The Discovery And Functional Analysis Of A Chlorophyll A Oxygenase 1 Gene ZmCAO1 Contributes To Chlorophyll B Biosynthesis In Maize

The leaves are one of important tissues for photosynthesis in crops.The photosynthetic products are involved in crop yields.Therefore,the identification and isolation of genes associated with photosynthesis is necessary to improve crop yield.In this study,we discovered a yellow-green leaf?ygl?mutant.The YGL gene was isolated from ygl mutant.The main research results as follows:1. Mutant ygl displayed decreased yield.We found a ygl mutant?#222?in the B73inbred line in a field.Its leaf color gradually changed from yellow to yellow green,instead of green,during development;the yellow-green leaf phenotype persisted during many years of observation.In growth chamber,ygl seedlings also exhibited yellow-green leaves under a 16 h light/8 h dark condition.The growth period of ygl was134.20±0.98 d,while 144.90±1.45 d in the control.The growth period in ygl was delayed about 10 d.compared to that of the control.In addition,several traits in ygl,such as the plant height,ear height,100-grain weight,ear length,ear thickness,grain length,grain width,grain thickness,and yield per ear all,were significantly reduced compared to those of the control.Our results indicates that the development of ygl may be stunted.2. The content of chlorophyll b in mutant ygl is reduced.Compared to the wild-type control,the photosynthetic rate,stomatal conductance,intercellular CO₂concentration and transpiration rate in ygl were significantly reduced at the seedling stage,jointing stage and heading stage.The total chlorophyll content,chlorophyll a,chlorophyll b,and carotenoid content in ygl leaves were also dramatically reduced.Interesting,in the jointing stage,the content of chlorophyll b in ygl was reduced more than 10 times,as well as over 4 times at the heading stage.By contrast,the content of ALA and Pchlide were significantly increased in ygl leaves.SDS-PAGE results showed that the abundance of light-harvesting pigment proteins?LHC?and LHC??in in ygl was significantly reduced compared to that of the control.3. Fine mapping and isolation of the YGL gene.In a Chang7-2×ygl F₂population,341 plants showed green leaves,and yellow-green leaves was 116.The rato of green leaf plants to yellow leaf plants was 3:1?X²<3.84?,which indicated that the yellow-green leaves in ygl mutants were caused by a recessive mutations of single genes.The BSA was carried out,and the YGL gene mapped into 17.18-Mb region on the long arm of chromosome 8.In mapped region,19 SNP markers were selected,for fine mapping of the YGL gene.The YGL gene waslocated into the region between 161.51 to162.56-Mb in Chr.8.In this region,there are 51 known genes,and 5 of 51 genes were involved in leaf color and chloroplast development.PCR was performed to obtain about1.5-Kb promoter and full-length CDS sequence of five genes.The results showed that a51-bp insertion in the fourth exon of GRMZM2G038487 in ygl,while not in the control.Sequence analysis showed that 51-bp insertion was the Popin transposon.the rest four genes,no sequence difference was found between Chang7-2 and mutant ygl.GRMZM2G038487 encodes a chlorophyll a oxygenase,and was selected as a candidate gene.4. The mechanism of the zmcao1 mutation leading to a decrease in yield.RNA-seq results showed that the expression levels of 50 photosynthesis-related genes were reduced in ygl,of which 9 Ribosomal protein or Ribosomal subunit protein?RPS??AY109733,bnl3.04,IDP1437b,IDP245,uaz251b,RPS10,RPS11,RPS12a,RPS13?,3Rubisco Activase?RCA??RCA1,RCA2,RCA3?,2 Sucrase Phosphate Synthase?SPS??SPS1,SPS2?genes were down-regulated in ygl.In addition,17 Golden2-like?GLK?genes were also down-regulated in ygl.56 genes related to seed development were down-regulated in ygl,and expression levels of 6 Empty Pericarp?EMP?genes and 6Basal Endosperm Transfer Layer?BETL?decreased in ygl.The expression levels of key genes of endosperm development EMP11 and EMP12 in ygl were significantly reduced.5. The ygl mutant was sensitive to waterlogging.Upon 2 days after waterlogging treatment,the leaves of ygl began to wilt,while not in the control.After 6 days post-treatment,ygl mutant died.Therefore,ygl mutant was sensitive to the waterlogging stress.Under waterlogging,the contents of O₂^-,and MDA in ygl leaves were significantly higher than the control.In addition,the enzyme activities in the leaves,such as SOD,and CAT were similar between ygl mutant and the control.6. Molecular mechanism of zmcao1 mutation leading to decreased stain resistance.RNA-seq results showed that the expression levels of 11 ERF,30 MYB and 23 NAC transcription factors in ygl were reduced,but the reduction level was not twice.In contrast,the expression levels of 54 WRKY genes decreased,and 18 of them decreased more than twice.In addition,24 MAPK genes in ygl were down-regulated,and MAPK is an upstream regulator of WRKY.These results indicate that zmcao1 causes ygl to fail to induce the MAPK cascade response,and activates the downstream WRKY gene in response to waterlogging stress,resulting in a reduced ability to withstand staining.