Photosynthetic Characteristics in Two Near Isogenic Lines of Winter Wheat

Guirong HUANG Xinying ZHANG Yajing WANG Fu FENG Xurong MEI Xiuli ZHONG

Abstract The development of near isogenic lines （NILs） is an important tool for physiological dissection of drought resistance in wheat. To better understand the potential for improving grain yield， a splitplot experiment was conducted under a mobile rain shelter using NILs of winter wheat with significant differences in the photosynthetic rate： the 908120 line with a high photosynthetic rate and the 908206 line with a low photosynthetic rate. The results indicated that the net photosynthesis rate （Pn）， stomatal conductance， and transpiration rate in flag leaves in line 908120 were significantly higher than that in line 908206 under uniform water treatments during the reproductive phase in replicated pooledculture trials. The maximum quantum yield Fv/Fm value and ribulose1，5bisphosphate carboxylase/oxygenase （RuBPCase） activity value were higher in line 908120 than that in line 908206， whereas the intercellular CO2 and cell membrane permeability in line 908120 were lower than that in line 908206. Higher leaf Pn， transpiration rate， and RuBPCase activity and lower membrane ion leakage rate ensured the robustness of line 908120 during times of irrigation and drought， which contributed to the maintenance of its high grain yield. Drought had a negative effect on these factors， resulting in decreased yield， and the photosynthesis rate of flag leaves markedly affected the yield in NILs of winter wheat. NILs are an important strategy for wheat adaptation to drought stress， but might not be the only mechanism causing the significant grain yield increase. Collectively， the characteristics of line 908120 resulted in a higher grain yield compared with that of line 908206. Further dissection of the drought avoidance mechanisms in wheat， as well as analysis and identification of the genes involved may be necessary.

Key words NILs; Yield; Winter wheat; Drought; Photosynthesis

Crop production of wheat， maize， and rice has increased worldwide in the past decades. However， crop yields cannot currently meet the demands of the increasing population and biofuel consumption on a global scale. Although agriculture can be conducted under relatively stable conditions， agronomic practices now need to adapt to climate change[1]， and thus， crop grain yields are slowly increasing. Wheat （Triticum aestivum L.） was first cultivated during the Neolithic Revolution approximately 10 000 years ago[2]. Wheat accounts for 20% of the caloric input of the worldюs population[3].

Water is an important limiting factor in agriculture， and drought has a strong impact on plant production worldwide. Water stress plays a crucial role in plants at several spatial and temporal scales during ontogenesis. It has been estimated that approximately 20-60 Mha of farmland in heavily irrigated areas will be forced to convert to rainfed management because of freshwater limitations[4]. In the North China Plain， the rainfall is inadequate for evapotranspiration during the winter wheat growing season[5-6]. In the coming decades， the shortage of water for food production will be an important limitation to global food security[7]. Meeting the increasing demands of a growing population is challenging for agricultural productivity under the background of climate change. Due to a trend toward decreased cropland for agricultural production， increasing the available land for agricultural intensification and improving the yield on existing agricultural land have become issues of concern[8-10]. As a global food crop， wheat is crucial to food security[11-15]. Realizing the goal of a projected 70% rise in wheat demand by 2050 will be difficult considering the low rates of yield increase.

The current ecological focus is to produce food using fewer resources under the background of global food security and population growth. Biotechnology is an essential technique to provide longterm solutions to these issues. Selection of highly droughttolerant wheat varieties is essential to protect crops from the detrimental effects of drought[16-17]. Notable increases in yield potential have been achieved in the past half century， and improving the efficacy of photosynthesis is essential for further yield increases[18]. Ribulose1，5bisphosphate carboxylase/oxygenase （RuBPCase） in flag leaves is one of the enzymes responsible for the low photosynthesis rate associated with drought stress[19]. In addition， RuBPCase is a key enzyme that regulates plant photosynthesis and respiration. RuBPCase and phosphoenolpyruvate carboxylase play important roles in adaptation mechanisms during the initial carboxylation step of wheat in response to water and salt stresses[20].

