Physiological traits contributed to the recent increase in yield potential of winter wheat from Henan Province,China

Bangwei Zhou,álvaro Sanz-Sáez,Abdelhalim Elazab,Tianmin Shen,Rut Sánchez-Bragado,Jordi Bort,Maria Dolors Serret and José Luis Araus

1Unit of Plant Physiology,Faculty of Biology,University of Barcelona,Barcelona 08028,Spain,2Henan Tianmin Seed Company,Lankao County,Kaifeng 475300,China,3Department of Plant Biology,University of Illinois,Urbana-Champaign,Illinois 61801,USA.＊Correspondence:alvaro_ssj@hotmail.com

INTRODUCTION

As the most populous country,answers to the question“Who will feed China?”have been sought by scientists and politicians since the 1990s.The rising Chinese population and the rapid growth of the Chinese economy have resulted in an increasing demand for wheat(Triticum aestivum L.)supplies,and this trend will continue for the following decades.The planting area in China is shrinking,because the spring wheat cultivated region is being reduced due to the economic competition with other crops and by the inherent low yield of spring wheat caused by its short growth cycle.Moreover,in spite the high yields achieved in the winter wheat cultivated region,which comprises approximately 94%of China’s total wheat production,winter wheat has suffered a slight decrease in planting area(USDA 2012).Henan Province,located in the east of Yellow and Huai Valleys winter wheat zone(YHVWWZ),is the primary winter wheat producer of China.It produces up to 25%of the national production with a planting area of approximately 5.3 million ha,and a production of approximately 30.8 million tons(NBS 2010).

The total wheat production and grain yield per unit area in Henan Province increased 8.8 and 7.1 times,respectively,from 1949 to 2007(Ye et al.2007).This enhancement in total production and yield of Chinese winter wheat is mainly due to the development of new crop management techniques such as new fertilizers or pesticides,and due to the breeding of new wheat cultivars that have continuously increased the yield potential of wheat(genetic gain)from 1960 to 2010(Zheng et al.2011;Xiao et al.2012).

Worldwide,different breeding strategies have been proposed to speed genetic gains in the next decades.In that context,proper phenotyping is paramount both for conventional breeding as well as to take full advantage of the powerful molecular techniques developed in recent times(Araus et al.2008;Cabrera-Bosquet et al.2012).Thus,different phenotyping traits and tools have been proposed,including:(i)plant agronomic traits associated with the past increase in grain yield(Xiao et al.2012);(ii)high photosynthetic capacity and/or efficiency(Reynolds et al.2009;Parry et al.2011),including that of the ear(Araus et al.1993a);(iii)higher efficiency in the use of resources such as water through the stable isotope abundances in plant tissues;and(iv)the optimization of source-sink balance to allow an enhancement of yield potential(Pérez et al.1989;Schnyder 1993;Zhang et al.2010,2012;Aranjuelo et al.2013).

In the traditional breeding strategy,grain yield components such as kernel number per unit area and/or average individual grain weight have been selected among different genotypes to reach a high yield(Slafer et al.1996).Meanwhile,some authors have shown that there are other yield components largely associated with the enhancement of yield,such as harvest index(HI),shoot biomass,and kernel number per spike(Yoshida 1972;Rebetzke et al.2002;Zhou et al.2007).High HI was not only the immediate result of the introgression of dwarf Rht genes in the middle of the 20th century(Reynolds et al.2009),it was also due to the continued selection of genotypes with reduced height and increased grain number per unit area in the post-Green Revolution period(Sharma et al.1985;Zheng et al.2011).However,Reynolds et al.(2005)showed that the genetic progress of HI in bread wheat has stalled since the mid-1980s.According to Parry et al.(2011),the selection of genotypes with increased biomass and photosynthetic efficiency will go beyond the HI limitation to achieve an increasing harvestable grain number and high yield rates.At the same time,it has been reported for Henan Province that breeding since 1970 to the present has not only reduced plant height and increased HI but has resulted in increases in spike number per unit area and shoot biomass(Zheng et al.2011).However,changes in other traits such as the rate of photosynthesis are unclear(Hawkesford et al.2013).Moreover,breeding changes have not necessarily been uniform across these four decades(Parry and Hawkesford 2010;Zheng et al.2011;Reynolds et al.2012;Xiao et al.2012).

Photosynthesis is one of the most highly integrated and regulated metabolic process to maximize the use of available light,optimizing the use of limited C and N resources(Paul and Foyer 2001).There is evidence that photosynthesis improvement,simultaneously with well-balanced grain sink strength,was associated with an improvement in yield potential(Fischer et al.1998;Reynolds et al.2011).The photosynthetic capacity and efficiency of shoot organs(flag leaf and spike)could be considered as the most important factors among total crop photosynthesis.It has been proposed that increasing leaf net photosynthesis leads to an increase in shoot biomass and hence grain yields(Parry et al.2011).In particular,under slight stress conditions,a number of authors have reported the positive correlations between grain yield and flag leaf photosynthesis in bread wheat(Shimshi and Ephrat 1975;Richards 2000).In recent years,many studies have revealed that the contribution of spike photosynthesis to grain filling(Araus et al.1993a;Tambussi et al.2007)should be considered parallel or even more relevant than the contribution of the flag leaf during the post-anthesis period.In that sense,spike traits that contribute to grain filling and ways to increase grain yield need to be further studied by plant biologists and breeders.However,a lack of correlation between photosynthesis and plant yield has been frequently reported among bread wheat genotypes(Makino 2011;Xiao et al.2012),probably due to the fact that modern cultivars have been bred for photosynthesis.One trait that could be selected to improve the photosynthetic response and then increase yield is the ribulose-1,5-bisphosphate carboxylase oxygenase(RuBisCo)content and/or activity(Parry et al.2011).During plant growth,especially under stomatal closure,RuBisCo abundance and kinetics would be the key limitation to photosynthetic CO2assimilation(Parry et al.2011,2013).

