Exploiting Key Genes Regulating Leaf Stomata Formation By Genome-Wide Association Analysis In Tomato

Tomato?Solanum lycopersicum?is an important vegetable crop and as a model plant for plant science.Stomata is an important botanical trait,which is an important contributor on photosynthesis,transpiration and adversity stress of plants.Stomata control gas and moisture exchanges between the plant and the external atmosphere.However,studies on stomatal development in tomato is still limeted.Therefore,it is of great significance to study the molecular mechanism of stomata formation in plants.In this study,based on the previous genome-wide association analysis?GWAS?in this lab of more than 500+tomato resequencing accessions,S1GMDH and SlALMT15 are thought to be candidate genes significantly correlated with the stomatal?density/Unit?of tomato leaves.We integrated gene sequence analysis,transgenic verification experiments,transcriptional regulation and adversity stress text to explore the regulatory mechanism of S1GMDH and SlALMT15 on stomatal number in tomato.The key research findings are as follows:1.Analysis of stomata number in leaves of more than 500 tomato resequenced materials by microscopic observation showed that stomata number in tomato leaves was rich in diversity.At 100 times the field of view,the number of stomata ranged from a minimum of 11 to a maximum of 81.2.Genome-wide association analysis analysis identified two genes that regulate stomatal number?density/Unit?in tomato leaves.GWAS was performed on the stomatal number?SN?of leaves with 4.1M SNPs obtained by resequencing,and the key genes SlGMDH and SlALMT15 regulating stomatal number of tomato leaves were identified on chromosome 3 and chromosome 11,respectively.GMDH is a mannose dehydrogenase and ALMT15 is a member of the aluminum-activated malate transporter family.3.Candidate genes have mutation sites in promoter regions.We sequenced the genomic region of the two genes,including that corresponding to the upstream promoter sequence.Sequence analysis showed that there were no amino acid changes in the ORF of SlGMDH and SlALMT15,but 6 polymorphisms were identified in the promoter region of SlGMDH,resulting in 18 cis-acting elements.SlALMT15 also had 18 polymorphisms in the promoter region,resulting in a difference of 36 cis-acting elements.4.The two candidate genes were expressed differently in leaves with different stomatal densities.qPCR experiment showing that SlGMDH and SlALMT15 were constitutive expression genes,but SlGMDH was highly expressed in young leaves and stems,while SlALMT15 was only highly expressed in stems.5.Transgenic function verified that candidate genes could regulate stomatal density of leaves.To elucidate how SlGMDH regulates stomatal number,we mutated SlGMDH in vivo using CRISPR/Cas9 in the high-SN accession Ailsa Craig?TS-9?.Less leaf stomatal number were observed in the three gmdh mutants compared with that in wild-type AC.Similarly,SlALMT15 knock-out lines exhibited reduced stomata number than the control.However,it should be noted that the no significant increase of stomata number were observed in SlGMDH or SlALMT15 overexpression lines?TS-55 background with low stomatal number?.6.Possible regulatory mechanism of two candidate genes.To identify regulation pathway of stomata development in tomato,the expression levels of tomato homologous genes of published genes in Arabidopsis thaliana regulated transcription facrtors were detected by Q-PCR experiments in the knock-out lines of SlGMDH and SlALMT15,respectively.EPF1 and FAMA were up-regulated and POLAR was down-regulated during stomatal development in SLGMDH-CR transgenic lines.During stomatal development in SLALMT15-CR transgenic lines,EPF1,EPF2,ER,TMM,SCRM,SPCH,MUTE and FAMA were up-regulated.7.Two candidate genes may also regulate drought resistance of plants.Stomata in the epidermal tissues of leaves are valves through which passes CO₂ and H₂O,as such they influence on photosynthesis,transpiration and adversity stress of plants.To evaluate whether knock-out SlGMDH or SlALMT15 in tomato can increase tolerance to drought stress,40-days old CR-gmdh or CR-almt15 transgenic lines and wild type?TS-9?were subjected to drought stress.The phenotypic observation and physiological indexes?photosynthetic rate,stomatal conductance,transpiration rate and MDA?of the drought-treated lines were determined.These results demonstrate that SlGMDH and SlALMT15 affect stomata development facilitating increased drought stress by reduce water loss.