Environmental and Experimental Botany 174 (2020) 104031

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

Photosynthetic plasticity allows blueberry (Vaccinium corymbosum L.) plants T

to compensate for yield loss under conditions of high sink demand
Antonios Petridisa,1, Jeroen van der Kaaya,2, Julie Sungurtasb, Susan R. Verrallc,
Susan McCalluma, Julie Grahama, Robert D. Hancocka,*
a
Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK
b
Ecological and Biochemical Sciences, The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK
c
Information and Computational Sciences, The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK

A R T I C LE I N FO A B S T R A C T

Keywords: In some growing environments blueberry (Vaccimium corymbosum L.) yields exhibit excessive annual variation
13
CO2-labelling associated with poor photosynthetic performance. The purpose of this study was to determine how photo-
Blueberry synthesis may be affected by demand and to define the mechanisms underpinning photosynthetic plasticity.
Carbon assimilation Manipulation of source-sink ratios revealed that yields were maintained following 50 % defoliation. This was
Carbohydrate metabolism
associated with an adaptive increase in photosynthetic capacity mediated via changes in stomatal physiology,
Source-sink
photosynthetic electron transport and CO2 assimilation. Transcripts encoding enzymes of the Calvin-Benson
Starch
Sugars cycle including the Rubisco large subunit, Rubisco activase, phosphoribulokinase and plastid-localised glycer-
aldehyde phosphate dehydrogenase 3 were more abundant in leaves from partially defoliated plants relative to
control plants. Short-term 13CO2 labelling experiments suggested that partial defoliation did not incur an as-
similation penalty although the accumulation of sugars and starch in some organs was reduced. Metabolite
profiles of leaves from partially defoliated plants exhibited some differences from those of control leaves,
however, no changes in the diurnal content of leaf sugar and starch were observed between treatments. The data
highlights the mechanisms by which blueberry leaves adapt to increased demand and demonstrate that pho-
tosynthetic plasticity can compensate for significant loss of the photosynthetic area.

1. Introduction
Photosynthesis occurs in the leaves and encompasses the light-de-
pendent conversion of inorganic carbon dioxide into carbohydrates
(photoassimilates) through a process known as carbon fixation or as-
similation (Jones et al., 2013). Once formed, photoassimilates are
transferred (mainly as sucrose) through the phloem into sink organs,
where they can either be used immediately for growth and development
or stored as starch. During the day, photosynthesis supports the export
of photoassimilates from source leaves to sink organs and in addition
supports the accumulation of starch in the chloroplasts and sucrose in
the vacuole to be remobilised during the night to support growth and
maintenance of sink organs over the diurnal cycle (Bihmidine et al.,
2013; Ludewig and Flügge, 2013; Smith and Stitt, 2007). This is a
simplified model that describes how leaves supply sink organs with
photoassimilates; in fact, source-sink interactions are complex and fi-
nely tuned to maintain the balance between source supply and sink
demand. Such interactions include feedforward and feedback mechan-
isms controlling photosynthesis, changes in the expression of photo-
synthetic genes, and hormonal or sugar signals enabling the commu-
nication between source and sink organs (McCormick et al., 2008; Duan
et al., 2016; Glanz-Idan et al., 2020; McCormick et al., 2006; Yu et al.,
2015).
Crop yield depends not only on the production of photoassimilates
through photosynthesis (source capacity), but also on the utilisation of
photoassimilates in sink organs (sink capacity or strength) (Sonnewald
and Fernie, 2018). Experimental manipulations that alter either source
capacity (e.g. elevated CO2, defoliation, shading, etc.) or sink strength

⁎
Corresponding author.
E-mail addresses: apetridis@food.au.dk (A. Petridis), jeroenvanderkaay@gmail.com (J. van der Kaay), julie.sungartis@hutton.ac.uk (J. Sungurtas),
susan.verrall@hutton.ac.uk (S.R. Verrall), susan.mccallum@hutton.ac.uk (S. McCallum), julie.graham@hutton.ac.uk (J. Graham),
rob.hancock@hutton.ac.uk (R.D. Hancock).
1
Present address: Department of Food Science, Aarhus University, Agro Food Park 48, 8200 Aarhus N, Denmark.
2
Present address: Xelect Ltd., Horizon House, Abbey Walk, St. Andrews, Fife, KY16 9LB, United Kingdom.