Based on a good understanding of the physiological and biochemical constraints to yield performance,utilization of key tools that associate wheat physiology traits can provide important information to wheat breeding and agronomy.Carbon stable isotope composition(δ13C)has been proposed as an indirect selection criterion for transpiration efficiency and grain yield in wheat(Araus et al.2002).The use of δ13C affords an easy way of screening for grain yield,dry matter,water status,and even photosynthesis(Condon et al.1990;Araus et al.1993a).The relationship between δ13C and grain yield varies from strongly positive(Condon et al.2002)to strongly negative(Araus et al.1998)under drought conditions.In many regions such as the Mediterranean Basin,Australia,and China,it was found that δ13C of the flag leaf and mature kernels correlated negatively with grain yield across genotypes under rain-fed conditions(Araus et al.1993a;Araus et al.2002;Rebetzke et al.2002;Xu et al.2007).However,other studies have illustrated that the correlation between δ13C and grain yield is low,in optimal conditions(Fischer et al.1998;Araus et al.2003;Monneveux et al.2005;Serret et al.2008).

The study of yield potential under field conditions is usually difficult because of natural biotic and abiotic stresses,such as drought and plagues,that limit the plant yield potential.In order to avoid interference from this stress on yield potential,it is more reasonable to estimate the yield potential of wheat under controlled and favorable conditions.The objective of this study was to study the agronomic and physiological traits responsible for increasing yield potential of winter wheat that have been released in Henan Province of China during the last two decades,and to that end,cultivars and advanced lines were included.

RESULTS

The individual traits influencing yield in the set of 10 Chinese bread wheat genotypes from Henan Province included in this study were analyzed(Table 1).The ANOVA showed that most of the traits were significantly affected by genotype differences(Tables 2,3).

Grain yield and yield components

Grain yield of different genotypes varied significantly,ranging from 5.55 Mg/ha for Yumai 35 released in 1995 to 14.84 Mg/ha for the advanced line Lankao 282.The mean grain yield of all genotypes was 9.12 Mg/ha(Table 2).Yield was positively correlated with the year of release(r2=0.60,P≤0.01),with the three advanced lines showing higher grain yield than the varieties already released(Figure 1A).The HI showed a highly significant difference between genotypes,while aboveground biomass showed a slight,albeit significant increase(Table 2).In fact,the year of release was significantly and positively correlated with grain yield,biomass,and HI(Figure 1A–C)as well as with kernel number per square meter(r2=0.79,P≤0.001)(data not shown).Spike weight,kernel weight per spike,and thousand kernel weight(TKW)also revealed clear differences between genotypes(Table 2),but they did not show a clear improvement trend according to the year of release(data not shown).Plant height of commercial genotypes showed a decreasing trend from the oldest to the newest genotypes,except for the dwarf genotype Lankao Aizao 6(Table 2).However,the advanced lines showed a significantly greater height than the commercial genotypes,also showing higher yield as a consequence of a general increase in plant biomass(Table 2).

Table 1.Name,year of release,and pedigree of 10 Chinese winter wheat genotypes from Henan Province,China,assayed at the Experimental Fields of the University of Barcelona

Table 2.Mean values and sum of squares type III combined with ANOVA for grain yield and agronomic yield components of seven winter wheat cultivars and three advanced lines from Henan Province,China

In order to test which yield components were better related to grain yield,a regression test between grain yield and yield components was performed(Table 4).The highest significant correlation was observed between grain yield and HI(r=0.90,P≤0.001),followed by kernel number per square meter,aboveground biomass,and kernel number per spike(r=0.80,P≤0.001;r=0.78,P≤0.001;r=0.71,P≤0.001,respectively).The linear relationships of spike length and spike weight with grain yield were also significant,but lower(r=0.62,P≤0.001;r=0.50,P≤0.01,respectively).Plant height affected grain yield weakly(r=0.44,P≤0.05),while TKW did not influence it significantly(Table 4).

Grain yield and physiology traits among organs

Organ dry weight and N content

At anthesis,there were significant differences across genotypes for spike and flag leaf dry weight,and total N content and N concentration(Table 3).Total N content of spikes(15.22 mg)was approximately 30%higher than that of the flag leaf(11.94mg)in spite of the much lower N percentage in the spike(2.04%)compared with the flag leaf(4.96%).Grain yield was negatively correlated with spike N concentration(r=-0.40,P≤0.05)(Table 5).However,grain yield was not correlated with the dry weight and N concentration of the flag leaf(Table 6).The kernel N content per spike was highly and positively correlated with the spike N content;however,flag leaf N content was not correlated with kernel or spike N content(Tables 5,6).

Carbon isotopes composition

For the set of genotypes assayed,δ13C values varied significantly among genotypes and organs(Tables 3,S1,S2).Significant differences among plant parts were observed for δ13C,with values increasing from the older genotypes to the advanced lines(Tables 3,S1,S2).In fact,grain yield was significantly and positively linearly associated with the δ13C of kernels(r=0.56,P≤0.001),spikes(r=0.52,P≤0.01),and flag leaves(r=0.50,P≤0.01)(Tables 5,6).Moreover,the δ13C of spikes showed a highly significant and positive correlation with spike dry weight(r=0.69,P＜0.001)(Table 5).In addition,kernel dry weight was positively correlated with δ13C of kernels(r=0.54,P≤0.01)(Table 6).Moreover,kernel δ13C increased linearly with the year of release(r2=0.55,P≤0.05)(Figure 1D).However,flag leaf and spike δ13C did not show a linear relationship with the year of release(data not shown).

Net carbon exchange and stomatal conductance

The stomatal conductance(gs)of the spike showed a significant difference among genotypes,whereas flag leaf gsdid not.Moreover,gross carbon exchange per unit area(GCE/m2)of the flag leaf did not change among genotypes while spike GCE/m2did(Tables 3,S2).Although the spike and flag leaf gsdid not display a linear tendency with grain yield,positive linear correlations between GCE/m2and gsin both organs were found(r=0.58,P≤0.001 for the flag,and r=0.79,P≤0.001 for the spike)(Tables 5,6).Meanwhile,GCE per whole organ(GCE/organ)when measured in the flag leaf showed significant differences between genotypes and did not correlate with grain yield(Tables 3,6).In contrast,GCE per whole spike was positively correlated with grain yield in spite of no significant difference among genotypes(r=0.40,P≤0.05)(Tables 3,5).Flag GCE per kernel unit was negatively correlated with grain yield(r=-0.59,P＜0.001),while spike GCE per kernel unit was not(Table 5).Spike and flag GCE per kernel unit correlated negatively with kernel N content per spike(r=-0.47,P≤0.01;r=-0.48,P≤0.01,respectively).

