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Histology and cell wall biochemistry of stone cells in the physical defense of
conifers against insects
Article in Plant Cell and Environment · October 2015
DOI: 10.1111/pce.12654

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Plant, Cell and Environment (2016) 39, 1646–1661 doi: 10.1111/pce.12654

Original Article

Histology and cell wall biochemistry of stone cells in the

physical defence of conifers against insects
1 1 2 3 1
Justin G. A. Whitehill , Hannah Henderson , Mathias Schuetz , Oleksandr Skyba , Macaire Man Saint Yuen , John
4 2 3 1,2,5
King , A. Lacey Samuels , Shawn D. Mansfield & Jörg Bohlmann
1
Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4,
2
Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada, V6T 1Z4,
3
Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada, V6T 1Z4, 4British
Columbia Ministry of Forests, Lands, and Natural Resource Operations, Victoria, BC, Canada, V8W 9C2 and 5Department
of Forest and Conservation Sciences, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada, V6T 1Z4

ABSTRACT Northwest rainforest ecosystem. Because of its high susceptibil-ity

Conifers possess an array of physical and chemical defences
against stem-boring insects. Stone cells provide a physical
defence associated with resistance against bark beetles and
weevils. In Sitka spruce (Picea sitchensis), abundance of
stone cells in the cortex of apical shoots is positively
correlated with resistance to white pine weevil (Pissodes
strobi). We identified histological, biochemical and molecular
differences in the stone cell phenotype of weevil resistant (R)
or susceptible (S) Sitka spruce genotypes. R trees displayed
significantly higher quantities of cortical stone cells near the
apical shoot node, the primary site for weevil feeding. Lignin,
cellulose, xylan and mannan were the most abundant
components of stone cell secondary walls, respectively. Lignin
composition of stone cells isolated from R trees contained a
higher percentage of G-lignin compared with S trees.
Transcript profiling revealed higher transcript abundance in the
R genotype of coumarate 3-hydroxylase, a key monolignol
biosynthetic gene. Developing stone cells in current year
apical shoots incorporated fluorescent-tagged monolignol into
the secondary cell wall, while mature stone cells of previous
year apical shoots did not. Stone cell development is an
ephemeral process, and fortification of shoot tips in R trees is
an effective strategy against insect feeding.
Key-words: bark beetle; plant insect interaction; plant resis-
tance; secondary cell wall; spruce; lignin; fluorescent-
tagged coniferyl alcohol.
INTRODUCTION
Conifers of the pine family (Pinaceae) comprise a diverse group of
ecosystem defining and economically important species that have
colonized habitats around the world ranging from sub-artic to
tropical. They include some of the dominant species of boreal and
coastal forests and are of critical impor-tance for global carbon
fixation (Potter 1999). Sitka spruce (Picea sitchensis) is a
characteristic native conifer of the Pacific
Correspondence: J. Bohlmann. e-mail: bohlmann@msl.ubc.ca
to the white pine weevil (Pissodes strobi), reforestation planting of
this species has dropped by 90% in recent decades (King & Alfaro
2009). Beginning in the early spring, adult weevils feed on stems
of young trees, without causing substantial damage. However,
during late spring and early summer, mated females oviposit in the
cortex of the previous year’s apical shoot (PYAS; Fig. 1). Hatching
of larvae coincides with the time when current year apical shoots
(CYAS) begin to grow and expand. Larvae feed within the cortex
and phloem of the PYAS, which destroy both the PYAS and CYAS
in a successful weevil attack and suppresses apical growth.
Genotypes of different levels of weevil resistance have been
identified through screening of Sitka spruce accessions in repli-
cated clonal trials from across its native geographic range (King
& Alfaro 2009; King et al. 2011). Metabolite profiling of these
trees revealed an association between weevil resistance and
the terpenes (+)-3-carene, dehydroabietic acid and terpinolene
(Robert et al. 2010). Phenotypic differences in (+)-3-carene
levels were controlled by variations within a small family of
(+)-3-carene synthase genes at the genome, transcriptome, pro-
teome and biochemical levels (Hall et al. 2011; Roach et al. 2014).
This previous work successfully used two clonally prop-agated
Sitka spruce genotypes of contrasting phenotypes: the highly
resistant (R) genotype H898, originating from a high weevil hazard
area in British Columbia, and the highly suscep-tible (S) genotype
Q903, originating from the weevil-free Haida Gwaii Islands located
northwest of Vancouver Island (King & Alfaro 2009; Robert et al.
2010; Hall et al. 2011). Clonal propagation of these two genotypes
facilitated experi-ments that required a large number of individual
trees. Insect behavioural studies demonstrated that weevils were
deterred from feeding on R trees when given a choice of R and S
trees, and female weevils showed delayed ovary development and
reduced reproductive success when feeding on R trees in no-
choice experiments (Robert & Bohlmann 2010). Recent work also
identified presence of cortical stone cells (sclereids) as the
phenotypic trait with the highest predictive power of weevil
resistance in a Sitka spruce population originating from a high
weevil hazard region (King et al. 2011). Stone cells are a type of
sclerenchyma cells with extremely thick and highly

1646 © 2015 John Wiley & Sons Ltd

 Stone cells and defence against insects 1647

Figure 1. Overall distribution of stone cells in previous year apical shoots (PYAS) of Sitka spruce genotypes susceptible (Q903) or resistant (H898) to spruce
weevil. Longitudinal sections were stained with phloroglucinol-HCl and toluidine blue. (a) Location of samples and longitudinal sections along the axis of the
PYAS. (b) Magnified PYAS in longitudinal section showing location of different tissues: Pd, periderm; Cx, cortex; Ph, phloem; C, cambium; Xy, xylem.