https://doi.org/10.1016/j.envexpbot.2020.104031
Received 31 January 2020; Received in revised form 5 February 2020; Accepted 6 February 2020
Available online 19 March 2020
0098-8472/ © 2020 Elsevier B.V. All rights reserved.
A. Petridis, et al.
(e.g. sink removal) indicate that these two processes are tightly co-
ordinated, so that changes in the source affect sink activity and vice
versa. For example, defoliation usually increases photosynthesis in re-
maining leaves to maintain source activity and support sink growth
(Blanusa et al., 2006; DaMatta et al., 2008; Eyles et al., 2013; Kasai,
2008; Jorquera-Fontena et al., 2016). By contrast, reduction in sink
demand leads to accumulation of carbohydrates in the leaves and
subsequent feedback downregulation of photosynthesis (Lawlor and
Paul, 2016; Oszvald et al., 2018); however, other mechanisms, which
are independent of end-product accumulation might also account for
the sink-mediated control on photosynthesis, such as alterations in
stomatal conductance that affect the availability of CO2 (DaMatta et al.,
2008; Quentin et al., 2011) or shifts in leaf temperature that damage
the structure of photosystem II (Li et al., 2007).
Resource allocation provides sink organs with energy and carbon
skeletons to support their ecological and physiological functions, in-
cluding growth and development (Ludewig and Flügge, 2013). Usually,
resources are not equally distributed among organs, but their dis-
tribution changes according to the developmental stage of the plant. For
example, many fruit crops, increase allocation of photoassimilates to
developing fruit to ensure continuous growth, but when the fruit ripens,
they redirect carbon resources to storage pools, such as roots and stems,
to support growth of new vegetation and reproductive structures in the
following year (Da Silva et al., 2014; Gauci et al., 2009; Hancock et al.,
2007). It is conceivable that any factor that reduces source capacity,
such as environmental stresses and pathogen attack, will increase
competition between organs for resources, restrict their growth and
eventually reduce yield. Despite the importance of whole plant carbo-
hydrate partitioning to crop productivity, especially for woody per-
ennials, little information exists about how changes in source-sink
balance influence the distribution of photoassimilates to different sink
organs. Indeed, most studies focused mainly on the interaction between
leaves and fruits, neglecting other sink organs that could potentially
lower the capacity of fruits to import photoassimilates. Therefore, a
detailed examination of carbohydrate partitioning at a whole plant
level, following manipulation of source-sink balance, could provide
useful information on the antagonistic relationship between organs and
inform techniques for supplying fruits with more carbohydrates.
Highbush blueberry (Vaccinium corymbosum L.) is a native species of
North America and over the last two decades its production has ex-
panded worldwide, primarily due to the assumed health benefits asso-
ciated with its consumption. Recent evidence suggests that many
blueberry-producing countries experience significant yield instability,
arising from subtle year-to-year environmental variation (FAO, 2016;
Petridis et al., 2018). Our previous work, focusing on the mechanistic
understanding of the yield instability trait in blueberries, established
that: (i) fruit growth depends primarily on the daily production of
carbohydrates by leaves and there is little evidence for remobilisation
of storage reserves for fruit development, (ii) photosynthetic capacity is
restricted by stomatal conductance and downstream biochemical pro-
cesses, (iii) the developing fruit is not a particularly strong sink with
photoassimilates evenly distributed to fruit and vegetative sink organs,
and (iv) most of the newly assimilated carbon is retained in leaves
(Petridis et al., 2018). The above findings raise the question whether
blueberry yield is restricted from the limited utilisation of photo-
assimilates by the fruit (sink limitation), the limited production of
photoassimilates by the leaves (source limitation) or perhaps both. On
one hand, high retention of assimilated carbon in the leaves might in-
dicate that the yield is sink-limited. In this scenario, low demand for
carbohydrates could lead to accumulation of non-structural carbohy-
drates in the leaves, which in turn would result in feedback down-
regulation of photosynthesis. On the other hand, yield might be limited
by the lack of non-structural carbohydrates to support fruit develop-
ment due to low carbon assimilation. In that case, resource limitations
may lead to fruit losses, reduction of fruit sink strength and increased
competition between sinks.
2
Environmental and Experimental Botany 174 (2020) 104031
Here, we manipulated source-sink balance, through controlled de-
foliation or complete defruiting, to examine the mechanisms controlling
source-sink relations in blueberries and to assess whether their yield is
source- or sink-limited. This study reveals that blueberry plants can
maintain yield under conditions of high sink demand through an
adaptive increase of their photosynthetic capacity (photosynthetic
plasticity). Our work suggests that breeding programmes aiming to
improve yield should focus on increasing the fruit pool size and the
delivery of photoassimilates from leaves to fruits.
2. Materials and methods
2.1. Plant material, growth conditions and treatments
Experiments were carried out at the experimental site of the James
Hutton Institute (Dundee, Scotland, 56˚27ʹ25ʹʹN, 3˚4ʹ11ʹʹW) during the
2018 growing season. Three-year-old blueberry plants (Vaccinium cor-
ymbosum L. cv. ‘Liberty’) were grown under a tunnel in 10 L pots filled
with substrate suitable for blueberry production (Levington Advance
CNS Ericaceous, pH 4.4–5.0; ICL, Ipswich, UK) and were fertigated
daily.
For source-sink manipulation experiments, plants were selected
based on their uniformity in size and fruit load. Three treatments were
applied at the whole plant level at the beginning of fruit set: (1) un-
treated plants (control), (2) 100 % fruit removal (FR), and (3) defo-
liation (50 % leaf removal, LR). For 50 % leaf defoliation, alternate
leaves were removed from the entire plant. The experimental design
was a randomised block and each treatment consisted of six replicate
plants. Three out of the six replicates were used for photosynthetic
analysis, 13C isotope labelling, and examination of sugar and starch
dynamics, while the remaining replicates were used to measure the
expression of Calvin cycle genes and the amount of rubilose-1,5-bi-
sphospate carboxylase oxygenase (Rubisco) protein. Harvest and in
planta measurements were conducted at ripening stage, i.e. when more
than 75 % of fruit had obtained blue colour.
2.2. Photosynthetic gas exchange and chlorophyll fluorescence
measurements
Gas exchange and chlorophyll fluorescence measurements were
conducted on the first fully expanded leaf of ‘Liberty’ shoots (4th-5th
node). All measurements were conducted on three independent re-
plicate plants. Net carbon assimilation rate (A), stomatal conductance
(gs) and intercellular CO2 concentration (Ci) were measured using a
CIRAS-2 portable photosynthesis system (PP Systems, Amesbury, MA,
USA) equipped with a PLC6 (U) 2.5 cm2 leaf cuvette, which provided
light through an integrated LED light unit. Leaf temperature was
maintained at 25 °C and relative humidity at 60 %. Except for A/Ci
curves, where CO2 was increased at regular intervals, CO2 concentra-
tion was supplied at 400 μmol mol−1 with a gas flow rate of 200 mL
min−1.
To generate light response curves, leaves were acclimated at 1000
μmol m−2 s-1 photosynthetic photon flux density (PPFD) for 45 min.
After acclimation, light irradiance was decreased using the following
PPFD sequence: 1000, 600, 400, 300, 200, 150, 100, 50 and 0 μmol
m−2 s-1. Before recording gas exchange parameters, leaves were equi-
librated at each irradiance for 4 min.
Chlorophyll fluorescence parameters (Genty et al., 1989; Maxwell
and Johnson, 2000) were measured concomitantly with light response
curves using the chlorophyll fluorescence module (CFM) of CIRAS-2.
Prior to generating light response curves, leaves were dark-adapted for
40 min. Then, a saturating red-light pulse (0.8 s; 6000 μmol m−2 s-1)
