TANG Chan-juan ,LUO Ming-zhao ,ZHANG Shuo,JlA Guan-qing,TANG Sha,JlA Yan-chao,ZHl Hui,DlAO Xian-min

Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

Abstract Chlorophyll (Chl) content,especially Chl b content,and stomatal conductance (Gs) are the key factors affecting the net photosynthetic rate (Pn).Setaria italica,a diploid C4 panicoid species with a simple genome and high transformation efficiency,has been widely accepted as a model in photosynthesis and drought-tolerance research.The current study characterized Chl content,Gs,and Pn of 48 Setaria mutants induced by ethyl methanesulfonate.A total of 24,34,and 35 mutants had significant variations in Chl content,Gs,and Pn,respectively.Correlation analysis showed a positive correlation between increased Gs and increased Pn,and a weak correlation between decreased Chl b content and decreased Pn was also found.Remarkably,two mutants behaved with significantly decreased Chl b content but increased Pn compared to Yugu 1.Seven mutants behaved with significantly decreased Gs but did not decrease Pn compared to Yugu 1.The current study thus identified various genetic lines,further exploration of which would be beneficial to elucidate the relationship between Chl content,Gs,and Pn and the mechanism underlying why C4 species are efficient at photosynthesis and water saving.

Keywords: photosynthetic capacity,chlorophyll content,stomatal conductance,EMS mutant variation,Setaria italica

Biomass synthesizedviaphotosynthesis is the primary determinant of plant productivity.Improving photosynthetic performance,therefore,is of great importance to drive necessary gains in crop yield (Ortet al.2015;Chabrandet al.2016;Nowickaet al.2018;Faralli and Lawson 2020).Chlorophyll (Chl)aandbare the most abundant pigments in the light-harvesting antenna system of higher plants;they play a central role in absorbing light and transmitting energy (Bjornet al.2009;Wang and Grimm 2015;Gitelsonet al.2016;Kumeet al.2018).In higher plants,the light-harvesting antenna system consists of a core antenna complex and several peripheral antenna complexes.Chlaoccurs universally in all antenna complexes (including photosystem I(PSI) and PSII core complexes and peripheral antenna complexes),whereas Chlbis found nearly exclusively in the light-harvesting Chla/b-protein complexes (LHCs)that form the peripheral antenna complexes (the PSI core and PSII core almost completely lacks Chlb) (Kunugiet al.2016;Kumeet al.2018).Changes in antenna size largely depend on LHCs abundance.For the amount of core complex per reaction center is fixed,whereas the organization of LHCs and peripheral antenna complexes is flexible among different photosynthetic organisms and under different solar conditions.According to Jiaet al.(2016),levels of LHCII and antenna size were highly correlated with the accumulation of Chlb.Plants with increased Chlbaccumulation assembled more LHCII complexes and had a larger antenna size (Tanaka and Tanaka 2011;Jiaet al.2016).Chlbcontent and the Chla/bratio,therefore,are useful as an index to indicate the LHCII abundance and antenna size of plants and are key parameters that determine light absorbing efficiencies(Baileyet al.2001;Jiaet al.2016;Kumeet al.2018).

Tanakaet al.(2001) reported that overexpression of chlorophyllide a oxygenase (CAO) increased the synthesis of Chlbfrom Chla,reducing the Chla/bratio from 2.85 to 2.65 and resulting in at least a 10–20%increase in the antenna size inArabidopsisthaliana(Tanaka Ret al.2001).Genetic modification of the biosynthesis of Chlbin tobacco (Nicotiana tabacum)also revealed that increased Chlbsynthesis in CAOoverexpressing plants increased antenna size,CO2assimilation,and dry matter accumulation,suggesting that engineering plants for larger antennae with higher Chlbcontent and lower Chla/bratio might have the potential for increasing photosynthesis,especially under low-light conditions (Biswalet al.2012).However,higher plants,in which large arrays of LHCs are assembled as peripheral components of PSI and PSII,are highly efficient at absorbing and transferring photons,an arrangement that allows them to operate under dim light conditions but which can lead to excess energy accumulation in full sunlight (Rooijenet al.2015;Chmeliovet al.2016;Kromdijket al.2016).The excess energy should then be released as heatvianonchemical quenching mechanisms to protect the plant from photo-induced damage (Horton and Ruban 2005;Chmeliovet al.2016;Kromdijket al.2016).Thus,a concept was proposed by Kirstet al.(2017) to develop a truncated light-harvesting chlorophyll antenna (TLA) to alleviate excess absorption of sunlight and the waste of excitation energy consumed by nonphotochemical quenching in order to improve productivity.Prior research inChlamydomonashas provided early evidence that a truncated light-harvesting antenna size could increase photosynthetic productivity in highdensity cultures under bright sunlight conditions (Polleet al.2003;Kirstet al.2012;Mitraet al.2012;Kirst and Melis 2014;Jeonget al.2017).Subsequent research on tobacco reported a TLA material,in which Chlaandbcontents were lowered to about 57 and 23% of wild type,respectively,and the Chla/bratio was elevated from about 3:1 to 8:1,which showed a 25% increase in stem and leaf biomass accumulation (Kirstet al.2017).Slatteryet al.(2017) reported that Chl deficiency in a soybean mutant line,Y11y11,led to greater rates of leaf-level photosynthesis per absorbed photon early in the growing season when mutant Chl content was～35% of the wild type.Although research on reducing Chl content to increase light-use efficiency and yield improvements has not yet been seen in cereal crops,it becomes increasingly clear that optimizing light-harvesting management,such as modifying the antenna size and Chl biosynthesis,should be a focus of research for future improvement in photosynthesis and crop productivity (Ortet al.2011;Songet al.2017).