Previous studies reported contradictory results regarding the relationship between photosynthetic and physiological function and grain yield[21-24]. Drought stress induces succulence， resulting in decreased leaf area and an increased number of chloroplasts[25]， which can increase the photosynthetic capacity. The photosynthetic characteristics of flag leaves have the most influence on the final grain yield of wheat， as they affect the net photosynthetic assimilation. Additionally， drought stress after anthesis can accelerate the senescence of leaves and thus shorten the duration of photosynthesis[26-27].

The varieties of winter wheat differ in terms of the constraints limiting their grain yield under various conditions[28]. Prolonged periods of drought cause significant losses in relative water content， leaf chlorophyll content， photosynthetic efficiency， and activities of antioxidant enzymes[29]. Photosynthesis （PS） II activity is not considerably reduced except in dry soil， in which the leaf water potential can be -2.5 MPa[30]. The method of fast fluorescence kinetics with high timeresolution has been applied to investigate the in situ acclimation of fieldgrown winter wheat under a harmful environment， and a low Fv/Fm ratio was found because of decreased Fm and increased Fo[31].

Stomatal conductance tends to decrease and the rate of photosynthesis is consequently reduced under mild drought stress conditions[32]. Drought stress also induces changes in cell membranes[33]. In addition， the selection for high yields was accompanied by an increase in photosynthetic productivity through unintentional improvement of leaf anatomical and biochemical traits[34]. High harvest index （HI） and biomass are considered as positive factors for yield， and when the HI potential reaches a relatively stable peak， biomass potential can be further developed[35-36]. However， the HI and biomass yield values vary among cultivars. Previous data suggest that the percentage of biomasscontributing yield has much more influence on yield than does HI. Current evidence suggests that the photosynthetic production of the flag leaf after anthesis contributes greatly towards biomass yield in wheat. The ultimate product of photosynthesis is dry matter[37].

The effect of drought on yield in winter wheat is difficult to determine because of the diversity in genetic backgrounds among cultivars， and the underlying mechanism remains unclear. In the present study， we assessed the poolculture performance of the NILs with respect to grain yield and photosynthetic characteristics in water regimes in an effort to determine the mechanisms causing differences in performance and effective strategies for improving the yield of winter wheat during the reproductive stage under drought stress.

Materials and Methods

Experimental site

The experiment was conducted at the Hengshui Dryland Watersaving Farming Experimental Station （37°54′N， 115°42′E） at the Hebei Academy of Agriculture and Forestry Sciences （Hengshui， Hebei Province， China） in the central zone of the Huang （Yellow River）-Huai （Huai River）-Hai （Hai River） Plain of China. There is extensive winter wheatsummer maize cropping in this region. The mean rainfall in the region is 497.1 mm， of which approximately 70% falls between July and August. Experiments were conducted during the 2013-2014 and 2014-2015 winter wheat growing seasons and performed under a mobile rain shelter to prevent natural rainfall. Each experimental plot trough measured 3.3 m ≠ 2.0 m with loam and was surrounded on four sides by concrete slabs to prevent water seepage. The levels of soil organic carbon， total nitrogen， and available phosphorus in the top20cm layer were 9.26， 0.67 and 4.82 g/kg， respectively. The levels of available potassium， pH， and the soil bulk density were 88.9 mg/kg， 8.11， and 1.36 g/cm3， respectively. At the time of sowing， 112.5 kg/hm2 of nitrogen， 75 kg/hm2 of phosphorus pentoxide， and 75 kg/hm2 of potassium oxide were applied to the soil. The winter wheat was planted at a density of 6.6≠106 plants/hm2 on October 18， 2013 and October 19， 2014. Thinning was done by hand at 7 d after emergence to attain a final population density of 3.3≠106 plants/hm2. Winter wheat was harvested on June 2， 2014 and June 3， 2015. The contrasting near isogenic lines （NILs） （line 908120 and line 908206） of wheat were selected based on the net photosynthetic rate （Pn） differences from recurrent winter wheat Jinmai 47 ≠ Jing 411. Line 908120 exhibited a high rate of photosynthesis， and line 908206 exhibited a low rate of photosynthesis.