was applied to determine the maximum quantum yield of photosystem
II. Following acclimation at 1000 μmol m−2 s-1 for 45 min (as described
above), gas exchange and chlorophyll fluorescence parameters, in-
cluding electron transport rate (ETR), quantum yield (ΦPSII) and non-
A. Petridis, et al.
photochemical quenching (NPQ), were recorded simultaneously.
A/Ci curves were generated at 1200 μmol m−2 s-1 light irradiance
using the following stepwise gradients: 400, 200,100, 50, 400, 600,
1000 and 1200 μmol mol-1.
2.3. Determination of soluble sugars and starch
Soluble sugars and starch were quantified in leaves harvested at
different time points within a day as well as in different organs of
blueberry plants following destructive harvest at the fruit ripening
stage. For time-course experiments, four leaves per plant, located at the
4th or 5th node, were harvested at 6.00, 7.00, 8.00, 10.00, 12.00,
14.00, 17.00, and 19.00 h, snap frozen in liquid N2 and stored at −80
°C until analysis. For analysis of carbohydrate distribution within
blueberry plants, three replicate plants that were at the fruit ripening
stage were transferred to the laboratory and the above-ground organs
(leaves, fruits, current-year shoots and old wood stems) were separated
and immediately frozen in liquid N2. Roots were washed to remove soil
and then placed in liquid N2. Following snap freezing, samples were
stored at -20 °C for one day until freeze-drying (Martin Christ
Gefriertrocknungsanlagen GmbH, Osterode, Germany). Dried samples
were milled to a fine powder (Cyclone Mill Twister, Retsch, Haan,
Germany) and stored at −20 °C until analysis.
Soluble sugars and starch were extracted from different blueberry
organs and quantified by high performance anion exchange chroma-
tography with pulsed amperometric detection (HPAEC-PAD) as pre-
viously described (Petridis et al., 2018).
2.4. 13
C isotope labelling
Whole plants were enclosed in transparent plastic bags at 10 a.m.
and labelled with 13CO2 for 2 h. To generate 13CO2, 3 mL of 70 % lactic
acid were injected into glass vials, containing 1 g of NaH13CO3 (13CO2,
treated plants) or NaH12CO3 (12CO2, control plants). The glass vials
were mounted on the pots before covering the plants with the bags.
Following 13CO2 labelling, the bags were removed and 13C was chased
for 24 h before harvesting above- and below-ground organs.
The stable carbon isotopic composition (δ13C) and carbon content of
lyophilised powdered material were analysed by OEA Laboratories Ltd
(Cornwall, UK) using a dual-pumped Sercon 20–20 isotope ratio mass
spectrometer (IRMS, Sercon Ltd, Crewe, UK) coupled to a Thermo
EA1110 elemental analyser (Thermo-Fisher Scientific, Waltham,
Massachusetts, USA). For each sample, approximately 1.15–1.2 mg of
tissue were weighed, and four standards were used to calibrate the data
(USGS40 L-glutamic acid, USGS41a L-glutamic acid, IAEA-CH6 sucrose
and NBS1577b bovine liver). Excess δ13C (‰) of a given organ was
calculated by subtracting δ13C values after 24 h of chasing from δ13C
values of control plants.
2.5. RNA extraction and quantitative RT-PCR
Ten fully developed leaves, located at the 4th or 5th node of blue-
berry shoots, were collected at 10 a.m. to measure transcript abundance
of key genes involved in CO2 assimilation. Leaves were immediately
snap frozen in liquid N2, ground to a fine powder with mortar and
pestle, and stored at −80 °C until analysis.
Total RNA was extracted from blueberry leaves using the cetyl-tri-
methyl-ammonium bromide (CTAB) method (Jaakola et al., 2001) with
minor modifications. The extraction buffer contained 2% CTAB, 100
mM Tris HCl (pH 8.0), 25 mM EDTA, 2 M NaCl and 0.15 g g−1 poly-
vinylpolypyrrolidone. Prior to extraction, 900 μL of extraction buffer
containing 2% β-mercaptoethanol were added into 2 mL micro-
centrifuge tubes and placed in a heating block at 65 °C for 10 min to
reach the indicated temperature. While waiting for the extraction buffer
to heat, 4 × 100 mg powdered leaf material of each sample was
weighed and placed into 2 mL microcentrifuge tubes that were kept in
3
Environmental and Experimental Botany 174 (2020) 104031
liquid nitrogen. A batch of 3 biological replicates was extracted each
time, resulting in a total number of 12 samples (3 replicates × 4
samples per replicate). When weighing was completed, the tubes con-
taining the sample were filled with warm extraction buffer and vor-
texed briefly. Then, the samples were placed for 10 min in a heating
block at 65 °C with periodic vortexing every 2 min. Following incuba-
tion, samples were centrifuged at 12,000×g for 10 min at 4 °C and the
supernatant (∼650 μL) was transferred into new 2 mL microcentrifuge
tubes. The sample supernatant was then extracted with an equal volume
of chloroform:isoamyl alcohol (24:1) and mixed vigorously by inver-
sion. Phase separation was achieved by centrifugation at 12,000×g for
10 min at 4 °C and the upper aqueous phase (475 μL) was reextracted
with an equal volume of chloroform:isoamyl alcohol (24:1). Following
centrifugation (12,000×g, 10 min, 4 °C), 0.25 volumes (75 μL) of 8 M
LiCl were combined with the supernatant (300 μL), the solution was
mixed gently by inversion, and RNA was precipitated overnight at 4 °C.
The next day, the RNA pellet was recovered by centrifugation
(12,000×g, 20 min, 4 °C) and washed with 500 μL of ice-cold 75 %
ethanol by centrifuging at 7500×g for 5 min at 4 °C. The RNA pellet
was then dissolved in 100 μL SSTE buffer containing 1 M NaCl, 0.5 %
SDS, 10 mM Tris-HCl and 1 mM EDTA (pH 8.0). At that point, the 4
dissolved RNA pellets of each replicate were united into a micro-
centrifuge tube. The combined samples were extracted once with an
equal volume (400 μL) phenol:chloroform:isoamyl alcohol and once
with an equal volume (300 μL) of chloroform:isoamyl alcohol. During
both extractions, phase separation was achieved by centrifugation at
12,000×g for 10 min at 4 °C. Then, 2.5 volumes (500 μL) of ice-cold
absolute ethanol were added to the supernatant (200 μL) and RNA was
precipitated at −20 °C for 2 h. The precipitated RNA was pelleted by
centrifugation at 18,000×g for 20 min at 4 °C and the RNA pellet was
washed with 500 μL of ice-cold 75 % ethanol by centrifuging at 7500×g
for 5 min at 4 °C. The RNA pellet was air dried for 5−10 min and
resuspended in 20 μL of diethyl pyrocarbonate (DEPC)-treated water.
To remove genomic DNA, the extracted RNA was immediately treated
with DNase using the DNA-free™ Kit (Invitrogen, Thermo-Fisher Sci-
entific, Waltham, Massachusetts, USA) according to the manufacturer’s
instructions. RNA concentration and purity were checked using ND-
1000 UV NanoDrop spectrophotometer (Thermo-Fisher Scientific,
Waltham, Massachusetts, USA) and then stored at −80 °C until cDNA
synthesis.
cDNA was synthesized from a 500 ng aliquot of total RNA using the
iScript™ Advanced cDNA kit (Bio-Rad Laboratories Ltd, Watford, UK).
Subsequently, qPCR was performed in a mixture comprising 5 μl of
SsoAdvanced™ SYBR® Green Supermix (Bio-Rad Laboratories Ltd,
Watford, UK), 1.5 μl of each gene-specific primer (diluted to 5 nM), and
2 μl (10 ng μL−1) of cDNA template. The amplification was monitored
on an Applied Biosystems StepOnePlus™ Real-Time PCR System
(Thermo-Fisher Scientific, Waltham, Massachusetts, USA), set to pro-
vide an initial denaturation of 95 °C for 30 s, followed by 40 cycles of 95
°C for 15 s and 60 °C for 30 s. For each treatment, three biological and
three technical replicates were used. The raw threshold cycle values
(CT) for all samples were normalized against the signal produced by the
constitutively transcribed gene GADPH2 (glyceraldehyde 3-phosphate
dehydrogenase 2). Primer specificity was assessed by inspection of the
melting curve after 40 cycles. The primer sequences were designed
using the QuantPrime primer design tool (Arvidsson et al., 2008) and
are listed in Supporting Information Table S1.
2.6. Protein extraction and SDS-PAGE analysis
To extract proteins for SDS-PAGE analysis, we used a method based
on phenol extraction coupled with ammonium acetate precipitation, as
described by Isaacson et al. (2006) with some modifications related to
the weight of starting material and volume of solvents used during
extraction. One hundred milligrams of frozen leaf powder and 10 mg
(10 % w/w) of PVPP were placed into a 2 mL microcentrifuge tube.
A. Petridis, et al. Environmental and Experimental Botany 174 (2020) 104031