lignified secondary cell walls (Evert 2006; Franceschi et al.
2005), which in conifers appear as individual cells or
clusters of cells in the primary cortex or secondary phloem.
Previous work in Sitka spruce and Norway spruce (Picea abies)
described stone cells that were randomly distributed in the mature
secondary phloem (Franceschi et al. 2005; Hudgins et al. 2004;
Hudgins & Franceschi 2004; Li et al. 2007; Wainhouse et al.
1990). It was suggested that stone cells are derived from
polyphenolic parenchyma cells in the secondary phloem as the
tree ages (Franceschi et al. 1998; Franceschi et al. 2005; Hudgins
et al. 2004; Hudgins & Franceschi 2004). Stone cells in the
secondary phloem of Norway spruce and Sitka spruce have been
implicated in resistance against the great spruce bark beetle
(Dendroctonus micans) (Wainhouse et al. 1998; Wainhouse et al.
1990). In contrast to previous observations in conifers, weevil
resistant Sitka spruce geno-types contain high quantities of stone
cells in the cortex rather than the secondary phloem (King et al.
2011). The cortex is a heterogeneous tissue produced during stem
primary develop-ment and is comprised of parenchyma cells,
cortical phenolic parenchyma cells, stone cells, cells containing
calcium oxalate crystals and resin canals (Franceschi et al. 2005).
The cortex is
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
thought to be a protective barrier of the growing shoot tips and
branches, which is eventually shed as a result of secondary
growth and replaced by the secondary phloem and bark as a
lasting outer defensive barrier (Franceschi et al. 2005). It is
also the primary site for weevil oviposition and feeding by
adult weevils and their larvae. Thickness of the primary cortex
is an important factor in the weevil life cycle as it affects
oviposition behaviour and supports developing larvae. The
thickest section of the primary cortex in Sitka spruce is located
along needle ridges on the upper portion of the PYAS tip,
which is the primary site for weevil feeding and oviposition in
the field (Fig. S1) (Manville et al. 2002). Highly weevil resistant
Sitka spruce genotypes contain significantly higher quantities
of stone cells in the cortex when compared with susceptible
trees (King et al. 2011).
Ultrastructural, immunohistochemical and metabolite analy-ses
of stone cells have been difficult in spruce due to their irregular
patterns of distribution in mature secondary phloem and often low
abundance (Hudgins et al. 2004). In addition, due to limited
knowledge of the onset and progression of stone cell
development, prior work has focused on the description of
presence or absence of fully developed mature stone cells
1648 J. G. A. Whitehill et al.
(Hudgins et al. 2004). Information about the biochemical
composition of the cell-type defining thick cell walls of isolated
conifer stone cells is missing. However, some prior knowledge is
available from analyses of complex mature secondary phloem
tissue, including stone cells, of Sitka spruce and Norway spruce
resistant to the great spruce bark beetle. High levels of lignin in
these tissues were attributed to stone cells being completely
lignified (Wainhouse et al. 1990). Furthermore, Li et al. (2007)
reported the presence of minute quantities of two
phenylpropanoids, the stilbene astringin and the dihydroflavonol
dihydroxyquercetin 3′-O-β-D-glucopyranoside, as part of the
soluble small molecule composition of stone cells isolated from the
secondary phloem of Norway spruce. In a different system, the
fruits of pear (Pyrus spp.), stone cells are described as being
primarily comprised of cellulose and lignin (Tao et al. 2009).
Here, we describe the contrasting stone cell phenotype of
weevil resistant and susceptible Sitka spruce genotypes. The high
abundance and consistent spatial and temporal patterns of
occurrence of stone cells in the cortex of apical shoots of R trees
permitted their isolation and detailed characterization ex situ, in
addition to in situ comparison of stone cell variation between R
and S trees. We used a combination of confocal and electron
microscopy, immunohistochemical staining, me-tabolite analysis,
transcript profiling of monolignol biosynthesis genes and
fluorescence-tagged monolignol incorporation as-says to
document the biochemical and molecular characteristics that
differentiate stone cells in the R and S genotypes.
MATERIALS AND METHODS
Plant materials and source of stone cells
Origins and growing conditions of clonally propagated Sitka
spruce genotypes resistant and susceptible to the weevil were
previously described (Robert & Bohlmann 2010). In brief, H898
(R) originates from a high weevil hazard region of the lower Fraser
Valley, British Columbia, Canada (49°14′N: 122°36′W); Q903 (S)
originates from the Haida Gwaii Islands (53°55′N: 132°05′W) with
no history of weevil attack. Grafted saplings were produced in
2008 and maintained in pots in an out-side nursery at the
University of British Columbia. Characteriza-tion of stone cells was
performed with PYAS tips or CYAS tips collected from intact
saplings (Fig. 1). PYAS tips are the primary site of weevil
oviposition in the late spring and early summer. Larvae hatch, feed
and pupate in the phloem and cortex of the PYAS. We used PYAS
that had completed expansion growth starting from an apical shoot
bud in the previous year. The PYAS went through overwintering
dormancy, which was completed when the terminal bud began to
swell and the CYAS started to expand. This stage of the PYAS
defines the conditions encountered by ovipositing adult weevils
and young feeding larvae. Stone cells were formed in expanding
CYAS tips. Stone cells in the fully expanded PYAS tip were
defined as mature. Stone cells in the cortex from the top 2 cm of
the PYAS tip were used for most of the analyses presented in this
paper, unless otherwise noted. For metabolite analyses, cortex
tissues were harvested, stone cells isolated and analyses
performed on both isolated stone cells and complex cortex
tissues.
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
Histology to determine stone cell abundance and
distribution
Distribution of stone cells in the cortex was measured in PYAS of
four R and four S trees collected on May 21, 2014 [486 growing
degree days (GDDs)]. The average PYAS length was 26.6 ± 2.2
cm (R) and 34.4 ± 2.0 cm (S). To compare stone cell abundance,
we standardized relative location along the length of each PYAS
by dividing each shoot into three equal sections (top, middle and
bottom; Fig. 1). For each tree, the top 2 cm of each of these three
sections were used for histology. Each 2-cm-long shoot section
was split longitudinally into two equal half sections, placed into
FAA fixative at 4 °C for 1 week. Samples were submitted to the
Wax-It service laboratory (Vancouver, BC, Canada) for
dehydration in an ethanol series and paraffin embedding. Paraffin-
embedded samples were thin-sectioned (20 μm), sections stained
with toluidine blue and counterstained with phloroglucinol-HCl.
Images were captured using an Aperio ScanScope CS digital
slidescanner (Aperio, Vista, CA, USA) for quantification of stone
cell abundance relative to total cortex area using the Aperio
ImageScope software. Regions identified as cortical resin canals
were subtracted using the Aperio negative pen tool. The Aperio
positive pixel count algorithm v. 9 was used to quantify tissue
staining (phloroglucinol red). The number of positive pixels (weak
to strong) was normalized to the number of total pixels (positive +
negative) to account for variations in the size of the region
sampled. Colour thresholds were established to detect the
phoroglucinol-HCl stain (cortical stone cells) as positive pixels and
background staining as negative pixels (normal cortical
parenchyma cells). The resulting colour markup was manually
inspected for each sample. Values for % stone cells, % cortical
parenchyma and % resin canals were compared among genotype
and location using two-way analysis of variance (ANOVA).
Following significant F-tests, means were separated post hoc
using the LSD test (α = 0.05). All data were analysed using IBM
SPSS Statistics v. 19 (SPSS Inc.).
Transmission electron microscopy of mature stone
cells
For transmission electron microscopy (TEM), stone cell sam-
ples were collected from the top 2 cm of PYAS tips collected
on July 2, 2013 (923 GDD). A cross section was cut from the
middle of the top 2 cm section (~1 mm width). A small block of
cortex (~1–2 mm wide × ~1–2 mm long × ~1–2 mm depth)
was then placed in fixation buffer [50 mM PIPES pH 7.2,
1.25% w/v gluteraldehyde, 2% w/v paraformaldehyde
(Karnovsky’s fix)]. Samples were placed under vacuum for 2 h,
stored overnight at 4 °C in Karnovsky’s fix and placed again
under vacuum for an additional 2 h. Samples were removed
from Karnovsky’s fix, washed 3× for 10 min each with 0.1 M
sodium cacodylate (pH 7.2) under vacuum, transferred into
fresh 0.1 M sodium cacodylate and placed under vacuum for 1
h. Samples were postfixed with 1% w/v OsO4 in 0.1 M sodium
cacodylate, pH 7.2. Samples were washed in distilled water
and dehydrated under vacuum in an ethanol series. Samples
were left under vacuum for 1 h at each dehydration