Increased stomatal conductance (Gs) has been a selective trait over decades in breeding for high-yielding varieties.As stomata govern the diffusion of CO2from the atmosphere to the leaf interior,it greatly influences CO2fixation in the mesophyll tissue (Lawson and Blatt 2014;Faralliet al.2019).Over the long term and under steady conditions,increasedGsalways strongly correlate with higher photosynthetic performance (Wonget al.1979;Faralliet al.2019).However,stomata also govern the diffusion of H2O from the leaf interior to the atmosphere.Under low soil water conditions,crops tend to reduceGsto reduce water consumption,thus leading to lower net photosynthetic rate (Pn) and depressed productivity(Gilbertet al.2011).Therefore,it is important to balance gains inPnwith performance under drought stress(Chabrandet al.2016;Golecet al.2019).It is reported that both C3and C4plants are suffering from a decline inGsand thus decreased intercellular CO2concentration(Ci) in the early phase of water stress (Israelet al.2022).However,as C4plants are featured by an efficient CO2-concentrating mechanism which enables them to saturate C4photosynthesis under relatively lowCi,C4plants thus may not decline CO2assimilation rates under the limitedGsas C3plants (Israelet al.2022;Tayloret al.2018).Pintoet al.(2014) reported that minor reductions of photosynthesis were produced in C4plants relative to C3and C3–C4species under a lower CO2concentration.And according to Pintoet al.(2014),the initial decline ofCito 50% to that of the control would not reduce CO2assimilation rates during the early phases of water stress in maize and amaranthus.A C4photosynthetic model described by Siebke (2002) also indicated that little influence would have on CO2assimilation rates even ifCideclined down to 50 Pa.It is therefore concluded that the efficient CO2-concentrating mechanism is key for C4plants to maintain relatively higher photosynthesis under water stress.Moreover,screening for mutants with few reductions in photosynthesis under restrictedGsin C4plants should be brought into focus for research on acclimating crops to drought conditions and improving crop yields.

Setariaitalica,a diploid C4panicoid species with small stature,a simple genome of 430 Mb,and high transformation efficiency,has been a useful tool for functional gene mining and research,especially for genes involved in photosynthesis and drought resistance (Brutnellet al.2010;Li and Brutnell 2011;Liet al.2016;Diaoet al.2014;Huanget al.2014;Tanget al.2017;Luoet al.2018;Doustet al.2019;Nguyenet al.2020;Pegleret al.2020;Soodet al.2020).In the current study,the Chl content of 28 leaf-colorSetariamutants and the photosynthetic performance of those mutants plus a further 20 mutants were characterized,aiming at providing a reference for modulating Chl content andGsto improve drought tolerance and photosynthetic efficiency in major crops.

2.1.Materials and growth conditions

Plant materials used for analyzing Chl and photosynthesis were derived from an ethyl methanesulfonate (EMS)-derivedSetariaitalicamutant library as previously described (Luoet al.2018).Among the 2 709 M3/M4 lines,28 were identified as leaf color mutants using a microscope,as described by Luoet al.(2018),and were compared with the wild-type Yugu 1.In addition to the 28 leaf color mutants,another 20 mutants (a total of 48 lines)were identified that might have variations in photosynthetic performance,as described by Luoet al.(2018),and therefore were subjected to photosynthesis analysis.

