Functional Study Of Tomato Atypical BHLH Transcription Factor SlPRE6-like In Plant Growth And Development

bHLH（basic Helix-Loop-Helix）transcription factor plays an important regulatory role in plant growth and development,stress resistance,light signal transduction and hormone synthesis.Plant transcription factor PREs are members of the atypical bHLH subfamily,and are mainly involved in the regulation of brassinosteroid signal transduction,cell elongation,light signal transduction and other pathways in model plants such as Arabidopsis and Rice.However,there are relatively few reports on the functions of PREs transcription factors in tomato.Therefore,it is particularly necessary to analyze the functions of tomato PREs family members.This study retrieved the SlPRE6-like gene with unknown function from the tomato SGN database,which belongs to the tomato PREs transcription factor family.Using wild-type tomato（Ailsa Craig）as research materials,through overexpression technology and CRISPR/Cas9 gene editing system,real-time fluorescent quantitative PCR technology（q RT-PCR）,plant hormone content determination,plant tissue sections,transcriptome deep sequencing analysis,yeast two-hybrid and other experimental techniques explored the function of SlPRE6-like gene in tomato,and the main results obtained are as follows:（1）The SlPRE6-like gene was cloned from tomato,and it was found that the SlPRE6-like gene only contained twoα-helix domains through the analysis of homologous sequences in bioinformatics,which was an atypical member of the bHLH subfamily;Promoter analysis showed that the upstream promoter region of SlPRE6-like gene contains multiple light signal and abscisic acid response elements,as well as a mesophyll cell differentiation element.These results indicated that SlPRE6-like may be closely related to light signal transduction,hormone signal transduction and mesophyll cell differentiation pathways.（2）Analysis of the tissue-specific expression pattern of SlPRE6-like using q RT-PCR technology showed that SlPRE6-like had the highest transcription accumulation level in the young leaves of tomato,followed by the expression level in flowers and calyxes at different stages,and the expression level was also higher in mature leaves,but the expression level was lower in fruits at different stages.It showed that SlPRE6-like may play an important role in tomato leaves.（3）Overexpression and CRISPR/Cas9 gene editing techniques were used to construct the overexpression and knockout vectors of the SlPRE6-like gene.After Agrobacterium-mediated genetic transformation,the overexpression and knockout lines of the SlPRE6-like gene were obtained respectively;The overexpression lines with higher expression efficiency of SlPRE6-like target genes were screened by q RT-PCR;Homozygous knockout lines were screened by sequencing;Phenotypic observation and statistics of overexpression lines showed that the plant height increased,Leaf color became lighter,leaf curled,compound leaves became longer;In contrast to the elongated leaf phenotype of the overexpression line,the leaf shape of the SlPRE6-like knockout mutant became more rounded than that of the wild type.These results indicated that SlPRE6-like affects tomato leaf morphogenesis.（4）The GA₃ content in mature leaves of the SlPRE6-like overexpression line was determined,and it was found that the GA₃ content in mature leaves increased by about50%compared with wild-type leaves;By measuring the water loss rate of the young and mature leaves of the SlPRE6-like overexpression line,it was found that the water loss rate of the mature leaves was significantly lower than that of the wild type,but the opposite was true in the young leaves;Microscopic observation of the dorsal and abdomen of mature leaves of SlPRE6-like overexpression lines showed that the stomata opening degree and density were significantly lower than those of the wild type;Compared with the wild type,the palisade tissue arrangement of mature leaves of the SlPRE6-like overexpression line became loose,the sponge tissue gap increased,and the leaf structure was loose;In addition,the iodine-potassium staining experiment of leaves found that the starch content of the SlPRE6-like overexpression lines’s leaves were lower than that of the wild type.These results indicated that SlPRE6-like affects the GA accumulation,microstructure and starch content of tomato leaves.（5）Through transcriptome comparison analysis of mature leaves of wild-type tomato and SlPRE6-like overexpression lines,a total of 3674 differential genes were found.In SlPRE6-like overexpression leaves,there were a total of 2577 up-regulated genes and 1097 down-regulated genes.Among these differential genes,we noticed that the expression levels of genes related to the gibberellin signal transduction pathway have been significantly changed.For example,the forward regulator of gibberellin SlGID1b-2 was up-regulated by 2.29 times,and the key enzyme in the third stage of the gibberellin synthesis pathway SlGA2ox2 was up-regulated by 4.05 times and SlGA20ox2 was down-regulated by 1.42 times.The q RT-PCR technology found that the expression levels of SlGID1b-2,SlGA2ox2 and SlGA20ox2 are similar to the transcriptome sequencing results.This indicated that the increased height phenotype of SlPRE6-like overexpression plants may be related to the expression changes of these gibberellin-related genes.At the same time,a large number of down-regulated chlorophyll a/b binding proteins in the differential genes were obtained from the transcriptome comparative analysis,such as Cab6,Cab12,CBP2,and Cab1D by 1.53,3.81,3.83,and 2.81 times,respectively,indicating that the SlPRE6-like gene is down-regulated.Overexpression may negatively regulate the accumulation of chlorophyll during the growth and development of tomato leaves.In addition,among the differentially expressed genes,SlSGR1 was up-regulated by 2.14 times and SlRbc S-3B was down-regulated by 1.46 times in the overexpression lines,which indicated that the overexpression of SlPRE6-like might change the leaf color of the overexpression lines by affecting the expression of the above genes.In addition,the rate limiting enzyme of starch synthesis,SlAGPase L3,was also down-regulated in the SlPRE6-like overexpression lines,indicating that SlPRE6-like may also change starch content by affecting the expression of starch synthesis related genes.（6）The yeast two-hybrid technique was used to detect the interaction of SlPRE6-like protein with light morphogenesis-related factor SlPAR1 and cell elongation regulator SlAIFs.The results showed that SlPRE6-like protein can interact with SlPAR1,SlAIF1 and SlAIF2,suggesting that SlPRE6-like may interact with At PREs in a similar manner to regulate cell expansion by interacting with target proteins.In conclusion,the function and action mode of SlPRE6-like,an atypical bHLH transcription factor in tomato,were preliminarily studied in this study,which laid a solid foundation for the comprehensive analysis of the function and action mechanism of PREs family genes in tomato.