Wheat plant selection for high yields entailed improvement of leaf anatomical and biochemical traits including tolerance to non-optimal temperature conditions

Abstract

Assessment of photosynthetic traits and temperature tolerance was performed on field-grown modern genotype (MG), and the local landrace (LR) of wheat (Triticum aestivum L.) as well as the wild relative species (Aegilops cylindrica Host.). The comparison was based on measurements of the gas exchange (A/ci, light and temperature response curves), slow and fast chlorophyll fluorescence kinetics, and some growth and leaf parameters. In MG, we observed the highest CO2 assimilation rate [Formula: see text] electron transport rate (Jmax) and maximum carboxylation rate [Formula: see text]. The Aegilops leaves had substantially lower values of all photosynthetic parameters; this fact correlated with its lower biomass production. The mesophyll conductance was almost the same in Aegilops and MG, despite the significant differences in leaf phenotype. In contrary, in LR with a higher dry mass per leaf area, the half mesophyll conductance (gm) values indicated more limited CO2 diffusion. In Aegilops, we found much lower carboxylation capacity; this can be attributed mainly to thin leaves and lower Rubisco activity. The difference in CO2 assimilation rate between MG and others was diminished because of its higher mitochondrial respiration activity indicating more intense metabolism. Assessment of temperature response showed lower temperature optimum and a narrow ecological valence (i.e., the range determining the tolerance limits of a species to an environmental factor) in Aegilops. In addition, analysis of photosynthetic thermostability identified the LR as the most sensitive. Our results support the idea that the selection for high yields was accompanied by the increase of photosynthetic productivity through unintentional improvement of leaf anatomical and biochemical traits including tolerance to non-optimal temperature conditions.

Full text

Vol.:(0123456789)
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Photosynthesis Research
https://doi.org/10.1007/s11120-018-0486-z
ORIGINAL ARTICLE
Wheat plant selection forhigh yields entailed improvement ofleaf
anatomical andbiochemical traits includingtolerance tonon-optimal
temperature conditions
MarianBrestic1· MarekZivcak1· PavolHauptvogel2· SvetlanaMisheva3· KonstantinaKocheva3· XinghongYang4·
XiangnanLi5· SuleymanI.Allakhverdiev6,7,8,9,10
Received: 2 January 2018 / Accepted: 23 January 2018
© Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract
Assessment of photosynthetic traits and temperature tolerance was performed on field-grown modern genotype (MG), and
the local landrace (LR) of wheat (Triticum aestivum L.) as well as the wild relative species (Aegilops cylindrica Host.). The
comparison was based on measurements of the gas exchange (A/ci, light and temperature response curves), slow and fast
chlorophyll fluorescence kinetics, and some growth and leaf parameters. In MG, we observed the highest CO2 assimilation
rate
(
ACO
2),
electron transport rate (Jmax) and maximum carboxylation rate
(
VCmax
)
. The Aegilops leaves had substantially
lower values of all photosynthetic parameters; this fact correlated with its lower biomass production. The mesophyll conduct-
ance was almost the same in Aegilops and MG, despite the significant differences in leaf phenotype. In contrary, in LR with
a higher dry mass per leaf area, the half mesophyll conductance (gm) values indicated more limited CO2 diffusion. In Aegilops,
we found much lower carboxylation capacity; this can be attributed mainly to thin leaves and lower Rubisco activity. The
difference in CO2 assimilation rate between MG and others was diminished because of its higher mitochondrial respiration
activity indicating more intense metabolism. Assessment of temperature response showed lower temperature optimum and
a narrow ecological valence (i.e., the range determining the tolerance limits of a species to an environmental factor) in
Aegilops. In addition, analysis of photosynthetic thermostability identified the LR as the most sensitive. Our results support
the idea that the selection for high yields was accompanied by the increase of photosynthetic productivity through uninten-
tional improvement of leaf anatomical and biochemical traits including tolerance to non-optimal temperature conditions.
Keywords Wheat· Landrace· Aegilops· Photosynthesis· Mesophyll conductance· Heat stress
Marian Brestic and Marek Zivcak have contributed equally to this
work.
