Leaf color and photosynthesis are important factors for rice growth and development.Hence,improving the photosynthetic rate is an effective approach for increasing rice yield.We isolated a gene,chlorophyllide-a oxygenase 1(OsCAO1),which characterized a rice near-isogenic line namedfgl(faded green leaf).Compared with its recurrent parent,Zhefu 802(ZF802,an early-seasonindicarice variety),fglhad a faded green leaf color and lower chlorophyll (Chl) content,especially Chl b content.Furthermore,genetic and physiological analysis confirmed that leaf color was affected by the novel alleleOsCAO1,which had a two-base pair deletion in the ninth exon.Subsequently,we over-expressed theOsCAO1gene in ZF802.Interestingly,whileOsCAO1over-expression (OX) increased Chl b synthesis,the OX plants had a higher photosynthetic rate and heavier 1000-grain weight than ZF802.In conclusion,the novel alleleOsCAO1was involved in the positive regulation of leaf color,photosynthetic rate and rice yield.

Approximately 90% of crop biomass comes from photosynthetic products (Makino,2011).Similarly,more than 2/3 of the dry matter in rice grains is obtained by photosynthesis (Wang et al,2008).Thus,improving photosynthesis is an effective strategy for rice productivity.Rice photosynthetic rate is affected by multiple factors,including light intensity (Gu et al,2017),rubisco enzyme (Perdomo et al,2017),chlorophyll content (Tanaka et al,2022),stomatal conductance (Fu and Wang,1994),and CO2concentration (Takai et al,2013).Chl is an essential component of photosynthetic apparatuses (light-harvesting complexes,LHCs) and plays a critical role in rice photosynthesis (Fromme et al,2003;Jung et al,2021;Si et al,2022).Alternatively,leaf is the main tissue responsible for photosynthesis and a cardinal source organ of rice.Changes in rice leaf color can indirectly influence rice yield to a certain extent (Zhang et al,2021).Therefore,leaf color is closely related to its photosynthetic assimilation abilities.A plant’s leaf color is determined by the leaf’s content,proportion,and type of pigments.Specifically,rice leaves’ two main pigment types are Chl and carotenoids.Chl consists of Chl a and Chl b,whereas Chl b is synthesized from Chl a by chlorophyllide-a oxygenase (CAO).Therefore,the expression ofCAOregulates the synthesis of Chl b and the ratio of Chl a to Chl b with different light intensities through the Chl cycle (Tanaka and Tanaka,2005).Furthermore,Biswal et al (2012) have reported that over-expressing full-lengthAtCAOinto tobacco improved Chl content and the ratio of Chl a to Chl b,changing and finally enhancing carbon dioxide assimilation,starch content and dry matter accumulation,especially in high-light growing conditions.

The near-isogenic line,fgl,was bred from the backcross of a leaf color mutant and ZF802 by 10 generations (Dong et al,1994;Zeng et al,2003).The leaf colors of ZF802 andfglwere different (Fig.1-A).Furthermore,Chl content analysis indicated that the contents of Chl a and Chl b infglwere significantly lower than those in ZF802,especially for Chl b(Fig.1-B1 and -B2).Therefore,the total Chl content offglwas much less than that of ZF802 (Fig.1-B3).However,the ratio of Chl a to Chl b offglwas nearly 4.7 times higher than that of ZF802 (Fig.1-B4).The difference of Chl content was closely related to the development and structural changes in rice leaf chloroplast (Zhou et al,2017;Yu et al,2019).Additionally,we further compared the chloroplast ultrastructure using transmission electron microscopy to reveal the difference in chloroplast development between the two genotypes.Although both showed clear chloroplast boundaries and intact external structures,the morphologies of the grana thylakoid and stroma lamella inside the chloroplast offglwere curled more significantly than those of ZF802,and the number of starch granules offglhad markedly increased in comparison with that of ZF802 (Fig.S1).These data indicated that a tremendous difference in chlorophyll content existed betweenfgland ZF802,related to the abnormal development of the chloroplast.An elevated photosynthetic rate is one proposed approach to boost intrinsic yield (Gibson et al,2011).Wang et al (2008)reported that the senescence offglwas slower than that of ZF802,and the decreasing rates of chlorophyll content,leaf area index and photosynthetic rate offglwere slower than those of ZF802 under natural light intensity.To further determine whetherfglhad better yield traits,we measured the yield-related agronomic traits between ZF802 andfgl.Results showed that the plant height,1000-grain weight,and grain weight per plant had significantly increased infglcompared to ZF802,and there was no significant difference between ZF802 andfglin the number of grains per panicle and seed-setting rate (Fig.1-C to-H).