PEPC and RuBisCo activity

The phosphoenolpyruvate carboxylase(PEPC)activity per unit dry matter of glumes and the flag leaf showed substantial differences among genotypes(Table 3).For almost all genotypes,the flag leaf PEPC activity per dry matter was higher than that in the glumes(Tables S1,S2).In addition PEPC activities of the flag leaf and glumes were negatively correlated with the dry weight of those organs(r=-0.52,P≤0.01 for the flag;r=-0.39,P≤0.05 for the spike).In the flag leaf,the PEPC activity was positively influenced by the N concentration(r=0.51,P≤0.05);however,this trend was not observed in the spike(Tables 5,6).On another aspect,the genotypic effect was significant for RuBisCo activity on a dry weight basis of the flag leaf and glumes(Table 3).RuBisCo activity per dry matter in glumes showed far lower values than the flag leaves.However,the RuBisCo activity of glumes was positively correlated with spike N concentration(r=0.47,P≤0.01),while flag leaf RuBisCo activity was not correlated with flag leaf N concentration(Tables 5,6).

Overall genotypic difference

A stepwise regression was performed to study which physiological traits influenced grain yield as independent variables for the 10 genotypes.Thus,the independent variables analyzed were δ13C,N concentration,gs,GCE/organ,GCE/m2,RuBisCo activity per unit dry matter of the flag leaf and the spike,and PEPC activity per unit dry matter of the flag leaf and spike(Table 7).The first trait selected was kernel δ13C,while flag GCE/m2was ranked next.The δ13C of the spike was selected as the last parameter,while flag leaf δ13C was not chosen.

DISCUSSION

Due to an increasing human population and decreasing cropping area,improvement of grain yield is the ongoing primary objective of the Chinese wheat breeding program(Zheng et al.2011;Xiao et al.2012).The present study,which focused on recent breeding advances on grain yield of wheats from Henan Province,showed a generally increasing trend of grain yield with the year of release(Figure 1A).A genetic advance in yield during the past decades has been reported for different regions of China(Zheng et al.2011;Xiao et al.2012).In addition,a positive relationship between grain yield and year of release has been also found in Australia,the UK,and USA for different cereals(Araus et al.2008).In the present authors’results,the advanced lines developed during the year 2012 showed a remarkably higher grain yield than the older genotypes(Figure 1A,Table 2),revealing that the yield potential could be further enhanced by breeding strategies.

Figure 1.Linear regression on year of release for grain yield(A),harvest index(HI)(B),aboveground biomass(C),kernel isotope composition(D),GCE per whole organ(GCE/organ)of spikes and flag leaves(E),and GCE per kernel of flag leaves and spikes(F)for the set of 10 Chinese wheat genotypes from Henan Province,China,released from 1995 to 2012The genotypes released in 2012 correspond to advanced lines.Determination coefficient(r2)and probabilities are given.ns,not significant.*P＜0.05;**P＜0.01.

The present result indicated that the increase in grain yield depended mainly on HI,kernel number per square meter,and aboveground biomass(Table 4),which agrees with previous reports for wheat in China(Zhou et al.2007;Xiao et al.2012).In addition,the HI and aboveground biomass showed an increasing trend with the year of release(Figure 1B,C).Contrary to this,Zheng et al.(2011)found that grain yield was directly attributed to an increase in TKW,which also contributed to the significant increase in HI.These results were different from ours,which showed that grain yield was closely related to kernel number per square meter but was not related to TKW(Table 4).Such differences observed in traits that had an influence on yield potential could be due to the different strategies related to agronomic and physiological advances employed during the last decades(Hawkesford et al.2013).However,Foulkes et al.(2011)suggested in a review analyzing the main targets for improving yield potential that the main objectives for breeders should be enhancement of HI through an increase in grain/kernel number per unit area.In the current work,HI showed the highest correlation coefficient with grain yield,reflecting that HI could be the first trait responsible for genetic advances in yield potential.At the same time,kernel number per square meter was the second trait in importance for influencing grain yield(Tables 2,4)and it could be the cause of the enhancement of the HI and grain yield,as has been pointed out by a number of authors(Fischer et al.1998;Fischer 2008;Foulkes et al.2011).Aboveground biomass was the third parameter best correlated with grain yield(Table 3)and showed a significant positive correlation between aboveground biomass and the year of release(Figure 1C).Therefore,it could be an important aim for future breeding to increase the biomass production while maintaining or even increasing the HI(Foulkes et al.2007).Although the Chinese breeders have reached a great achievement in their breeding strategy to increase HI(Zhou et al.2007),the advanced lines assayed in the present study showed higher HI than current cultivars,suggesting that a further increase in plant partitioning towards grain yield may be possible(Tables 2,4).In fact,the theoretical maximum HI of 0.62(Austin 1980)is still far above the experimental values achieved.

Table 7.Stepwise analysis for grain yield for the whole set of 10 genotypes of Chinese wheat