* Marian Brestic
marian.brestic@uniag.sk
* Suleyman I. Allakhverdiev
suleyman.allakhverdiev@gmail.com
1 Department ofPlant Physiology, Slovak Agricultural
University, Tr. A. Hlinku 2, 94976Nitra, Slovakia
2 National Agricultural andFood Centre, Research Institute
ofPlant Production, Piešťany, Slovakia
3 Institute ofPlant Physiology andGenetics, Bulgarian
Academy ofSciences, Acad. Georgi Bonchev Street,
Bldg. 21, 1113Sofia, Bulgaria
4 State Key Laboratory ofCrop Biology, Shandong Key
Laboratory ofCrop Biology, Shandong Agricultural
University, Tai’an, China
5 Northeast Institute ofGeography andAgroecology, Chinese
Academy ofSciences, Changchun130102, China
6 Institute ofPlant Physiology, Russian Academy ofSciences,
Botanicheskaya Street 35, Moscow, Russia127276
7 Institute ofBasic Biological Problems, Russian Academy
ofSciences, Pushchino, MoscowRegion, Russia142290
8 Department ofPlant Physiology, Faculty ofBiology,
M.V. Lomonosov Moscow State University, Leninskie Gory
1-12, Moscow, Russia119991
9 Moscow Institute ofPhysics andTechnology, Institutsky lane
9, Dolgoprudny, MoscowRegion, Russia141700
10 Institute ofMolecular Biology andBiotechnology,
Azerbaijan National Academy ofSciences, Matbuat Avenue
2a, 1073Baku, Azerbaijan

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Abbreviations
A
CO
2
CO2 assimilation rate
ca Reference CO2 concentration
ci Intercellular CO2 concentration
F0 Basal fluorescence
Fv/Fm Maximum quantum yield of photosystem II
photochemistry
gm Mesophyll conductance
Jmax Electron transport rate
LMA Dry mass per leaf area
LR Local landrace
MG Modern genotype
PPFD Photosynthetic photon flux density
PSII Photosystem II
RH Relative air humidity
(
VCmax
)
Maximum carboxylation rate
VPD Vapor pressure deficit
WR Wild relative
Introduction
Photosynthesis is the key process necessary for plant pro-
duction. Therefore, many crop scientists have believed that
enhancing photosynthesis at the level of the single leaf
would increase yields (Makino 2011). Several studies have
used a series of wheats to examine the changes in photosyn-
thetic capacity that have occurred with domestication and
the increase in ploidy. Environmental stress may decrease
the rate of photosynthesis not only because of detrimental
effects on cell biochemistry, but also because of changes
in the diffusion of carbon dioxide (CO2) from the atmos-
phere to the site of carboxylation (Flexas etal. 2008; Warren
2008). Under high CO2 levels, the rate of diffusion of CO2
into the cell increases due to a high concentration gradient;
consequently, the uptake of CO2 through stomata and sto-
matal conductance increases (Reynolds etal. 2010). Even
under normal conditions, the slow diffusion of CO2 to the
site of carboxylation in the chloroplast can significantly limit
photosynthesis (Flexas etal. 2008; Warren 2008; Zhu etal.
2010). The diffusion of CO2 between intercellular airspaces
and the Rubisco enzyme is usually described by a param-
eter called internal or mesophyll conductance (gm). Different
species or genotypes show substantial variation in gm, and
the limiting effects of gm on photosynthesis are close to that
of stomatal conductance (Flexas etal. 2008; Warren 2008;
Barbour etal. 2010).
In wheat evolution two major periods were recognized.
The first period represents a thousand years of coinciden-
tal improvement before the nineteenth century from wild
wheat ancestors to local landraces (LRs), well adapted, but
generally low-yielding. The systematic breeding starting
later led to gradual improvement of key traits, resulting in
release of recent high-yielding modern genotypes (MGs).
The history of wheat cultivation is very old. About 10,000
BP, hunter-gatherers began to cultivate wild emmer. Sub-
conscious selection gradually created a cultivated emmer
(T. dicoccum, 2n = 4x = 28, genome AuAuBB) that sponta-
neously hybridized with another goat grass (Ae. tauschii,
2n = 2x = 14, genome DD) around 9000 BP to produce an
early spelt (T. spelta, 2n = 6x = 42, genome AuAuBBDD)
and in next period also another wheat taxa (Kihara 1944;
McFadden and Sears 1946; Kerber 1964; Kislev 1980;
Dvorak etal. 1998; Matsuoka and Nasuda 2004).
As a modern bread wheat (Triticum aestivum L.) culti-
vars, we denote the semidwarf cultivars carrying Rht1 and
Rht2 genes varieties. Release of these genotypes after 1965
led to significant increase of wheat production worldwide,
causing that most of cultivated varieties have partly similar
genetic background (Smale etal. 2002). By introduction of
these new, modern varieties, the harvest index was improved
and higher yield potential of new genotypes was obtained. In
addition, the breeding was focused on traits such as resist-
ance to pathogens, technological quality of grains, etc. How-
ever, the conventional selection practiced by the majority
of breeders has not considered directly the physiological or
biochemical traits and certainly not the photosynthetic traits.
Anyway, it is well known that the yield-based breeding, even
in the period before the green revolution, led to the uninten-
tional improvement of many traits.
On the other hand, there are hundreds of thousands of lan-
draces, the local cultivars within the T. aestivum L. species
not belonging to the group of MGs that constitute the wheats
of the world. Most of them are not cultivated in present for
agricultural use and they are concentrated in genebanks,
which protect them as a heritage. They represent genetic
resources of enormous diversity, potentially useful as donors
of important traits (Hoisington etal. 1999).