Fig. 1. Productivity and photosynthetic performance of ‘Liberty’ plants with altered source-sink balance. (a) Yield, (b) net CO2 assimilation rate (A), (c) stomatal
conductance to water vapour (gs), and (d) internal CO2 concentration (Ci) were measured at fruit ripening stage. Photosynthetic measurements were undertaken on
fully expanded leaves at 25 °C using a fixed cuvette CO2 concentration of 400 μmol mol−1 and a light intensity of 1200 μmol m-2 s-1. Data were subjected to one-way
ANOVA and significant differences (P ≤ 0.05) between means were determined using Tukey’s test. In all plots, data are represented as mean ± SE, n = 3 and values
that were significantly different between treatments are indicated by different letters. FR, 100 % fruit removal; LR, 50 % leaf removal.

Samples were suspended in 500 μL of ice cold extraction buffer (0.7 M
sucrose, 0.1 M KCl, 50 mM Tris−HCl (pH 7.5), 0.5 mM EDTA, 2% β-
mercaptoethanol and 10 μL mL−1 protease inhibitor cocktail) and
vortexed vigorously to solubilise proteins. Then, an equal volume (500
μL) of phenol saturated with Tris-HCl (pH 8.0) was added and the
sample vortexed briefly prior to mixing for 30 min at 4℃ using a blood
rotator. Following centrifugation (5,000×g, 30 min, 4℃), the upper
phenolic phase (350 μL) was collected carefully and transferred into
another microcentrifuge tube. An equal volume of extraction buffer was
added to the collected phenolic phase, followed by brief vortexing,
shaking for 30 min at 4℃, and centrifugation (5,000×g, 30 min, 4℃).
The same procedure was repeated once more, but this time with 200 μL
phenolic phase and extraction buffer. After shaking and centrifugation,
the upper phenolic phase (150 μL) was mixed with 5 volumes (750 μL)
of ice-cold 0.1 M ammonium acetate in methanol and the mixture was
stored at -20℃ for 1 h for protein precipitation. Following centrifuga-
tion at 5,000×g for 30 min at 4℃, the supernatant was discarded, and
the pellet was washed twice with 2 volumes (300 μL) of ice-cold me-
thanol to remove ammonium acetate, phenol, lipids and pigments. At
each washing step, samples were centrifuged at 5,000×g for 10 min at
4℃. Then, the pellet was washed twice with 2 volumes (300 μL) of ice-
cold acetone to replace methanol and achieve faster and more effective
drying. Finally, the pellet was left to dry for 10 min and resuspended in
a protein resuspension buffer appropriate for downstream analytical
applications, containing 150 μL Tris-HCl buffer (100 mM Tris-HCl pH
7.5, 10 mM EDTA, 5 mM DTT, 10 μL mL protease inhibitor cocktail)
and 150 μL NuPAGE LDS sample buffer (4×) at a ratio 1:1. Total
protein concentration was measured spectrophotometrically using the
Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories Ltd, Watford,
UK).
To determine the amount of Rubisco, aliquots of protein extracts
plus loading buffer, corresponding to 7 μg, were loaded onto a 12 %
Bolt™ Bis-Tris Plus Gels (Thermo-Fisher Scientific, Waltham,
Massachusetts, USA) and separated by SDS-PAGE at a constant voltage
(200 V) for 35 min. When electrophoresis finished, the gels were in-
cubated in 100 mL staining solution (0.1 % Coomassie R-250 in 40 %
ethanol and 10 % acetic acid) for 45 min. The gels were then trans-
ferred to a container containing 100 mL of destain solution (10 %
ethanol and 7.5 % acetic acid) and were shaken overnight at room
temperature on an orbital shaker. After achieving the desired back-
ground, the gels were scanned with a G:box F3 imaging system
(Syngene, Cambridge, UK) and the relative amount of both large and
small Rubisco subunits was determined by densitometry using the
GeneTools analysis software (Syngene, Cambridge, UK).
2.7. Gas Chromatography – Mass Spectrometry (GC–MS) analysis of
primary metabolites
Polar and non-polar metabolites were extracted, derivatised and
analysed by GC–MS according to Dobson et al. (2008). Metabolite
profiles were acquired using a GC–MS (DSQII, Thermo-Finnigan, Hemel
Hempstead, UK) system equipped with a DB5-MSTM column (15 m
×0.25 mm ×0.25 μm; J&W, Folsom, CA, USA) as described by Foito
et al. (2013).
2.8. Statistical analysis
Data were subjected to one-way analysis of variance (ANOVA).
Significant differences (P ≤ 0.05) between means were determined
using the Tukey’s test. Statistical analysis was performed using IBM
SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY).
3. Results
3.1. Blueberry plants exhibit photosynthetic plasticity in response to
variation in sink demand
To examine how changes in source-sink balance influence