step (10% 100% v/v EtOH with 10% increments). At 100%,
samples were left under vacuum for 1 h and placed into fresh
100% EtOH and repeated three times. After the final 100% EtOH
dehydration wash under vacuum, samples were left in fresh 100%
EtOH and stored at 4 °C overnight after which samples were
transferred to 100% acetone for 1 h under vacuum to remove
excess water. Samples were washed three times with acetone
under vacuum for 1 h for each dehydration wash. Samples were
transferred from 100% acetone into three sequential infiltration
steps of a 3:1, 1:1 and 1:3 acetone to a Spurr’s resin mix for 1–2 h
for each infiltration step. Following the final infiltration, samples
were transferred to a 100% Spurr’s resin mix and placed under
vacuum for 1 h. Samples were then transferred into fresh resin
three times and held under vacuum for 1 h at each step. Following
the final resin wash, samples were placed into freshly made
Spurr’s resin and placed on an orbital shaker at room temperature
(RT) overnight. The following day, samples were placed in freshly
made Spurr’s resin and arranged individually in size ‘00’ flat
bottom embedding capsules (Electron Microscopy Sciences,
Hatfield, Pennsylvania, USA), covered with fresh Spurr’s resin and
cured overnight at 60 °C. Stone cell samples were thin sectioned
(~70–90 nm) with a Leica EM UC6 ultramicrotome using an ultra
35° diamond knife (Diatome, Biel, Switzerland) and mounted on
100-mesh 0.5% Formvar-coated copper grids. Sections were
stained with 2% w/v uranyl acetate in 70% v/v methanol for 20 min
followed by Reynold’s lead citrate for 10 min. A Hitachi H7600
TEM was used to examine the stained samples, and images were
taken with an AMT Advantage (1 megapixel) CCD camera
(Advanced Microscope Technologies).
Light microscopy and immunohistochemistry
Samples for light microscopy (LM) and immunohistochemistry
(including LM10, anti-mannan and Pontamine Fast Scarlet 4B
Staining) were collected on June 27, 2012 (~750 GDD) from R
and S PYAS tips. Samples were fixed overnight at 4 °C in
Karnovsky’s fix, dehydrated (no postfixation in OsO 4) in an
ethanol series and embedded in London Resin (LR) White
acrylic resin. Cross sections and radial longitudinal sections
were thin sectioned (500 nm) with a Leica EM UC6
ultramicrotome and a histo 45° diamond knife (Diatome, Biel,
Switzerland) and transferred to Teflon multiwell slides (EMS
cat # 63424-06) coated with poly-L-lysine. For routine LM,
sections were stained with Stevenel’s blue (del Cerro et al.
1980) and imaged on a Leica model LMD 6000 Laser
Microdissection Microscope with a Hitachi analog 3 CCD cam-
era. For immunohistochemistry, sections were covered with
100 μL of 5% BSA in TBST (10 mM Tris-buffer pH 7, 0.25 M
NaCl, with 0.1% Tween 20) buffer and incubated for 1 h at RT
with slight agitation. Sections were washed with TBST buffer
and incubated with 1:36 dilution (36 μL/well) of anti-β-(1–4)-D-
mannan monoclonal antibody (Biosupplies Australia Pty Ltd.,
Melbourne, Australia; cat # 400-4) or anti-xylan LM10 antibody
(www.plantprobes.net) for 4 h, followed by three washes with
TBST buffer (100 μL/well) for 1 min each to remove free
antibody. Sections were incubated with 1:100 dilution of
secondary antibody (Alexa 488 – 36 μL/well) in
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
Stone cells and defence against insects 1649
TBST buffer for 1 h at RT in the dark. Sections were washed
twice in TBST buffer, resuspended in 100 μL/well of TBST
buffer and transferred to slides, mounted in TBST buffer, and
covered with a coverslip for imaging. Fluorescent signals were
detected with a Perkin-Elmer UltraView VoX spinning disk
confocal mounted on a Leica DMI6000 inverted microscope
and a Hamamatsu 9100-02 CCD camera. Samples were
mounted in water and imaged using a Leica glycerol
immersion 40× objective (Plan-Apo, NA 1.4). A 488 nm laser
was used for excitation and 525 nm emission filter for imaging
mannan/xylan localization. Autofluorescence under blue light
was used as a counter image to mannan/xylan localization
(Franceschi et al. 1998). Pontamine Fast Scarlet 4B (Sigma)
was used to stain sections (500 nm) prepared as described in
the preceding texts. Sections were stained for 10 min in S4B
staining solution [0.1% S4B (w/v) in ½ strength MS media
(Murashige & Skoog 1962)], washed and mounted in ½
strength MS media and mounted onto microscope slides. A
561 nm laser was used for excitation and 595 nm emission
filter for imaging Pontamine Fast Scarlet 4B-stained cellulose.
Time course analysis of stone cell development
Stone cell development was tracked in the cortex of R trees.
GDDs, calculated using the average method (Herms 2004), were
employed as the reference system for time points during the
growing season. A developmental base threshold tempera-ture of
5 °C was used for GDD calculations (Alfaro et al. 2000). GDDs
were calculated from daily maximum and minimum temperatures
measured at Vancouver International Airport
(http://climate.weather.gc.ca) with January 1 as the starting date
for a given year. Beginning March 8, 2012 (~90 GDD) the top 2
cm of the CYAS tips were sampled from individual trees in bi-
weekly or weekly intervals once the terminal bud began to
expand. Sections were split longitudinally, stained with
phloroglucinol-HCl and viewed at 40× magnification under a
dissecting microscope. Nascent stone cells were first observed in
the cortex on June 27, 2012 (~750 GDD) (Fig. S2). Subsequent
characterization of stone cell secondary cell walls focused on the
period of 675 to 850 GDD.
Stone cell isolation for cell wall analyses
Tissues for carbohydrate and lignin analyses were collected on
June 20, 2014 (780 GDD) from R and S trees. Analyses of lignin
monomers (thioacidolysis), lignin and carbohydrates were per-
formed with triplicate trees of each genotype. Stone cells were
isolated from bark (including periderm, cortex, secondary phloem,
cambium) of the top 2 cm of PYAS tips and lateral branches from
the same whorl to obtain sufficient amounts of isolated stone cells.
In addition, needles, bark and xylem were harvested from PYAS
for cell wall analyses. Materials were stored at 80 °C until
processing. For stone cell isolations, bark samples of 2–5 g (FW)
were homogenized in liquid N2 using a mortar and pestle, washed
twice with 50 mL double-distilled (dd)H 2O, twice with 50 mL 100%
(v/v) EtOH and twice with 50 mL ddH 2O. Following each washing
step, samples were spun at 500g for 1 min and the supernatant
discarded. Following the
1650 J. G. A. Whitehill et al.
final ddH2O wash, pellets were washed twice with acetonitrile.
Pellets were suspended in fresh acetonitrile and left overnight at 4
°C, then centrifuged (1000g) and acetonitrile discarded. Pellets
were washed once with ddH 2O, resuspended in ddH2O, sonicated
twice for 90 s, centrifuged (1000g) and supernatant removed.
Pellets were then incubated for 20 min at RT in tert-butyl-methyl
ether (TBME) to remove oleoresin and other hydrophobic
constituents, after which the supernatant was discarded. Pellets
were left at RT to allow the TBME to evapo-rate overnight in a
fume hood. Dried pellets were washed four times with ddH 2O. The
resulting samples consisted of individual stone cells, stone cell
clusters, xylem fibers and cellular debris. To further enrich for
stone cells, samples were subjected to a glyc-erol gradient (100,
75 and 50%) in a 50 mL centrifuge tube and spun at 100g for a
single 5 to 10 s burst. The bottom layer contain-ing stone cells
was removed with a pipet and transferred to a new tube. The top
layer was transferred to a new gradient and the process repeated.
The bottom layer from the second gradient was combined with the
first bottom fraction, and the combined stone cell fraction was
subjected to a new glycerol gradient. This process was repeated
as necessary until a highly enriched stone cell isolate was
attained. The final stone cell-enriched isolate was washed three
times (to remove any remaining glycerol) and resuspended in
MilliQ H2O. Purification and enrichment of stone cells were
monitored using a dissecting microscope. Final clean-up of the
stone cells was performed in a Petri dish under a dissecting
microscope where stone cells were manually sepa-rated from non-
stone cell material using fine point tweezers and a pipet.
Collections of R stone cells (~200 mg DW for each of three
biological replicates) took 1–2 weeks; collections of S stone cells
(~200 mg DW for one replicate) took 1.5 months and consisted of
stone cells pooled from ~80 separate trees. Each biological
replicate was collected in a 1.5 mL microcentrifuge tube and were
filled to just above 0.5 mL mark (~200 mg DW) and stored at 4 °C
in MilliQ H2O until processing for cell wall analyses.
Klason analysis
Soluble and cell wall-bound phenolics were removed from
plant materials prior to cell wall analyses according to Bonello
& Blodgett (2003). Plant tissues were ground to a fine powder,
and Klason analyses were performed as described in Coleman et
al. (2008). Because of the very small amounts of stone cells in S
trees, we could not compare variation among biological replicates
of S stone cells. Instead, we analysed a single sample of pooled S
stone cells isolated from ~80 separate individual trees (~100 mg
DW). For all other samples, data obtained for carbohydrates, acid-
soluble and acid-insoluble lignin and total lignin were compared
among genotype and tissue type using two-way ANOVA for
needles, bark and xylem. Following significant F-tests, means
were separated post hoc using the protected LSD test (α = 0.05).
All data were analysed using IBM SPSS Statistics v. 19 (SPSS
Inc., 2010).
Thioacidolysis analysis
Following removal of soluble and cell wall-bound phenolics,
samples of stone cells, needles, bark and xylem were processed
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
according to previously published methods (Robinson & Mansfield
2009). Due to the very small amounts of stone cells in S trees, we
could not compare variation among biological replicates of S stone
cells. Instead, we analysed and compared S stone cells in two
separate approaches. First, stone cells iso-lated from three
biological replicates were pooled and analysed. Second, to
statistically compare lignin composition of S stone cells, three
additional technical/pseudo replicates (~10 mg DW) were
analysed from a pooled S stone cell collec-tion that consisted of
stone cells isolated from ~80 separate individual trees. All tissues
were ground to a fine powder and oven-dried at 70 °C overnight. A
revised thioacidolysis procedure was then performed on each
sample according to Robinson and Mansfield (2009). Lignin
proportions were com-pared among genotype and tissue type
using two-way ANOVA. Lignin data did not meet assumptions of
normality and homogeneity of variance, even after several
transformations. Thus, non-parametric methods were used to
compare samples. Lignin proportions were rank-transformed and
analysed using the Kruskal–Wallis test to identify significant
differences among all treatment and species combinations. The
Kruskal–Wallis test was also used post hoc to separate treatment
and species combinations in pairwise comparisons (α = 0.05). All
data were analysed using IBM SPSS Statistics v. 19 (SPSS Inc.,
2010).
Fluroescent-tagged monolignol incorporation
assays
γ-Nitrobenzofuran (NBD)-tagged coniferyl alcohol (NBD-CA)
(Tobimatsu et al. 2011; Tobimatsu et al. 2013) feeding experi-
ments were performed using the top 2 cm of resistant CYAS
tips collected on June 28, 2013 (810 GDD) to correspond with
the time when stone cell secondary walls are normally
developing. Longitudinal cross sections of CYAS tips were
sectioned (70–100 μm) using a Bausch and Lomb sliding
microtome. Sections were transferred to a 6-well cell culture
plate and immersed with ½ MS media. Labelling of cell wall
lignin using NBD-CA and imaging of samples was carried out
as described in Schuetz et al. (2014).
qRT-PCR analysis
CYAS and PYAS from R and S trees were collected on June 27,
2012 (750 GDD). Bark (periderm, cortex, secondary phloem,
cambium and some secondary xylem) was ground to a fine
powder, and total RNA was isolated using the Purelink® Plant
RNA Reagent (Life Technologies). Total RNA concen-tration was
determined using a Nanodrop 1000 (Thermo Scien-tific), and RNA
integrity was assessed using Bioanalyzer 2100 RNA Nano chip
assays (Agilent). Equal RNA amounts were used for cDNA
synthesis with the Maxima First Strand cDNA Synthesis kit (cat #
K1671, Thermo Scientific) and random hexamer oligonucleotides.
Sequences for monolignol pathway genes were identified in the
Sitka spruce expressed sequence tag (EST) and full-length cDNA
(FLcDNA) database (Ralph et al. 2006) using reference
sequences from BRaunschweig EN-zyme Database (BRENDA)
(Schomburg et al. 2002; Table S1).
 Stone cells and defence against insects 1651