Experiments were conducted between July and September 2016 in the greenhouse of the Institute of Crop Sciences,Chinese Academy of Agricultural Science,Beijing,China (40°N,116°E).The growth temperature of the greenhouse was in a range of 30 to 38°C.The artificial light was turned on at 8 a.m.and turned off at 6 p.m.Plants were grown in 3 m×0.9 m plots with a row space of 0.3 m and a space in the rows of 5 cm,using standard agronomic practices (e.g.,irrigation,weeding,and pest control).

2.2.Chlorophyll analysis

Leaf tissues were taken from fully developed flag leaves about 5 d before flowering (50 d after seeding).A hole puncher was used to obtain 100 pieces of tissue of 3-mm diameter from the middle zone of the flag leaves.Foilwrapped 10 mL centrifuge tube and carton were used to keep samples in the dark.For each material,six replicates were made.The leaf tissues were placed into 80% acetone and shaken for 24 h until the tissue color had completely faded.Finally,the solutions were placed in a UV-1800 ultraviolet/visible light spectrophotometer to measure their absorbance at 663 and 645 nm.The contents of Chlaand Chlbwere then calculated using the formula described by Lichtenthaler (1987) :

where V is the volume of the 80% acetone for each replicate and S is the area of the leaf tissue for each replicate.

2.3.Photosynthesis analysis

The photosynthetic performance of the 48 mutants was measured using the LI-6400 photosynthesis system with a red-blue light source and 6-cm2cuvette (LI-6400XT,LI-COR,USA).For each line,we used at least 10 flag leaves of 50-day-old plants with the same height and orientation that allowed them to receive full light.Measurements were taken from 9:30 to 11:30 a.m.during 8–13 August.Cuvette conditions were: photosynthetic photon flux density,1 500 μmol m?2s?1;sample CO2concentration,400 μmol mol?1;flow rate,500 mL s?1,and the cuvette fan was set to fast.The cuvette was placed in the middle leaf zone.Before logging the measurement,the criteria for stability are that parameters in line a are stable,parameters in line c are normal,fluctuation of ΔCO2in line b<0.2 μmol mol–1,and one digit behind the decimal point of Photo is stable.

To minimize the effect of varied light,temperature,and vapor pressure deficit (Vpd) on the photosynthetic parameters obtained at different times,we carefully controlled the environmental conditions that might affect photosynthetic performance.Specifically,we chose sunny days with comparable solar radiation intensity,temperature,and humidity to do measurements and recorded photosynthetic parameters only when leaf temperature and Vpd reached 39 to 41°C and 4.5 to 5.5 kPa.

To make the photosynthetic parameters obtained at different times comparable to each other,at each measurement period (from 9:30 to 11:30 a.m.),we measured wild-type Yugu 1 at the same time with the mutant lines.Thus,the parameters of Yugu 1 were used as a reference to normalize the photosynthetic parameters of the mutants obtained at different measurement periods.The normalizing formula was as follows:

whereYXis the photosynthetic data of Yugu 1 at a specific time;Ymis the mean value of the photosynthetic data of Yugu 1 over all time periods;TXis the photosynthetic data of the mutant at a specific time period;T is the normalized photosynthetic parameter of the mutant.

Except for Chl content,photosynthetic parameters presented in our text,such asPn,Gs,andCi,were all normalized data.

2.4.Data analysis

For statistical analysis,one-way ANOVA followed by an LSDt-test was performed to evaluate the difference between Yugu 1 and the mutant lines.Correlation coefficients between all possible pairs of measured parameters were analyzed by Pearson’s correlation test.Data analyses were performed with SPSS 17.0.Box and scatter plots were constructed with OriginPro 2019 and R Studio.

3.1.Characterization of chlorophyll content of the leaf color mutants

Firstly,as shown in Fig.1-A,the Chlacontent of the 28 mutants presented an obvious right-skewed distribution,varying from 1.46 to 2.02 mg dm?2(Appendix A).The Chlacontent of 18 out of the 28 mutants (64.2%) was significantly lower (P<0.05) than that of the wild-type Yugu 1(2.02 mg dm?2).Among the 18 mutants with reduced Chlacontent,15 mutants (53.6%) showed Chlacontent slightly reduced to 1.7–1.9 mg dm?2,while three mutants(t53,t68,and t104) showed more markedly reduced Chlacontents of 1.45–1.65 mg dm?2.No mutant presented significantly increased Chlacontent (Fig.1-A;Appendix A).

