Study On The Mechanism Of SD1 Regulating Tomato Stem Development

Stems play important roles in the growth and development of plants,e.g.stems can not only support plant architecture,but also act as a channel for nutrient transport,and stems can even serve as vegetative explants for asexual reproduction.In Arabidopsis thaliana and Populus,shoot apical meristem and secondary growth have been studied intensively.Tomato（Solanum lycopersicum）is an important model plant in horticultural study.At present,there are few studies on its stem development.Therefore,it is of great importance to deeply explore the molecular mechanism of tomato stem development.In this study,the tomato stem diameter gene SD1 was identified previously from GWAS.The molecular mechanism of SD1 regulating tomato stem development was explored by functional characterization in transgenic plants,gibberellin response,tissue specific expression and regulatory elements analysis.The main findings of this study are as follows:1.According to the SNP and InDel variations of 360 tomato accessions,SD1 was classified into 4 categories and 8 haplotypes.During the process of domestication and improvement from PIM（Solanum pimpinellifolium）to CER（Solanum lycopersicum var cerasiforme）and to BIG（Solanum lycopersicum）,SD1 was evolved from haplotypes V and VI to the other six haplotypes,accompanied by an increase in stem diameter.The expression analysis of SD1 in stem diameter extreme accessions showed that SD1 expression in extreme accessions with thick stem were higher than those with thin stem,indicating that SD1 expression could affect stem diameter.2.When SD1 expression was suppressed in AC by RNA interfering and gDNA of SD1 from thick stem genotype was overexpressed in LA1589,SD1 expression in RNAi lines were significantly lower than AC and the stem diameter were significantly less than AC;SD1 expression in overexpression lines were slightly higher than LA1589 and the stem diameter were significantly larger than LA1589,indicating that SD1 could positively regulate stem diameter.In addition,SD1 exhibited impact on fruit transverse diameter,vertical diameter and single fruit weight.When overexpressing gDNA of SD1 from thick stem genotype as well as thin stem genotype in TS18,SD1 expression in both kinds of overexpression lines were slightly higher than TS18 and the stem diameter were significantly larger than TS18.It was concluded that SD1 from both thick stem genotype(SD1^TK)and thin stem genotype(SD1^TN)could regulate stem diameter.3.The promoter of SD1^TK harbored an additional gibberellin responsive cis-element due to the 11 bp deletion.After 100μM GA₃ treatment on SD1 RNAi and overexpression plants,SD1^TK and SD1^TN showed different gibberellin response in range and frequency.4.In SD1 RNAi and overexpression lines,expression of genes in gibberellin signal transduction pathway were analyzed,and results showed that SD1 could affect the expression of some genes in this pathway such as RGL1-1、RGL1-2 and GAI,their expression were all up-regulated in RNAi lines,but the mechanism remains elusive.The expression analysis of genes in gibberellin biosynthesis pathway showed that SD1 did not affect the expression of genes in this pathway.The expression analysis of genes related to stem growth and development showed that SD1 could affect the expression of some genes in shoot apical meristem and secondary growth activities.The expression of WUS was down-regulated in overexpression lines and the expression of WOX4 as well as KNAT2 were both up-regulated in RNAi lines,but the mechanism remains elusive.5.When staining flowers,young fruits,green and red ripe fruits of SD1Pro^TK::GUS and Pro^TN::GUS transgenic plants,it was found that SD1 was primarily expressed in anthers and seeds.In situ hybridization of the cross section of LA1589mature stem indicated that SD1 was mainly expressed in the cambium,with less expression in phloem and xylem.6.Yeast one hybrid experiments showed that PARB could bind to the 11 bp InDel（200 bp）region of SD1^TK promoter with weak binding capacity;RR6 could bind to SD1^TK promoter（2 kb）.