Molecular Mechanism Of SD16 In Controlling Rice Plant Height

Semi-dwarfing of rice is an important agronomic trait closely related to photosynthetic efficiency and yield.It is significant to study in-depth and elucidate the molecular regulation mechanism of plant height for the construction of ideal plant architecture in rice breeding.Here we report a rice semi-dwarf mutant named semi-dwarf16（sd16）obtained by the gene knock-out via CRISPR/Cas9 system.The obtained mutants were studied using cytology,physiology,genetics and molecular biology methods.And the main results are as follows:（1）The semi-dwarf of sd16 is caused by the first exon of SD16 was edited by the CRISPR/Cas9 system and an insertion mutation occurred,resulting in loss of gene function.（2）According to the heterozygous mutants showed normal plant heights,and it’s self-pollinated progeny produced a 3:1 segregation ratio of normal plant height to semi-dwarf,demonstrating that SD16 is a dominant gene.（3）The plant height of sd16 is between dwarf and normal plants,so it is classified as semi-dwarf.sd16 also has variations on other agronomic traits,such as with increased number of tillers,shortened and narrowed leaves,shorter panicle length,reduced grains number,decreased seed-setting rate,reduced tgrain weight,reduced germination rate.The shortening of each internode is the reason for the semi-drawfing of sd16.（4）Paraffin sections of the second internode of wild type and sd16 were observed.The result of transverse section shows that the medullary cavity is larger than that of wild type,and the number of parenchyma cells is less in sd16.The result of longitudinal section shows that the decrease in cell number is the decisive cause of the shortening of the internode of sd16.（5）The result of real-time RT-PCR shows that SD16 has higher expression in stem and root.（6）Two transient expression vectors P35S::SD16-eGFPN and P35S::SD16-eGFPC were constructed and co-transformed with nuclear-localized P35S::TDR-RFP vector into protoplasts of rice.The result shows that SD16 is a nuclear protein.（7）Under dark culture conditions,both wild type and sd16 seedlings grew mesocotyls,indicating that sd16 is not a BR-associated mutant.The transcriptome analysis of seedlings between sd16 and wild type found that the difference in gene expression of the diterpene biosynthetic pathway was most obvious.In this pathway,it was found that part of the metabolic enzymes of GGPP and ent-CDP（a raw material for synthetic GA）are up-regulated in sd16,and oxidases that degrade bioactive GA are upregulated in mutants,indicating that the semi-dwarfing of sd16 is closely relate to the GA synthesis pathway.（8）Differences of the contents of 17 kinds of GAs were detected in seedlings between sd16 and wild type.SD16 affects the early 13-hydroxylation pathway and the contents of bioactive GA₁ and GA₃ were lower in sd16 than in the wild type.By analyzing the relative contents of various types of GAs in the early 13-hydroxylation pathway,it was found that the relative content of GA₂₀ in sd16 was increased,while the relative content of GA₁decreased significantly.It was speculated that there was a blocking site in the step of GA₂₀synthesis to GA₁.Starting from the synthesis of the first GA(GA₁₂),sd16 has a lower GA content than the wild type.（9）SD16 is predicted to encode an E3 ubiquitin ligase.Phylogenetic tree analysis shows that SD16 may have a conserved function in the grass family.Bioinformatics analysis of the promoter region of SD16 shows that SD16 may be regulated by light signal,photoperiod,abiotic stress（low temperature,drought,hypoxia）and hormone signal（ABA,GA,MeJA）,and in certain tissues（mesenchyme,endosperm,seed）has specificity in expression.In summary,the semi-dwarf phenotype of sd16 is presumed to be due to the loss of a nuclear E3 ubiquitin ligase in wild type plants,affecting the GA biosynthetic pathway directly or indirectly,leading to a decrease in the synthesis of bioactive GAs and extending cell division cycle,reducing the cell numbers in various tissues and organs of the plants and eventually leading to a semi-dwarf phenotype.In this study,we have studied a new molecular mechanism that controls the plant height of rice through a variety of experimental methods,laying the foundation for finding the unknown genes regulated by SD16 and elucidating the mechanism of plant height regulation.