4
A. Petridis, et al.
productivity of blueberry plants, fruit yield was measured at the end of
the growing season when most fruits had ripened (more than 75 % of
fruit had obtained blue colour). Defoliated plants lacked 50 % of their
leaves yet fruit yield was not significantly different from control plants
(Fig. 1a). Our previous analysis revealed that blueberry yields are
highly dependent on photosynthetic CO2 assimilation and that blue-
berry plants lack carbohydrate reserves to support continuous fruit
development under conditions of insufficient CO2 assimilation (Petridis
et al., 2018). We therefore tested the hypothesis that defoliated plants
maintained fruit development via an adaptive increase of photo-
synthetic CO2 assimilation. Infra-red gas exchange analysis revealed
that the light saturated carbon assimilation rate (A) was two-fold higher
in plants subjected to partial defoliation relative to control plants
(Fig. 1b). Increased A was associated with a significantly higher rate of
stomatal conductance (gs) in partially defoliated plants (Fig. 1c). On the
contrary, plants that had been subjected to defruiting showed no
change in light-saturated assimilation rate or gs. Although there were no
significant differences in leaf internal CO2 (Ci) concentrations between
the control leaves and either of the treatments, Ci was significantly
lower in leaves from partially defoliated plants relative to plants from
which fruit had been removed at the start of the season (Fig. 1d).
Correlation analysis indicated a high positive relationship (r =
0.78, P ≤ 0.01) between A and gs, suggesting that stomatal con-
ductance represents a significant factor limiting CO2 assimilation in
blueberry leaves. Nevertheless, photosynthesis is a complex process and
when source-sink relations change, other factors, related to leaf pho-
tochemistry, may also account for changes in carbon assimilation (Rivas
et al., 2007; Urban et al., 2004). To determine how changes in source-
sink balance influence PSII photochemistry in blueberry leaves, light
response curves were generated and chlorophyll fluorescence para-
meters were recorded under different ambient light regimes. Light re-
sponse curves confirmed and extended the results observed for light
saturated measurements of CO2 assimilation. Fruit removal resulted in
the saturation of assimilation at lower light intensity than in control
plants or plants that were partially defoliated (Fig. 2a). Plants from
which fruits were removed showed the lowest maximal rate of assim-
ilation while those that had been partially defoliated exhibited the
highest maximal rate. Photosynthetic electron transport mirrored CO2
assimilation with leaves from plants lacking fruit exhibiting the lowest
light saturated electron transport rate while those from partially defo-
liated plants exhibited the highest electron transport rate (Fig. 2b).
Whereas all leaves exhibited a decline in the quantum yield of PSII as
light intensity increased, the quantum yield of leaves from partially
defoliated plants was greater than that from control or defruited plants
at all light intensities (Fig. 2c). No consistent impact of treatment on
non-photochemical quenching was observed (Fig. 2d). Taken together
these data suggest that partial defoliation resulted in increased effi-
ciency in the conversion of light energy to electron transport in partially
defoliated plants, but also suggest a greater electron demand. Further
experiments were therefore conducted to examine the biochemical
factors that might influence electron demand.
Chlorophyll content was similar among treatments (Fig. 3a), sug-
gesting that changes in electron transport and carbon assimilation are
unrelated to the capacity of leaves to capture light. Nevertheless, under
identical light intensity, defoliated plants had higher carbon assimila-
tion rate compared with control and defruited plants at equivalent in-
ternal leaf CO2 concentrations as indicated from A/Ci curves (Fig. 3b).
This suggests that defoliation resulted in enhanced carboxylation effi-
ciency, presumably due to greater carbon flux through the Calvin cycle.
To determine whether this was related to leaf Rubisco content, ex-
tracted leaf proteins were subjected to SDS-PAGE followed by Coo-
massie staining to estimate the relative amount of both large and small
Rubisco subunits. The results indicated that the amount of the large, but
not the small, subunit was marginally higher in leaves of partially de-
foliated plants compared with control leaves (Supporting Information
Fig. S1). However, leaves from defruited plants also exhibited a slightly
5
Environmental and Experimental Botany 174 (2020) 104031
higher (although not significant) amount of Rubisco compared with
control leaves suggesting that Rubisco content was unlikely to be a
significant factor in the greater biochemical efficiency of leaves from
partially defoliated plants.
3.2. Defoliation upregulates the transcription of key Calvin-Benson cycle
genes
To define whether enhanced CO2 assimilation in defoliated plants
was influenced by biochemical determinants beyond Rubisco content,
the abundance of transcripts encoding enzymes participating in the
Calvin-Benson cycle namely Rubisco large subunit, Rubisco activase
(RCA), fructose-1,6-bisphosphatase (F-1,6-BPase), glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK)
were measured. qRT-PCR revealed that defoliation increased the
abundance of most of the examined transcripts with the exception of
those encoding F-1,6-BPase and GAPDH1 (Fig. 4). GAPDH1 is described
as a cytosolic isoform that participates in glycolysis (Serrato et al.,
2009), while in photosynthetic tissues F-1,6-BPase has both cytosolic
and plastidal isoforms where the former participates in sucrose meta-
bolism and the latter in the Calvin cycle (Daie, 1993). Searches for
available blueberry sequences identified only a single transcript that
could be confidently identified as F-1,6-BPase. This sequence (accession
number CV090224) had high homology to cytosolic sequences from
other species suggesting that its function was associated with sucrose
metabolism. The abundance of transcripts encoding Rubisco large
subunit were two-fold higher in leaves from defoliated plants relative to
control (Fig. 4a), consistent with increased leaf Rubisco content.
However, transcripts encoding RCA were five-fold greater in leaves
from partially defoliated plants than in leaves from control plants
(Fig. 4b). This result is consistent with the observation that leaves from
partially defoliated plants exhibited improved carboxylation efficiency
(Fig. 3b). Transcripts encoding plastidial GAPDH3 (Fig. 4d) and PRK
(Fig. 4f) were 3–4 fold more abundant in leaves from partially defo-
liated compared with leaves from control plants suggesting not only
improved carboxylation efficiency but also increased capacity and flux
through the Calvin cycle. No differences in transcript abundance were
found between control and defruited plants, consistent with the find-
ings from gas exchange analysis.
3.3. Alteration of source-sink relationships impacts the leaf metabolome
To obtain further understanding regarding leaf metabolic response
to altered carbon demand, an untargeted metabolome profile was ob-
tained from control leaves or leaves of plants that had an altered source-
sink balance. A total of 88 chromatographic features were quantified in
leaf extracts comprising 55 features in the polar fraction and 33 in the
non-polar fraction. In the polar fraction a total of 40 features were
identified based upon their mass spectra and coelution with authentic
standards while 25 features were identified in the non-polar fraction
(Supporting Information Table S2).
Principal components analysis based upon all of the chromato-
graphic features revealed a clustering of replicate samples from the
same treatment and a clear separation of control leaves and leaves from
plants that had been 50 % defoliated (Supporting Information Fig. S2).
Leaves from plants that had undergone sink removal also exhibited
separation from control leaves and particularly from leaves collected
from plants that had been partially defoliated.
Analysis of variance revealed eighteen components that showed
significant variation in abundance dependent on source-sink manip-
ulation. Five amino acids and the polyamine putrescine exhibited sig-
nificant differences between treatments (Fig. 5a). Glycine, serine and
glutamate were all more abundant in leaves of plants from which fruit
had been removed than in leaves of control plants or those that had
been partially defoliated. In contrast, lysine, proline and putrescine was
less abundant in leaves from partially defoliated plants than from leaves
A. Petridis, et al. Environmental and Experimental Botany 174 (2020) 104031

Fig. 2. Photochemical responses of ‘Liberty’ blueberry leaves to alterations in source-sink balance. Light response curves of (a) net CO2 assimilation rate (A), (b)
electron transport rate (ETR), (c) quantum yield of PSII photochemistry (ΦPSII), and (d) non-photochemical quenching (NPQ). Measurements were undertaken on
fully expanded leaves at 25 °C using a fixed cuvette CO2 concentration of 400 μmol mol−1. In all plots, data are represented as mean ± SE, n = 3. ●, control; , 100
% fruit removal (FR); △, 50 % leaf removal (LR).

of plants in other treatment groups. Similarly, the levels of inositol and
quinate were lower in leaves of partially defoliated plants than in
control leaves while the levels of the phenolic acids caffeate and
chlorogenate (caffeoyl quinate) were higher (Fig. 5b). Two unidentified
polar compounds exhibited similar behaviour to the phenolic acids
while a third unidentified polar compound was marginally higher in
leaves from defruited plants than leaves from plants in the other
treatment groups (Fig. 5c). Alterations in source-sink ratios also af-
fected non-polar compounds in leaves, particularly long chain fatty
alcohols where fruit removal or partial defoliation resulted in a lower
abundance of docosanol (C22), tetracosanol (C24) and heptacosanol
(C26) relative to control leaves (Fig. 5d). On the contrary partial de-
foliation alone increased the concentration of nonacosanol (C29) in
leaves.
3.4. Changes in non-structural carbohydrate pools due to manipulation of
source-sink balance
Plants can monitor their carbohydrate status and adjust the balance
between carbon supply and demand in response to source-sink im-
balances by regulating transcriptional, translational and post-transla-
tional processes (White et al., 2016). For example, high carbon status in
the leaves downregulates photosynthesis, while high sink demand up-
regulates it. In this regard, the observed differences in photosynthesis
between defoliated and control plants might be related to differences in
leaf non-structural carbohydrate pools that arise from manipulation of
source-sink balance. To check this possibility, we quantified soluble
sugar and starch levels in leaves at different time-points within a day, to
gain insights into the diurnal fluctuation of non-structural

Fig. 3. Biochemical responses of ‘Liberty’ blueberry leaves to alterations in source-sink balance. (a) Chlorophyll content, (b) A-Ci curves generated using a photo-
synthetic photon flux density (PPFD) of 1200 μmol m−2 s-1 and leaf temperature of 25 °C. In all plots, data are represented as mean ± SE, n = 3. FR, 100 % fruit
removal; LR, 50 % leaf removal.

