Study On The Functions Of RALF Signal In The Regulation Of Growth And Cold Tolerance Of Tomato

Peptide signals play an important role in cell-to-cell communication,and many peptides are involved in various growth and development processes as well as environmental responses.Rapid alkalinization factor(RALF)is a class of plant polypeptide families.RALFs are involved in a variety of physiological processes,such as root elongation,plant-microbe interactions,pollen development,pollination and fertilization.The number and function of RALFs among different species are diverse,while the function of the RALF gene in tomato(Solanum lycopersicum)is unclear.At present,the research on RALFs peptides mainly focuses on growth and development,but the role in environmental stress is still unclear.In this paper,we identified RALF genes with conserved structures in tomato,studied its transcriptional expression patternby using GUS expressing line,and explored the function of RALFs in tomato growth and development and cold tolerance using Virus Induced Gene Silence(VIGS)technology.The main findings are as follows1.The RALF genes of tomato were identified and found that the RALF4 gene plays an important role in regulating root development.The phylogenetic tree was used to analyze the homology between the RALF gene in tomato and the AtRALF gene in Arabidopsis,and four RALF genes were selected according to the conserved structural features of RALF.Their expression patterns in different organ tissues were determined,and the results indicated that RALF may be involved in the development of different organs.RALF4 was mainly expressed in stems,leaves and flowers,but its expression in roots is much lower than other genes;RALF5 was mainly expressed in roots and flowers,especially in roots;RALF6 was mainly in roots,stems,leaves and flowers;RALF7 was mainly expressed in leaves,while expression in roots was as low as RALF4.RALF4,RALF5,RALF6,and RALF7 genes were silenced by VIGS technology.The results showed that compared with the control TRV plants,RALF4-silenced plants were significantly dwarfed,while the growth of RALF5-silenced plants were promoted,and RALF6-and TRALF7-silenced plants showed no significant difference.The results showed that although RALF4,RALF5 and RALF6 had high homology,their functions were quite different.Compared with TRV plants,root development of RALF4-silenced plants was significantly inhibited,indicating that RALF4 may play a role in the specific developmental stages of roots.The above results indicated that RALF4 plays an important role in root development,while RALF5 inhibits plant growth.2.We defined the expression pattern of RALF4 gene in roots and clarified that RALF4 did not regulate lateral root development by affecting auxin signaling.We found that exogenous RALF4 did not significantly inhibit root elongation under low concentration,but it still had the biological activity of RALF that increases the pH of the growth medium.In the transgenic plant pRALF4::GUS,the expression of the RALF4 gene in the root was found to be specifically expressed in the lateral root primordia and the base of the lateral root,indicating that RALF4 may not participate in the development of the main root,but affect the growth of the lateral root.Auxin is the most important hormone in root development.It was found that silencing RALF4 resulted in increased apical auxin response and increased auxin synthesis.However,after exogenous treatment of RALF4,the response of auxin in roots was not altered.At the same time,the treatment of NAA promoted lateral root development but did not induce the expression of RALF4.The rulf4 knock out mutant was constructed using CRISPR/Cas9 technology,further clarifying the role of RALF4 in lateral root development.Therefore,the loss of RALF4 leads to the inhibition of root development,and its regulation process may be independent of the auxin signal.3.RALF5 negatively regulated cold tolerance.The expression levels of RALF4,RALF5 and RALF6 were transiently up-regulated under low temperature stress.The three genes of RALF4,RALF5 and RALF6 were silenced by VIGS,and the silenced plants were treated with low temperature,and the tolerance of the target gene-silenced plants were found to be stronger than that of the control TRV plants.Plants silenced with RALF5 is the most tolerant.The gene expression of CBF1 and COR47 in CBF anti-cold pathway was determined.It was found that the gene expression level of RALF5 silenced plants was significantly up-regulated,and especially for CBF1.In addition,RALF5-silenced plants had the highest accumulation of ABA content as compared to the control TRV plants.This ABA accumulation was associated with an increase in the expression level of the ABA-synthesis gene NCED1.In addition,BR synthesis gene DET2 was also significantly up-regulated in RALF5-silenced plants,suggesting that BR may also be involved in cold tolerance.The above experiments showed that RALF5 negatively regulated plant low temperature tolerance.