We isolated the gene affecting thefglphenotype.First,map-based cloning was performed using a segregated population of 1 682 F2plants generated from a cross between YT17 (ajaponicavariety) andfgl,among which 1 279 plants exhibited normal phenotype,and 403 plants exhibitedfglphenotype.Then,the corresponding gene-regulating leaf color fromfglwas mapped to an interval between markers RM3451 and RM4771 on chromosome 10.We further fine-mapped the locus of the candidate region to a 52.6-kb interval between InDel markers ID2 and ID3,containing 11 open reading frames (ORFs) (Fig.1-I).Genomic DNA sequencing revealed a two-base-pair (GA,residues 2 973-2 974) deletion at the ninth exon ofLOC_Os10g41780infglcompared with ZF802.The deletion resulted in two amino acid substitutions at 522-523 (DA to RH)and a non-synonymous change at 524 of isoleucine (I) to a stop code,leading to the premature termination of translation (Fig.1-I and -J).Therefore,FGLis a novel allele ofOsCAO1.Subsequently,we identified and confirmedOsCAO1’s function by constructing an over-expression vector and introducedOsCAO1into the ZF802 background to verify whether theOsCAO1was responsible forfglcharacterization.Two OX lines (OX-1 and OX-2),with higherOsCAO1expression levels,were selected (Fig.1-K and -L).The total Chl contents differed slightly between OX plants and ZF802 (Fig.1-M1),with the Chl b contents of OXs increased and the ratio of Chl a to b decreased compared with those in ZF802 (Fig.1-M2 and -M3).Furthermore,OX plants showed higher net photosynthetic rate and stomatal conductance than ZF802 (Fig.1-N and -O).

Feng et al (2020) indicated that strengthening the antioxidant capacity could remove hydrogen peroxide (H2O2) and alleviate the inhibition of stomatal conductance by abscisic acid (ABA),enhancing leaf photosynthesis.Next,we determined the mechanism responsible for higher photosynthetic rate in OX plants by measuring H2O2and ABA contents,and antioxidant capacities [catalase (CAT) and peroxidase (POD)].Results showed that OX plants had higher CAT and POD activities than ZF802,while ABA and H2O2contents markedly decreased(Fig.1-P to -S).All data measurements for experimental materials fit the growth trends,showing that the function ofOsCAO1affected photosynthetic rate.LHCs in photosystem II regulate glutathione content in the guard cells,and increase the sensitivity to ABA,resulting in stomatal closure (Jahan et al,2016).Hence,we propose thatOsCAO1affects LHCs in photosystem II in response to the ABA content in plants,thereby affecting its photosynthesis.

Dry matter accumulation increased inCAOOX plants(Biswal et al,2012).We measured the yield-related agronomic trait to investigate whetherOsCAO1over-expression increases photosynthesis,including whether this increase could benefit the grain yield.Investigations showed that plant heights in OX plants were higher than that in ZF802 (Fig.1-K and -T).Furthermore,OX plants had the maximum number of tillers.(Fig.1-U).We also found that the panicle length of ZF802 was shorter than that of OX (Fig.1-V),but there was no significant difference in the number of grains per panicle (Fig.1-W).Moreover,the 1000-grain weight and grain weight per plant of ZF802 were significantly lower than those of OX plants (Fig.1-X and -Y),and compared to ZF802,OX plants had a much lower seed-setting rate (Fig.1-Z).Due to the obvious difference in grains weight,we further explored the variety of grain phenotypes (Fig.S2).Grain length and thickness were markedly increased in OX plants compared with ZF802,but grain width was unvaried.Furthermore,the ratio of length to width of all the three genotypes was different.Therefore,we could infer that the higher expression ofOsCAO1was responsible for agronomic traits’ improvement.