Fig.1 Chlorophyll (Chl) content of selected 28 leaf-color mutants grown in the field.A,Chl a.B,Chl b.C,Chl a/b ratio.D,total Chl content.Insets in the lower right corner of A–D showed frequency distribution histograms of the selected 28 mutants for Chl a content,Chl b content,Chl a/b,and total Chl content.X-axis was grouped value range of the parameters;Y-axis was counts of mutant lines in the value range.Blue arrows indicate the position of wild-type Yugu 1 in the distribution.Values are mean±SE(n=6).Asterisks below the X-axis denote significant differences between the indicated mutant and Yugu 1: ＊＊,P<0.01;＊,P<0.05.

Changes in Chlbcontent of the 28 mutants were more striking than those of Chla,and the content varied from 0.41 to 1.38 mg dm?2in what appeared to be a bimodal distribution (Fig.1-B).The Chlbcontent of wild-type Yugu 1 (0.95 mg dm?2) was very close to the median of the distribution (0.89 mg dm?2).Ten mutants performed significantly higher Chlbcontent than Yugu 1,and 14 mutants performed significantly lower Chlbcontent than Yugu 1.Three mutants,t53,t68,and t104,showed greatly reduced Chlbcontent to half that of Yugu 1,at 0.4575,0.4723,and 0.4087 mg dm?2,respectively(Fig.1-B;Appendix A).The total Chl content of t53,t68,and t104 also decreased substantially (Fig.1-D;Appendix A).Changes in the Chla/bratio were highly correlated with Chlbcontent.As shown in Fig.1-C,mutants with increased Chlbcontent all presented decreased Chla/bratio,and mutants with decreased Chlbcontent all presented an increased Chla/bratio.

3.2.Characterization of photosynthetic performance of the mutants

As shown in Fig.2,abundant variations were found among the selected mutants.Compared with the wild type Yugu 1 (in whichPnwas 18.24 μmol CO2m?2s?1),23 out of 48 mutants exhibited significantly increasedPn(P<0.05),with thePnof nine mutants (t53,t34,t57,t54,t17,t58,t103,t39,and t45) exceeding 25 μmol CO2m?2s?1,and thePnof a further nine mutants (t27,t114,t50,t41,t105,t42,t24,t28,and t118) as high as 23–25 μmol CO2m?2s?1(Fig.2-A;Appendix B).In contrast,12 mutants showed significantly reducedPn(P<0.05),among which seven mutants (t65,t91,t69,t72,t64,t6,and t66)hadPnlower than 13 μmol CO2m?2s?1(Fig.2-A).ThePnof the remaining 13 mutants showed no significant difference compared with Yugu 1.

Fig.2 Net photosynthetic rate (Pn) (A),stomatal conductance (Gs) (B),and internal CO2 concentration (Ci) of selected 48 mutants grown in the field.Insets in the upper left corner of A–C showed histograms of the frequency distribution of Pn,Gs,and Ci of the selected 48 mutants.X-axis was grouped value range of the parameters;Y-axis was counts of mutant lines in the value range.Blue arrows indicated the position of wild-type Yugu 1 in the distribution.Error bars represent standard error (n=10).Asterisks below the X-axis denote significant differences between the indicated mutant and Yugu 1: ＊＊,P<0.01;＊,P<0.05.

As shown in Fig.2-B,Gsof the selected 48 mutants were in a range of 0.08 to 0.26 mol H2O m–2s–1.Eleven mutants,namely t6,t14,t20,t89,t10,t95 t86,t11,t8,t50 and t97 performed decreasedGsthan that of Yugu 1((0.119±0.052) mol H2O m–2s–1).Moreover,23 mutants(t81,t58,t30,t68,t66,t69,t17,t26,t93,t105,t41,t28,t27,t114,t34,t24,t118,t23,t54,t42,t103,t45,and t39) behaved with increasedGswhen compared of that of Yugu 1.Remarkably,according to the distribution histogram ofGsinserted in Fig.2-B,Gsof most mutant lines were in a range of 0.08 to 0.26 mol H2O m–2s–1.Two mutants (t45 and t39) behaved with distinct increases inGsat 0.227 and 0.2530 mol H2O m–2s–1.Ciof the selected 48 mutants ranged from 30 to 210 μmol CO2mol–1.Moreover,as shown in Fig.2-C,12 mutants (t50,t14,t20,t95,t11,t10,t97,t58,t17,t87,t53,and t31)performed decreasedCi,and 21 mutants (t71,t118,t81,t24,t114,t45,t27,t67,t26,t93,t72,t68,t64,t91,t66,t69,t39,t30,t65,t42,and t23) performed increasedCiwhen compared with Yugu 1.

