| Optimizing carbohydrate partitioning not only helps to improve crop yield,but also enhances crop adaptation to various abiotic stresses.Therefore,cloning carbohydrate partitioning related genes and elucidating their mechanisms are of great theoretical and practical value.In this study,chl3（chlorotic leaf3）,a leaf chlorosis mutant in maize caused by defective carbohydrate partitioning,was used as material.The causal gene ZmPHOH（Zea mays Starch Phosphorylase）underlying chl3 was isolated by BSR-Seq and map-based cloning,and the mechanism of ZmPHOH regulating maize carbohydrate partitioning was clarified.The main results are as follows:1.chl3 is a carbohydrate partitioning defective mutant.Under normal conditions,chl3in seedlings did not differ significantly from the wild-type,but its mature leaves began to develop chlorosis from the apical to the basal petals after the 5-leaf stage.Moreover,a short-term low temperature treatment at the seedling stage was able to induce the chl3chlorosis phenotype to appear before the 5-leaf stage.Further analysis showed that excessive starch was accumulated in chl3 bundle sheath chloroplasts,resulting in severely impaired chloroplast function,and the starch hyperaccumulation was due to abnormal transitory starch metabolism,suggesting that chl3 is defective in carbohydrate partitioning.2.Mutations in ZmPHOH resulted in the chl3 phenotype.The F2 segregating population was constructed by crossing chl3 with maize inbred line Mo17,and genetic analysis revealed that the leaf fading green trait of chl3 was controlled by a single recessive locus(χ2=1.32<χ02.05,1=3.84,P<0.05).Zmchl3 was mapped in an 820-kb interval on chromosome 3 by a BSR-Seq combined with a map-based cloning strategy.The gene in the interval,Zm00001d042842,encodes cytosolic-type starch phosphorylase harboring a 141‐bp insertion in chl3,which caused a shift mutation and premature termination.It was therefore used as a candidate gene for Zmchl3,named ZmPHOH.Both two allelic mutants of ZmPHOH had leaf chlorosis phenotypes,and these alleles cannot complement chl3,which confirmed that ZmPHOH is the mutated gene in chl3.3.ZmPHOH regulates carbohydrate partitioning and affects maize grain yield.The inactivation of ZmPHOH in chl3 leaves impaired maltose metabolism,which in turn reduced the efficiency of transitory starch degradation and ultimately led to increased leaf starch content and altered carbohydrate metabolic patterns.RNA-Seq further showed that the elevated carbohydrate content in chl3 leaves inhibited the expression of photosynthetic genes and reduced the carbohydrate partitioning efficiency.chl3 had significantly lower kernel weight,but the carbohydrate content in the kernels was not significantly different from that of the wild type（P>0.05）,and the association analysis result indicated that ZmPHOH was significantly correlated with 100-kernel weight.Taken together,these results suggest that ZmPHOH can regulate carbohydrate partitioning efficiency and affect maize grain yield.4.ZmPHOH affects the adaptation of maize to low temperatures.When chl3 seedlings were treated at 8°C for two days and then restored to normal temperature,it was found that the soluble sugar content in chl3 leaves was significantly increased after cold treatment and could not be catabolized at the end of the cold treatment,indicating that ZmPHOH may be involved in the catabolism of soluble sugar during post-cold recovery.Photosynthetic characterization showed that the damage caused by the cold treatment to the photosynthetic capacity of chl3 could not be repaired at the end of the cold treatment.RNA-Seq revealed that the excessive soluble sugar level during the post-cold recovery inhibited the expression of photosynthetic genes in chl3 young leaves,which not only led to the early appearance of chlorosis phenotype but also adversely affected the later growth and development.These results suggest that ZmPHOH affects the adaptation of maize to low temperature stress. |