Positive correlations between grain yield and the δ13C of the flag leaf,spike,and kernel were observed(Tables 5,6).These correlations indicated that genotypes with higher grain yield(the newest ones)showed an enhancement of13C isotope content.This result differed from those obtained in other field studies where grain yield was negatively correlated with the δ13C of upper(penultimate and flag)leaves and kernels(Fischer et al.1998;Araus et al.1998;Merah et al.2001).However,Craufurd et al.(1991)obtained similar results as in this experiment,showing a positive correlation between δ13C and grain yield in barley grown under well-watered field conditions.According to previous reports,the increase in δ13C may be due to stomatal closure(low stomatal conductance,and consequently less CO2diffusion)due to drought and/or a higher photosynthetic capacity(Farquhar and Sharkey 1982;Ehdaie et al.1991).In fact,most of the negative relationships between δ13C and grain yield reported in the published work refer to field studies where the plants suffer some degree of water stress,even if under irrigation,and they grow directly in the soil allowing different genotypes to explore water resources in the soil in a different manner(Condon et al.1987).In other words,negative relationship between δ13C and grain yield is probably sustained by genotypic differences in water use instead of water use efficiency(Blum 2009).Nevertheless,as described in the Material and Methods section,the plants were fully irrigated depending on their needs.Moreover,the estimated yields as well as the values of analyzed δ13C were well in the range of reports for well-watered genotypes(Craufurd et al.1991;Zheng et al.2011;Xiao et al.2012).In addition,the gsof the flag leaves(Table 3)ranged similarly to that reported in a previous study with Chinese wheat under well-watered conditions(Lu et al.1998).Therefore,the increase in δ13C of more recent genotypes was not due to the emergence of drought stress.In the same sense,the lack of a relationship of the δ13C of flag leaves and spikes with their gs,revealed that genotypic variability in δ13C was due to intrinsic differences in photosynthetic capacity(Farquhar and Richards 1984).In that sense,newer genotypes should exhibit higher intrinsic photosynthetic capacity.This was despite the fact that the newer genotypes exhibited lower N concentration in the photosynthetic organs,which may eventually result in decreasing δ13C(Shangguan et al.2000).However,no differences in RuBisCo activity were found among genotypes in spite of the lower N concentration of most recent genotypes.In fact,a positive effect on photosynthesis of a larger sink had been reported(Kaschuck et al.2010;Aranjuelo et al.2013)and the dry weight and size of the spike could be considered as an index of sink strength(Schnyder et al.2003);therefore,the genotypes with bigger spikes had higher sink strength,displaying an increased photosynthesis that was shown by the positive relationship of GCE per spike with grain yield and spike dry weight(Table 5).

The δ13C also showed different values among plant organs;spikes had a higher δ13C than the flag leaves,possibly due to the lower gsof spikes in comparison to flag leaves.Moreover,glumes can re-fix carbon released from respiration of kernels(Araus et al.1993a).In fact,for each plot,the value of kernel δ13C was in between the δ13C of the spike and flag leaf,but usually closer to the value of one of the two organs(Table 3),indicating which photosynthetic organ contributed more to grain filling.However,in an experiment conducted by Aranjuelo et al.(2011)using13C labeling,it was shown that during the beginning of post-anthesis,the C fixed by the flag leaf was stored as structural C compounds,starch,and soluble sugars,and then respired.Only a small amount of those soluble sugars arrived at the spike.On the other hand,the C synthesized in the spike was directed towards grain development.

In agreement with previous results(Austin 1994;Fischer et al.1998;Xiao et al.2012),flag leaf GCE per unit area did not change significantly among genotypes(Tables 3,S1).However,spike GCE per unit area showed differences between cultivars(Tables 4,S2)possibly due to differences in respiration(data not shown).Moreover,flag leaf and spike GCE per organ changed among genotypes due to the variable leaf area and spike size among different cultivars(Tables 2,S1,S2).In this study,GCE per whole organ when measured in the flag leaf was not correlated with grain yield(Table 6;Figure 1E),suggesting that the GCE of this organ has a minor role in grain filling(Aranjuelo et al.2011).Nevertheless,the GCE per organ when measured in the spike showed a positive correlation with grain yield,possibly due to the fact that grain yield per spike was largely associated with the size of the spike(Tables 3,5,Figure 1E).A larger spike may exhibit not only a higher sink size but also an increased photosynthetic capacity.Thus,spike photosynthesis has an important contribution to grain filling,with the awns being the main photosynthetic organ(Tambussi et al.2007).As has been explained,grain yield depends on the spike size and/or number of kernels per spike(sink)and the availability of assimilates fixed by photosynthetic organs(source)to fill these grains(Zhang et al.2010).Thus,GCE of the flag leaf and of the spike expressed per unit kernel(flag leaf GCE/kernel or spike GCE/kernel)may represent the balance between source(photosynthesis or GCE)and sink strength(kernel).Flag leaf GCE/kernel decreased in the newest cultivars and was negatively correlated with grain yield and year of release.However,the spike GCE/kernel did not show any linear relationship with grain yield or year of release(Table 6,Figure 1F).This could mean that,in spite of their higher yield,newer genotypes showed a balanced source-sink relationship in terms of spike photosynthesis,while the contribution of the flag leaf assimilates to the kernels had further decreased in more recent genotypes.Beside the above considerations,the increase in grain yield caused by an enhanced sink capacity could be the reason for the decrease in the N concentration in both the spikes and the flag leaves of the newer genotypes,whereas no differences occurred in the N concentration of kernels(Tables 3,S1,S2).

In recent years,Zhang et al.(2008,2012)had shown that PEPC may be involved in protein biosynthesis during grain development and that it could have an important role in regulating C and N metabolism in the spike of wheat.This enzyme synthesizes oxaloacetate by the carboxylation of phosphoenolpyruvate(PEP)and it is also a key step in the synthesis of aspartate and malate,used for N accumulation and transport by anaplerotic CO2fixation in crops(Smith et al.1989;Araus et al.1993b).Therefore,PEPC may have a particular role in the spike,contributing to glume re-fixation of the CO2respired by growing grains(Araus et al.1993b)as well as N partitioning from the spike tissues into the kernels(Mi et al.2000).In the present authors’results,the PEPC activity was significantly and negatively correlated with dry weight of flag leaves and spikes,and positively correlated with N concentration of the flag leaves but not with that of the glumes(Tables 5,6).Moreover,in the present authors’study,the newest genotypes,which had larger spikes and grain yield,did not exhibit higher PEPC activity,possibly due to a dilution phenomenon as had been observed for the N concentration(Table 5).Although the activities of PEPC and RuBisCo enzymes showed significant variations between genotypes in different parts of plants,they were,however,unsuitable for assessing genotypic yield performance due to the lack of correlation with grain yield(Tables 5,6).Moreover,there were no relationships between year of release and activity of either of these enzymes.In the same sense,there were no genotypic differences in the GCE rates of the flag leaf on an area basis.By contrast,the carbon stable isotope compositions were more appropriate for genotype differentiation and for higher grain yield prediction.This statement was confirmed by the stepwise analysis of grain yield as dependent of the physiological traits.In this analysis,kernel δ13C and spike δ13C were the best traits explaining the genotypic differences in grain yield(Table 7).The GCE per square meter when measured in the flag leaf was also included but with a negative sign,meaning that those genotypes with higher yield exhibited lower photosynthetic rates per unit leaf area.In fact,a tendency to lower photosynthetic rates per unit leaf area in the modern cultivars has been reported(Austin 1989;del Blanco et al.2000).Similar to the present authors’study,these studies showed a trend associated with a lower N concentration in photosynthetic tissues due to a dilution effect.

