Molecular Mechanism Of Photoperiod-Thermo-Sensitive Dwarf 1 In Controlling Rice Plant Height

Suitable plant height contributes greatly to rice grain yield.It is significant to elucidate the molecular mechanism controlling plant height for the construction of ideal plant architecture in rice breeding.Here we report a dominant rice dwarf mutant named Photoperiod-Thermo-sensitive Dwarf 1?PTD1?,which arose from somaclonal variation.Compared with the wild type gene ptd1,PTD1 was previously found to have a base pair deletion and a base pair substitution,which caused frameshift in the C-terminal of the coded protein and thus the PTD1 dwarf.ptd1 was predicted to encode a non-specific Lipid Transfer Protein?nsLTP?.The members of nsLTPs family are characterized with a conserved Eight Cysteine?Cys?Motif?8 CM?.The last two Cys of the 8 CM motif were lost due to the frameshift in PTD1 C terminal.In this study,the responses of PTD1 to photoperiod and temperature were detected by Quantitative RT-PCR and Western Blotting analyses;A gene editing method based on CRISPR/Cas9 system was also used to reveal the function of ptd1/PTD1;Furthermore,to uncover the molecular mechanism underlying PTD1,the effects of protein structure alterations on plant height were studied using transgenic plants with ptd1 carrying modified C-terminal and plants with ptd1 bearing site-directed mutagenesis of conserved Cys in the 8 CM.The main results are as follows:1.The ptd1 CRISPR/Cas9 knock-out plants showed a wild type phenotype,indicating that ptd1 is not a necessary gene for rice plant height development.The PTD1 knock-out plants recovered a normal plant height,reconfirming that PTD1 is the causal gene and functions in a protein level for the dwarfism.2.The results of treatments of PTD1 under different combinations of temperature and photoperiod showed that temperature is a major factor and its effect is greater than that of photoperiod on controlling PTD1 plant height.3.The analyses of PTD1 treated under different combinations of temperature and photoperiod also revealed that the expression of PTD1 was affected by temperature and photoperiod.PTD1 showed severe dwarf phenotype in long day and low temperature conditions,consistent with the highest expression levels of mRNA and protein of PTD1;while the plant height was almost completely restored in short day and high temperature conditions corresponding to the lowest expression levels of mRNA and protein of PTD1.4.A site-directed mutagenesis in Cys56 and Cys76 of ptd1(forming the 3^rd and 4^th disulfide bonds)caused the transgenic plants a mimic dwarf phenotype,demonstrating that loss of the two disulfide bonds in ptd1 is crucial for the dwarfism.In contrast,the transgenic plants with a site-directed mutagenesis in Cys31 and Cys41 of ptd1(forming the 1^st and 2ⁿdd disulfide bonds)showed wild type phenotype,indicating that loss of the 1^stt and 2^nd disulfide bonds in ptd1 cannot lead to dwarfism.5.Based on the analysis of a series of transgenic plants with modified C-terminal of ptd1/PTD1,it was revealed that the alteration of C-terminal structure has serious impact on dwarfism.In summary,the prerequisites for gain-of-function of PTD1 in inhibiting cell division and elongation are:loss of the 3^rd and 4^thh disulfide bonds in the 8 CM of ptd1 combined with an appropriate length of C-terminal.This study revealed a novel molecular mechanism for regulating plant height,as well as provided new genetic resources for molecular breeding in rice.