To further explore the relations amongPn,Gs,andCi,scatter plots ofPnvs.Gs,Civs.Gs,andPnvs.Ciwere made (Fig.3).Fig.3-A,D,and G presented scatter plots ofPnvs.Gs,Civs.Gs,andPnvs.Ci,respectively,of the 11 mutant lines with decreasedGs.Fig.3-B,E,and H presented scatter plots ofPnvs.Gs,Civs.Gs,andPnvs.Ci,respectively,of the 14 mutant lines with unchangedGs.Fig.3-C,F,and I presented scatter plots of the 23 mutant lines with increasedGs.Among the 23 mutants with significantly increasedGs(P<0.01),17 (t17,t23,t24,t26,t27,t28,t34,t39,t41,t42,t45,t54,t58,t103,t105,t114,and t118) also showed increasedPnperformance(Fig.3-C,blue).However,three mutants (t30,t81,and t93) exhibited no significant increase inPnperformance(Fig.3-C,red “Δ”).Three mutants (t66,t68,and t69)showed substantially decreasedPnperformance despite their increasedGs(Fig.3-C,greenblue “+”).Interestingly,Ciwas significantly increased in these six mutants (t30,t81,t93,t66,t68,and t69) compared with Yugu 1 (Fig.3-F).

Fig.3 Scatter plots of net photosynthetic rate (Pn) vs.stomatal conductance (Gs),internal CO2 concentration (Ci) vs.Gs,Pn vs.Ci of the 48 mutant lines.A–C,scatter plot of Pn vs.Gs with mutant lines exhibiting decreased Gs (A),unchanged Gs (B),and increased Gs (C) when comparing with that of Yugu 1.D–F,scatter plot of Ci vs.Gs with mutant lines exhibiting decreased Gs (D),unchanged Gs (E),and increased Gs (F) when comparing that of Yugu 1.G–I,scatter plot of Pn vs.Ci with mutant lines exhibiting decreased Gs (G),unchanged Gs (H),and increased Gs (I) when comparing that of Yugu 1.Blue represented mutants with increased Pn by comparing with that of Yugu 1;greenblue “+” represented mutants with decreased Pn by comparing with that of Yugu 1;red “△”represented mutants with unchanged Pn by comparing with that of Yugu 1;red dotted line in A–C marked the Pn value of 13 μmol CO2 m?2 s?1;red line in D–F marked the Ci value of Yugu 1 at 107 μmol CO2 mol–1.

TheGsof 14 mutants,namely t31,t35,t53,t54,t64,t65,t67,t71,t72,t82,t84,t87,t91,and t104,did not differ significantly from theGsof Yugu 1 (Fig.2-B).Thus,the opportunity for the mesophyll cells to capture CO2for photosynthesis was equivalent in these mutants and in Yugu 1,and therefore the differences inPnperformance between these mutants and Yugu 1 were largely attributable to non-stomatal factors,such as CO2capture and assimilation efficiency.Among the 14 mutants,five mutants (t53,t57,t82,t87,and t104) showed increasedPnperformance (Fig.3-B,blue),five mutants (t64,t65,t67,t72,and t91) showed decreasedPnperformance(Fig.3-B,greenblue “+”),and four mutants (t31,t35,t71,and t84) showed no difference inPnperformance when compared with Yugu 1 (Fig.3-B,red “Δ”).The five mutants with decreasedPnperformance showed significantly increasedCivalues (P<0.05),whereas the five mutants with increasedPnperformance showed decreasedCivalues (Fig.3-E).These results suggested thatCiandPnare negatively correlated under consistentGsconditions (Fig.3-H).

Furthermore,compared with Yugu 1,there were 11 mutants,namely t6,t8,t10,t11,t14,t20,t50,t86,t89,t95,and t97,with significantly decreasedGs(P<0.05)(Fig.2-B).ThePnperformance of these mutants differed from the wild type (Fig.3-A).Three mutants (t6,t86,and t89) exhibited decreasedPnin line with their decreasedGs.However,seven mutants (t8,t10,t11,t14,t20,t95,and t97) showed no change inPnperformance despite their decreasedGs.Remarkably,one mutant,t50,showed significantly increasedPn(P<0.01) despite its decreasedGs.Interestingly,theCiof these seven mutants (t10,t11,t14,t20,t95,t97,and t50) was also significantly decreased compared with Yugu 1 (Figs.2-C and 3-D).These results indicated that mutant t10,t11,t14,t20,t95,t97,and t50 had increased capacity for capturing CO2under decreasedGs.

Moreover,according to Fig.3-G,H,and I,PnandCiwere negatively correlated in most cases.However,such a correlation did not exist among the mutant lines with dramatically increasedGs,such as t103,t39,t42,and t23(Fig.3-I).

