.30).

Seasonal differences in photosynthetic gas exchange

While winter photosynthesis of all species was significantly less than summer

values on both cold and mild days (p < 0.0001 for both), on the warmer days (air

temperature > 16°C) some species were able to photosynthesize at rates that did not

significantly differ from summer rates (green species: V. minor p = 0.09; red species: G.

urceolata p = 0.52; Fig. 4). The remaining species photosynthesized significantly below

summer levels on warm winter days (p < 0.01). As a group, the mean percent seasonal

decline in photosynthesis relative to summer values exhibited by red leaves (30%) did not

128
significantly differ from that of green species (41%) (p = 0.53). At all temperatures

measured, all ten species exhibited significantly reduced conductance during the winter

relative to the summer (p < 0.0001).

Fluorescence

Winter Fv/Fm values increased significantly (p < 0.0001) with temperature for all

species (Fig. 1; r2 values > 0.56 for all green species; r2 values > 0.41 for all red species).

Fv/Fm increased with temperature along the same slope (m = 0.0158) for both red and green

groups (p = 0.733). Fv/Fm values of red species were consistently higher (~0.15 on

average) than those of green species across the temperature range measured, and Y-

intercepts of regressions were significantly higher for red species as a group compared to

green species (p = 0.016). On cold, mild, and warm winter days, red-leaf species had

consistently higher (~0.15 units on average) mean Fv/Fm than green species (p < 0.0001 for

all; Figs 1 & 2).

Pigments

Species with red winter leaves had significantly higher total chlorophyll (24% on

average), and anthocyanin content than green species during the winter (p < 0.0001 for

both; Table 2). Red species also had significantly (27%) lower chlorophyll a/b ratios than

green species on average (p < 0.0001; Table 2).

129
Discussion

The hypothesis that species with red winter leaves would be characterized by lower

photosynthetic capacity during winter than green-leaf species was not supported in this

study. Mean maximum photosynthesis of red-leaf species during winter did not differ from

green-leaf species on cold, mild, or relatively warm winter days (Figs 1 & 2). In fact, the

two species with the highest photosynthesis values during winter were both red-leaf species

(L. japonica and the red R. azalea sp.). Mean winter declines in photosynthesis relative to

summer values (Fig. 4) also did not significantly differ between red-leaf species relative to

green-leaf species, with some members of both groups having Asat on warm winter days

near values observed during the summer, and others exhibiting more drastic declines (i.e. <

50% summer photosynthesis).

Fluorescence measurements of PSII were consistent with a photoprotective function

by anthocyanin. Red-leaf species had consistently higher maximum light-capture

efficiency of PSII (Fv/Fm) than green-leaf species across all measured temperatures during

winter days (Figs 1 & 2); similar trends were also observed for pre-dawn measurements

during the winter (data not shown). Because Fv/Fm is correlated with sustained non-

photochemical quenching (with lower values corresponding with increased retention of

photoprotective antheraxanthin and zeaxanthin), these results suggest that red-leaf species

rely less on sustained xanthophyll-mediated photoprotection than green-leaf species during

the winter, most likely due to anthocyanin’s attenuation of absorbed sunlight reducing the

need for additional photoprotection (Demmig-Adams et al. 1996). However, it is also

possible that red-leaf species are utilizing more the rapidly-reversible component of NPQ

(i.e. pH-dependent conversion of violaxanthin into antheraxanthin and zeaxanthin), which

130
is lost during dark adaptation; this is unlikely, however, because ΦPSII (non-dark adapted

quantum efficiency of PSII) was also consistently higher in red species than green (data not

shown), by roughly the same order of magnitude as Fv/Fm.

Anthocyanin’s shading effect was also evidenced in the relatively shade-adapted

light response curves of red compared to green leaves (Fig. 3). Under red and blue LED

light (which does not overlap with the absorbance spectrum of anthocyanin), red-leaf

species had significantly steeper slopes for the linear part of the light response curves than

green-leaf species, and higher photosynthesis under low PAR (i.e. < 500 µmol m-2 s-1)

accordingly. The two groups did not differ in these respects during the summer, when all

leaves were green. Higher photosynthesis at low PAR is a common feature of more shade-

adapted plants, reflecting either decreased respiration rates and/or a greater emphasis on

light capture relative to light processing (Larcher 2003). Because dark respiration values

were not significantly different between the red-and green-leaf groups during the winter,

increased Asat at low PAR was likely due to greater light capture capacity. Consistent with

this explanation, total chlorophyll content was significantly higher and chlorophyll a:b

ratios significantly lower in red-leaf species compared to green (similar to results reported

in Manetas et al. 2003, Lee et al. 2003, Hughes et al. 2005; Table 2).