Experimental design

Experiments were performed using a randomized design with three replicates： each line was planted in five rows of length 2 m， with 20 cm between rows， under irrigation and drought conditions. For both irrigation and drought treatments， the soil was fully watered before sowing to ensure a high rate of seedling emergence. Water was applied from a pump outlet using plastic pipes， and the amount of irrigated water was measured using a flow meter. A total of 225 mm and 75 mm of water was applied for the irrigation and drought treatments， respectively， in two consecutive experiments. At sowing time， 75 mm of water was applied for both the irrigation and drought treatments. In the irrigation experiment only， 75 mm of water was applied at the stem elongation and grainfilling stages. At maturity， the average gravimetric soil moisture content （0-160 cm in depth） was 13.7% and 13.4% （irrigation） and 11.2% and 11.3% （drought） in the 2013-2014 and 2014-2015 growing seasons， respectively.

Measurements

They were observed that 112 d after sowing， the jointing stage began， and 141 d after sowing， the filling began for two NILs in two growing seasons. The time of flowering period was earlier than the time of grainfilling period by 4-5 d.

Measurement of the photosynthetic gas exchange parameters of flag leaves： The Pn， intercellular CO2 （Ci）， transpiration （Tr）， and stomatal conductance （Gs） in intact flag leaves were measured with an LICOR 6400 portable photosynthesis system （LICOR， Inc.， Lincoln， NE， USA） between 9：00 and 11：00 a.m. Measurements were made at a saturating photosynthetic photon flux density （PPFD） of 1 200 mol/（m2·s） and atmospheric CO2 concentration. The temperature inside the leaf chamber was set to （26÷1）→ with approximately 70% ambient humidity. Measurements were made using 10 leaves for each replicate of each treatment.

Measurement of Fv/Fm： Prior to detection， the flag leaves were darkadapted for 20 min on a sunny day at 12：00 a.m. The photochemical efficiency of PSII was measured using a chlorophyll fluorescence meter （FIM1500， ADC， Hoddesdon， UK） and expressed as maximum quantum yield of photosystem II photochemistry （Fv/Fm）.

Measurement of RuBPCase activity： RuBPCase activity was measured according to the method of Lilley and Walker[38]. Flag leaves were picked on a sunny day between 9：00 and 11：00 am， wrapped with tinfoil paper， then immediately frozen in liquid nitrogen for 30 min and stored at -80 until uniform laboratory measurement. Leaf segments （0.5 g） were homogenized using a pestle and mortar with 2.5 ml of extraction buffer （0.1 M TrisHCl pH 8.0， containing 10 mM MgCl2， 1 mM EDTA， 7 mMmercaptoethanol， 5% glycerol （v/v） and 1% PVP） followed by centrifugation at 10 000 g for 10 min at 4. The clear supernatant was decanted slowly and used as the crude RuBPCase source. Before quantification， the crude extract was activated with 200 M NaHCO3 and 1 M MgCl2， at 25 for 10 min. The reaction mixture contained 50 mM MgCl2， 0.21 mM NADH， 5 mM DTT， 5 mM phosphokinase， 15 units of phosphoglyceric phosphokinase， five units of glyceraldehydephosphate dehydrogenase， and 10 mM NaHCO3. Reactions were initiated by addition of 0.5 mol of RuBP. The activity of RuBPCase was determined according to the oxidation rate of NADH at 340 nm.

Measurement of cell membrane permeability： Membrane ion leakage rate was measured as described by Welti et al.[39]using a conductivity meter （SevenExcellenceTM; Zurich， Switzerland）. Briefly， after selection of winter wheat showing a consistent growth pattern， three flag leaves were collected， inserted into a ziplock bag， and placed in a precooling incubator. The leaves were washed with tap water to remove dust， rinsed with deionized water， and placed in 50 ml plastic centrifuge tubes. After addition of 25 ml of deionized water， the tubes were placed in a 23 water bath for 1 h and shaken at a frequency of 120 s1. A conductivity meter was used to measure the initial conductance value. The centrifuge tube was placed in a constant temperature water bath at 100 for 10 min and cooled to 23 to determine the conductance value. The membrane ion leakage rate was calculated as the initial conductance value/total conductance ≠ 100%.

Measurement of grain yield： When the winter wheat had attained maturity， 1m lengths of 3 rows were harvested manually and air dried.