3.3.Correlation analysis between photosynthetic parameters and Chl content

To explore the correlation relationships between the photosynthetic parameters and Chl content,we analyzed the correlations between all possible pairs of measured parameters using Pearson’s correlation test.The correlation and scatterplot matrix with the 28 leaf color mutants are shown in Fig.4.Gsperformed the strongest correlation withPn,with a correlation coefficient of 0.63(P<0.001).Chlbcontent and Chla/bratio showed a weak correlation withPn,with coefficients of 0.32 (P<0.1)and–0.31 (P<0.1),respectively.The correlation betweenCiandGs(0.60) was significant (P<0.001).Correlations between Chlaandb(0.56),Chlaand Chla/bratio (0.62),and Chlband Chla/bratio (0.99) were also significant.

Fig.4 Correlation and scatterplot matrix.The upper triangular matrix showed a Pearson correlation coefficient between each pair of the measured parameters.The larger font size indicated a stronger correlation.The lower triangular matrix showed the pairwise scatter plots between net photosynthetic rate (Pn),stomatal conductance (Gs),internal CO2 concentration (Ci),Chl a,Chl b,Chl a/b ratio,and total Chl content (CA).Each circle represented a mutant line;red line was the fitting curve of scatter plots;diagonal labels showed the parameters.＊＊＊,P-value<0.001;＊＊,P-value<0.01;,P-value<0.1.

To further explore the effect of different levels ofGsand Chlbcontent onPn,another two scatter plots were constructed in which mutants were grouped into two clusters by different levels ofGsand Chlbcontent,respectively.As shown in Fig.5-A,blue circles represented mutants with decreasedGsby comparing with Yugu 1.Greenblue plus-signs represented mutants with increasedGswhen compared with Yugu 1.Yugu 1 was denoted as a red triangle.It could be concluded that a clear positive linear relationship exists betweenPnandGswithin the mutants that behaved with higherGsthan Yugu 1.However,such a linear relationship did not exist among the mutants with decreasedGs.As shown in Fig.5-A,four mutant lines,t10,t11,t14,and t95,which behaved with significantly decreasedGscompared to Yugu 1,exhibited comparablePnperformance with Yugu 1.Remarkably,one mutant,t50,behaved with significantly decreasedGsbut increased Pn compared with Yugu 1.

Fig.5 Grouped scatter and box plots.A,scatter plot of net photosynthetic rate (Pn) vs.stomatal conductance (Gs).Blue represented mutants with decreased Gs by comparing with Yugu 1;greenblue “+” represented mutants with increased Gs by comparing with Yugu 1.Yugu 1 was denoted as a red “Δ”.B,scatter plot of Pn vs.Chl b.Blue represented mutants with decreased Chl b content by comparing with Yugu 1;greenblue “+” represented mutants with increased Chl b content by comparing with Yugu 1.Yugu 1 was denoted as a red “Δ”.

In fact,among the 23 mutants with significantly increasedPn,17 (73.9%) showed increasedGs(Fig.3-A–C);among the 23 mutants with increasedGs,17 (73.9%)had increasedPn(Fig.3-C).However,among the 12 mutants with significantly decreasedPn,only four (33.3%)had decreasedGs,five (41.7%) showed no significant change inGs,and the rest three (25%) had increasedGs(Fig.3-A–C).In addition,among the seven mutants with markedly decreasedPn(lower than 13 μmol CO2m?2s?1),only one (14.3%) exhibited decreasedGs,four (57.1%)had no change inGs,and two (28.6%) had increasedGs(Fig.3-A–C).These results suggested that,although increasedPnwas mostly attributable to increasedGs,the decreasedPnof most mutants was not attributable to decreasedGsin our study.

Fig.5-B presented a scatter plot in which mutants were grouped into decreased Chlbcontent cluster and increased Chlbcontent cluster by comparing that of Yugu 1.Pnof most of the mutants with decreased Chlbcontent was lower than that of the mutants with increased Chlbcontent.Interestingly,five mutants,t45,t50,t53,t104,and t105,had decreased Chlbcontent but performed significantly higherPnwhen compared with Yugu 1.As t45,t50,and t105 also exhibited variations inGs(Fig.2-B),it was hard to determine which factor led to the increasedPn.Thus,two mutants (t53 and t104) were finally identified to perform dramatically decreased Chlbcontent but significantly increasedPn(with no changes onGs).