Higher photosynthesis at lower light levels may also explain why red-leaf species

were able to photosynthesize at saturated values under ambient sunlight, despite the

shading effect of anthocyanin. Pietrini and Massacci (1998) estimated that anthocyanins at

concentrations near the maximum values reported here (i.e. ~4.4 mol/g) may attenuate

~43% incoming PAR in vivo. Thus, under full sunlight, the amount of unabsorbed

irradiance remaining should still be high enough to saturate photosynthesis (as all red-leaf

131
species photosynthetically saturated near 25% full sunlight; Fig. 3). Perhaps anthocyanins

reduce PAR transmittance sufficiently to curtail photoinhibition, but still transmit enough

to allow the leaf to attain light saturation of photosynthesis, maximizing sunlight capture

and utilization during the winter.

Because red and green-leaf species measured here did not differ in seasonal

photosynthesis, we conclude that red coloration in winter leaves does not appear to

correspond with diminished photosynthetic capacity. Instead, the comparable seasonal

photosynthesis of red leaves compared to green suggests that photoprotection by

anthocyanin represents an alternative (rather than additive) photoprotective strategy

employed by some plants during the winter. Perhaps up-regulation of anthocyanin

compensates for species-specific deficiencies in other means of photoprotection (e.g. NPQ,

antioxidants). Research is currently underway by the authors to investigate this possibility.

Alternatively, it is also possible that red-leaf species are indeed limited in photosynthetic

capacity during the winter, but are able to photosynthesize at rates similar to green species

due to anthocyanin’s photoprotective effect. However, this would be difficult to determine

without, for example, utilizing anthocyanin-less mutants of red-leaf species.

132
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135
Table 5.1

Detailed descriptions of species used in this study. An asterisk by species name denotes

species which synthesize anthocyanin in winter leaves

136
Table 5.1

Species Family Plant form Origins Anthocyanic tissues

*Galax urceolata (Poir.) Diapensiaceae Forb/herb Native Winter rhizomes and

Brummitt petioles, winter, juvenile
and injured leaves

*Leucothoe fontanesiana Ericaceae Shrub Native Winter stems, winter,

(Steud.) Sleumer juvenile, and injured leaves

Runners, winter leaves

*Lonicera japonica (Thunb.) Caprifoliaceae Vine Introduced

*Hexastylis shuttleworthii Winter leaves, winter

(Britten & Baker) Aristolochiaceae Forb/herb Native petioles, flowers

*Rhododendron spp. Flowers, new growth stems

(horticultural azalea) Ericaceae Shrub Introduced senescing leaves

Kalmia latifolia (L.) Juvenile and injured leaves,

Ericaceae Shrub Native stems, flowers,

Rhododendron maximum (L.) Glands on juvenile leaves

Ericaceae Shrub Native and stems, developing
stems, flowers
Vinca minor (L.)
Flowers
Apocynaceae Vine/ Introduced
Rhododendron catawbiense groundcover
(Michx.) Flowers, senescing leaves
Ericaceae Shrub Native
Rhododendron spp.
(horticultural azalea) Flowers, new growth stems
Ericaceae Shrub Introduced senescing leaves

137
Table 5.2

Winter photosynthetic and non-photosynthetic pigment content, expressed per gram of

fresh weight. Gaultheria procumbens was used as a red representative instead of H.

shuttleworthii due to limited availability of H. shuttleworthii leaves for destructive analyses.

138
Table 5.2

Total Chl Chl a/b Anthocyanin

(mg/g) (mol/g)
Green spp. K. latifolia 0.886 4.63 -
(0.22) (0.88)

R. azalea spp 0.810 4.45 -

(0.20) (0.62)

0.987 4.29 -
R. catawbiense (0.13) (0.75)

0.793 3.61 -
R. maximum (0.16) (0.34)

0.857 2.11 -
V. mino (0.14) (0.30)

Red spp. L. fontanesiana 1.08 3.28 1.04

(0.29) (0.34) (0.32)

L. japonica 0.623 3.10 2.61

(0.087) (0.30) (0.34)

G. urceolata 1.61 2.21 3.27

(0.26) (0.33) (1.0)

0.978 2.29 5.3

R. azalea spp (0.19) (0.28) (1.4)

1.10 3.07 1.05

G. procumben (0.11) (0.71) (0.55)

139
Figure 5.1

Winter maximum light capture efficiency of PSII (Fv/Fm), light-saturated photosynthesis

(Asat), and leaf conductance (g) for red (dashed lines) and green (solid lines) broadleaf

evergreen species measured across a range of ambient temperatures.

