Mechanism Of Small Signaling Peptides RALF Regulating The Growth And Development Of Root In Tomato

Small signaling peptides,as a new kind of phytohormones,are important cellular messengers for short distance communication,and are involved in plant developmental processes and environmental response.The RAPID ALKALINIZATION FACTOR(RALF)family of small signaling peptides have been well-studied and linked to multiple physiological and developmental processes,such as root elongation,pollen germination and pollination fertilization.The small signaling peptide RALFs family has been found in different species,but the number and their function are various.And the current research mainly focuses on model plant Arabidopsis thaliana.However,tomato(Solanum lycopersicum)as an important horticulture crop,the role of RALFs in the growth and development of tomato is still unclear.In our study,tomato was used as materials.We selected several RALF genes specifically expressed in tomato root through RALF family tree phylogenetic evolution and gene expression analysis.And we observed the expression models of RALFs by GUS reporter gene,and constructed mutant materials to observe phenotype by CRISPR-Cas9 technology.And thus we explored the role of small signaling peptide RALF function in tomato growth and development.The main results are as follows:1.We have elucidated that RALF4,RALF5 and RALF6 are significant in tomato root development.RALF was first found to regulate root development in tobacco.We identified RALF4,RALF5 and RALF6 in tomato,the homolpogous of Nt RALF in tobacco,through the amino acid sequence alignment and phylogenetic tree.We detected the expression pattern of RALF4,RALF5 and RALF6 in different plant tissues,and found these RALF genes might be involved in root development.RALF4 was the least expressed in the root,and RALF6 was the second,while RALF5 was the most expressed in the root,which was several hundred times higher than that in other parts.The expression of the three genes in the root had a certain specificity.We then used wild-type treated with exogenous small signaling peptides RALF4,RALF5 and RALF6,and found that the elongation of main root was inhibited when treated with exogenous small signaling peptides.While the inhibition was not obvious when peptides were in low concentration compared with high concentration,and it could still cause the alkalinization of culture medium,suggesting the importance of small signaling peptide RALF in the root system.We further collected RNA from these treatments and applied reverse transcription to detect related RNA transcription level,and found auxin synthesis genes,auxin polar transport carrier protein-related genes and reactive oxygen species(ROS)-related genes were induced when treated with peptides,but the expression pattern of RALF6 was different to RALF4 and RALF5,which might be due to functionally differentiated.2.We have illuminated the expression pattern of tomato RALF4 which might enroll in the process of lateral root breaking through epidermis.It was showed that RALF4 was specifically expressed on the surface and base of the lateral root primordia of tomato when it did not break through the epidermis using p RALF4::GUS material,indicating that small signaling peptide RALF4 was involved in the growth and development of the lateral root.The elongation of primary roots of wild-type plants also showed inhibition when treated with exogenous small signaling peptide RALF4,and the number of lateral roots increased.Subsequently,we constructed ralf4 mutant,and there was no difference in the phenotype of the primary root,but the number of the lateral roots decreased,further indicating that RALF4 may play a role in the development of the lateral roots.Different from the up-regulation of FZY4 induced by exogenous RALF4,the expression of FZY4 gene was down-regulated in the mutant,and the expression of DR5-GUS in auxin response was decreased.We then applied exogenous NAA,and found the expression of RALF4 was induced at the junction of the main lateral root in p RALF4::GUS plant,while the RALF4 expression on the surface of the lateral root primordia showed little change.We also found the elongation of primary root was inhibited,the number of lateral roots showed no difference and the total length of lateral roots increased in ralf4 mutant when treated with NAA,which were consistent with that of the wild-type.These results suggested that RALF4 may regulate lateral root germination by affecting auxin.3.We have demonstrated that tomato RALF5 might participated in the development of lateral roots with FER2 as a receptor,and RALF6 also might be involved in this process but they had functional differentiation.The plant height and primary root length were inhibited in ralf5 mutant,and the number of lateral roots and total length of lateral roots were shortened.When wild-type and ralf5 mutant plants were treated with exogenous peptide RALF5,the number of lateral roots increased and the total length of lateral roots increased.While there was no difference in plant height in ralf6 mutant,primary root length was shortened,lateral root number was increased and their total length was no different,the plant height and root length of OE-RALF6 were inhibited,and the root biomass was also decreased.These results indicated that both of RALF5 and RALF6 were involved in the growth and development of lateral roots,but showed functional differentiation.We subsequently constructed fer2 and fer3 mutants.Plant height,root length,number of lateral roots and total length of lateral roots were decreased in fer3 mutant compared with wild-type.While in fer3 mutant,there was no difference in plant phenotype compared with the wild-type.The phenotype of fer2 mutant was similar with ralf5 mutant,indicating that FER2 may be the receptor of RALF5.We then applied exogenous 5μM small signaling peptide RALF5,and found fer2 mutants were not sensitive to small signaling peptide RALF5.But in fer3 mutants,plant height and primary length were shortened,the number of lateral roots and total length of lateral roots were increased.These results suggested that FER2 may act as a receptor for small signaling peptide RALF5 and FER3 may play a role in other growth and development processes.