4.1.Chl b is an active parameter for photosynthetic modulation

Chlbis synthesized from Chlathrough the activity of chlorophyllideaoxygenase (CAO),but increased CAO activity would not increase Chlbcontent at the expense of decreasing Chlacontent (Biswalet al.2012).In contrast,overexpression ofAtCAOin tobacco significantly increased not only Chlbcontent but also Chlacontent and other intermediates involved in Chl biosynthesis(Biswalet al.2012).Other studies in tobacco also showed that overexpression of some of the genes involved in the chlorophyll biosynthesis pathway co-modulated the expression of several other chlorophyll biosynthetic genes(Shalygoet al.2009).In the current study,the Chl content of the 28Setariamutants was characterized.And a total of 18,24,and 24 mutants behaved with variations in Chla,Chlb,and Chla/bratio,respectively.Chlaandbcontents were positively correlated,suggesting that there was also parallel regulation of Chlaandbbiosynthesis in monocot cereals,such asSetaria(Fig.4).It has been reported that Chl content,especially Chlbcontent,and the Chla/bratio are the key factors that determine the light-harvesting antenna size (Baileyet al.2001).The current study found a small change in Chlacontent and a more dramatic change in Chlbcontent among theSetariamutants.Moreover,as shown in Fig.4,weak correlations were found betweenPnand Chlbcontent,while such correlations were not found betweenPnand Chlacontent,suggesting that Chlbcontent might be a more responsive indicator than Chlato estimatePnperformance.

4.2.Decreased Chl content mostly leads to decreased Pn,with some exceptions

Generally,increased Chlbcontent represents an enlarged light-harvesting antenna size and thus promotes photosynthesis (Tanaka and Tanaka 2011;Jiaet al.2016).Previous studies reported that increased Chlbsynthesis led to an increased photosynthetic performance in tobacco andArabidopsis(Tanakaet al.2001;Biswalet al.2012).Hu (2009) also reported that there were significant correlations between Chl content and photosynthetic rate in recombinant inbred lines of rice in well-watered conditions (Huet al.2009).In the current study,we also found a weak correlation between Chlbcontent andPn(Fig.4).Pnof most mutants with decreased Chlbcontent(compared with Yugu 1) was lower than that of the mutants with increased Chlbcontent when compared with Yugu 1 (Fig.5-B).We speculate that the photosystem of these mutants might resemble those of thesistl1andsistl2mutants ofSetaria,which had defects in chloroplast development and chlorophyll biosynthesis (Zhanget al.2018;Tanget al.2019),thus leading to a significant decrease in photosynthesis.However,we also found two mutants (t53 and t104) that had decreased Chlbcontent and unchangedGsbut performed significantly higherPnwhen compared with Yugu 1.Previous reports on green algae,tobacco,and soybean showed the prospect of reducing Chl content to improve photosynthesis and yields (Kirst and Melis 2014;Kirstet al.2017).However,similar reports had not yet been seen in other crops.Our study thus provides new evidence that reduced Chl content could also promote photosynthesis in the C4cropSetaria.

Our study identified various mutants with modulated Chlbbiosynthesis andPn.Further investigations on them would ultimately elucidate the relationship between Chlbcontent and photosynthetic performance.Such research would also underpin efforts to modify Chl biosynthesis and the antenna size to improve photosynthesis inSetariaand other economically important cereals.

4.3.Promising future for elucidating the high photosynthetic efficiency and water-saving mechanism of C4 plants

Gsis one of the key factors that greatly affect photosynthesis.Generally,increasedGsstrongly correlate with higherPn(Wonget al.1979;Faralliet al.2019).In this study,a total of 35 mutants were found that had significant variations inPn,and we also found significant positive correlations betweenGsandPn.Interestingly,correlations between decreasedGsand decreasedPnwere found not as evident as the correlation between increasedGsand increasedPn(Fig.5-A),indicating that decreasedGswould not necessarily lead to decreasedPninSetaria.This might be becauseSetariais a C4plant and thus more tolerant to low CO2concentrations and capable of keeping relatively high rates ofPnunder restrictedGs.

Drought tolerance is one of the key factors that determine crop productivity (Nowickaet al.2018;Golecet al.2019).Generally,crops tend to reduceGsto reduce water loss under low soil water conditions,leading to lowerPnand depressed productivity (Gilbertet al.2011).Balancing photosynthetic performance whenGsis restricted is thus a desirable target when breeding drought-tolerant materials (Golecet al.2019).In this study,seven mutants (t50,t10,t11,t14,t20,t95,and t97) had decreasedGsbut not decreased photosynthetic performance,indicating that it is possible to develop water-savingSetariacultivars with no evident loss ofPnby balancingGsandPn.Our results indicate thatSetariais a valuable model crop for elucidating why C4species are generally drought-tolerant and water-saving (Diaoet al.2014),and the mutants characterized in this study provide fundamental materials for such research.