Measurement of drought tolerance and stress susceptibility inde： TOL and SSI were calculated according to Fischer et al.[40]and Rosielle et al.[41]for winter wheat as follows：

Drought tolerance （TOL）=Control yield-Yield under drought conditions（1）

The stress susceptibility index （SSI） =（1-Yield under drought condition/Control yield）/（1-Mean yield under drought condition/ Mean control yield）（2）

Statistical analysis

Microsoft Excel 2010 was used for data processing and to generate the figures. SAS 9.2 was used to determine significant differences among the treatment means at a level of significance of 0.05. Multiple comparisons were performed using the LSD （Least Significant Difference） test at =0.05.

Results and Analysis

Grain yield

There were significant differences in the grain yields of the two wheat NILs under different conditions and during the two growing seasons （Fig. 1）. Drought conditions significantly decreased the grain yield of winter wheat of both lines. In the 2013-2014 winter wheat growing season， the yield of line 908120 was 10% and 15% higher than that of line 908206 under drought and irrigation conditions， respectively. In the 2014-2015 winter wheat growing season， the yield of line 908120 was 12% and 18% higher than that of line 908206 under drought and irrigation conditions， respectively. The tolerance （TOL） of line 908120 was 1 065 kg/hm2 and 1 282 kg/hm2 in the 2013-2014 and 2014-2015 winter wheat growing seasons respectively; that of line 908206 was 753 kg/hm2 and 877 kg/hm2， respectively， in these seasons. The stress susceptibility index （SSI） value was 1.1 for line 908120 and 0.9 for line 908206 in both growing seasons. This indicates a significant difference in yield and droughtresistance between the two NILs. The high photosynthetic rate of line 908120 exhibited lower TOL and higher SSI compared with the low photosynthetic rate of line 908206.

Photosynthetic characteristics of flag leaves during the anthesisfilling stage

Winter wheat varieties show different photosynthetic characteristics of the flag leaves and different responses to water stress. Photosynthetic characteristics after anthesis are important for grain production， and highly efficient Pn results in a high yield during the grainfilling stage. In the 2013-2014 and 2014-2015 winter wheat growing seasons， Pn was significantly different between the NILs under both irrigation and drought conditions. However， there was no significant variation in the Pn of each NIL between the two growing seasons because of their individual variety characteristics （Fig. 2）. The average Pn value of flag leaves after anthesis of line 908120 was 11.3% and 11.9% higher than that of line 908206 in the 2013-2014 and 2014-2015 winter wheat growing seasons， respectively.

Net photosynthetic rate

The Pn of line 908120 was significantly higher than that of line 908206 during the anthesisfilling stage （Fig. 3）. The Pn values of both NILs were the highest after anthesis. The peak Pn values of lines 908120 and 908206 were observed at 7 and at 14 d after anthesis， respectively. The highest and lowest Pn values of line 908120 were both less than those of line 908206. The Pn values of both NILs were higher under the irrigation condition than that under the drought condition in both winter wheat growing seasons， suggesting that drought reduced the Pn， and that line 908206 was more sensitive to drought.

Stomatal conductance

There was a trend toward lowhighlow stomatal conductance （Gs） in both NILs during the measurement period （Fig. 4）. The peak Gs values of lines 908120 and 908206 were found at 7 and at 14 d after anthesis. Irrigation promoted an increase in Gs for the exchange of gas and moisture and the production of carbohydrate. The Gs of line 908120 was higher than that of line 908206 during the grainfilling stage. The Gs of line 908120 was higher than that of line 908206 by 24.3% and 21.5% under drought and irrigation conditions， respectively， in the 2013-2014 winter wheat growing season， and by 12.3% and 19.5%， respectively， in the 2014-2015 winter wheat growing season. The Gs measured under drought conditions rapidly decreased during the late grainfilling stage.

Intercellular CO2 （Ci）

The value of the parameter Ci， which is used in photosynthetic physiology and ecology， depends on the concentration of CO2 in the air， Gs， mesophyll conductance， and the photosynthetic activity of mesophyll cells. The Ci of line 908120 showed the lowest value on day 7 after anthesis， whereas line 908206 showed the lowest Ci on day 14 after anthesis （Fig. 5）. The Ci of the flag leaves was higher under the drought condition in both NILs， and that of line 908206 was significantly higher than that of line 908120. Since the concentration of CO2 in the air was constant， the change of Ci was associated with Gs， mesophyll conductance， and the photosynthetic activity of mesophyll cells in the plant. The Ci was closely related to Gs， and the two factors showed an inverse trend during the anthesisfilling stage.