In addition to Triticum accessions, the second group of
resources useful in wheat breeding represents wild relatives
(WRs). The closest relative of wheat growing in Central
Europe is the tetraploid jointed goatgrass (Aegilops cylin-
drica Host.). As in the case of other polyploid Triticeae,
Ae. cylindrica is an amphiploidy (genome CCDD), resulting
from hybridization between the diploids Ae. caudata (CC)
and I (DD). There are numerous local forms in Central, but
mostly and South Europe and in the Middle East (Guadag-
nuolo etal. 2001).
Recently, the advanced technical tools for rapid meas-
urements and diagnostics of plants are available. The gas
exchange systems enable to perform exact measurements
even in field conditions. Moreover, the more precise pho-
tosynthetic models were developed, based mainly on the
Farquhar–von Caemerrer–Berry Model (Farquhar etal.
1980), which enable to identify the main limitations of

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CO2 assimilation (Long and Bernacchi 2003; Flexas etal.
2004). Recently, the gas exchange measurements are sup-
ported by continuous measurements of chlorophyll fluo-
rescence that provide quite a precise estimation of elec-
tron transport rate (Flexas etal. 2007; Zivcak etal. 2013).
Moreover, chlorophyll fluorescence represents a unique
tool for diagnostics of plant health status, photosynthetic
performance as well as effects of plant stress on plants
(Zivcak etal. 2008; Brestic etal. 2016; Kalaji etal. 2017)
and assessment of plant stress tolerance (Li etal. 2014;
Kalaji etal. 2016), for review on measuring stress effects
on crops, see Brestic and Zivcak (2013).
The aim of our experiments was to apply modern tech-
nical and methodical approaches to identify differences in
photosynthetic performance, temperature tolerance, and
limitations of photosynthetic assimilation (particularly to
limitations by mesophyll conductance) in plant material
representing different stages of wheat improvement. The
MG represents the result of recent breeding programs; it
is characterized by low height, favorable canopy architec-
ture and high yields. The LR represents the results of the
long period of domestication and intuitive, unconscious
selection in local conditions, recently available only in
gene banks. Moreover, a secondary WR of genus Aegilops
was used, which represents the material coming from wild
populations, being similar to the status before beginning
of wheat cultivation in the past.
Materials andmethods
Experimental setup
Experiments with field grown (field trials, Genebank of
PPRC-RIPP in Piešťany, Slovak Republic) modern wheat
variety ‘Astella’, LR ‘Diosecka’ (both T. aestivum L.) and
its WR species Ae. cylindrica Host. were realized in the
regular growing season. The seeds were sown in autumn
directly to the soil manually into experimental plots with
the area 1.5m2. Previous crop was field peas (Pisum sati-
vum spp. arvense). In the autumn, plants were fertilized
with 330kgha−1 NPK (15-15-15), 150kg ha−1 AMOFOS
fertilizer (12% N, 52% P2O5) and 120 kg ha−1 and potas-
sium chloride (60% K2O). Spring fertilization was real-
ized in the dose of 110kg ha−1 of ammonium nitrate with
limestone (27% N). Also, the autumn and spring spray
against weeds were realized in optimal doses. Average
temperature in the monitored period was 17.1°C; the
average soil temperature was 18.3°C with an average
daily sum of rainfalls 3.3mm; soil humidity 32.5%; air
humidity 44.8%.
Analyses ofgrowth andleaf traits
The growth and leaf traits were analyzed regularly during
the season. Ten plants of each genotype were taken, meas-
ured, and analyzed in laboratory. Only the results recorded
after anthesis showing the maximum height, dry mass level
and traits of fully developed flag leaves are shown in this
paper. Dry mass and leaf area were analyzed for leaves and
stems individually, the flag leaf of the main stem was meas-
ured separately. The average maximum height, total plant
dry mass, flag leaf area, and dry mass per leaf area (LMA)
are presented. LMA was calculated as the ratio of flag leaf
dry weight and flag leaf area (g m−2) according to Radford
(1967) and Hunt (1978).
Gas exchange measurements
Measurements of photosynthetic gas exchange were realized
with utilization of the open infrared gas analyzer (CIRAS-2;
PP-Systems, Hitchin, UK). The net CO2 assimilation rate
(
ACO
2)
, stomatal conductance (gs), and intercellular CO2
concentration (ci) were estimated. All parameters were
measured on the flag leaf 3–12 days after anthesis. The
measurements were done in laboratory conditions, imme-
diately after cubic intact block of soil with plants was dug
out with care, minimizing disturbance to the main part of
the root system up to 30cm depth. The plants in the cen-
tral position within the block were measured. To avoid the
influence of plant removal on the results of gas exchange
measurements, only the results obtained on the plants able
to fully open the stomata were used for analyses.
Light photosynthetic response curves were measured at
leaf temperature set on 25°C and reference CO2 concentra-
tion (ca) set to 380 µmol mol−1 (ppm) of CO2. Photosynthetic
photon flux density (PPFD) during light curve was 50, 100,
150, 200, 300, 400, 600, 800, 1000, and 1200 µmol (pho-
tons) m−2 s−1.