Sitka spruce accessions with ≥90% amino acid identity to
previously characterized conifer orthologs were selected for
qRT-PCR (Table S2). The full-length PsCCR sequence (NCBI
GenBank under accession KR758750) was generated by
identifying the clone in our cDNA library glycerol stocks and
sequencing the cDNA insert from both ends (Ralph et al.
2006). Putative gene models for monolignol pathway genes
were identified via exonerate [protein2genome model, version
2.2.0, (Slater & Birney 2005)] by aligning Sitka spruce
sequences to the white spruce genome (Birol et al. 2013;
Warren et al. 2015). Gene-specific primers were designed to
cover a <200 nt region unique to the gene of interest and
bridged an intron–exon boundary (Table S3). Reference gene
selection was performed using geNorm (Vandesompele et al.
2002) included in the qbase+ software (Hellemans et al.
2007). Relative transcript abundance was calculated based on
the geometric mean of ELF1a and Cdc2 (Table S3) with the
comparative cycle threshold (∆∆Ct) method using triplicate
measurements with four biological replicates. Efficiencies for
all primer pairs were calculated using LinRegPCR (Ruijter et
al. 2009). qRT-PCR reactions were performed on a Bio-Rad
CFX96 real-time system using the SsoFast kit (www.bio-rad.
com). Target gene specificity was confirmed by sequence
verification of amplicons. Calibrated normalized relative
quantities (CNRQs) were compared among genotype and
tissue type using two-way ANOVA. Following significant F-
tests, means were separated post hoc using the LSD test (α =
0.05). All data were analysed using IBM SPSS Statistics v. 19
(SPSS Inc., 2010). Sequences were analysed in CLC Bio
Main Workbench 7.6 (CLC bio, Århus, Denmark).
parenchyma cells were the opposite to cortical stone cells with
the top location (F2,23 = 40.785; P < 0.001) having the overall
least amount of cortical parenchyma cells. R trees had lower
amounts (F1,23 = 25.076; P < 0.001) of cortical parenchyma at
the top PYAS location than S trees (Fig. 3b). The percentage
of resin canals did not differ between the two genotypes
(F1,23 = 1.607; P = 0.221), location (F 2,23 = 0.050; P = 0.951) or
the genotype × location interaction (F2,23 = 1.960; P = 0.170)
(Fig. 3c). Significant interaction between genotype and location for
percent abundance of stone cells (F2,23 = 23.550; P < 0.001) and
percent abundance of cortical parenchyma (F2,23 = 10.811; P <
0.001) was due to the differences in stone cell abundance
between R and S trees at the top location.
Anatomical and ultrastructural characteristics of
stone cells in R and S genotypes
The cortex of Sitka spruce (Fig. 4) consists of parenchyma cells,
cortical phenolic parenchyma cells, stone cells and resin ducts
(Franceschi et al. 2005). In the R genotype, stone cells are highly
abundant in cross sections of the cortex, appearing as large, densely
packed clusters separated from one another by parenchyma cells or
cortical phenolic parenchyma cells (Fig. 4a,c). Occasionally, cortical
phenolic parenchyma cells may be found within these clusters of stone
cells (Fig. 4c,e). In the low-stone cell S genotype, stone cells appear
mostly as isolated clusters

