The Mechanism Of MELONGENODIA And HELICAL Genes Regulating Fruit Elongation And Plant Spiral Growth In Tomato

Appearance quality is an important commodity trait of vegetables.As a typical fruit vegetable,tomato has abundant fruit shape types,which is a model species for fleshy fruit study.Tomato fruit is developed and expanded from the single ovary.The fruit shape of tomato is protruding or sagging in different directions based on the spherical shape after selection and domestication,which is caused by single or multiple gene mutations.Several genes regulating longitudinal and horizontal cell growth have been cloned,such as SUN,OVATE,LC,FAS,etc.The cell morphology in the process of fruit longitudinal growth depends directly or indirectly on the dynamics of cytoskeleton,such as microtubules.However,the regulation mechanism that cytoskeleton involved in tomato fruit shape is still unclear,which needs to be improved by revealing the new genes.In addition,we also focus on the plant archetecture to study the gene regulation of helical growth in tomato.This paper consists of two parts.Part I,we identified the MELONGENODIA?Mel?via map-based cloning,which encodes a palmitoyltransferase?PAT?,and the fruit shape regulation function of this gene is confirmed by transgenic analysis.Furthermore,we found for the first time that the function of palmitoylation on tomato fruit shape.This study revealed the molecular mechanism of the fruit isotropic growth,which was regulated by the palmitoylation of microtubule-associated protein that mediated by Mel.In part II,the HELICAL?Hel?was cloned and identified,and the mechanism of helical growth in tomato was elucidated,which was regulated by the microtubule-associated protein that encoded by Hel.The main results are as follows:?.Cloning and identification of tomato fruit shape regulatory gene Mel and its molecular mechanism1.The elongated fruit of mel mutant is caused by the elongation of longitudinal cells,especially the placenta cell of ovary.The fruit shape was determined before anthesis.The mel mutant was crossed with LA1589?S.pimpinellifolium?to construct F₂and BC₁F₂segregation populations for map-based cloning.Mel was fine-mapping at chromosome 5by BSR-seq and molecular markers analysis,which was between molecular markers5D-19 and Ch5-20.The physical distance was about 159 kb.The annotation information of the tomato reference genome showed that the region contained 19 putative ORFs?ORF1-19?.The sequencing and sequence alignment analysis confirmed that ORF9 in the mel mutant had 1 bp deletion was found in 1320 bp downstream of the ATG,which was lead to a premature stop codon.Therefore,we considered ORF9 as the Mel candidate.2.Knockout of ORF9 in the wild-type LA2838A by CRISPR approach result in the round fruit was transformed into oblong fruit.Expression ORF9 in the mel mutant driven by the native promoter of ORF9 was complemented oblong fruit shape into round fruit.On the contrary,the overexpression of ORF9 in the mel mutant driven by CAMV-35S promoter was complemented oblong fruit shape into a flat round.These results indicated that ORF9 is Mel.3.Mel encodes a palmitoyl acyl transferase?PAT?,which is a membrane protein located in the plasma membrane.Tissue expression analysis showed that Mel was highly expressed in the active growing parts of tomato.Histochemical analysis of the GUS driven by the native promoter of Mel indicated that Mel was mainly expressed in shoot apexs and the placenta of fruit.4.The mutation was introduced in the DHHC conserved domain led to abolish palmitoyltransferase activity of Mel,namely Mel ^C209S,was transgenic into mel mutant driven by CAMV-35S promoter.The phenotypic observation found that the transformants could not complement oblong shape into a flat round,indicating that Mel palmitoyltransferase activity is essential for the isotropic growth of fruits.Exogenous palmitoylation inhibitor 2-BP leads to elongated fruit shape in LA2838A,which indicated that palmitoylation is indispensable for the regulation of fruit isotropic growth.The palmitoylation levels of proteins from LA2838A and mel-cr lines were identified by acyl-biotinyl exchange assay and label-free quantitative proteomics.A total of 26 down regulated proteins in mel-cr lines were identified,such as Calmodulin 1?CAM1?.A total of 78 proteins in the Mel protein mixture that might interact directly or indirectly by forming a complex with the Mel protein were identified by IP-MS approach,including CAM1 and a microtubule-associated protein MAP65-1 that regulates microtubule bundles.5.The direct protein-protein interacting between MAP65-1 and Mel was proved by CO-IP.Knockout of MAP65-1 by CRISPR led to an elongated fruit in LA2838A,but not as obvious as mel-cr lines.Furthermore,the overexpression of MAP65-1^C17Ain map65-1-cr lines which was introduced an invalid mutations in the predicted palmitoylation site of MAP65-1,had the same effect on restore wild-type fruit shape as MAP65-1-OE,indicating that the function of MAP65-1 in fruit elongation did not depend on palmitoylation.There was no significant difference in fruit shape index between map65-1-cr/mel-cr double mutant and the mel-cr mutant,indicating that they might involve in the same pathway of regulating fruit longitudinal elongation,and Mel had epistatic effect on MAP65-1.Similarly,CO-IP showed that MAP65-1 and CAM1 had direct protein-protein interaction,but MAP65-1 could not interact with CAM1^C27S,which was introduced an invalid mutations in the predicted palmitoylation site of CAM1,suggesting that the protein-protein interaction between MAP65-1 and CAM1 is palmitoylation depended.Based on these results,we speculated that Mel could involve in the protein-protein interaction between CAM1 and MAP65-1 through the palmitoylation of CAM1,and then regulates the microtubules dynamic and determined the isotropic growth of cells.Moreover,Mel,MAP65-1 and CAM1 interacts to each other to regulate the morphology of tomato fruit.?.Identification of Hel in helical growth of tomato1.The helical growth of hel mutants was caused by twist and helical arrangement of cell.Hel was fine-mapping at the long arm of chromosome 4 by BSR-seq and molecular markers analysis,which was located in the approximately 390 kb physical distance between SNP4-3 and SNP4-4.2.The region between SNP4-2 and SNP4-6 encompassed 13 putative ORFs,only a27 bp fragment replacement was found in the ORF2 between the hel mutant and wild type,which resulting in a premature stop codon in ORF2.Knockout of ORF2 by CRISPR in wild-type plants produced a helical growth phenotype similar to the hel mutant.The hel phenotype was complemented by ORF2 overexpression.These prove that ORF2 is Hel.3.Hel encodes a microtubule-associated protein,CELLULOSE SYNTHASE INTERACTION PROTEIN,which is homologous with CSI1 in Arabidopsis.CSI1functions as an interacting factor between the cellulose synthase complex and cortical microtubule.The hel mutant is significantly different from the csi1 mutant in Arabidopsis.Thus,there may be unknown regulatory mechanism that need further study.