Temperature photosynthetic response curves were meas-
ured at reference CO2 concentration (ca) set to 380 µmol
mol−1 (ppm) of CO2, PPFD was 1000µmol (photons)
m2s−1. Leaf temperature was controlled by leaf chamber,
measured by IR sensor and the photosynthetic rates were
measured at 10, 15, 20, 25, 30, 35, and 40°C after 10min
at each temperature.
Air humidity was set to approximate value 60% of RH. In
a range of 10–25°C, air humidity was maintained between
55 and 65% (VPD 0.35–1.0kPa). At high temperature, the
air humidity in the chamber was decreasing; therefore, the
air entering the system was humidified. Nevertheless, the
relative air humidity at high temperature levels was decreas-
ing below the set value of 60%; for example, at 40°C the RH
in measuring chamber was ~ 45% (VPD 3.3kPa). Anyway,
as the tested plants were acclimated to high temperature and

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low air humidity in field conditions, the high VPD at high
temperature level was not causing significant stomata clo-
sure within the time intervals of the measurements at high
temperature levels.
A/ci photosynthetic response curves were measured at leaf
temperature set on 25°C and PPFD 1000 µmol (photons)
m−2 s−1. Reference CO2 concentration (ca) was set to 50,
100, 150, 200, 250, 300, 380, 500, 800, 1000, 1500, and
1800 µmol mol−1 (ppm) during A/ci photosynthesis response
curve. Relative air humidity was ~ 60% (VPD ~ 0.9kPa).
To analyze photosynthetic limitations and capacities, the
data were analyzed using the A/ci Curve Fitting 10.0 utility
available at http://landfl ux.org/Tools .php. This utility uses
the curve-fitting equations described by Ethier and Living-
ston (2004), based on the kinetic parameters of Farquhar
etal. (1980) and Bernacchi etal. (2001). From the model,
following parameters were analyzed:
(
VCmax
)
—maximum
carboxylation rate; Jmax—CO2-saturated electron transport
rate of the thylakoids reactions which ultimately supply the
necessary energy in the form of ATP and NADPH for the
regeneration of RuBP; kC—carboxylation efficiency equal
to the change in assimilation with respect to a change in
chloroplast CO2
(
kC=dACO
2
∕dcc
)
; gm—leaf mesophyll
(internal) conductance between intercellular spaces (substo-
matal cavity and the chloroplast); gm,t—total mesophyll con-
ductance, i.e., the total conductance between intercellular
spaces and chloroplast, (rm,t = rm + rc; rm,t = 1/gm,t; rm = 1/gm;
rc = 1/kC;
k
C
=
dACO
2∕
dc
c
).
Test ofPSII thermostability
The test of PSII thermostability was realized according to
Brestic etal. (2012). The heat treatment was performed in
darkness; cuttings from the middle part of the leaves of app.
50mm length were placed into small sealable polyethylene
bags. The bags were closed and then, they were completely
submersed into the water in water bath with precisely con-
trolled water temperature for 30min. The time of exposure
was found as sufficient after previous analyses using differ-
ent exposure times. After 30min of exposure, the bag was
pulled from the bath, kept still in darkness, and after cooling
to laboratory temperature (5–10min) the sample was meas-
ured. In a single temperature experiment, the temperature
level of 42°C was used.
Chlorophyll fluorescence measurements
The Chl a fluorescence measurements were carried out
using Handy-PEA (Hansatech Instruments Ltd, UK); the
leaf samples were illuminated with continuous red light
(the wavelength in peak 650nm; the spectral line half-
width 22nm). The light was provided by an array of three
light-emitting diodes. The light pulse intensity used was
3500µmolm−2s−1; duration of the light pulse was 1s. The
fluorescence signal was recorded with maximum frequency
105 points s−1 (each 10µs) within 0–0.3ms, after that the
frequency of recording gradually decreased collecting all
together 118 points within 1s. Measurements on leaf seg-
ments were performed in the middle part of a leaf blade,
out of the main leaf vein, after 30min of dark adaptation,
using leaf clips.
Statistical analyses
The majority of reported data represent the weighted
mean ± standard error. Statistical analysis was performed
using analysis of variance (ANOVA) followed by the post
hoc Tukey HSD test using software Statistica version 9.0
(Statsoft Inc., Tulsa, Oklahoma, USA). The numbers of
repetitions used in gas exchange and growth analyses were
six to ten. In the PSII thermostability, usually 20 leave sam-
ples of each genotype were measured before and after heat
treatment.
Results anddiscussion
As expected, species and genotypes differed in growth and
leaf parameters (Fig.1). Compared to modern variety, the
landrace (LR) was much taller, but it had the same plant dry
mass and the same flag leaf area, but substantially higher
LMA, indicating thicker leaves or leaves with a greater den-
sity. In contrary, Aegilops, the WR species, was character-
ized by the smallest plants, small and thin leaves (low LMA)
and substantially lower dry mass production compared to
both wheat cultivars.