Left column: representative green species (K. latifolia), middle column: representative red

species (G. urceolata), right column: best fit lines of all red (dashed) and green (solid)

species measured. Red species: L. japonica (A), R. azalea spp. (B), G. urceolata (C), L.

fontanesiana (D), H. shuttleworthii (E). Green species: R. catawbiense (F), K. latifolia (G),

R. azalea spp. (H), R. maximum (I), V. minor (J).

140
Figure 5.1

141
Figure 5.2

Cold, mild, and warm winter day measurements of Asat, g, and Fv/Fm for green-leaf and

red-leaf species. Bars represent means of 5-15 replicates ± SD. Cold winter day

measurements were taken on January 29, 2007 (low -9°C , high temp -3°C); mild, February

7, 2006 (low -3°C, high 10°C); warm, December 15, 2006 (low 8°C, high 20°C); summer

measurements taken on June 8, 2006 (low 12°C, high 23°C).

142
Figure 5.2

143
Figure 5.3

Seasonal light response curves for red and green broadleaf evergreen species. Top row:

summer; bottom row: winter. Left column: representative green species (K. latifolia),

middle column: representative red species (G. urceolata), right column: best fit lines of all

red (dashed) and green (solid) species measured. Red species: L. japonica (A), R. azalea

spp. (B), G. urceolata (C), L. fontanesiana (D), H. shuttleworthii (E). Green species: R.

catawbiense (F), K. latifolia (G), R. azalea spp. (H), R. maximum (I), V. minor (J).

144
Figure 5.3

145
Figure 5.4

Percent decrease in maximum observed winter Asat relative to summer values.

White bars represent green species, grey bars represent red species. Asterisks denote

significant declines in maximum winter photosynthesis relative to summer values (p <

0.05).

146
Figure 5.4

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 CHAPTER VI

CONCLUSIONS AND FUTURE DIRECTIONS

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 The results presented here are generally consistent with a photoprotective

explanation of anthocyanin pigments in photosynthetic tissues; however, unresolved issues

still remain. As described in Chapters II and III, abaxial anthocyanin pigments were

demonstrated to be of functional importance in photoprotection. In the case of high-light

species with vertical leaf orientations and/or in environments characterized by high

substrate albedo (Galax urceolata in Chapter II), the abaxial anthocyanic layer directly

intercepted irradiance incident on the more light-sensitive abaxial leaf surface, effectively

reducing photoinhibition of photosynthesis. In species adapted to the deep shade (Begonia

heracleifolia in Chapter III), abaxial anthocyanins also appeared to function in

photoprotection, though not through direct interception of incident light, but rather, through

attenuation of internally-scattered green light transmitted through the adaxial leaf surface.

This latter adaptation would presumably function to buffer internal light levels during brief

to extended periods of high light (e.g. sunflecks or sun-patches) in species adapted to the

deep shade, without interfering with light interception during periods of low light (as

adaxial anthocyanins might). Despite the experimental support drawn from these studies

on abaxial anthocyanin, however, there still remain cases of abaxial pigmentation for which

neither of these explanations seems appropriate. For example, some species of water lilies

exhibit abaxial coloration in floating leaves. Clearly, because of their horizontal orientation

against the water surface, abaxial surfaces of water lilies are not directly exposed to high

light (as was the case with G. urceolata), and so direct interception of incident light seems

an improbable function. Furthermore, these species are almost continuously exposed to

high adaxial irradiance, and so, adaxial anthocyanins would seem more appropriate for

photoprotection than abaxial (in contrast with understory species). There is currently no

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adaptive explanation for abaxial coloration in these floating leaves. Another interesting,

yet unresolved, case of abaxial pigmentation is that of abaxial red/green variegation (e.g.

Figure 3.1), which is usually observed in understory plants. Although an abaxially

red/green variegated plant was used in Chapter III to test the photoprotection hypothesis, it

is still unclear why this species (and others) would evolve pigmentation in only portions of

leaf tissues. Herbivory avoidance seems one likely explanation, as leaves may appear

smaller or damaged compared to fully green leaves. This hypothesis, however, has not yet

been tested. It is also worth noting that in Begonia heracleifolia (Figure 3.1) it is the

tissues most distal to the major veins that exhibit pigmentation. Perhaps the increased

vulnerability to dehydration stress exacerbates oxidative damage, rendering distal tissues in

greater need of photoprotection. This hypothesis too has not been tested.

