| Rice is one of the most important crops in the world. As the world population is continually increasing whereas the farmland is rapidly decreasing, there is an urgent need to increase grain yield. Plant height is crucial for grain yield. In the1960s and1970s, wide application of rice varieties with semi-dwarf phenotype substantially increased grain yield, which was called’green revolution’of rice. Dwarf mutants are ideal materials for studying of the mechanism of dwarfism in rice. Up to date, dozens of dwarf mutants have been reported in rice, however, most of which are semi-dwarfs and studies on the genetic and molecular mechanism of severe dwarfs are rare. In this study, we isolated and characterized a severe dwarf mutants of rice, chromosome segment deleted dwarf1(csddl). Map-based cloning and functional analysis of the csddl mutant leaded to the understanding of genetic and molecular mechanism that caused the severe dwarf phenotype of csddl. We also studied the csddl in physiology and cell morphology. These results promoted our knowledge of the molecular mechanisms of dwarfism in rice, and should be useful for application of rice dwarfs in breeding and production.The csddl was isolated from the progeny of kitaake, a japanica cultivar, with tissue culture-induced mutation. The csddl showed severe dwarfism with erect and twisted dark-green leaves, unelongated internodes and sterility. Light microscope observation revealed that cells in the unelongated internodes of csddl failed to elongate and form well-organized cell files along the longitudinal axis, which resulted in the unelongated internodes of csddl. Compared with wild type culms, the stems of csddl were wrapped with all the leaf sheathes. Cross sections of wild-type and csddl internodes showed that csddl had much more layers of cells compared with wild type. These results could explain thicker culms of csddl than wild-type plants. Cross sections of csddl blades showed that csddl had structural defects in the midrib; and bulliform cells in the csddl blades were fewer and smaller compared with that of wild type. The structural defects of midrib and bulliform cells may cause the twisted morphology of the csddl leaves. Pigments content and electron microscope analysis showed that the dark-green leaves of csddl plants are caused by increased content of chlorophyll, morphological and structural changes in chloroplasts.Genetic analysis revealed that csddl mutant phenotype is controlled by a single recessive gene. BC2F2mapping population is constructed by crossing csddl/+with an indica variety,9311. The csddl locus was mapped to a219-kb interval on the short arm of chromosome1with2332BC2F2mutant individuals. We further found that a168-kb chromosome segment was deleted in csddl within the mapping region. Twenty three putative genes were located in the deleted segment predicted by rice data base. Expression analysis of the twenty three genes by RT-PCR revealed that fifteen genes were expressed in wild-type plants, which suggests that candidate gene of csddl may be in the expressed genes.Further analysis of the fifteen expressed genes leaded to the identification of a candidate gene of csddl, CYP90D2/D2, which encodes a brassinosteroid (BR) biosynthetic enzyme. BR are a group of plant steroids which promotes cell division and elongation, and regulates plant height, suggesting CYP90D2/D2could be the responsible gene of csddl. We screened various rice mutant collections, and isolated two T-DNA insertion lines of D2gene, which displayed severe dwarfism and resembled csddl strikingly. In the darkness, these mutants showed a typical’de-etiolated phenotype’of BR mutants, which suggests that they are BR-related mutants. BR treatment rescued the severe dwarf phenotype of csddl, which demonstrates that the csddl mutant is defective in BR biosynthesis. Co-segregation analysis of T2mutant plants revealed that the T-DNA insert co-segregated with the severe dwarf phenotype in the two T-DNA lines. Expression analysis by RT-PCR revealed that the D2gene was not expressed in csddl and the two T-DNA mutants, suggesting that they are null alleles of D2gene. So, we suspected that D2is the candidate gene of csddl.Introduction of wild-type D2gene into csddl rescued the severe dwarf phenotype of csddl. Overexpression of D2gene in csddl also rescued the severe dwarf phenotype of csddl. However, transgenic plants transformed with six other candidate genes in the deleted segment of csddl did not rescue the csddl phenotype. We also performed RNAi analysis of D2gene in wild type. Some T0D2-RNAi plants displayed severe dwarfism, and expression of D2gne was not detected in these severe dwarf plants. These results confirmed that the severe phenotype of csddl was caused by the deletion of the D2gene.Analysis of phylogenetic relationships among CYP90D2/D2and related BR-biosynthetic cytochrome P450proteins (CYPs) showed that CYP90D2/D2gene belongs to the CYP90D gene subfamily. Expression pattern analysis revealed that D2gene preferentially expresses in the tissues in which cells are actively dividing and elongating, which is consistent with the function of BR that regulate cell division and elongation. Expression of D2was promoted by darkness and suppressed by light and BR treatment, suggesting that expression of D2gene is negatively regulated by light and controlled by a feedback mechanism of BR. Expressions of several BR signaling and BR-responsive genes were decreased in csddl and T-DNA mutants. These results may be caused by deficiency of endogenetic BR and decreased BR response in these mutants.The d2-1and d2-2are two mild dwarfs with single base mutation in D2gene. The d2-l allele showed unelongated second internode, and d2-2allele showed partly shortened second internode. The csddl, and T-DNA lines, d2-3and d2-4, are null alleles of D2gene. They showed severe dwarfism and resembled each other strikingly in different genetic background, and csddl allele in T65or9311background still showed severe dwarfism. These results demonstrated that the phenotypic differences between severe dwarfs (csddl, d2-3and d2-4) and mild dwarfs (d2-l and d2-2) in d2alleles are not caused by the differences of genetic backgrounds but by the different levels of severity of lesions in the D2gene. Allelism test between csddl and d2-1revealed that d2-1is dominant to csddl. RNAi analysis of D2gene in d2-1showed that some To RNAi plants displayed severe dwarfism and transcripts of D2was significantly decreased or not detectable in such plants, which reveales that d2-1is not a null allele. These results donmonstrate that the mutant D2gene in d2-1still retains some functions, which results in the mild dwarf phenotypes of d2-1. |
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