Transpiration rate

During the measurement period， the transpiration rate （E） of the two NILs increased and then decreased as the winter wheat growing process progressed. The peak E values of lines 908120 and 908206 were highest at 7 and 14 d after anthesis， respectively （Fig. 6）. Drought conditions had a negative effect on the E of the two NILs in both winter wheat growing seasons. The E of both NILs rapidly decreased after reaching their highest points under the drought condition. The E of line 908120 was higher than that of line 908206 by 6.1% and 10.1% under drought and irrigation conditions， respectively， in the 2013-2014 winter wheat growing season. The E of line 908120 was higher than that of line 908206 by 11.9% and 11.8% under drought and irrigation conditions， respectively， in the 2014-2015 winter wheat growing season. These changes suggest a close relationship between the growing stage and the environment in two different cultivars.

Intercellular CO2 （Ci）

The value of the parameter Ci， which is used in photosynthetic physiology and ecology， depends on the concentration of CO2 in the air， Gs， mesophyll conductance， and the photosynthetic activity of mesophyll cells. The Ci of line 908120 showed the lowest value on day 7 after anthesis， whereas line 908206 showed the lowest Ci on day 14 after anthesis （Fig. 5）. The Ci of the flag leaves was higher under the drought condition in both NILs， and that of line 908206 was significantly higher than that of line 908120. Since the concentration of CO2 in the air was constant， the change of Ci was associated with Gs， mesophyll conductance， and the photosynthetic activity of mesophyll cells in the plant. The Ci was closely related to Gs， and the two factors showed an inverse trend during the anthesisfilling stage.

Changes in the Fv/Fm

Drought had a marked effect on the two NILs， and the different lines had different maximum photosystem ？quantum yield （Fv/Fm） values （Fig. 8）. Under drought conditions， the Fv/Fm of line 908120 decreased from 0.808 to 0.721 after anthesis in the 2013-2014 winter wheat growing season. Under irrigation conditions， the Fv/Fm of line 908120 decreased from 0.811 to 0.795， followed by an increase to 0.806 and a decrease to 0.736 at the grainfilling stage in the 2013-2014 winter wheat growing season. This result suggests that supplemental irrigation can contribute to improving metabolism before injury. The Fv/Fm of line 908120 was higher than that of line 908206 under both conditions in the two winter wheat growing seasons.

RuBPCase activity

RuBPCase is a crucial regulatory enzyme of photosynthetic carbon metabolism. As shown in Fig. 9， RuBPCase activity decreased sharply at 7 d after anthesis in line 908120 and at 14 d after anthesis in line 908206. Water stress may decrease the rate of growth and delay the peak of RuBPCase activity. RuBPCase activity in flag leaves was higher in line 908120 than in line 908206 after anthesis， suggesting that line 908120 had a higher RuBPCase capacity.

Discussion and Conclusions

Wheat cultivation has been ongoing for centuries worldwide， and highyielding genotypes are gradually selected in wheat evolution， mainly due to improvement of photosynthesis. The C3 cycle and C4 cycle and crassulacean acid metabolism are pathways of photosynthesis， and the C3 cycle is the fundamental pathway of carbon assimilation in natural environments. Ribulose1，5bisphosphate carboxylaseoxygenase （RuBPCase） is critical because it catalyzes carboxylation and oxygenation reactions. However， RuBPCaseassociated bottlenecks of the C3 cycle can be overcome by transgenic plants with higher photosynthetic rates that also utilize the enzymes of the bacterial glycolate pathway. There are eight enzymes in the regeneration project of CO2 acceptor molecule RuBP （Fig. 10）， contributing to the increase in photosynthesis and yield with the C3 cycle[43]. The goal is to synergistically increase the nitrogen and water use efficiency with productivity.

Photosynthesis has facilitated improvement of yield potential over the past 50 years， while constraints for photosynthesis have been increasingly explored. Photosynthesis indicates that the efficiency of conversion of the intercepted light into biomass and both leaf photosynthesis and yield have similar increases. It takes 2-3 years to proceed with the regeneration of CO2 acceptor molecule RuBP by genetic and molecular techniques and 10-15 years by conventional breeding. It is controversial to conclude that increased leaf area may reduce the photosynthetic rate because of selfshading， and photosynthesis may be limited by either sink capacity or source capacity. Therefore， the adoption of the same genotype may be advisable to conduct relevant analyses. Although carbohydrates are important for plant function， photorespiration consumes approximately 30% carbohydrate produced by the C3 pathway[44-45]， whereas the maximum Rubisco activity and rate of regeneration of RuBP decide the CO2 uptake rate of the C3 pathway.