The gas exchange measurements showed significantly
higher steady-state net assimilation rate (Fig.2a). Interest-
ingly, the lower photosynthetic rate in LR or WR compared
to MG was not associated with lower intercellular CO2 con-
centration (ci), but we observed the opposite trend (Fig.2b).
Moreover, similar mean values of stomatal conductance (gs)
were observed in all three genotypes (Fig.2c). The unequal
A
CO
2∕
c
i
ratio indicates differences in efficiency by which
the photosynthetic apparatus uses the CO2 entering the leaf
(Fig.2d). These results suggest that the differences in CO2
assimilation were not caused by stomatal limitations of CO2
diffusion. The highest mitochondrial respiration per leaf area
measured in darkness (Fig.2e) was found in modern vari-
ety, significantly lower was in LR and in Aegilops. How-
ever, when the respiration was recalculated per dry mass
unit (Fig.2f), we observed similar values in the LR and
the Aegilops, but significantly higher dark respiration in a
modern variety.

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In general, the results presented in our study confirm
that the modern, high-yielding genotype of wheat was bet-
ter in all traits related to the leaf photosynthesis compared
to old LR or wild ancestor. Despite the photosynthesis had
never been the direct and conscious selection criterion, the
photosynthetic improvement was obviously a necessary by-
product of a long-term selection aimed at yield increase.
The relationship between the photosynthetic performance
and yield is not straightforward, with an accumulation
capacity of the sink being extremely important (Peet and
Kramer 1980; Borrás etal. 2004); anyway, the studies ana-
lyzing releases of crop genotypes through several decades
show that yield increases were obtained through increases
in harvest index until the 1980s, after which the increase
in biomass accumulation and photosynthesis became more
important. This indicates a transition from sink to source
limitation in two of our major C3 crops (Shearman etal.
2005; Hubbart etal. 2007). Many other research findings
strongly support the hypothesis that a sustained increase in
leaf photosynthesis can lead to increase in total production
of biomass that would be necessary to gain further increase
in crop yield (Long etal. 2006; Parry etal. 2007; Zhu etal.
2010; Evans 2013). The scientific dogma about generally
oversized capacity of crop photosynthetic apparatus, broadly
accepted in 1980s and 1990s, was broken up after the pub-
lication of results of series experiments with increased CO2
in different conditions, documenting a significant increase
of grain yield because of increase of leaf photosynthesis
(Drake etal. 1997; Mitchell etal. 1999; Bender etal. 1999;
Ainsworth etal. 2002; Ainsworth and Long 2005). Moreo-
ver, analysis of photosynthetic rate in Australian bread wheat
genotypes with different date of release indicated that the
selection for higher grain yield led to unconscious selection
for higher photosynthetic rate (Watanabe etal. 1994). Simi-
larly, our results comparing the photosynthetic and the leaf
parameters of high-yielding wheat (T. aestivum L.) genotype
with a WR species of the same group used as a gene donor
in wheat breeding (Ae. cylindrica Host.) or LR supports this
idea (Fig.1). These data indicate that the WR species of
the same group as wheat, having almost the same vegeta-
tion period and canopy development as the observed geno-
type of winter wheat, is much less productive, it has smaller
and thicker leaves. Moreover, the leaves of Aegilops dem-
onstrated significantly lower metabolic activity, as shown
by the rate of mitochondrial respiration per dry mass unit
(Fig.2).
Further analysis of A/ci curve by Farquhar–von Caemer-
rer–Berry model (Farquhar etal. 1980) adjusted by Ethier
and Livingston (2004) enabled to uncover the individual
limitations of CO2 assimilation process (Fig.3). Whereas
the trend of the maximum carboxylation rate
(
VCmax
)
was
very similar to CO2 assimilation (Fig.3a), in case of the
maximum electron transport rate (Jmax) we did not observe
Fig. 1 Comparison of selected growth parameters measured in a
modern genotype (MG), landrace (LR), and in a wild relative (WR).
The measurements were done after anthesis when the maximum
plant height was achieved. a The average aboveground dry mass of
plants; b average plant height; c leaf area of a flag (upper) leaf; d
the dry mass of flag leaf per unit of leaf area (the dry mass per leaf
area, LMA). The columns represent weighted mean values (n = 10).
The error bars represent standard error of mean. Small letters above
the bars (a, b, c) indicate significance of mean values of individual
parameters, where bars with the different letters indicate significant
difference of parameter values at P = 0.05, but bars with the same let-
ters indicate no significant difference of parameter values among MG,
LR, and WR (by ANOVA and post hoc Tukey HSD test)

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significant differences between the LR and the modern
wheat variety (Fig.3b). Anyway, in Aegilops, Jmax was low.