In addition to the influence ofGs,internal features including the Kranz anatomy and the CO2concentrating mechanism ofSetariamean it is a model with high photosynthetic efficiency (Li and Brutnell 2011;Diaoet al.2014).In this study,14 mutants did not exhibit significant differences inGscompared with Yugu 1.Five mutants (t64,t65,t67,t72,and t91) showed decreasedPnwhile another five mutants (t53,t57,t82,t87,and t104) had increasedPnperformance.These mutants,thus,might have variations in the Kranz anatomy and the CO2concentrating mechanism that led to differentPnperformances.In addition,six mutants (t30,t81,t93,t66,t68,and t69) had increasedGsbut not increasedPn,indicating that these mutants might have defects in the Kranz anatomy and CO2concentrating mechanism,thus leading to decreasedPndespite more CO2flowing into the leaf interior.

In summary,according to the photosynthetic parameters obtained by Li6400 measurements,17 mutants (t17,t23,t24,t26,t27,t28,t34,t39,t41,t42,t45,t54,t58,t103,t105,t114,and t118;Table 1,category I)behaved with increasedPnand increasedGs,and four mutants (t6,t8,t86,and t89;Table 1,category IX) had decreasedPnand decreasedGs.Eleven mutants had negative variations in photosynthetic characteristics.For example,mutants t30,t81,and t93 showed increasedGsbut unchangedPn(Table 1,category II);t66,t68,and t69 had increasedGsbut decreasedPn(Table 1,category III);t64,t65,t67,t72,and t91 had unchanged Gs but decreasedPn(Table 1,category VI).Five mutants had positive variations in photosynthesis,namely t53,t57,t82,t87,and t104,which had unchangedGsbut increasedPn(Table 1,category IV).Remarkably seven mutants behaved with highPnunder restrictedGsconditions.For example,t50 had decreasedGsbut increasedPn(Table 1,category VII) and t10,t11,t14,t20,t95,and t97 had decreasedGsbut unchangedPn(Table 1,category VIII).Further investigations on these mutants would be beneficial to elucidate the mechanism of high-efficiency photosynthesis in C4plants.

Table 1 Categories of the 48 mutants defined by photosynthetic parameters

4.4.Perspectives of Setaria as a model in photosynthesis research

Factors that strongly limit photosynthesis include the inability to efficiently use midday high light intensities as well as low-intensity light and inefficiency of the catalytic rateof the carboxylation enzyme Rubisco (Ortet al.2015).To date,numerous strategies have proposed modulating Chl biosynthesis to improve light-use efficiency,some of which have shown positive results in tobacco andArabidopsis(Biswalet al.2012;Kirstet al.2017;Ruban 2017).However,similar results in other crops have not yet been reported.As there appears to be little prospect of improving the kinetic and catalytic features of native Rubiscos,other strategies aimed at improving CO2uptake and conversion by increasing stomatal and mesophyll conductance and introducing highly efficient carboxylation systems,like the C4photosynthetic pathway,have been proposed for improving photosynthesis (Ortet al.2015).However,because of the complex relationship between Chl biosynthesis,antenna size,and light-use efficiency,as well as the complexity of carbon uptake and conversion mechanisms,many difficulties remain to be overcome in improving photosynthesis,and so further research is required.

Setariaitalica,a diploid C4panicoid species with small stature,a simple genome,high transformation efficiency,and excellent drought tolerance,has been a useful tool for functional gene mining and research (Diaoet al.2014;Huanget al.2014;Doustet al.2019;Nguyenet al.2020;Pegleret al.2020).As it uses the highly efficient C4mechanism of photosynthesis,it has been regarded as a useful model for C4photosynthesis research(Brutnellet al.2010).Screening for mutants inSetariawith a wider range of variation in different photosynthetic characteristics would add benefit to elucidating the mechanisms of C4photosynthesis and generating breeding materials with higher photosynthetic rate and water-use efficiency.

The current study identified abundant mutants in Setaria with multiple variations in chlorophyll content,stomatal conductance and net photosynthetic rate to provide materials for further investigation,ultimately aiming to establish a drought-tolerant and more efficient photosynthesis mechanism for major crops.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32241042 and 31771807),the National Key R&D Program of China (2021YFF1000103),the China Agricultural Research System (CARS-06-04),and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.We thank Dr.Huw Tyson,from Liwen Bianji,Edanz Group China (www.liwenbianji.cn/ac),for editing the English text of a draft of this manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.10.014

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