In Chapter IV, the coordination between anthocyanin decline and photosynthetic

maturation (and thus, decreased need for photoprotection) was demonstrated using

developing leaves of three deciduous-tree species. This was the first study to

quantitatively describe the association between relative need for photoprotection and

anthocyanin concentration. It should be noted, however, that this relationship was purely

correlative, and could also be attributed to other functions of anthocyanin in addition to

photoprotection. For example, because young leaves are more vulnerable to herbivory

than mature leaves (due to a thinner lamina and cuticle), red coloration may function as a

camouflage to insects lacking a red photoreceptor. This explanation is consistent with the

correlation between loss of redness and attainment of 80% leaf thickness, though it does

not necessarily explain the correlation with development of leaf structural anatomy or

photopigments. Additionally, juvenile leaves might also require anthocyanin as an

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antioxidant during development, for the same reasons it would be beneficial as a light-

attenuating molecule (i.e. increased vulnerability to photo-oxidative damage during

development). This study did not discern whether anthocyanins were functioning as one

or the other, but rather, simply correlated their presence with need for photoprotection.

Future studies should aim to discern between these two functions.

In the study presented in Chapter V, it was hypothesized that red-leafed species

required anthocyanins because of intrinsic limitations to photosynthetic capacity during

winter, which would increase need for photoprotection accordingly. However, results

showed that red-leafed species did not differ in photosynthetic capacity relative to green-

leafed species during either summer or winter. Anthocyanins did appear to function in

photoprotection though, as red-leafed species exhibited significantly reduced sustained

photoinhibition of photosynthesis compared to green-leafed species, and also showed

symptoms of shade adaptation. Regardless, it remains unclear why certain evergreen

species would synthesize anthocyanin in winter leaves while others would not. One

possibility is that red-leaf species are indeed limited in photosynthetic capacity during the

winter, but are able to photosynthesize at rates similar to green species due to

anthocyanin’s photoprotective effect. However, this would be difficult to determine

without, for example, utilizing anthocyanin-less mutants of red-leaf species. Another

possibility is that anthocyanin simply represent an alternative (rather than additive)

photoprotective strategy employed by some plant species during the winter, perhaps

compensating for species-specific deficiencies in other modes of photoprotection (e.g. NPQ,

antioxidants). I have begun to test this hypothesis in a study not presented in this

dissertation, and have thus far found no significant difference in low molecular weight

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antioxidants between red and green-leafed species; xanthophyll analyses are currently

underway. Finally, a third possibility is that anthocyanins are synthesized to alleviate

oxidative stress resulting from low water potentials, either through acting as an antioxidant

or through light-attenuation. According to this hypothesis, species which synthesize

anthocyanin during winter would correspond with those exhibiting the most extreme

declines in daily water potentials (i.e. drought tolerators). This experiment is also currently

underway.

In conclusion, although unanswered questions remain regarding the photoprotective

role of anthocyanins pigments in photosynthetic tissues, the studies presented in this

dissertation were successful in strengthening the evidence for an important photoprotective

function.

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 Nicole Michelle Hughes
Wake Forest University
Department of Biology
Winston-Salem, NC 27109
(336) 414-4116
Email: hughnm5@wfu.edu

EDUCATION B.S. Biology and Education, 2001, Stetson University

M.S. Biology, 2004, Appalachian State University
Advisor: Dr. Howard S. Neufeld
Ph.D. Candidate, Biology, Wake Forest University
Expected graduation: May 2009
Advisor: Dr. William K. Smith

TEACHING

Certification: 6-12 Biology, State of Florida Teaching License

Lecture Positions: Current Topics in Science Research (2004-2005)

Young Middle Magnet School of Math, Science, and Technology;
Tampa, FL

Biology (2001-2002)
Deltona High School; Deltona, FL

Integrated Science (2001-2002)

Deltona High School; Deltona, FL

7th Grade Science (2001)

DeLand Middle School; DeLand, FL

Laboratory Classes Taught:

Plant Systematics (2008); Wake Forest University

Plant Ecophysiology (2006) ; Wake Forest University
Comparative Physiology (2005-2007); Wake Forest University
General Biology I (2002-2004); Appalachian State University
General Biology II (2002-2004); Appalachian State University
General Biology I (2003); Caldwell Community College

Guest Lecturer: Plant Physiological Ecology, Wake Forest University (2007)

Environmental Science, R.J. Reynolds High School (2008)

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RESEARCH

Publications

Hughes NM, Neufeld HS, Burkey KO. 2005. Functional role of anthocyanins in high-light
winter leaves of the evergreen herb Galax urceolata. New Phytologist 168: 575-587.