A transient expression system was constructed with rice protoplasts isolated from young rice plant stalks to locate the OsCAO1.Subcellular localization was observed by the fusion protein signal,OsCAO1-GFP (green fluorescent protein),localized to chloroplast.However,the signal of free GFP distributed in the cytoplasm was not located in the chloroplast as in a control (Fig.S3).Therefore,to further determine the region of signal target,including a rough estimate of the position and length of the signal protein,we constructed and transformed four GFP-fused expression vectors: OsCAO11-30-GFP,OsCAO11-60-GFP,OsCAO130-541-GFP and OsCAO160-541-GFP,respectively.The results showed that only the fluorescence signals of OsCAO11-30and OsCAO160-541were undetected in the chloroplast,while the GFP fluorescence signals of the other fusions at the 30th-60th amino acid sequences of N-terminal were detected in the chloroplast (Fig.S3).The 30th-60th amino acids in the N-terminal,as the localization signal of OsCAO1,are thus necessary for OsCAO1to target the chloroplast.

To dissect the new haplotype ofOsCAO1,we further analyzed the coding sequences ofLOC_Os10g41780in 147 cultivated rice varieties (Wu et al,2021).Investigations showed that Zhaiyeqing 8,Zhenguiai 1 and Qingguiai 5 had 14 single bases replaced in the ninth exon,only two amino acids exchanged for the other amino acids by sequence alignment(Fig.S4-A).Subsequently,we measured their photosynthesis characteristics and showed that the stomatal conductance of the three varieties were significantly higher than that of the control(Guichao 2,a conventional rice variety which had been promoted more than 6.67 × 106hm2in China).Although the photosynthesis rates were increased,the increase was insignificantly (Fig.S4-B and -C).Therefore,we inferred that this change in the ninth exon ofOsCAO1played a positive role in photosynthetic rate.

This study isolated a novel allele,OsCAO1,which has variations in the ninth exon ofLOC_Os10g41780(Fig.1-I and-J),from the near-isogenic line,fgl,with lower Chl b content and better agronomic traits infgl(Fig.1-B to -H).Over-expressedOsCAO1in ZF802 background showed an increased photosynthesis rate.According to the literature,the over-expression ofCAOresults in increased Chl b content(Tanaka et al,2001;Tanaka and Tanaka,2005).Biswal et al(2012) over-expressed full-lengthCAOfromArabidopsisin tobacco,resulting in an increased Chl b synthesis and a decreased of ratio between Chl a and Chl b.These results suggest that the up-regulation of Chl b biosynthesis can increase the electron transport rates and enhance carbon dioxide assimilation,starch content,and dry matter accumulation (Biswal et al,2012).Lee et al (2005) reported two homologousCAOgenes in the rice genome,and suggested that onlyOsCAO1play a major role in Chl b biosynthesis,whileOsCAO2function in the dark.Hence,in this study,we over-expressedOsCAO1,and then transformed it into ZF802,and found that compared with ZF802,OX plants had higher Chl b content and lower ratio of Chl a to Chl b.In addition,the main agronomic traits of OX plants were also improved (Fig.1-T to -Y).Consequently,we deduced that over-expression ofOsCAO1in rice played the same role as the influence by over-expressingCAOin tobacco.

ACKNOWLEDGEMENTS

This study was supported by the Shenzhen Science and Technology Program,China (Grant No.KQTD2016113010482651),Natural Science Foundation of Zhejiang Province in China (Grant No.LR20C130001),and Hainan Yazhou Bay Seed Laboratory,China(Grant No.B21HJ0219).We thank Professor TIAN Zhixi (Institute of Genetics and Developmental Biology,Chinese Academy of Sciences) for helping to analyze genome sequencing,and Dr.FU Guanfu (China National Rice Research Institute) for supporting the measurement of photosynthetic parameters.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science;http://www.ricescience.org.

File S1.Methods.

Fig.S1.Transmission electron microscopy of chloroplasts in Zhefu 802 andfglleaves.

Fig.S2.Grain characterization of ZF802 and over-expressing lines.

Fig.S3.Subcellular localization of OsCAO1 and truncated OsCAO1s in rice protoplasts.

Fig.S4.Identification and characterization of new haplotype in varieties.