Map-based Cloning And Functional Study Of Rice Green-Revertible Yellow Mutant Gene Gry340

Chloroplasts are important organs for photosynthesis,which is connected with the photosynthesis and synthesis of photosynthetic pigments.Rice?Oryza sativa L.?is a model plant of monocotyledon in gene function research.The leaf-color mutants is a common phenotype of rice,it played an important part in the study of synthesis and degradation of photosynthetic pigments,development and structures of chloroplasts,mechanism of photosynthesis and photomorphism.Our group obtained a green-revertible yellow leaf mutant gry340 through ethyl methanesulfonate?EMS?mutagenesis.In this study,through phenotype characterization,map-based cloning,and gene functional analysis,ultrastructure of chloroplasts and chondriosomes,complementation analysis,subcellular loacalization,qRT-PCR analysis,the ispE ispF double mutant was constructed by crossing gry340 with 505ys mutant deeply studied,and the main results were as following.The gry340 mutant displayed a yellow leaf phenotype before the three-leaf stage,but gradually turned green with development,and recovered normal green after the nine-leaf stage.The days to heading of gry340 mutant was increased 9 days.At the mature period,its plant height,number of productive panicles per plant,number of spikelets per panicle,panicle length,seed-setting rate and 1000-grain weight significantly reduced with the wild type,respectively.To characterize the green-revertible yellow leaf phenotype of the gry340mutant,we measured its photosynthetic pigment contents at different stages.The result showed that the mutant had significant reductions of 67.6%,83.3%,71.7%and 52.1%in Chl a,Chl b,total Chls and carotenoids at the three-leaf stage,respectively,relative to the wild type.With plant development,leaves of gry340 gradually turned green and its photosynthetic pigment contents also gradually increased,and almost reached to the level of the wild-type at nine-leaf stage and booting stage.The gry340 mutant displayed a yellow leaf phenotype under the 23?and 30?,respectively.The gry340 mutant had significant reductions in Chl a,Chl b,total Chls and carotenoids under the 23?and 30?relative to the wild type.The above indicates that the photosynthetic pigment changes was the direct cause of the mutant phenotype in the gry340 mutant,and less affected by the temperature.We observed the ultrastructure of chloroplasts in yellow leaves of gry340 at the three-leaf stage using transmission electron microscopy.As a result,compared with the well-developed thylakoids in the wild-type chloroplasts,gry340 mutant chloroplasts had not obvious grana stacks,and lacked well-structured thylakoid membranes.In addition,we aslo observed the abnormality of mitochondria morphology,and rarefaction and vacuoles of mitochondrial structure in the mutant,compared with those in the wild type.After the gry340 mutant turn green,the chloroplasts had obvious grana stacks and well-structured thylakoid membranes,and the mitochondrial matrix was electron-dense,finely granular and relative homogenous.The genetic analysis suggested that the yellow leaf mutant phenotype of gry340 is controlled by a single recessive nuclear gene.Mapping of the gry340 locus was performed using F₂ population from the cross between gry340 and Minghui 63.Finally,the gry340locus was narrowed down to an 84 kb region between two InDel makers C4 and C5,respectively.16 putative genes had been annotated within the above 84-kb region in Rice Genome Annotation Project.The sequencing analysis showed that a single nucleotide C-to-T substitution occurred at position 2554 in LOC_Os01g58790?corresponding to position 575 in its cDNA?in gry340 mutant,which resulted in an amino acid change from Ala-192 to Val in the encoded protein.LOC_Os01g58790 gene encodes a putative GHMP kinases ATP-binding protein,belongs to IspE superfamily and is predicted as4-diphosphocytidyl-2-C-methyl-D-erythritol kinase.Therefore,LOC_Os01g58790 gene was identified as the candidate gene of gry340,and designated tentatively as OsIspE.OsIspE is a single-copy gene in rice genome.The sequencing results showed that total lengths of OsIspE genomic and cDNA sequences are 4834 bp and 1206 bp,respectively,and encodes a 401-amino acid protein with a molecular mass of approximately 43 kD.Phylogenetic analysis showed that OsIspE is more closely related to IspE proteins of barley,maize and sorghum than those of other species.This subcellular localization suggested that OsIspE protein is really localized in the chloroplast.The pCAMBIA2300-OsIspE plasmid containing full-length cDNA sequence of the wild-type OsIspE gene under control of the actin1 promoter was introduced into the gry340 mutant by Agrobacterium-mediated transformation.The complementation experiments showed normal green leaves as the wild-type leaves,and their photosynthetic pigment contents were almost restored to the wild-type level.These results suggested that a single base pair mutation in OsIspE gene is responsible for the mutant phenotype of gry340.To analyze the expression pattern of OsIspE gene at seedling stage and booting stage by qRT-PCR,respectively.The results showed that OsIspE was constitutively expressed in all tissues,including roots,stems,leaf blades,leaf sheaths and young panicles.However,leaf blades had the highest expression,followed by leaf sheaths,but stems,young panicles and roots had remarkably lower expressions.In addition,we examined the OsIspE expression in leaf blades at different stages?including the three-,six-,nine-leaf stages and booting stage?,and found that its expression levels in leaf blades remarkably increased with development,which peaked in the ninth leaf and only declined about a half in the flag leaf at booting stage.Subsequently,we further analyzed expression change of OsispE in the gry340 mutant during the process of turning green from yellow.The results showed that transcript level of OsispE in the mutant was significantly lower than that of OsIspE in the wild type at the three-leaf stage.Then,OsispE expression was gradually increased with development,and almost reached expression level of OsIspE in the wild type at the nine-leaf stage and booting stage.In this study,we produced an ispE ispF double mutant by crossing gry340 with 505ys plants.As a result,the ispE ispF double mutant plants displayed more severe yellow phenotype and grew slower than gry340 mutant,and finally they all died at the three-leaf stage in the F₂ population.The result suggested that simultaneous mutation of single amino acids in both IspE and IspF proteins in in the double mutant could completely abrogate the function of MEP pathway for isoprenoids biosynthesis.To analyze differences of gene expression between the double mutant and its wild-type Nipponbare,we examined expression levels of the related 30 genes by qRT-PCR,including seven MEP-pathway genes,six MVA-pathway genes,eight photosynthetic genes and nine mitochondrial genes.As a result,in the 21 genes for MEP-and MVP-pathways and photosynthesis,only psbA remained constant,ispE,HMGR1,HMGR2 and psbP were dramatically up-regulated,and other 16 genes were all significantly down-regulated.By contrast,in nine mitochondrial genes,cox2 and atp8 were significantly down-regulated and other seven genes were dramatically up-regulated.Meanwhile,we found that most of the related 30 genes in the gry340 mutant had similar expression with those in the ispE ispF double mutant,except ispE,MPDC1,psbP,cox2 and apt8.In a word,these data suggested that functional defect of a MEP-pathway gene not only could affect expressions of other MEP-pathway genes,but aslo could influence expressions of MVA-pathway genes,photosynthetic genes and mitochondrial genes.Previous studies showed that chloroplast development status may alter the expression of nuclear genes via retrograde regulation.In addition,chloroplasts and mitochondria are highly interdependent in many biochemical pathways.Thus,it is likely that retrograde signals derived from the impaired mutant chloroplasts and mitochondria may affect the expression of the mentioned-above genes.