Therefore,the present authors can conclude that bread wheat breeding advances during recent decades in Henan Province,China,have been achieved through an increase in HI,kernel number per square meter,and aboveground biomass.A higher δ13C seems also to be involved in these advances,which suggests a progressive improvement in constitutive water use efficiency not associated with a trend towards lower stomatal conductance in the most recent genotypes.However,genetic advance does not appear related to changes in photosynthesis rates on an area basis when measured in the flag leaf or the spike,but only to a higher,whole-spike photosynthesis.In the same sense,RuBisCo and PEPC activities on a dry matter basis did not change with breeding advances.Results also indirectly support the concept that under potential yield conditions,the spike contributed more than the flag leaf to kernel formation.

MATERIALS AND METHODS

Plant material and experimental design

Ten bread wheat genotypes,including seven milestone cultivars widely cultivated in Henan Province during the past two decades,along with three advanced lines bred for high yield potential in Henan Province,were assayed(Table 1).The selected milestone cultivars had proved their high yield performance in the YHVWWZ,and represent the advancement in wheat breeding in Henan Province from 1995 to 2010.The three advanced lines were selected by Tianmin Henan Seed Company among 10,000 genotypes for their high yield in a preliminary field assay in Henan Province during the 2010/2011 cropping season(www.seedinfo.cn;http://en.tian-min.com).

The experiment was conducted outdoors in the Experimental Field Facilities of the Faculty of Biology,University of Barcelona(Barcelona,Spain),from 10 Dec.2011 to 14 Jun.2012.Seeds of the 10 genotypes were germinated in Petri dishes at 5°C over 15 d.After that,20 seeds of each genotype were planted in polyvinyl chloride bags filled with an artificial substrate(20 L/bag)following agronomic practices of 400 seeds/m2.The substrate was a mix of gravel(1:5),sand(2:5),and peat(2:5)(v/v),and a slow release fertilizer was added before planting(40 mg of NPK fertilizer for each bag).The experiment was carried out in a randomized complete block design with three blocks.Each genotype was assayed with five bags(five replicates)to end up with 150 bags in total(three blocks×10 genotypes×5 replicates=150 bags).The plants were irrigated with a half-strength Hoagland nutrient solution by drip irrigation to reach the optimum level of nutrition and irrigation.A fungicide(NIMROD,active component:bupirimate,BRAVOAG,Mexico DF,Mexico)was applied,at the flag leaf emergence,spike emergence,and the milky stage,to prevent powdery mildew and yellow rust.

Analysis of total N content and carbon isotope composition

One week after anthesis,three spikes and three flag leaves from each plot were randomly collected,and at physiological maturity,another three spikes were sampled for kernel analysis.All samples were dried at 60°C and the kernels were separated from the spike in order to obtain the samples for the kernel analysis.After that,all the samples were ground into a fine powder for the ratio of C13/C12and total N content analyses.For each analysis,approximately 1 mg samples of kernels and spikes,and 0.7 mg flag leaves were packed into tin capsules.Measurements were conducted at the Scientific Service Facilities of the University of Barcelona.Total N concentration was measured by an elemental gas analyzer(Flash 1112 EA;ThermoFinnigan,Breman,Germany).The same EA coupled with an isotope ratio mass spectrometer(Delta C IRMS,ThermoFinnigan,Bremen,Germany)operating in continuous flow mode was used to determine the stable carbon(13C/12C).The13C/12C ratios were expressed in δ notation,which was determined according to Farquhar et al.(1989):

where sample referred to plant material and standard to the Pee Dee Belemnite calcium carbonate standard.International isotope secondary standards of known13C/12C ratios(IAEA CH7polyethylene foil,IAEA CH6sucrose,and USGS 40 L-glutamic acid)were used for calibration with a precision of 0.1‰.

Gas exchange

Gas exchange(GE)parameters were measured on sunny days approximately 1 week after anthesis in the central segment of flag leaf blades and the entire spike using a LI-COR 6400 portable photosynthesis system(LICOR Biosciences,Lincoln,NE,USA).For each plot,the flag leaves and spikes of the main shoots of three plants were measured from 10.00 to 15.00 hours(solar time).Net carbon exchange(NCE)was evaluated in ambient CO2conditions(～400 μmol/mol CO2)with the GE chamber maintained at 25°C and 50%relative humidity(RH).In the case of the flag leaves,the standard GE chamber was used for measuring photosynthesis at light saturation conditions(1,800 μmol/m2per s photosynthetic photon flux density(PPFD)),whereas the spikes were placed in a conifer chamber(LICOR Biosciences)for measuring photosynthesis under natural light conditions(～1,700 μmol/m2per s PPFD).Following this,dark respiration was assessed by covering the GE chamber with a piece of heavy black cloth until stabilization.GCE was calculated adding the dark respiration to the net photosynthesis rate.GCE values were expressed per square meter(μmol CO2/m2per s),per whole organ(nmol CO2/s per organ),or per kernel(nmol CO2/kernel per s).After GE measurement,the measured flag leaf,was cut and immediately scanned(PIXMA/MP140 scanner;Canon,Melville,NY,USA)to calculate the area using commercial software,Digimizer 3.7(2009;MedCalc Software,Ostend,Belgium).Spike area was calculated by measuring the length and width of the four faces of each spike with a millimeter ruler.