RESULTS
R and S genotypes differ in stone cell abundance at
the top of the apical shoot tip
Stone cells develop in the cortex of the Sitka spruce CYAS tip
between 600 and 750 GDD (Fig. S2). Longitudinal sections of the
PYAS tip revealed larger, more abundant stone cell clusters in the
R genotype, compared with the S genotype (Fig. 1a). The
distribution of stone cells in the PYAS was restricted to the cortex
underlying the periderm (Fig. 1b). Phloroglucinol-HCl staining
showed overall higher abundance of lignified stone cells in the top
2 cm of the PYAS tip of the R genotype (Fig. 2a,c) when
compared with the same region of the S geno-type (Fig. 2b,d).
Interestingly, in R trees, stone cells were found throughout the
cortex (Fig. 2a), while in S trees, stone cells were almost
exclusively found in close proximity to vascular tissues (Fig. 2d). In
a quantitative analysis, stone cells were significantly (F 1,23 =
31.215; P < 0.001) more abundant in cortex from the top 2 cm
section of the PYAS tip of R trees compared with S trees (Fig. 3a).
When comparing the abundance of stone cells along the length of
the PYAS (Fig. 1), in both genotypes, stone cells were significantly
(F2,23 = 65.248; P < 0.001) more concentrated in the top location,
but stone cell abundance was not significantly different between
Figure 2. Distribution of stone cells near the apical bud of the PYAS
the middle and bottom locations within or between genotypes (Fig. tips in R and S trees. Longitudinal sections were stained with
3a). Differences in the pattern of distribution and abundance of phloroglucinol-HCl and toluidine blue showing differences in stone cell
cortical abundance and morphology in R (a, c) and S trees (b, d).
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
1652 J. G. A. Whitehill et al.
near needle fascicle vascular bundles (Fig. 4b,d). These clusters
may also include cortical phenolic parenchyma cells (Fig. 4d,f).
Stone cells at the periphery of a cluster may be in contact with ray
parenchyma originating within the vascular bundle (Fig. 4h). Stone
cells in S trees appear to be almost completely
Figure 3. Percent area of cortex occupied by stone cells, resin canals and
cortical parenchyma. The abundance (% total area of the cortex) of stone
cells, cortical parenchyma and resin canals were compared in the cortex of
R and S genotypes at three defined locations along the length of the PYAS
(top, middle and bottom; Fig. 1). Quantities of (a) stone cells was
significant for genotype (P < 0.001), location (P < 0.001) and genotype ×
location (P < 0.001); (b) cortical parenchyma was significant for genotype
(P < 0.001), location (P < 0.001) and genotype × location (P < 0.001) and
(c) resin canals did not differ for any of the factors analysed. Error bars
represent the standard error of the mean. Differences between
genotype and location were analysed using two-way analysis of
variance (ANOVA). Different letters indicate significantly different
means (analysed within tissue type) separated post hoc following
significant F-tests by the protected LSD test (α = 0.05).
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
filled in with secondary walls and have very little to no
remain-ing cell lumen (Fig. 4f), compared with R stone
cells, which typically have a larger lumen (Fig. 4e,g).
Transmission electron microscopy of adjacent stone cells
within a cluster showed the primary cell wall (PCW) as an
electron-dense border outside of the secondary cell wall layers
and a compound middle lamellae (CML) that separate
individual cells (Fig. 5a,b). The secondary cell wall of R and S
stone cells are comprised of tightly packed regular concentric
layers (SL) interrupted by pit canals (Fig. 5c,d). Examination of
the ultrastructure at higher magnification revealed the
boundary between concentric layers containing fibrils that are
arranged in opposing heliocoidal bow-shaped arcs (Fig. 5e,f).
In both R and S stone cells, secondary cell wall organization of
stone cells is consistent between genotypes, with multiple
thickened wall layers.
Imaging of cellulose and hemicelluloses in cell
walls of stone cells
To characterize the composition of the thickened secondary cell
wall layers of stone cells, cellulose staining was performed using
Pontamine Fast Scarlet 4B (Anderson et al. 2010). The secondary
cell walls of stone cells fluoresced brightly, in contrast to the low
background fluorescence of the PCWs of the cortex. The variable
shape of the resistant stone cells and their dense secondary cell
walls with abundant pits were clearly revealed by the cellulose
stain (Fig. 6a). This was in contrast to the compact, more rounded
shape of the stone cells in suscep-tible trees (Fig. 6b). Although
the stone cells had different overall architecture, cellulose-rich cell
wall layers and pits crossing the thick secondary wall were
common features.
Xylans and mannans are the main secondary cell wall-
specific hemicellulose constituents of conifer tracheids, in the
form of glucuronoarabinoxylan and galactoglucomannan
(Ebringerová et al. 2005; Nanayakkara et al. 2009; Scheller
& Ulvskov 2010; Donaldson & Knox 2012). To determine if
these same hemicellulosic components are present in stone
cell walls, both xylan and mannan epitopes were examined
using immunofluorescent labelling. Xylan distribution was
probed using anti-xylan LM10 antibody, which is specific for
the xylose backbone of the glucoronoxylan (xylan) hemicellu-
lose (McCartney et al. 2005). LM10 labelling demonstrated the
presence of xylan in the secondary cell walls of mature stone
cells in both genotypes but not in the adjacent primary walls
(Fig. 7a,b). In addition, we used BGM C6 that recog-nizes
beta-1,4 mannan epitopes (Pettolino et al. 2001), such as
galactoglucomannan found in conifer secondary cell walls
(Kim et al. 2011). Anti-mannan labelling was similar to that
observed for xylan using LM10 antibodies and was restricted
to the secondary cell walls of stone cells (Fig. 7c,d). Thus, the
secondary cell walls of stone cells from both genotypes are
made up of cellulose and hemicelluloses that are similar to
those of tracheids of common gymnosperms. Morphological
features detected in these histochemical studies (Figs. 2 & 4),
such as stone cells with open lumens and multiple pits, were
consistently observed.
 Stone cells and defence against insects 1653

Carbohydrate and lignin cell wall metabolites of
stone cells in R and S trees
Following imaging of the cell wall components for lignin (Fig.
2), cellulose (Fig. 6) and hemicelluloses (Fig. 7), we performed
quantitative metabolite analyses of cell wall composition of
xylem and bark, as well as needles from the PYAS of R and S
trees using Klason analysis (Table 1). We also performed
quantitative chemical analyses of isolated stone cells from R
and S trees, including statistical analysis of
variation of stone cell composition from multiple trees representing
biological replicates of R trees. Because of the very limited
amounts of stone cells present in S trees, it was not possible to
determine variation among biological replicates of S stone cells.
With regard to cell wall carbohydrates, stone cells from both
genotypes had higher quantities of xylose than any of the other
tissue types. This is surprising given that gymnosperms are
known to be mannan-rich and that the xylan-based hemicelluloses
are only minor components. Between the two genotypes, R stone
cells had higher amounts of glucose