The estimation of internal (mesophyll) conductance (Fig.3c)
suggests substantially lower gm values in LR compared to
the modern variety and Aegilops. It is probably due to the
different leaf properties, also shown by values of high LMA
in LR (Fig.1d). The total internal conductance gm,t (Fig.3d)
was considerably higher in a modern variety compared to
LR and Aegilops (Fig.3d); in the case of LR, the decrease
was caused mainly by low mesophyll conductance as well
as by moderately lower kC. In Aegilops, the dominant com-
ponent of total internal resistance was low kC, as the meso-
phyll conductance of thin and soft leaves was rather high.
Although the results of our experiments, compared to pre-
viously mentioned studies, have not such a high potential
to provide conclusions with a general validity, the meas-
urements brought several interesting details uncovering the
specific mechanisms leading to photosynthetic improve-
ments in MGs. Although the higher leaf thickness in wheat
compared to Aegilops seems to be extremely important, the
comparison of MG with a LR shows that the higher LMA
does not necessarily lead to higher photosynthesis. The esti-
mates of mesophyll conductance confirmed our expectations
that the mesophyll limitation in thin and soft Aegilops leaves
will be very low. On the other hand, the mesophyll conduct-
ance was significantly lower in leaves of LR with thick and
hard leaf. The mesophyll conductance represents the con-
ductance for CO2 flowing from the intercellular airspaces
to the carboxylation site in the chloroplasts, which includes
barriers consisting of the cell wall, plasma membrane, chlo-
roplast envelope, and stromal thylakoids; both in air and liq-
uid phases (Evans etal. 2009). In previous years, it was
shown that gm plays a crucial role in the regulation of pho-
tosynthesis, representing up to 40% of the CO2 diffusional
Fig. 2 Photosynthetic parameters obtained from gas exchange meas-
urements in steady state realized in a modern variety (MG), landrace
(LR) and in wild relative species (WR). The measurements were done
on flag leaf during anthesis and the short time after anthesis. a The
average CO2 assimilation rate
(
ACO
2)
measured at 380ppm of CO2;
b intercellular CO2 concentration (ci); c leaf stomatal conductance
(gs) d CO2 assimilation rate to intercellular CO2 concentration ratio
(
ACO
2
∕ci
)
; e dark respiration (absolute value of
A
CO
2
measured in the
dark) per leaf area unit; f dark respiration (absolute value of
A
CO
2
measured in the dark) recalculated per dry mass unit. The conditions
in leaf chamber were set as follows: leaf temperature 25°C, PPFD
1000µmol (photons) m−2 s−1, reference CO2 concentration 380ppm,
RH ~ 60%. The columns represent weighted mean values (n = 10);
the error bars represent standard error of mean. Small letters above
the bars (a, b, c) indicate significance of mean values of individual
parameters, where bars with the different letters indicate significant
difference of parameter values at P = 0.05, but bars with the same let-
ters indicate no significant difference of parameter values among MG,
LR, and WR (by ANOVA and post hoc Tukey HSD test)

Photosynthesis Research
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limitations (Warren 2008). Especially, an increase of meso-
phyll limitations was observed in non-optimum conditions,
such as drought stress (Flexas etal. 2004; Olsovska etal.
2016), high temperature (Bernacchi etal. 2002; Allakhver-
diev and Murata 2004; Allakhverdiev etal. 2008), and some
other environmental factors limiting the photosynthesis
(Allakhverdiev etal. 1997; Bernacchi etal. 2002; Nishiy-
ama etal. 2006; Mohanty etal. 2007; Murata etal. 2007;
Flexas etal. 2008; Allakhverdiev 2011). In our experiment
(Fig.3), we used the estimation of mesophyll conductance
using the model of Ethier and Livingston (2004), which is
based on analysis of the shape of A/ci curve. Similar to other
methods, this model has also some limitations (Pons etal.
1999), especially when it is used in non-optimum conditions.
Therefore, the results represent only the rough estimate and
possible subtle variances cannot be reliably recognized.
However, in our case, we found relatively high differences
among observed samples. When mesophyll conductance is
high enough [> 0.6mol (CO2) m−2 s−1], it can be assumed as
“infinite” as the mesophyll limitation is relatively low com-
pared to other factors limiting CO2 assimilation. In wheat
LR, the values significantly < 0.6mol (CO2) m−2 s−1, indi-
cating an important mesophyll limitation even in optimum
conditions. Logically, the differences in leaf anatomy can be
expected as the main reason of the different gm. However,
the studies performed on species differing significantly with
a leaf thickness have shown the opposite trends: the higher
thickness, the lower mesophyll limitation. This was mainly
due to the higher surface area of the mesophyll cells (Hanba
etal. 1999). Moreover, it was shown that the direct effect
of leaf thickness is relatively low, especially in herbaceous
plants (Flexas etal. 2012). Kaminski etal. (1990) made the
comparison of anatomical parameters and photosynthetic
rates per unit leaf area on fully expanded flag leaves of Triti-
cum and Aegilops species at light saturation. They found
that wheats with thinner leaves, lower LMA, and chlorophyll
per unit area had the higher photosynthetic rate compared
to genotypes with more thick and dense leaves. The surface
area of the mesophyll cells per unit volume of mesophyll
tissue was similar in all ploidy levels, but the internal (i.e.,
mesophyll) conductance can be suggested to play the major
role in reaching the higher photosynthetic rate in genotypes
with thin leaves. Thus, they observed the similar trend to our
results, i.e., increase of mesophyll limitation with leaf thick-
ness, which was evident when comparing MG with Aegilops.