6
A. Petridis, et al.
carbohydrates and to check whether their levels differ between treat-
ments, especially at the beginning and/or the end of a day. Such time-
course analysis could provide information about how changes in
source-sink balance may alter the capacity of blueberry leaves to export
sugars (particularly sucrose) or to break down starch reserves at night.
7
Environmental and Experimental Botany 174 (2020) 104031
Fig. 4. Impact of alteration on source-sink
ratio on levels of transcripts encoding enzymes
of the Calvin cycle in ‘Liberty’ blueberry leaves.
Columns represent the mean relative abun-
dance of transcripts encoding Rubisco (a),
Rubisco activase (b), cytosolic glyceraldehyde-
3-phosphate dehydrogenase 1 (c), plastidal
glyceraldehyde-3-phosphate dehydrogenase 3
(d), fructose-1,6-bisphosphatase (e) and phos-
phoribulokinase (f) and bars represent stan-
dard error (n = 3). Abundance data were
normalised against the signal produced by the
constitutively transcribed gene GLYCERALDH-
YDE-3-PHOSPHATE DEHYDROGENASE 2
(GADPH2) and are represented relative to the
mean expression level observed in control
plants. Data were subjected to one-way
ANOVA and significant differences (P ≤ 0.05)
between means were determined using Tukey’s
test where lower case letters above columns
indicate statistical similarity or difference. FR,
100 % fruit removal; LR, 50 % leaf removal.
For all treatments, soluble sugar (sum of glucose, fructose and sucrose)
was present at around 90 mg g DW−1 at dawn. The sugar concentration
increased during the first two hours after dawn and then gradually
declined until dusk (Fig. 6a). No significant differences were observed
in leaf sugar content dependent upon source-sink manipulation. Leaves
Fig. 5. Impact of alteration of source-sink bal-
ance on metabolic profiles of ‘Liberty’ bluberry
leaves. Figures indicate metabolites that ex-
hibit significant differences between leaf sam-
ples and are clustered as amino acids and other
amides (a), miscellanous polar metabolites (b),
unknown polar metabolites (c) and non-polar
metabolites (d). Mean metabolite abundance is
represented relative to abundance in control
leaves ± SE (n = 3). Data were subjected to
one-way ANOVA and significant differences (P
≤ 0.05) between means were determined
using Tukey’s test where different letters above
bars denote differences between treatments. ◼,
Control; , 100 % fruit removal (FR); □, 50 %
leaf removal.
A. Petridis, et al.
were also observed to contain significant amounts of starch at dawn
which also increased over the first two hours before remaining rela-
tively constant until dusk (Fig. 6b) presumably to be turned over during
the night. Significant differences between treatments were only ob-
served in leaves sampled at noon. Taken together, these data indicate
that manipulation of source-sink ratios has little impact on leaf sugar
and starch content suggesting that carbohydrate export is closely mat-
ched to CO2 assimilation under all experimental treatments.
To determine how alteration of source-sink ratio affected the dis-
tribution of carbohydrates the distribution of soluble sugars and starch
in different organs of blueberry plants was quantified at the end of the
growing season immediately prior to fruit harvest. With the exception
of the fruit removal treatment, the majority of soluble sugars in the
plant were accumulated in fruits (Fig. 7a) followed by leaves (Fig. 7b),
stems (Fig. 7c, d) and roots (Fig. 7e). Large amounts of starch were
8
Environmental and Experimental Botany 174 (2020) 104031
Fig. 6. Diurnal changes in leaf soluble sugars
(a) and starch (b) in response to source-sink
manipulation in ‘Liberty’ blueberry leaves.
Leaves were sampled at regular intervals be-
tween dawn (6.00) and dusk (19:00). Each
data point represents the mean value of three
replicates ± SE. Data were subjected to one-
way ANOVA and significant differences (P ≤
0.05) between means were determined using
Tukey’s test. Different letters next to data
points indicate significant differences between
treatments and where letters are absent no
significant differences were observed.
recovered from leaves, stems and roots. While defruiting had no impact
on the accumulation of starch and sugars in leaves, 50 % leaf removal
resulted in a reduction in the total amount of sugars and starch re-
covered in that organ due to the reduced organ weight (Fig. 7b). Si-
milarly, a significant reduction in the quantity of sugars recovered from
fruits was observed following partial defoliation (Fig. 7a), driven
mostly by the lower (although statistically insignificant) fruit yield in
these plants (Fig. 1a). 50 % defoliation also resulted in a significant
reduction in starch accumulation in old stems compared to control
plants (Fig. 7d) although no differences were observed in starch or
sugar accumulation in current year shoots or stems (Fig. 7c, e). Taken
together these data suggest that photosynthetic efficiencies in partially
defoliated plants were insufficient to fully compensate for the loss of
photosynthetic area although they were sufficient to maintain yield.
Fig. 7. Whole organ soluble sugars and starch
of ‘Liberty’ plants with altered source-sink re-
lations. Charts indicate the total amount of
sugars ( ) or starch (□) accumulated in (a)
fruit, (b) leaves, (c) current year shoots, (d)
older stems and (e) roots. Data were normal-
ised relative to a plant with 100 g DW old stem
by dividing the mass of each organ in the plant
by the mass of the stem and multiplying by
100. Data were subjected to one-way ANOVA
and significant differences (P ≤ 0.05) between
means were determined using Tukey’s test.
Uppercase (for starch) and lowercase (for so-
luble sugars) letters denote differences be-
tween treatments. Bars are the mean value of
three replicates ± SE, n = 3. FR, 100 % fruit
removal; LR, 50 % leaf removal.
A. Petridis, et al.
3.5. Partitioning of newly assimilated carbon to fruit of partially defoliated
plants is marginally higher and most of it is retained in the leaves
To understand how changes in source-sink balance influence the
distribution of newly acquired photoassimilates, plants were labelled
with 13CO2 for 2 h at the ripening stage. 13C was chased for 24 h before
harvesting above- and below-ground organs. Despite the dark period
intervening between labelling and harvest, large amounts of 13C were
retained in leaves (Fig. 8) consistent with the observation that leaf
starch is not fully turned over in a single night (Fig. 6). Consistent with
gas exchange measurements, leaves from partially defoliated plants
exhibited a higher 13C concentration than leaves from control plants or
those from which fruit had been removed (Fig. 8a). However, when
organ mass was taken into consideration, the total 13C recovered from
leaves was similar in all treatments (Fig. 8b). The concentration of 13C
in the sink organs of defoliated plants was also higher than in control or
defruited plants with the exception of current year shoots (Fig. 8a),
probably as a result of greater total assimilation during the period of
exposure. However, when calculated on a per organ basis, only older
stems accumulated more 13C in partially defoliated plants compared to
those subjected to other treatments (Fig. 8b).
When 13C distribution was estimated on a per plant basis taking
account of the weights of individual organs, the current year shoots
were the greatest sink organ with between 55 and 75 % of the trans-
ported 13C accumulating in this organ (Fig. 8b). The remainder of the
transported 13C was evenly distributed between fruit, older stems and
roots highlighting the requirement for current photoassimilation to si-
multaneously support fruit development, vegetative growth and accu-
mulation of storage carbohydrates.
4. Discussion
Previous work has demonstrated that the blueberry crop exhibits
significant yield instability that is associated with limited capacity of
the plants to assimilate CO2 and accumulate storage carbohydrates in a
fluctuating light environment (Petridis et al., 2018). Measurements of
carbohydrate dynamics along with 13C-labelling has additionally re-
vealed that a large portion of newly assimilated carbon is retained in
the leaves, primarily in the form of sucrose and starch (Darnell, 1991;
Petridis et al., 2018). It has previously been found that high sugar and/
or starch accumulation in leaves leads to a feedback downregulation of
photosynthesis in a range if species including blueberry (Jorquera-
Fontena et al., 2016). This is associated with either limiting sink
strength or limiting photoassimilate export capacity at the leaf level