Figure 4. Anatomy of stone cells in Sitka spruce cortex cross sections from R and S genotypes. Samples were taken from the top 2 cm of the
previous year apical shoot tip, embedded in LR White resin, sectioned (500 nm) and stained with Stevenel’s blue. R (a, c, e, g) samples have more
cortical stone cells than samples from the same location on S (b, d, f, h) samples. CP, cortical parenchyma; CPC, cortical phenolic parenchyma cell;
R, ray cell; SC, stone cells/cluster; SCL, stone cell lumen; VB, vascular bundle.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
1654 J. G. A. Whitehill et al.
Figure 5. Transmission electron micrographs (TEMs) of stone cells in
R and S PYAS tips. Ultrastructure of R (a, c, e) stone cells is similar to
that found in S (b, d, f) stone cells. B, boundary between secondary
wall layers; CF, cellulose fibrils; CML, compound middle lamellae;
PCW, primary cell wall; Pi, pit; SL, secondary layer.
than S stone cells. Among tissue types, the carbohydrate
composition of stone cells appeared unique and most notably
differed from xylem tissues, which contained higher quantities
Figure 6. Cellulose in stone cell secondary cell walls. Pontamine S4B staining and fluorescence imaging of cellulose in secondary cell walls of stone
cells from R (a) and S (b) genotypes. Arrows in (a) and (b) point to pits in stone cell secondary walls. L, lumen.
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
of cellulose but less lignin. Lignin was the most abundant
chemical constituent of stone cells, and stone cells contained
more lignin than any other tissue type investigated (Table 1).
Xylem, a highly lignified tissue, contained less acid-insoluble
1
lignin and total lignin than S stone cells (~170 mg g DW) and
1
R stone cells (~110 mg g DW). Between genotypes, R stone
cells contained less acid-insoluble lignin and total lignin than S
stone cells. Overall, cell wall carbohydrates, lignin and total
recovered varied across genotype and tissue type and are
included for comparative purposes against stone cell tissues in
Table 1 and Table S5.
Using thioacidolysis analysis, we further compared the lignin
subunit (G/H) ratios between R and S genotypes and across
PYAS tissue types using the Kruskal–Wallis test statistic.
Although not significantly different, subunit ratios tended to
differ between genotypes (H = 104.000; P = 0.065; N = 24)
and tissue types (H = 6.887; P = 0.076; N = 24) (Table 2).
While not statistically significant based on three replicates, S
stone cells consistently had a higher proportion of H-lignin
subunits and lower proportion G-lignin compared with R stone
cells and to the other tissues analysed (Table 2).
In vivo cell wall incorporation of monolignol by
developing stone cells
Developing stone cells require substantial quantities of soluble
metabolites to assemble the thickened secondary walls, yet once
formed are considered senescent. To assess the capacity of
developing and mature stone cells to incorporate monolignols into
their cell walls in vivo, we performed feeding experiments with
NBD-CA (Tobimatsu et al. 2013) using the top 2 cm of R CYAS
tips at 810 GDD. This sample was chosen as it represents a time
point of active stone cell development as assessed by microscopic
analysis (Fig. S2). We detected the incorporation of fluorescence-
tagged coniferyl alcohol specifi-cally in CYAS tips developing
stone cells that may not have completed secondary cell wall
deposition (Fig. 8a,b). In contrast, mature stone cells of the PYAS
tips showed no fluorescence after incubation with NBD-CA
indicating that

Figure 7. Histo-immunochemical labelling of hemicellulose in mature cortical stone cells. Antibodies were used specific to the hemicelluloses xylan
(a, b) and mannan (c, d) and fluorescence detected in stone cell secondary cell walls of R (a, c) and S (b, d) stone cells. Autofluoresence of cortical
tissues is used as counter image (red).
the biochemical capacity to polymerize monolignols into the
secondary cell wall was no longer active. As a positive control,
we also examined NBD-CA incorporation into xylem cells from
CYAS and PYAS (Fig. 8e,f). Sitka spruce xylem tissue
incorporated the fluorescent-tagged monoliginol in a similar
manner as has been previously reported for other conifer
xylem tissue (Tobimatsu et al. 2013).
Transcript abundance of the monolignol
biosynthetic pathway
The results described in the preceding texts lead us to examine
the transcript abundance of a set of 10 genes representing eight
steps in the monolignol biosynthetic pathway in PYAS and CYAS
in the R and S genotypes. PsC4H, PsHCT, PsCCOMT2 and
PsCAD1 were expressed differentially between genotypes (Fig. 9
and Table S4). All genes were more highly expressed in the S
genotype. Overall, CYAS had higher transcript levels for all
monolignol biosynthetic pathway genes tested except PsC4H,
PsCAD1 and PsCAD2, which did not differ between CYAS and
PYAS. Interactions between genotype and tissue
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
Stone cells and defence against insects 1655
type were observed for PsCCR with PYAS from R trees
having lower transcript levels than all other comparisons (Fig.
9). PsC3H and Ps4CL had higher transcript levels in CYAS of
R than S genotypes. Conversely, transcripts of PsC4H and
PsHCT were more abundant in CYAS of S than R trees (Fig. 9
and Table S4). Expression of monolignol biosynthetic pathway
genes was more active in CYAS than PYAS, and higher
expres-sion of PsC3H in R CYAS indicates a difference in
regulation of G-lignin production between genotypes.
DISCUSSION
Stone cells are an important constitutive physical defence of coni-
fers against stem-feeding insects, but until now, little is known
about their histology, chemical composition and development.
Here, we characterized and compared cortical stone cells in
representative weevil resistant and susceptible Sitka spruce
genotypes with high and low quantities of cortical stone cells,
respectively. Clonally propagated trees of these genotypes were
previously established to allow for comparative studies that
require a large number of trees for replicated molecular and
1656 J. G. A. Whitehill et al.

biochemical analyses (Robert & Bohlmann 2010; Robert et al.

2010; Hall et al. 2011). In both genotypes, cortical stone cells were

NChemicalcompositionofPYAS(PreviousYearApicalShoot)tissuesfromhigh(R)andlow(S)stonecellcontainingSitkasprucegenotypesasdeterminedbyKlasonanalysis. = 1 for S stoneNcellsandisapooledsampleisolatedfrom~80clonalsprucetrees.Dataisincludedhereforcomparativepurposesbutwasnotanalysedstatistically(denotedwithasolidblackline).=3forallothersamples;standarderrorinparentheses.DifferentlettersindicatesignicantlydifferentmeansseparatedwithincolumnbytheprotectedLSDtest(=0.05).αfi

Total recovered
consistently located at the top most sections of apical shoots,
which is the primary location for oviposition and feeding by weevils
(Manville et al. 2002; Silver 1968). In the weevil resistant

93. (0.7) a025877514762267515Sxylem1.(0.1)cND4.(0.1)b38.(0.6)a7.(0.2)b9.(0.5)a0.(0.01)b30.(0.2)a31.(0.2)b92.(0.8)aRbark8.(0.6)a0.(0.1)a2.(0.1)d27.(1.0)bc4.(0.2)c3.(0.2)c1.(0.04)a18.(0.8)c20.(0.c66.(0.7)c3617237300Sbark9.(0.4)a0.(0.02)a2.(0.1)c29.(2.2)b3.(0.1)d3.(0.1)c1.(0.06)a16.(0.9)c18.(0.8)c66.(2.5)c07644376366282537961Rneedle3.(0.04)b0.(0.1)b1.(0.02)d28.(2.6)bc4.(0.1)c6.(0.1)b1.(0.05)a27.(0.5)b29.(0.6)b74.(1.9)bSneedle3.(0.1)b0.(0.1)b1.(0.02)d24.(1.0)c3.(0.03)d6.(0.1)b1.(0.03)a31.(1.1)a33.(1.1)a74.(0.9)b6296887520Rstonecells4.(0.2)0.(0.1)1.(0.03)30.(0.9)13.(0.8)5.(0.1)0.(0.2)40.(0.9)41.(0.9)96.(0.7)223401786303495301993Sstonecells4.0.1.24.12.4.1.46.47.95.

genotype, significantly higher quantities of cortical stone cells were
present at the top, but not lower portions, of the PYAS compared
with the S genotype. This indicates that fortifying the top of the
Total lignin

shoot tip with abundant stone cells is an effective strategy against

insect feeding. The stone cells from both geno-types had thick
secondary cell walls that were synthesized from the typical
components, namely cellulose, mannan and xylan and lignin,
Acid-insoluble

although the morphology of the R genotype included larger cells

with more open lumens. The composition of stone cell wall
carbohydrates and lignin differed between tissue type and
genotype, as did lignin subunit ratios. Transcripts of monolignol
biosynthetic pathway genes were generally more abundant in
Acid-soluble

CYAS compared with PYAS. Transcript abundance of p-

coumarate-3-hydroxylase (C3H), a key gene in monolignol
biosynthesis, was higher in CYAS of R than S genotypes. The
incorporation of a fluorescently tagged monolignol into develop-ing
cortical stone cells suggests that the enzymatic machinery for
Mannose

polymerization of lignin monomers is active only during the first

year of growth of the apical shoot, which indicates a narrow time
window for cortical stone cell lignification.
Previous reports on the distribution of stone cells in conifers
Xylose

found that they are typically scattered throughout the secondary

Lignin, mg/100 mg

phloem with no consistent pattern of occurrence (Hudgins et al.