Hughes NM, Smith WK. 2007. Attenuation of incident light in Galax urceolata
(Diapensiaceae): concerted influence of adaxial and abaxial anthocyanic layers on
photoprotection. American Journal of Botany 94: 784-790.

Hughes NM, Morley CB, and Smith WK. 2007. The coordination of anthocyanin decline
and photosynthetic maturation in developing leaves of three deciduous tree species. New
Phytologist 175: 675-685.

Hughes NM, Smith WK. 2007. Seasonal photosynthesis and anthocyanin production in ten
broadleaf evergreen species. Functional Plant Biology 34: 1072-1079.

Hughes NM, Vogelmann TC, Smith WK. 2008. Optical effects of abaxial anthocyanin on
absorption of red wavelengths by understory species: re-visiting the back-scatter hypothesis.
Journal of Experimental Botany 59: 3435–3442.

Smith WK, Hughes NM. 2008. Progress in coupling plant form and photosynthetic
function. Castanea (in press).

Hughes NM, Johnson DM, Akhalkatsi M, and Abdaladze O. 2008. Characterization of

seedling microsites at the alpine-treeline ecotone for Betula litwinowii, a deciduous treeline
species. Arctic, Alpine, and Antarctic Research (in press).

Archetti M, Döring T, Hagen S, Hughes N, Leather S, Lee D, Lev-Yadun S, Manetas Y,

Ougham H, Schaberg P, Tomas H. 2008. Adaptive explanations for autumn colours- An
interdisciplinary approach. Trends in Ecology and Evolution (accepted).

Manuscripts in Preparation

Hughes NM, Vogelmann TC, SmithWK. Attenuation of green light by abaxial

anthocyanin reduces photoinhibition under high-light in understory species.

Hughes NM, Burkey KO, Smith WK. Seasonal antioxidant and xanthophyll cycle
characteristics of broadleaf evergreen species differing in anthocyanin production during
winter.

Hughes NM, Reinhardt KS, Gierardi AR, and Smith WK. Comparative water relations
broadleaf evergreen species differing in production of anthocyanin in winter.

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Paper Presentations

Hughes NM, Smith WK. "Winter color change: why do some evergreen species synthesize
anthocyanin in winter leaves, while others don't?" Ecological Society of America, oral
presentation (Milwaukee, WI 2008).

Hughes NM, Smith WK. "Winter color change: the adaptive role of anthocyanin pigments
in broad-leaf evergreen species". Botanical Society of America, oral presentation
(Vancouver, BC 2008).

Hughes NM, Morley CB, Smith WK. "Red coloration in developing leaves: Coordination
of anthocyanin decline and photosynthetic maturation". Botanical Society of America,
poster presentation (Vancouver, BC 2008).

Hughes NM, Smith WK, and Vogelmann TC. "The adaptive significance of red abaxial
coloration in understory plants". Association of Southeastern Biologists, oral presentation
(Spartanburg, SC 2008).

Hughes NM, Smith WK, Reinhardt KS, and Burkey KO. "Winter color change in
'evergreen' leaves: The adaptive significance of anthocyanin pigments". Association of
Southeastern Biologists, poster presentation (Spartanburg, SC 2008).

Hughes NM. "Why leaves turn red: The functional significance of anthocyanin pigments in
photosynthetic adaptation". Wake Forest University departmental seminar (2008).

Hughes NM. "Winter color change: Functional role of anthocyanins in evergreen species".
Oxford University workshop on autumnal leaf colouration (2008).

Hughes NM, Smith WK, Vogelmann TC. "Abaxial anthocyanin: contrasting adaptive
function in high light and low light species". Ecological Society of America, oral
presentation (San Jose, CA 2007).

Hughes NM, Smith WK, Vogelmann TC. "Why undersurfaces of leaves are sometimes red:
contrasting the adaptive function of abaxial anthocyanin in high light and low light species
". Botanical Society of America, oral presentation (Chicago, IL 2007).