(Demmig-Adams et al., 2014). Here, we used a physiological, bio-
chemical and molecular approach to examine the mechanisms con-
trolling source-sink relations in blueberries and to determine whether
yield is limited by sink demand or leaf capacity to export photo-
assimilates. Our data indicates that blueberry yield is not significantly
reduced after 50 % defoliation associated with a compensatory increase
9
Environmental and Experimental Botany 174 (2020) 104031
Fig. 8. Newly assimilated carbon in individual
organs of ‘Liberty’ plants after manipulation of
source-sink balance. Plants were labelled for 2
h with 13CO2 and harvested after chasing for
24 h. 13C concentration was estimated in in-
dividual organs (a) and data were used to es-
timate the mean total 13C-assimilation in three
replicate plants normalised to 100 g DW older
stem (b). Data were subjected to one-way
ANOVA and significant differences (P ≤ 0.05)
between means were determined using Tukey’s
test. Different letters above bars denote differ-
ences between treatments for the same organ
and where letters are absent differences were
insignificant. Bars are the mean value of three
replicates ± SE, n = 3.
in photoassimilation driven by alterations at the photochemical, bio-
chemical and stomatal levels. In our experiments replication was
somewhat limited and it is likely that a statistical reduction in yield
may be observed if a higher number of replicated plants were used.
Nevertheless, the data suggest that enhanced photosynthetic rates fol-
lowing partial defoliation can at least in part compensate for potential
yield losses. On the contrary, complete fruit removal did not down-
regulate CO2 assimilation capacity. These data suggest that typical
source-sink feedback relationships influencing photosynthesis in blue-
berry are more complex than previously reported in other systems.
A major finding was that in defoliated plants an overall increase in
photosynthesis helped to maintained yield via, indicative of photo-
synthetic plasticity in response to altered sink-source balance. This in-
crease was manifested by greater stomatal conductance, electron
transport rate, carboxylation efficiency and expression of Calvin-Benson
cycle genes (Fig. 1–4).
The increase in photosynthesis in response to defoliation can be
partly attributed to the increase in stomatal conductance (Fig. 1b) that
enhanced CO2 availability for Calvin-Benson cycle reactions (Fig. 1b, c).
Many fruit-tree species alter stomatal responses in response to changes
in source-sink balance (Blanusa et al., 2006; DaMatta et al., 2008; Li
et al., 2007; Jorquera-Fontena et al., 2016) and it has been suggested
that changes in soil-to-leaf hydraulic conductance following partial
canopy removal may be a significant driver in tree species (Quentin
et al., 2011). In blueberries, previous work examining the effect of
source-sink balance on leaf traits found a positive correlation between
stomatal conductance and photosynthesis (Jorquera-Fontena et al.,
2016). This finding agrees with our results. The observation that there
was no difference in stomatal conductance between control and
defruited plants, implies that while stomatal behaviour can be influ-
enced by an increased relative sink demand, reduced sink demand does
not influence stomata.
The induction of photosynthesis due to defoliation was also related
to an increase in electron transport rate that is probably driven by the
higher electron demand for the reductive phase of the Calvin-Benson
cycle. Leaves from partially defoliated plants also exhibited a greater
quantum efficiency of PSII at higher light intensities (Fig. 2c). On the
contrary, NPQ was similar amongst all treatments at all light intensities
(Fig. 2d) indicating that greater quantum efficiency of leaves from de-
foliated plants was not the result of reduced dissipation of energy as
heat. These data suggest that enhanced quantum efficiency of leaves
from defoliated plants were probably driven by other mechanisms such
as alterations in thylakoid structure (Allen and Forsberg, 2001) and
alterations in the phosphorylation status of light harvesting complexes
and proteins associated with electron transport and downstream bio-
chemical processes leading to photosynthetic optimisation (Ruban,
2015; Schönberg et al., 2017).
Notably, defoliation upregulated the expression of all genes that
encoded proteins specific to the Calvin-Benson cycle while closely re-
lated genes encoding proteins with identical molecular functions in
A. Petridis, et al.
other pathways were not more strongly expressed (Fig. 4). Indeed, the
transcript levels of four genes, including RUBISCO, RUBISCO ACTIV-
ASE, PHOSPHORIBULOKINASE, and GLYCERALDEHYDE-3-PHOSPH-
ATE DEHYDROGENASE3, were more abundant in defoliated plants.
Rubisco is responsible for the carboxylation of CO2 and is the rate-
limiting enzyme of the cycle. It seems that defoliation increases the
carboxylation efficiency of Rubisco (Fig. 3b), which in turn results in
higher photosynthetic rate. This increase in carboxylation efficiency of
Rubisco is probably driven by Rubisco activase that enhances its acti-
vation state. This is further supported by the observation that the
quantity of Rubisco protein was only marginally higher in defoliated
plants compared to the other treatments. Our findings agree with those
of Kasai (2008), who found that the activation state of Rubisco in
soybean was higher in plants with low source:sink ratio. However,
while photosynthetic responses of soybeans to changes in source-sink
balance are linked to leaf carbohydrate status, there is no evidence here
for a sugar-mediated regulation of blueberry photosynthesis. Indeed,
although defoliated plants had significantly higher photosynthetic rate
compared with control and defruited plants, foliar carbohydrate con-
tent was identical in all treatments. This contrasts with previous data
that indicated that severe sink restriction caused by girdling and fruit
removal resulted in a significantly higher carbohydrate content in cv.
‘Brigitta’ leaves (Jorquera-Fontena et al., 2016). Generally, Rubisco
activase uses the energy generated from the hydrolysis of ATP to re-
model the conformation of Rubisco (Carmo-Silva and Salvucci, 2013)
and is able to adjust the rate of carbon assimilation to the rate of
electron transport through changes in Rubisco activation state (Salvucci
et al., 1985). It is thus possible that in blueberries, under conditions of
high sink demand, the increase in photochemical efficiency and pre-
sumably ATP supply promotes the induction of Calvin-Benson cycle
through an upregulation of Rubisco activase expression and subsequent
activation of Rubisco. However, there are also reports indicating that
the increase in photosynthesis due to changes in source-sink balance is
not accompanied by changes in Calvin-Benson cycle enzymes. For ex-
ample, DaMatta et al. (2008) found that in coffee tree the activities of
enzymes involved in photosynthetic pathways (Rubisco and GAPDH)
were unaffected by changes in source-sink balance. Besides Rubisco and
Rubisco activase, defoliation also increased the expression of chlor-
oplastic-GAPDH3 and phosphoribulokinase, which are involved in the
reduction and regeneration phase of the Calvin-Benson cycle, respec-
tively, indicating an overall upregulation of the cycle to sustain high
sink demand. Phosphoribulokinase is unique to photosynthesis and is
responsible for the regeneration of the initial C5 acceptor of CO2 by
phosphorylating ribulose-5-phosphate to form ribulose-1,5-bispho-
sphate (Jones et al., 2013). The increase of phosphoribulokinase and
Rubisco following defoliation suggests that under normal conditions
photosynthesis is limited by the rate of ribulose bisphosphate re-
generation and by the rate of carboxylation.
Defoliated plants demonstrated a capacity to significantly enhance
CO2 assimilation to meet the demands of developing fruit thereby al-
lowing them to maintain yield at levels statistically similar to those of
control plants (Fig. 1a, b). However, we have previously shown that the
blueberry crop continuously loses yield throughout the growing season,
suggesting that fruit development is not fully supported by available
photoassimilates in control plants (Petridis et al., 2018). Furthermore,
yield can be enhanced by the placement of mulches to reflect light back
into the canopy (Petridis et al., 2018), a phenomenon that is also as-
sociated with enhanced maximum CO2 assimilation rates (Petridis
et al., in preparation). This raises questions regarding the mechanisms