RuBisCo activity

Three flag leaf blades and three spikes per plot were harvested 1 week after anthesis and stored at–80 °C.Aliquots of 0.5 g of frozen leaves and glumes were ground in a cold mortar using an extraction buffer with 100 mmol/L Bicine-NaOH(pH 7.8),10 mmol/L MgCl2,10 mmol/L β-mercaptoethanol and 2%polyvinylpolypyrrolidone(PVPP)(w/v).The extract was clarified by centrifugation at 26,850 g for 10 min at 4°C.Enzyme activity was determined by measuring the absorbance at 340 nm with a Cecil CE 7200 spectrophotometer(Cecil Instruments,Cambridge,UK)as described previously by Lilley and Walker(1974).

PEPC activity

The extraction of PEPC was carried out according to the method of Sayre and Kennedy(1979)with slight modifications.Aliquots of 0.5 g of frozen leaves and glumes collected for the RuBisCo activity analysis were ground in a cold mortar using an extraction buffer with 100 mmol/L Bicine-NaOH(pH 7.8),10 mmol/L MgCl2,10 mmol/L β-mercaptoethanol,and 2%PVPP(w/v).The extract was clarified by centrifugation at 26,850 g for 10 min at 4°C.PEPC activity was estimated spectrophotometrically(using a Cecil CE 7200 spectrophotometer;Cecil Instruments)coupling the reaction with the oxidation of NADH by malate dehydrogenase(MDH)according to Blanke and Ebert(1992).One hundred microliters of enzyme extract supernatant were added to a 1 mL final reaction solution that contained 50 mmol/L Tris-HCl(pH 7.8),10 mmol/L MgCl2,0.25 mmol/L ethylenediaminetetraacetic acid,5.0 mmol/L NaHCO3,4 U MDH,0.1 mmol/L NADH,and 2.0 mmol of PEP.

Grain yield and yield components

All the plants were harvested at maturity from the three middle bags for measuring the agronomic traits.Before the harvest,plant heights were measured from the soil surface to the tip of the spikes excluding awns.Then,the plants were harvested and dried for 48 h at 60°C to obtain aboveground biomass and grain yield.For estimating other yield components,five samples of representative spikes per bag were collected randomly to calculate HI,TKW,kernel number per square meter,spike length,and kernel weight per spike.

Statistical analysis

The hypothesis of zero difference between means was tested with ANOVA,performed using the general linear model procedure to calculate the effect of the different studied genotypes on the measured parameters.Mean separation of genotypes for the measured parameters was done by a Tukeyb multiple comparison test(P＜0.05).In order to test the association between grain yield and physiological traits,linear stepwise models across genotypes were constructed,with P=0.05 as the criterion for variables to be either included or removed from the model.A series matrix of simple coefficient correlations was analyzed based on the yield performance,and agronomic and physiological traits of the different genotypes.All data were analyzed using the SPSS version 16 statistical package(SPSS,Chicago,IL,USA).Figures were created using SigmaPlot version 12.0 for Windows(Sysat Software,Point Richmond,CA,USA).

ACKNOWLEDGEMENTS

The present authors would like to acknowledge Hennan Tianmin Seed for providing germplasm and financial support to Bangwei Zhou.This work was supported by Breeding to Optimise Chinese Agriculture(OPTICHINA)(Coordination and support action:FP7-KBBE-2010-4,grant agreement number:266045)and by Improvement of Durum Wheat to Present and Future Mediterranean Conditions(Ministerio de Ciencia e Innovación:AGL2010-20180).álvaro Sanz-Sáez was the recipient of a research grant from the Department of Education,Linguistic,and Education of the Basque Country Government.

Aranjuelo I,Cabrera-Bosquet L,Morcuende R,Avice JC,Nogués S,Araus JL,Martínez-Carrasco R,Pérez P(2011)Does ear C sink strength contribute to overcoming photosynthetic acclimation of wheat plants exposed to elevated CO2?J Exp Bot 62:3957–3969

Aranjuelo I,Sanz-Sáez A,Jauregui I,Irigoyen JJ,Araus JL,Sánchez-Díaz EriceG(2013)Harvest index,a parameter conditioning responsiveness of wheat plants to elevated CO2.J Exp Bot 64:1879–1892

Araus JL,Amaro T,Casadesus J,Asbati A,Nachit MM(1998)Relationship between ash content,carbon isotope discrimination and yield in durum wheat.Aust J Plant Physiol 25:835–842

Araus JL,Bort J,Brown HR,Bassett C,Cortadellas N(1993b)Immunocytochemical localization of phosphoenolpyruvate carboxylase and photosynthetic gas exchange characteristics in ears of Triticum durum Desf.Planta 191:507–514

Araus JL,Brown HR,Febrero A,Bort J,Serret MD(1993a)Ear photosynthesis,carbon isotope discrimination and the contribution of respiratory CO2to differences in grain mass in durum wheat.Plant Cell Environ 16:383–392

Araus JL,Slafer GA,Reynolds MP,Royo C(2002)Plant breeding and water relations in C3 cereals:What should we breed for?Ann Bot London 89:925–940

Araus JL,Slafer GA,Royo C,Serret MD(2008)Breeding for yield potential and stress adaptation in cereals.Plant Sci 27:377–412

Araus JL,Villegas D,Aparicio N,del Moral LFG,El Hani S,Rharrabti Y,Ferrio JP,Royo C(2003)Environmental factors determining carbon isotope discrimination and yield in durum wheat under Mediterranean conditions.Crop Sci 43:180–190

Austin RB(1989)Genetic variation in photosynthesis.J Agric Sci 112:287–294

Austin RB(1994)Plant Breeding opportunities.In:Boote KJ,Bennett JM,Sinclair TR,Paulsen GM,eds.International Symposium on Physiology and Determination of Crop Yield.University of Florida,Gainesville,FL,USA.pp.567–586

Austin RB(1980)Genetic improvement in winter wheat yield since 1900 and associated physiological changes.J Agric Sci 94:675–689

Blanke MM,Ebert G(1992)Phosphoenolpyruvate carboxylase and carbon economy of apple seedlings.J Exp Bot 43:965–968