2004), which has made it difficult to monitor and investigate their
Glucose

Table 2. Lignin subunit proportions (H/G) of PYAS tissues isolated

from high (R) and low (S) stone cell containing Sitka spruce genotypes
as determined by thioacidolysis (Robinson and Mansfield 2009). S
Galactose

stone cells were analysed from two separate collections. Stone cells
isolated from three biological replicates are included for comparative
purposes and were isolated from the same tissues used in the other
analyses (denoted with a solid black line). S stone cells harvested from
~80 individual PYAS were pooled, analysed in triplicate and included
Rhamnose

for statistical analyses. Subunit ratios tended to differ between

genotype (P = 0.065) and tissue type (P = 0.076) following statistical
comparisons using the Kruskal–Wallis test statistic.

%H average %G average
PhenotypeandtissuetypeArabinose

Genotype and tissue type (±SEM) (±SEM)

R bark 5.0 ± 2.7 95.0 ± 2.7

Carbohydrates, mg/100 mg

S bark 6.2 ± 0.4 93.8 ± 0.4

R xylem 4.1 ± 0.8 95.9 ± 0.8
31. (0.4) b
30. (0.4) a

S xylem 4.6 ± 1.0 95.4 ± 1.0

0. (0.01) b

R needles 4.9 ± 2.4 95.1 ± 2.4

9. (0.5) a

S needles 6.1 ± 1.1 93.9 ± 1.1

7. (0.2) a

R stone cells 6.8 ± 1.1 93.2 ± 1.1

37. (1.0) a

a
S stone cells (pooled technical 12.2 ± 0.5 87.8 ± 0.5
5. (0.3) a

replicates)
ND
2. (0.2) c
Table1.

Rxylem

b
a

S stone cells 13.7 86.1

a
Technical replicates (three) of S stone cells isolated and pooled
ND= Not Detected

from ~80 clonal spruce trees.

b
Technical replicate (one) consisting of stone cells isolated from
three biological replicates harvested and collected from the other
tissues analysed for comparative purposes.
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
 Stone cells and defence against insects 1657

Figure 8. Monolignol incorporation in cell walls of developing stone cells. Incorporation of an NBD-tagged monolignol (coniferyl alcohol) probe into
Sitka spruce R stone cell and xylem tissues. Green fluorescence was observed in developing stone cells (a, b) and xylem (e, f) tissues of CYAS tips,
while no fluorescence was observed in mature stone cells of PYAS tips (c, d). Transmitted light images (b, d, f) are shown for comparison.

development and cell type-specific features. However, in the
weevil R Sitka spruce genotype characterized here, stone cells
are consistently located in the cortex of the apical shoot tip, which
uniquely facilitated the characterization of their distribution,
morphology, cell wall composition and biochemical activity of cell
wall monolignol incorporation. Specifically, cortical tissues found at
the top of PYAS tips in the weevil R genotype was comprised of
over 30% stone cells, while only approximately 10% of the cortex
of the S genotype are made up of stone cells.
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
Mechanisms that control stone cell formation in the cortex of
Sitka spruce, or any other plant tissue, are still unknown. Previ-
ous studies hypothesized that stone cells in the secondary phloem
were derived from polyphenolic parenchyma cells as the phloem
ages over several years (Franceschi et al. 1998; Franceschi et al.
2005). In the present study, stone cells develop in the current
season’s apical growth in the young tissues of Sitka spruce
cortex. The inverse relationship between stone cell abundance
and normal cortical parenchyma cells suggests
1658 J. G. A. Whitehill et al.

Figure 9. Transcript abundance of the monolignol biosynthetic pathway in Sitka spruce apical shoots. (a) Monolignol biosynthetic pathway in Sitka
spruce. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; C3H, p-coumarate 3-hydroxylase; HCT, hydroxycinnamoyl-CoA:
shikimate hydroxycinnamoyl-transferase; 4CL, 4-coumarate:CoA ligase; CCOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA
reductase; CAD, cinnamyl alcohol dehydrogenase. (b) Transcript analysis by quantitative real-time PCR (qRT-PCR) represented as the calibrated
normalized relative quantity (CNRQ) for the monolignol biosynthetic pathway in Sitka spruce current year (CYAS) and previous year (PYAS) apical
shoots from R and S genotypes. Error bars represent the standard error of the mean. Differences between genotype and tissue type were analysed
using two-way analysis of variance (ANOVA). Different letters indicate significantly different means (analysed within gene) separated post hoc
following significant F-tests by the protected LSD test (α = 0.05).

that stone cells are displacing these other cell types. Stone cells
co-occur with abundant cortical phenolic parenchyma cells, but
further characterization is required to test a developmental
relationship between these two cell types. The secondary walls of
stone cells are comprised of over 40% polymeric lignin, and
cortical phenolic parenchyma cells could contribute to this rich
lignin sink. We also observed occasional connectivity of stone cell
clusters with ray cells. Ray parenchyma have been interpreted as
a conduit for signal transduction between inner
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
and outer tissues of conifer stems (Hudgins & Franceschi
2004), and recently, it has been shown that lignification can
occur in a non-cell autonomous fashion in other plant species
(Smith et al. 2013). An association between ray parenchyma
and cortical stone cells suggests that the stone cells retain
symplastic contact with adjacent parenchyma throughout the
process of secondary cell wall formation.
Secondary cell walls of Sitka spruce stone cells revealed
a multilayered polylamellate ultrastructure, consistent with