driving source-sink communication to maintain an appropriate level of
photoassimilation to support sink development and why blueberry
plants limit photoassimilation under standard growing conditions. One
possibility is that increased photoassimilation is part of a wounding
response caused by extensive defoliation. Previous work has focussed
on photosynthetic responses to insect-induced defoliation within the
context of defence and tolerance. In the short-term (hours to days)
10
Environmental and Experimental Botany 174 (2020) 104031
insect damage to foliage generally reduces photosynthesis and photo-
synthetic gene expression via a genetically programmed response
(Bilgin et al., 2010; Kerchev et al., 2012). On the contrary, several
studies have indicated longer term (weeks) increases in photosynthesis
following insect infestation as a compensatory mechanism imparting
tolerance to herbivory (Kucharik et al., 2016; Zhao and Chen, 2012).
Therefore, some of the signalling pathways leading to increased pho-
tosynthesis several weeks or months after defoliation could be shared
with those associated with biotic defence responses. However, the ob-
servation that maximum photosynthetic rates can also be enhanced in
the absence of foliar damage by placing reflective mulch alongside
plants (Petridis et al., in preparation) suggests that signalling pathways
other than those associated with a damage response can influence
photosynthetic rates.
Whatever the signalling mechanisms, the enhancement of source
activity due to defoliation indicates that under normal conditions
photosynthesis is not operating at its full potential and implies that
photosynthesis operates as if plants have a high source:sink ratio. The
perception of high source-sink ratio may also explain why blueberries
have relatively low photosynthetic rate compared with other fruit-tree
species, such as apple, pear, peach and sweet cherry (Duan et al., 2016;
Gonçalves et al., 2005; Teo et al., 2006; Zhang et al., 2005). In general,
many woody perennials regulate their photosynthesis in response to
sink demand (Duan et al., 2016). Here, we show that blueberries can
also adjust their capacity for photosynthesis in response to an increased
sink demand for photosynthetic products. Defoliation decreases source
size, while sink demand remains constant. This increases the demand
for photoassimilates from remaining leaves which in many species re-
duces end-product inhibition and increases photosynthetic rate of the
remaining leaves (Eyles et al., 2013; Layne and Flore, 1995). On the
contrary, our data suggest that the upregulation of photosynthesis in
blueberries in response to elevated sink demand is unrelated to foliar
carbohydrate levels, since the concentration of non-structural carbo-
hydrates in leaves does not differ between treatments (Fig. 6a). If
photosynthesis in blueberries is not regulated by end-product inhibi-
tion, then a question that arises is whether there is an alternative me-
chanism to control photosynthetic responses due to changes in source-
sink balance. Recent work in tomato has proposed a role for cytokinins
in regulating sink-source relationships (Glanz-Idan et al., 2020). In this
case, partial defoliation resulted in an adaptive increase in photo-
assimilation that was accompanied by a rapid decrease in transcripts
encoding low and high affinity sucrose transporters responsible for
phloem loading leading to a redirection of photoassimilates into in-
creasing the size of the photosynthetic organ and away from fruit de-
velopment. However, further investigations will be required to reveal
whether cytokinin signalling is also involved in sink to source com-
munication in blueberry.
Following carbon fixation, the next step for supplying sink organs
with substrate for growth and maintenance is to export photo-
assimilates into the phloem (phloem loading). In general, there are
three main strategies for loading sugars to the phloem: apoplastic and
symplastic with or without polymer trapping (Ainsworth and Bush,
2011; Rennie and Turgeon, 2009). In apoplastic loading, the transport
of sucrose into the phloem involves the action of plasma membrane
transporters and requires energy provided by proton motive force. In
symplastic loading, which includes many trees and shrubs, sugars dif-
fuse along a concentration gradient form mesophyll cells to the phloem
through numerous plasmodesmata. In some plants, diffusion through
plasmodesmata is passive, while in other plants it may involve polymer
trapping (Rennie and Turgeon, 2009). Between symplastic and apo-
plastic loading strategies, the latter shows greater plasticity in exporting
photoassimilates under conditions favourable for photosynthesis (White
et al., 2016). CO2 enrichment experiments indicated that plants with
symplastic loading accumulated more non-structural carbohydrates
than those with apoplastic loading due to limitations in export capacity
(Körner, 1995). Vaccinium species have previously been described as
A. Petridis, et al.
possessing a minor vein structure described as primitive closed
(Gamalei, 1989) that is associated with apoplastic phloem loading
pathways (Gamalei, 1991). However, our findings suggest that further
examination of phloem loading mechanisms in blueberry is warranted.
Regardless of the mechanism that blueberries use to export photo-
assimilates to the phloem, it is evident that photoassimilate export is
tightly regulated and integrated with other metabolic processes to
maintain an appropriate partitioning of resource between the photo-
synthetic organs, other vegetative structures and the reproductive or-
gans. Our data indicates that leaves accumulate more non-structural
carbohydrates than the other organs, regardless of treatment (Fig. 7).
Furthermore, newly assimilated carbon is retained in the leaves and
only a small portion is distributed to sink organs after a single dark
period (Fig. 8). This contrasts with herbaceous plants where source
limited plants are almost completely devoid of leaf starch at dawn
(Smith and Stitt, 2007). These data indicate that blueberries operate a
conservative strategy where leaves retain much more carbohydrate, at
least in the short term than Arabidopsis plants. This may be related to
the ecological niche in which blueberries occur as understory plants
(Nestby et al., 2019) where light availability may vary dependent on
competition from neighbouring species which could change rapidly
dependent on growth or defoliation of competitors caused by biotic or
abiotic stresses. Such a lifestyle implies that light availability cannot be
guaranteed hence leaves may retain carbohydrate to provide a buffer
under such conditions.
5. Conclusions
We show that blueberries have the capacity to respond to changes in
photoassimilate demand via alterations in stomatal, photochemical and
biochemical parameters. Such alterations are able to at least partially
compensate for significant levels of defoliation. Given that under
standard growing conditions blueberry fruit yields are subject to lim-
itations imposed by photosynthetic assimilation this raises the question
of why photosynthetic capacity is limited under these conditions. Our
data indicates the potential to exploit perception of sink demand in
breeding strategies for improved germplasm with higher yields.
Funding information
This research was supported by Innovate UK (Grant/Award
Number: 102130) in collaboration with the Agriculture and
Horticultural Development Board, Soil Essentials, Marks and Spencer, S
&A Produce (UK) Ltd, Delta-T Devices, Thomas Thomson (Blairgowrie)
Ltd and Castleton Fruit Farm. The James Hutton Institute receives
support from the Rural and Environmental Science and Analytical
Services Division of the Scottish Government. Funders had no roles in
data collection, analysis or interpretation, or in preparation of the
manuscript. Funders approved manuscript submission.
CRediT authorship contribution statement
Antonios Petridis: Conceptualization, Methodology, Investigation,
Writing - original draft. Jeroen van der Kaay: Methodology,
Investigation. Julie Sungurtas: Formal analysis. Susan R. Verrall:
Formal analysis. Susan McCallum: Resources, Funding acquisition.
Julie Graham: Conceptualization, Supervision, Funding acquisition.
Robert D. Hancock: Conceptualization, Methodology, Writing - review
& editing, Supervision, Funding acquisition.
Declaration of Competing Interest
We have no conflicts of interest.
11
Environmental and Experimental Botany 174 (2020) 104031
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.envexpbot.2020.
104031.
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