Blum A(2009)Effective use of water(EUW)and not water-use efficiency(WUE)is the target of crop yield improvement under drought stress.Field Crops Res 112:119–123

Cabrera-Bosquet L,Crossa J,von Zitzewitz J,Serret MD,Araus JL(2012)High-throughput phenotyping and genomic selection:The frontiers of crop breeding converge.J Integr Plant Biol 54:312–320

Condon AG,Farquhar GD,Richards RA(1990)Genotypic variation in carbon isotope discrimination and transpiration efficiency in wheat leaf gas exchange and whole plant studies.Aust J Plant Physiol 17:9–22

Condon AG,Richards RA,Farquhar GD(1987)Carbon isotope discrimination is positively correlated with grain yield and dry matter production in field-grown wheat.Crop Sci 27:996–1001

Condon AG,Richards RA,Rebetzke GJ,Farquhar GD(2002)Improving intrinsic water-use efficiency and crop yield.Crop Sci 42:122–131

Craufurd PQ,Austina RB,Acevedob E,Hallc MA(1991)Carbon isotope discrimination and grain-yield in barley.Field Crops Res 27:301–313

del Blanco IA,Rajaram S,Kronstad WE,Reynolds MP(2000)Physiological performance of synthetic hexaploid wheat-derived populations.Crop Sci 40:1257–1263

Ehdaie B,Hall AE,Farquhar GD,Nguyen HT,Waines JG(1991)Water-use efficiency and carbon isotope discrimination in wheat.Crop Sci 31:1282–1288

Farquhar GD,Richards RA(1984)Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes.Aust J Plant Physiol 11:539–552

Farquhar G,Sharkey TD(1982)Stomatal conductance and photosynthesis.Annu Rev Plant Physiol 33:317–345

Farquhar GD,Ehleringer JR,Hubick KT(1989)Carbon isotope discrimination and photosynthesis.Annu Rev Plant Physiol Plant Mol Biol 40:503–537

Fischer RA(2008)The importance of grain or kernel number in wheat:A reply to Sinclair and Jamieson.Field Crops Res 105:15–21

Fischer RA,Rees D,Sayre KD,Lu ZM,Condon AG,Larque Saavedra A(1998)Wheat yield progress associated with higher stomatal conductance and photosynthetic rate,and cooler canopies.Crop Sci 38:1467–1475

Foulkes MJ,Slafer GA,Davies WJ,Berry PM,Sylvester-Bradley R,Martre P,Calderini DF,Griffiths S,Reynolds MP(2011)Raising yield potential of wheat.III.Optimizing partitioning to grain while maintaining lodging resistance.J Exp Bot 62:469–486

Foulkes MJ,Snape JW,Shearman VJ,Reynolds MP,Gaju O,Sylvester-Bradley R(2007)Genetic progress in yield potential in wheat:Recent advances and future prospects.J Agric Sci 145:17–29

Hawkesford MJ,Araus JL,Park R,Calderini D,Miralles D,Shen T,Zhang J,Parry MAJ(2013)Prospects of doubling global wheat yields.Food Energy Secur 2:34–48

Kaschuk G,Hungria M,Leffelaar PA,Giller KE,Kuyper TW(2010)Differences in photosynthetic behaviour and leaf senescence of soybean(Glycine max[L.]Merrill)dependent on N2fixation or nitrate supply.Plant Biol 12:60–69

Lilley RM,Walker DA(1974)An improved spectrophotometric assay of ribulose bisphosphate carboxilasa.Biochem Biophys Acta 385:226–229

Lu ZM,Percy RG,Qualset CO,Zeiger Z(1998)Stomatal conductance predicts yields in irrigated Pima cotton and bread wheat grown at high temperatures.J Exp Bot 49:453–460

Makino A(2011)Photosynthesis,grain yield,and nitrogen utilization in rice and wheat.Plant Physiol 155:125–129

Merah O,Deléens E,Souyris I,Nachit M,Monneveux P(2001)Stability of carbon isotope discrimination and grain yield in durum wheat.Crop Sci 41:677–681

Mi GH,Li TG,Zhang FS,Zhang JH(2000)Is nitrogen uptake after anthesis in wheat regulated by sink size?Field Crops Res 68:183–190

Monneveaux P,Reynolds MP,Trethowan R,González-Santoyo H,Pe?a RJ,Zapata F(2005)Relationship between grain yield and carbon isotope discrimination in bread wheat under four water regimes.Eur J Agron 22:231–242

NBS(2010)National Bureau of Statistics of China.Viewed 30 October,2012(on-line).http://datacenter.cngrain.com/IndexProduce.aspx?Flag=2&PId=10&TId=1

Parry MAJ,Andralojc PJ,Scales JC,Salvucci ME,Carmo-Silva AE,Alonso H,Whitney SM(2013)Rubisco activity and regulation as targets for crop improvement.J Exp Bot 64:717–730

Parry MAJ,Hawkesford M(2010)Food security:Increasing yield and improving resource use efficiency.Proc Nutr Soc 69:1–9

Parry MAJ,Reynolds M,Salvucci ME,Raines C,Andralojc PJ,Zhu XG,Price GD,Condon AG,Furbank RT(2011)Raising yield potential of wheat.II.Increasing photosynthetic capacity and efficiency.J Exp Bot 62:453–467

Paul MJ,Foyer CH(2001)Sink regulation of photosynthesis.J Exp Bot 52:1381–1400

Pérez P,Martinez-Carrasco R,Martín Del Molino M,Rojo B,Ulloa M(1989)Nitrogen uptake and accumulation in grains of three winter wheat varieties with altered source-sink ratios.J Exp Bot 40:707–710

Rebetzke GJ,Condon AG,Richards RA,Farquhar GD(2002)Selection for reduced carbon isotope discrimination increases aerial biomass and grain yield of rainfed bread wheat.Crop Sci 42:739–745

Reynolds M,Bonnett D,Chapman SC(2011)Raising yield potential of wheat.I.Overview of a consortium approach and breeding strategies.J Exp Bot 62:439–452

Reynolds M,Foulkes J,Furbank R,Griffiths S,King J,Murchie E,Parry M,Slafer G(2012)Achieving yield gains in wheat.Plant Cell Environ 35:1799–1823