previous descriptions of stone cells in a few angiosperms
(Parameswaran & Liese 1975; Parameswaran & Sinner 1979;
Reis et al. 1991; Reis et al. 1992; Reis & Vian 2004). Stone cells
in pear (Pyrus spp.) have a different arrangement of cell wall
layers when compared with conifer tracheids (Donaldson et al.
1999; Fromm et al. 2003; Jin et al. 2013). Varying degrees of
lignification distinguish the three-layered secondary wall (S 1, S2
and S3) of spruce tracheids (Donaldson et al. 1999; Fromm et al.
2003). We found Sitka spruce stone cells to have a simplified
structure compared with conifer tracheids more consistent with
observations of pear stone cells (Tao et al. 2009; Jin et al. 2013).
Sitka spruce stone cells have an electron-dense CML, followed
inwards by a PCW and second-ary wall. The secondary cell wall
typically has a different suite of hemicelluloses from the PCW
(Scheller & Ulvskov 2010), and the stone cells’ secondary cell wall
was robustly labelled with antibodies recognizing xylan and
mannan epitopes, which did not label the adjacent primary wall. In
contrast to the hemi-cellulose composition of conifer tracheids,
Sitka spruce stone cells contain almost three times the amount of
xylan compared with mannan. Xylan can act at the interface
between hydrophilic cellulose and hydrophobic lignin and enhance
adhesion of one component onto the other (Busse-Wicher et al.
2014). Elevated xylan abundance might be necessary to generate
increased quan-tities of lignin in stone cell walls.
Immunohistochemical labelling of stone cells showed more evenly
distributed signals for xylan than for mannan throughout the stone
cell secondary wall. However, xylan signals are absent in the
CML, primary wall and pit canals consistent with previous
observations of stone cells in other systems (Reis & Vian 2004).
These data provide support to the hypothesis that xylan can direct
the cellulose microfibrils in a heliocoidal array (Reis & Vian 2004).
The secondary cell wall of stone cells differs from tracheids in both
their heterogeneity and xylan composition in conifers.
Individual layers of the polylamellate secondary wall of
spruce stone cells appear to have a helicoidal construction as
has been observed for stone cells in other plant systems (Reis
& Vian 2004; Roland et al. 1989; Roland et al. 1987). This
helicoidal structure is the result of cellulose microfibrils being
synthesized around the cell as helices within each layer (Roland et
al. 1987). The electron-dense bands that separate the individual
layers are regions where cellulose microfibrils are in an orientation
that is parallel to the section surface (Roland et al. 1987). The
presence of cellulose in stone cell secondary walls was
investigated with Pontamine S4B, which has a primary binding
affinity to cellulose (Anderson et al. 2010; Thomas et al. 2012).
Labelling of radiata pine (Pinus radiata) tracheid cell walls with
S4B indicated that cellulose microfibrils will fluoresce more readily
when in a parallel orientation to the excitation light (Thomas et al.
2012). The bright staining (in relation to primary walls of
neighbouring parenchyma cells) of individual layers within Sitka
spruce stone cell secondary walls with S4B are in agreement with
our TEM observations of cellulose microfibrils running as a
heliocoidal construction within individual cell wall layers. R stone
cells had a higher proportion of cellulose, as measured in the form
of glucose in the Klason analysis, than S stone cells, which is
consistent with the larger, more abundant stone cell secondary
walls identified with
© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 1646–1661
Stone cells and defence against insects 1659
Pontamine S4B. Clearly, commitment to secondary cell wall cel-
lulose deposition is an underlying mechanism that generates in-
tense and less intense stone cell formation in the two genotypes.
Lignification of cell walls involves the biosynthesis of
monolignols in the cytosol followed by translocation to the cell wall
polysaccharide matrix and polymerization (Liu 2012). Conifer
lignin is derived from the monolignols p-coumaryl and coniferyl
alcohol but lacks syringyl units (Harris 2006; Wagner et al. 2012).
Quantification of lignin in the bark of Sitka spruce and Norway
spruce was previously used as an indirect measure for stone cell
abundance (Wainhouse et al. 1990). Highly lignified bark
contained increased quantities of stone cells and led to the
conclusion that spruce stone cells were com-posed primarily of
lignin (Wainhouse et al. 1990). While Sitka spruce stone cells also
contain substantial amounts of other sec-ondary wall components,
we found that lignin was indeed the most abundant constituent of
the secondary walls comprising over 40% (w/w) of the total dry
weight. Compared to R, S stone cells showed a higher percentage
of H-lignin units. Elevated levels of H-lignin are commonly found in
middle lamella and compression wood lignins inherent to conifers
and are produced in stress lignins found in cell cultures of P. abies
following elicitor treatment (Fukushima & Terashima 1991; Lange
et al. 1995; Nanayakkara et al. 2009; Terashima & Fukushima
1988). Increased heterogeneity in the monolignol composition of
lignin impacts the susceptibility of wood to degradation by cell
wall-degrading microorganisms (Faix et al. 1985; Skyba et al.
2013). It is possible that S stone cell walls may be less rigid
because of a higher abundance of H-lignin subunits, while the
higher proportion of G-lignin subunits may contribute to R stone
cells being potentially more resistant to degradation by feeding
insects.
Measurement of transcripts confirmed that the monolignol
biosynthetic pathway genes are differentially expressed
between CYAS and PYAS, and these differences may be
associated with the transient period of stone cell development
in CYAS. The activity of p-coumarate-3-hydroxylase (C3H)
leads to the production of methoxylated monolignol subunits
(Franke et al. 2002), and these transcripts were more
abundant in R CYAS than S suggesting a defining role in the
lignin composition of R stone cells. Future work will employ
laser microdissection and transcriptome analyses to
accurately identify genes involved in stone cell biosynthesis.
Following translocation into the cell wall polysaccharide matrix,
monolignols are oxidatively polymerized by cell wall-localized
laccasses and/or peroxidases (Vanholme et al. 2012). Sitka
spruce cortical stone cells develop during a short window of time
in actively expanding CYAS tips (between ~675 and 850 GDD).
Use of the fluorescently tagged monolignol analog NBD-CA
(Tobimatsu et al. 2011; Tobimatsu et al. 2013) permitted the in
vivo detection of monolignol incorporation in lignifying stone cells
and tracheids. We showed that lignification of stone cell walls is
an ephemeral event in the CYAS that occurs during early devel-
opment of apical shoot tips and not active in PYAS tips.
CONCLUSIONS
Stone cells in Sitka spruce R and S genotypes differ in abun-
dance, morphology, distribution and cell wall composition.
1660 J. G. A. Whitehill et al.
The highest density of stone cells at the top of the PYAS tip of
the R genotype co-localizes with the site of adult feeding,
oviposition and the first feeding activity of the freshly hatched
larvae. The high density represents a formidable physical
barrier, a consequence of their extended morphology, thick
lamellar secondary cell walls and high lignin content. Stone
cell development is completed in a short period of time in the
CYAS tip before the time of following year weevil attack. The
representative R genotype used in this and previous studies is
assumed to have evolved higher levels of natural resistance
because of increased selection pressure from the native
weevil, whereas the S genotype has no history of co-
appearance with this insect and therefore lacks co-evolved
defence mechanisms (King and Alfaro 2009). The new
information on stone cell composition and development will
enable future work on mechanisms that control stone cell
formation and ultimately assist in the selection and breeding of
Sitka spruce with increased stone cell abundance for improved
weevil resistance.
ACKNOWLEDGMENTS
We thank Dr. Carol Ritland and Ms. Karen Reid for project and
laboratory management support; Ms. Angela Chiang, Ms.
Agnes Yuen and Mr. Yaseen Mottiar for technical assistance;
Dr. Timothy R. Sexton for discussion of methods; Mr. Garnet
Martens, Mr. Kevin Hodgson, Mr. Derrick Horne and Mr.
Bradford Ross for assistance at the University of British
Columbia Bioimaging Facility; Dr. Christopher Keeling for
assistance with gene identification and Dr. John Ralph and Dr.
Yuki Tobimatsu for providing fluorescently tagged coniferyl-
alcohol (NBD-CA). The work was supported by funds to J.B.
from the Natural Sciences and Engineering Research Council
(NSERC) of Canada and funds to J.B. from Genome Canada,
Genome British Columbia and Genome Quebec in support of
the SMarTForests Project. JB is a UBC Distinguished
University Scholar.
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Received 13 July 2015; received in revised form 28 September
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
Figure S1. An adult white pine weevil female feeding and
ovipositing.
Figure S2. Time course of stone cell development.
Table S1. NCBI Accession numbers of amino acid
sequences from functionally characterized conifer
monolignol biosyn-thetic pathway genes used to query
Sitka spruce EST and FLcDNA resources.
Table S2. Monolignol biosynthetic pathway gene
oligonucleo-tides used in this study.
Table S3. Reference gene oligonucleotides used in this study.
Table S4. F-values, degrees of freedom (df), and associated
significance following two-way ANOVA analyses of genotype,
tissue type, and the genotype x tissue type interactions on the
calibrated normalized relative quantities (CNRQ) of lignin
biosynthetic pathway genes in first and second year leader
materials from R and S Sitka spruce genotypes. Significant
(α= 0.05) differences are bolded within row.
Table S5. F-values, degrees of freedom (df), and associated
significance following two-way ANOVA analyses of genotype,
tissue type, and the genotype x tissue type interactions of
carbohydrate and lignin composition of xylem, bark, and
needle tissues from R and S Sitka spruce genotypes. Signifi-
cant (α= 0.05) differences are bolded within row.