In our case, despite high gm, the Aegilops had lower photo-
synthesis due to metabolic limitations. More surprising are
relatively high differences between the values of gm indi-
cated in a modern wheat genotype and values in old LR.
They can be explained either by unequal thickness of cell
walls or in the surface areas of chloroplasts exposed to inter-
cellular air species (Niinemets etal. 2009). A lower dark
respiration per dry mass unit in LR compared to MG gives a
reason to believe that there is a much higher contribution of
Fig. 3 Model-derived parameters characterizing photosynthetic limi-
tations obtained using data from analyses of A/ci-curve within gas
exchange measurements realized in a modern variety (MG), landrace
(LR), and in a wild relative (WR). The measurements were done on
flag leaf during anthesis and the short time after anthesis. a Maxi-
mum rate of carboxylation
(
VCmax
)
; b maximum light driven elec-
tron flux (Jmax); c mesophyll (internal) conductance of leaf (gm); d
total conductance of leaf (gm,t). The columns represent weighted
mean values (n = 6–8); the error bars represent standard error of
mean. Small letters above the bars (a, b, c) indicate significance of
mean values of individual parameters, where bars with the different
letters indicate significant difference of parameter values at P = 0.05,
but bars with the same letters indicate no significant difference of
parameter values among MG, LR, and WR (by ANOVA and post hoc
Tukey HSD test)

Photosynthesis Research
1 3
extracellular structures (e.g., due to thicker or lignified cell
walls) in leaves of LR compared to MG, which may limit
the CO2 exchange between the gas and liquid phases. This
hypothesis, however, needs further investigation.
In addition to diffusion limitations, the catalytic efficiency
or the photosynthetic enzyme (especially of Rubisco) plays
a major role in reaching high carboxylation efficiency (Parry
etal. 2007), which is reflected also in crop breeding pro-
grams (Reynolds etal. 2011). Zhu etal. (2010) indicated
that the increase of catalytic efficiency would increase the
rate of photosynthetic fixation without need of additional
Rubisco. This would be beneficial, especially in high light
conditions, where the Rubisco enzymatic activity limits the
photosynthetic rate. In contrary, the increase in specificity
factor would increase net CO2 uptake in low light conditions,
where the electron transport rate limits the photosynthesis;
the electron transport chain would be directed away from
photorespiration into photosynthesis. Unfortunately, it was
shown that increase in specificity factor leads to decrease
in catalytic activity of Rubisco (Bainbridge etal. 1995).
Therefore, there are conflicting consequences at the level of
the canopy. The increased specificity factor would increase
light-limited photosynthesis, while the associated decrease
in catalytic efficiency would lower the light-saturated rate
of photosynthesis (Zhu etal. 2004). The analyses of A/ci
curve and light response curves (Fig.2) enabled to identify
a higher carboxylation efficiency and lower light compen-
sation point of CO2 assimilation (not shown here) in MG
compared to LR and wild species. It indicates that during
the selection, the priority was given at an efficiency of pho-
tosynthetic in high light at the expense of efficiency at light
limiting conditions. A higher photosynthetic performance
of modern wheat variety flag leaf went along with a higher
level of mitochondrial respiration (Fig.2). It can be consid-
ered as a logical consequence of higher metabolic (mainly
nitrogen) demand of high-yielding variety.
Based on the temperature response curve of CO2
assimilation rates (Fig.4), the Aegilops was found to be
more sensitive to high temperature, with a thermal opti-
mum at lower level as compared to wheats. Responses
of wheat genotypes were similar. Test of thermostability
at PSII level assessed using rapid fluorescence kinetics
record (Fig.5) showed LR to be more susceptible to heat
impairment as shown on significant increase of the basal
fluorescence (F0) and the maximum quantum yield of
PSII (Fv/Fm). In the fluorescent raw curves, there is an
evident local maximum in time of 0.3ms followed by a
subsequent decrease of fluorescence, which is denoted
as a K-step. The visual appearance of this step is an indi-
cator of serious damage at the level of oxygen evolving
complex (OEC) within photosystem II. The most pro-
nounced K-step increase followed by fluorescent decrease
was found in LR indicating the heat susceptibility of this
Fig. 4 The temperature response curve of CO2 assimilation rate
measured in leaves of the modern variety (MG), landrace (LR), and
in a wild relative (WR). The points represent the relative values of
CO2 assimilation (measured values divided by the maximum assimi-
lation, Amax). The values of maximum assimilation rates (Amax)
recorded in temperature response curves for each genotype are shown
on the small plot within the graph
Fig. 5 The results of PSII thermostability test at 42 °C in leaves of
the modern variety (MG), local landrace (LR), and wild relative
Aegilops (WR). The polyphasic curves represent the fast chlorophyll
a fluorescence kinetics plotted on the logarithmic time scale (average
curve for each genotype). The small bar plots inside the graph show
the values of basal fluorescence F0 and maximum quantum yield of
PSII photochemistry (Fv/Fm) before and after heat treatment

Photosynthesis Research
1 3
genotype. In contrary, the Aegilops was found to be the
most resistant to heat injury of PSII, as shown by the
values of Fo, Fv/Fm, and the fluorescence curve shape.