Reynolds M,Foulkes MJ,Slafer GA,Berry P,Parry MAJ,Snape JW,Angus WJ(2009)Raising yield potential in wheat.J Exp Bot 60:1899–1918

Reynolds MP,Pellegrineschi A,Skovmand B(2005)Sink-limitation to yield and biomass:A summary of some investigations in spring wheat.Ann Appl Biol 146:39–49

Richards RA(2000)Selectable traits to increase crop photosynthesis and yield of grain crops.J Exp Bot 51:447–458

Sayre RT,Kennedy RA(1979)Photosynthetic enzyme activities and localization in Mollugo veticillata populations differing in the levels of C3 and C4 cycle operation.Plant Physiol 64:293–299

Schnyder H(1993)The role of carbohydrate storage and redistribution in the source-sink relations of wheat and barley during grain filling.New Phytol 123:233–245

Schnyder H,Schaüfele R,L?tscher M,Gebbing T(2003)Disentangling CO2fluxes:Direct measurements of mesocosm-scale natural abundance13CO2/12CO2gas exchange,13C discrimination,and labelling of CO2exchange flux components in controlled environments.Plant Cell Environ 26:1863–1874

Serret MD,Ortiz-Monasterio I,Pardo A,Araus JL(2008)The effects of urea fertilization and genotype on yield,nitrogen use efficiency,δ15N and δ13C in wheat.Ann Appl Biol 153:243–257

Shangguan ZP,Shao MA,Dyckmans J(2000)Nitrogen nutrition and water stress effects on leaf photosynthetic gas exchange and water use efficiency in winter wheat.Environ Exper Bot 44:141–149

Sharma RC,Smith EL,McNew RW(1985)Stability of harvest index and grain yield in winter wheat.Crop Sci 27:104–108

Shimshi D,Ephrat J(1975)Stomatal behavior of wheat cultivars in relation to their transpiration,photosynthesis and yield.Agron J 67:326–331

Slafer GA,Calderini DF,Miralles DG(1996)Yield components and compensation in wheat:Opportunities for further increasing yield potential.In:Reynolds MP,Rajaram S,Mcnab A,eds.Increasing Yield Potential in Wheat:Breaking the Barriers,CIMMYT,Mexico.pp.101–113

Smith AJ,Rinne RW,Seif RD(1989)Phosphoenolpyruvate carboxylase and pyruvate kinase involvement in protein and oil biosynthesis during soybean seed development.Crop Sci 29:349–353

Tambussi EA,Bort J,Araus JL(2007)Water use efficiency in C3 cereals under Mediterranean conditions:A review of physiological aspects.Ann Appl Biol 150:307–321

USDA(2012)United States Department of Agriculture Foreign Agricultural Service Viewed October 30,2012(on-line).http://www.pecad.fas.usda.gov/highlights/2012/03/china%20wheat/

Xiao YG,Qian ZG,Wu K,Liu JJ,Xia XC,Ji WQ,He ZH(2012)Genetic gains in grain yield and physiological traits of winter wheat in Shandong province,China,from 1969 to 2006.Crop Sci 52:44–56

Xu X,Yuan HM,Li SH,Trethowan R,Monneveux P(2007)Relationship between carbon isotope discrimination and grain yield in spring wheat cultivated under different water regimes.J Integr Plant Biol 49:1497–1507

Ye YL,Yang SQ,Huang YF,Liu F(2007)Development and prospect of wheat production in Henan province.Chin Agric Sci Bull 23:199–199

Yoshida S(1972)Physiological aspects of grain yield.Ann Rev Plant Physiol 23:437–464

Zhang HP,Turner NC,Poole ML(2010)Source–sink balance and manipulating sink–source relations of wheat indicate that the yield potential of wheat is sink-limited in high-rainfall zones.Crop Past Sci 61:852–861

Zhang YH,Wang ZM,Huang Q,Shu W(2008)Phosphoenolpyruvate carboxylase activity in ear organs is related to protein concentration in grains of winter wheat.J Cer Sci 47:386–391

Zhang YH,Zhou SL,Huang Q,Leng GH,Xue QW,Stewart BA,Wang ZM(2012)Effects of sucrose and ammonium nitrate on phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase activities in wheat ears.Aust J Crop Sci 6:822–827

Zheng TC,Zhang XK,Yin GH,Wang LN,Han YL,Chen L,Huang F,Tang JW,Xia XC,He ZH(2011)Genetic gains in grain yield,net photosynthesis and stomatal conductance achieved in Henan Province of China between 1981 and 2008.Field Crop Res 122:225–233

Zhou Y,He ZH,Sui XX,Xia XC,Zhang XK,Zhang GS(2007)Genetic improvement of grain yield and associated with traits in the northern China winter wheat region from 1960 to 2000.Crop Sci 47:245–253

SUPPORTING INFORMATION

Additional supporting information can be found in the online version of this article:

Table S1.Mean values and sum of squares type III combined with ANOVA of different physiological parameters of seven winter wheat cultivars and three advanced lines from Henan Province,China

All the parameters are the means of three replicates of each genotype.Means followed by the same letter were not significantly different at P=0.05 by the Tukey-b test.(**P≤0.01 and***P≤0.001).δ13C,carbon isotope composition;gs,stomatal conductance;GCE/m2,gross carbon exchange per square meter,GCE/organ,gross carbon exchange per whole organ;GCE/kernel,gross carbon exchange per kernel;PEPC,phosphoenolpyruvate carboxylase.

Table S2.Mean values and sum of squares type III combined with ANOVA of different physiological parameters of seven winter wheat cultivars and three advanced lines from Henan Province,China

All the parameters are the means of three replicates of each genotype.Means followed by the same letter were not significantly different at P=0.05 by the Tukey-b test.(*P≤0.05;**P≤0.01;and***P≤0.001).δ13C,carbon isotope composition;gs,stomatal conductance;GCE/m2,gross carbon exchange per square meter,GCE/organ,gross carbon exchange per whole organ;GCE/kernel,gross carbon exchange per kernel;PEPC,phosphoenolpyruvate carboxylase.