Drought is a worldwide problem that threatens agricultural development and results in direct loss of crop production. Research on wheat drought tolerance is being conducted worldwide by scientists with the goal of selection and breeding strategies for varieties to improve drought tolerance. Reactive oxygen species （ROS） are induced by drought and lead to membrane lipidoxidation， enzyme inhibition， and finally cell death[46]. Under drought conditions， photosynthesis is important to maintain plant metabolism and performance， and more loss of water by transpiration is usually avoided by closing stomata[47]. In addition， drought stress exerts negative effects on PSII reaction centers and chlorophyll fluorescence， and studying different sensitivities under drought stress for wheat varieties can increase our understanding of the defense mechanism. The leaf relative water content （RWC） greatly decreases during later periods of ontogenesis under drought stress. The potential quantum yield of PSII（Fv/Fm ratio） in cv. Azamatli95 was maximal during the stalk elongation stage at the beginning of drought[29]. Soil water deficit causes the decline of activities of PS？and photosynthetic pigment content during ontogenesis， and different cultivars adopt respective defense systems to protect the photosynthetic apparatus.

The large difference in grain field among NILs with small genetic differences under drought stress is in full agreement with Venuprasad et al.[48]. In the present study of two winter wheat cultivars， the high photosynthetic rate of line 908120 showed a higher grain yield than line 908206 under irrigation and drought conditions during two growing seasons. Our findings confirm those of Wang et al.[49]， who observed that there were significant differences in Pn between the two wheat NILs， whether it was at the seedling stage or the flowering grainfilling stage. The rate of photosynthesis and chlorophyll fluorescence after anthesis was limited by drought in different degrees for the two NILs. In addition， drought gradually increased the cell membrane permeability， and irrigation had a relative compensatory function. RuBPCase activity was sensitive to stress during the grainfilling stage in the present study， similar to previous research results.

Evaporation proceeds in soil， leaf， and air under natural conditions. The transpiration rate is impacted by closed stomata， and the semipermeability of cell membranes is impaired under stress conditions. The dwarf mutant RhtB1c plants exhibited greater tolerance， membrane integrity， and osmoregulation and were less affected by photosynthesis under drought stress[50]. Stomata and trichome density， leaf area， and leaf thickness exhibited no differences between modern varieties and old historic varieties under a drought stress environment. There was a significant relationship between the amount of water loss， and the perimeter and dissection index. The stomata are nearly closed at relative water content values below 65%[51]. Cell membranes are impaired to a greater extent under drought stress， and leaf shape affects evaporation and water status， which then further affects the cell membrane stability. Change in the leaf shape is a mechanism used to adapt to specific environments[52]， and it is closely related to decreased evaporation and cell membrane integrity. Generally speaking， these findings suggest that growth conditions have different effects on the photosynthetic apparatus and cell membrane integrity on ontogenesis. Drought conditions could accelerate the selection of droughttolerant genotypes.

According to our experimental results， the grain yield was significantly higher in line 908120 than in line 908206 across two growing seasons under both irrigation and drought conditions. Near isogenic lines exhibited significant differences in the net photosynthetic rate， stomatal conductance， transpiration rate， Fv/Fm， and RuBPCase activity， as well as in the amount of intercellular CO2 and cell membrane permeability. Photosynthetic characteristics contributed to drought resistance in two NILs of winter wheat， and they were found to vary with water arrangements including irrigation treatment and drought treatment as the control group. There was more optimal performance of photosynthesis and cell membrane permeability in the two NILs under irrigation conditions than that under drought conditions， and line 908120 exhibited more optimal performance under both conditions compared with line 908206. Compared to stomatal conductance and transpiration rate and Fv/Fm， the intercellular CO2， cell membrane permeability， and RuBPCase activity were associated with a higher photosynthetic capacity at the leaf level during the growing season. Collectively， these characteristics of line 908120 resulted in a higher grain yield compared with that of line 908206.

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