Very interesting is also the difference between wheats
and Aegilops in optimum temperature for photosynthe-
sis (Fig.4). The decrease of photosynthesis at elevated
temperature is associated mostly with Rubisco deactiva-
tion, which represents a regulatory feedback from one
of the processes contributing to the RuBP regeneration
capacity (Kubien and Sage 2008). Galmes etal. (2015)
found that the optimum temperature (Topt) for Rubisco
catalytic activity correlated with specificity factor Sc/o
for land plants. Despite the lower temperature optima,
the Aegilops expressed a higher level of PSII thermo-
stability (Fig.5). Knight and Ackerly (2002) found that
photosynthetic thermotolerance is significantly different
between genera and species, highly plastic, and this plas-
ticity causes that plants adapted to warmer environments,
when measured in the field, have higher temperature tol-
erance compared to those adapted to moderate conditions.
In general, the relationship between temperature optimum
for CO2 assimilation and resistance of PSII to high tem-
perature is complicated, especially when comparing dif-
ferent ecological groups. For example, alpine species with
low temperature optima express a higher PSII thermo-
stability compared to many herbs living in the moderate
zone (Neuner and Pramsohler2006). On the other hand,
when comparing plants belonging to similar groups, e.g.,
field crops, the species with higher temperature optima
have usually a higher critical temperature. For example,
the damage of PSII in barley occurred at temperature by
6°C lower compared to maize (Havaux etal. 1990). In
addition, the differences in PSII thermostability were also
found in the genotypes of the same species. For example,
in collections of wheat grown in the same field trials,
it was found that the PSII thermostability and plasticity
were higher in genotypes coming from warmer environ-
ments (Brestic etal. 2012). Acclimation capacity to heat
stress is associated also with recovery from heat-induced
damage (Takahashi etal. 2004), which was shown also
in wheat (Kreslavski etal. 2009). As the accession of
Ae. cylindrica used in this study originates from a warm
and dry location, the high PSII thermostability, but also
a high temperature optimum can be expected. There-
fore, the lower temperature optimum compared to wheat
(Fig.4) seems to be not a consequence of adaptations to
low temperature, but the result of the overall setup of CO2
assimilation pathway, which is, however, not optimal for
the needs of high-yielding wheat genotypes, especially
taking into an account the gradual elevation of tempera-
ture observed worldwide and needs associated with an
increase of photosynthetic production in Climate Change
conditions.
Conclusions
In summary, our results indicated several differences in
photosynthetic traits between MG, LR, and WR species at
the different levels, such as leaf anatomy, morphology and
biochemistry, CO2 diffusion, carboxylation efficiency, light
use efficiency, and temperature optima. The better photo-
synthetic performance of modern wheat variety suggests
that the long-term wheat selection and breeding towards
high yield and optimum phenotype were associated with
selection towards optimization of photosynthetic processes.
Compared to wild wheat relative species Aegilops, we found
a higher robustness of the leaf photosynthetic apparatus in
wheat genotypes, leading to higher photosynthetic capacity.
Moreover, in wheat genotypes, we have identified a wider
range determining the optimum temperature limits compared
to Aegilops, which may contribute to higher photosynthetic
productivity across the growing season. On the other way,
we found that a lower photosynthetic capacity observed in
more robust leaves of old wheat LR was associated with a
significant mesophyll limitation of CO2 fixation. Thus, it
must be taken into an account that the use of wild ances-
tors and LRs in breeding may have some pleiotropic effects
related to introgression of undesirable photosynthetic traits
resulting in lower photosynthetic productivity.
Acknowledgements This work was supported by the projects VEGA-
1-0923-16, VEGA-1/0831/17, APVV-15-0721, APVV SK-BG-2013-
0029, and APVV SK-CN-2015-0005, and by the Grants from Russian
Foundation for Basic Research (Nos: 17-04-01289; 17-54-7819), and
by Molecular and Cell Biology Programs from Russian Academy of
Sciences.
Author contributions MZ, MB, and SIA wrote the paper. MZ con-
ducted the statistical analyses and analyses of photosynthetic param-
eters. PH provided unique biological material and led the field experi-
ments. SM, KK, XY, and XL contributed to experimental design and
interpretation of results, and helped to draft the manuscript. Neither
the manuscript nor any part of its content has been published or sub-
mitted for publication elsewhere. All authors read and approved the
final manuscript.
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