Hughes NM, Smith WK. "When are red juvenile leaves mature enough to be green? The
coordination of anthocyanin decline and photosynthetic maturation". Association of
Southeastern Biologists, oral presentation (Columbia, SC 2007).

Hughes NM, Smith WK. "Contrasting the adaptive function of abaxial anthocyanin in
leaves adapted to high-light and low-light environments". Ecological Society of America,
oral presentation (Memphis, TN 2006).

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Hughes NM, Smith WK. "The coordination of anthocyanin decline and photosynthetic
maturation in developing leaves of three deciduous tree species". Wake Forest University
Graduate Student Research day, poster presentation (2006).

Hughes NM. "Functional role of anthocyanin in high-light, winter leaves of the evergreen
herb, Galax urceolata". Appalachian State University departmental seminar (2004).

Hughes NM, Neufeld HS. "Ecophysiological and biochemical function of anthocyanins in

high-light winter leaves of Galax urceolata". Association of Southeastern Biologists, oral
presentation (Memphis, TN 2004).

Hughes NM. "Functional role of anthocyanin in high-light, winter leaves of the evergreen
herb, Galax urceolata". Sigma Xi, ASU chapter annual meeting invited seminar (2004).

Hughes NM. "Prevelance and frequency of temporomandibular joint disorders in musicians:

psychological and mechanical stress implications". Stetson University departmental
seminar (2001).

Hughes NM. "Prevelance and frequency of temporomandibular joint disorders in musicians:

psychological and mechanical stress implications". Stetson University Scholarship and
Performance Day (2001).

Awards

Physiological Section Award for Best Poster

-2008 Botanical Society of America meeting

Elton C. Cocke Award for Outstanding Graduate Student in Biology

-Wake Forest University (2008)

Best Student Paper Presentation In Botany

-2008 Association of Southeastern Biologists meeting

Best Student Poster In Botany

-2008 Association of Southeastern Biologists meeting

Euguene P. Odum Award for Best Student Paper Presentation in Ecology (runner-up)
-2008 Association of Southeastern Biologists meeting

Quarterman-Catherine Keever Award for best Student Poster in Ecology

-2008 Association of Southeastern Biologists meeting

Billings Award for Best Ecophysiological Paper Presentation (runner-up)

-2007 Ecological Society of America meeting meeting

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Conant Award for Botanical Imaging (third place)
-2007 Botanical Society of America meeting

Eugene P. Odum Award for Best Student Paper in Ecology

-2004 Association of Southeastern Biologists meeting

Outstanding Graduate Research in the Sciences

-ASU chapter of the Sigma Xi Scientific Research Society (2004)

Outstanding Graduate Teaching Assistant Award

-Appalachian State University (2004)

Biology Department’s Excellence in Research Award

-Stetson University (2001)

Outstanding Senior in the Department of Biology

-Stetson University (2001)

Randolph L. Carter Award- Outstanding Secondary School Intern

-Stetson University (2001)

Runner-up for best oral presentation at the 2001 Stetson Scholarship & Performance Day
-Stetson University (2001)

Award for outstanding contribution to music by a non-major

-Stetson University School of Music (2001)

Grants and Funding

Richter Fellowship for International Research

-Wake Forest University (2008)

Student Travel Award

-Association of Southeastern Biologists (2004, 2008)

Elton C. Cocke Travel Award

-Wake Forest University, Biology Department (2006, 2007, 2008)

Vecellio Award
- Wake Forest University (2006, 2007, 2008)

Alumni Travel Award

-Wake Forest University (2006, 2007, 2008)

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Professional Affiliations: Association of Southeastern Biologists
Botanical Society of America
Sigma Xi Scientific Research Society
Ecological Society of America
Beta Beta Beta biological honors society

FINE ARTS

Visual Art: A Minimalist Evening- Performance Art with Pianist Bruce Moser
-University of North Carolina, Greensboro (2008)

Wake Forest University Student Art Exhibition

-Charlotte and Philip Hanes Gallery, Wake Forest University (2008)

Artists on Liberty Gallery featured artist- Solo Show

-Artists on Liberty Gallery, Winston-Salem, NC (2008)

Science as Art Exhibition

-Chelsee’s Coffee Shop, Winston-Salem, NC (2008)

Music: Principal Bassoon, Stetson University Orchestra (1999-2001)

Principal Bassoon, Stetson University Symphonic Band (1997-2001)
Blue Banana Quintet, Stetson University (1998-2001)
Music Teacher, Day Spring Summer Camp (2000)

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