Study Of Molecular Mechanism Of MicroRNA1917 Mediating Tomato Pedicel Abscission

Ethylene is one of the most important phytohormones,which widely involved in a series of plant physiology and biochemistry processes,such as organ morphogenesis,senescence and abscission,flowering and fruit ripening and response to stresses.At present,hormone synthesis genes,receptors and signaling molecules have been improved to be targeted by miRNAs,suggesting miRNAs could take participate in hormone response and regulation in plants.So study of functions of miRNAs in ethylene-induced tomato pedicel abscission will provide the theoretical basis for definiting the regulation of miRNAs in tomato ethylene responses and molecular mechanism of tomato pedicel abscission.In this study,we constructed small RNA(sRNA)and degradome sequencing libraries with two separated groups of tomato pedicel explants collected at the early stage(0-4 h)and later stage(8-32 h)after flower removal to screen miRNAs and targets involved in tomato pedicel abscission,verified functions of key miRNA in over-expressing transgenic lines and analyzed spatio-temporal expression pattern of Sly-miR1917 and its targets.Our results provide insights into molecule mechanism of miRNAs participating in tomato ethylene responses,and further explore the significance of post-transcriptional regulation in ethylene signaling pathway,such as alternative splicing and miRNA-mediated degradation.The main results are as follows:1.Identification of miRNAs and targets involved tomato pedicel abscission,and screening of core miRNA.Our results showed that a total of 56 known and 11 novel miRNAs targeting 223 mRNAs were identified during tomato pedicel abscission.These targets were significantly enriched in not only intracellular,organelle-related biological processes but also in metabolic and hormone signaling pathways.The expression and regulatory patterns of selected miRNAs varied in different stages of abscission and influenced by exogenous ethylene,which indicated a complex regulatory network of miRNA-mediated target interaction during tomato pedicel abscission.2.Defining mechanism of Sly-miR1917 participating tomato ethylene reponses.Our results showed Sly-miR1917 and SlCTR4svs expression showed reverse relation in organ development,senescence and ripening.Overexpressing Sly-miR1917 lines lead to enhanced triple response in etiolated seedlings,exaggerated petiole curvature,accelerated pedicel abscission and fruit maturity.Expression of ethylene responsive genes showed a modification in 35S::Sly-miR1917 lines.Combined with the complement inhibited of accelerated pedicel abscission in overexpressing Sly-miR1917 lines by VIGS down-regulated EIN2 expression,these data suggested that Sly-miR1917 altered ethylene signaling via down-regulated SlCTR4svs to participate in the regulation of tomato ethylene responses.3.Identification of expression patterns of Sly-miR1917 and its targets during tomato pedicel abscission,and feedback regulation of targets on Sly-miR1917.Our results showed SlCTR4 gene possesses 3 different splice variants which contained Sly-miR1917 binding site due to exon skipping alternative 3' splice site in exon.The SlCTR4 isoforms generated from SlCTR4sv1-3 and S1CTR4 fall into a category,and shared high similarity with CTR proteins from another cultispecies of tomato Solanum pennellii,Solanum tuberosum and Capsicum annuum.There were three splice variants of SlCTR4 detected in tomato pedicel,each showed a unique expression pattern.Sly-miR1917 and SlCTR4sv3 showed a complementary spatio-temporal expression pattern during tomato pedicel abscission,and the localization of SlCTR4sv3 mRNA relied on special organization structure in pedicel AZ,suggested that SlCTR4sv3 may be a core target of Sly-iR1917 during pedicel abscission.The SlCTR4sv3 accumulation in 35S::SlCTR4sv3 lines were slightly higher than that in wild-type plants,while the expression of SlCTR4sv3 was significantly induced in 35S::mSlCTR4sv3 lines,which indicated the expression level of SlCTR4sv3 was strictly controlled by Sly-miR1917;and the increased expression level of SlCTR4sv3 induced Sly-miR1917 accumulation,especially in the 35S::mSlCTR4sv3 lines,implied the negative feedback regulation of SlCTR4sv3 on Sly-miR1917.4.Definiting mechanism of Sly-iR1917-based ethylene signaling involved in 2,4-D herbicide.Our results revealed that SlIAA15,SlIAA29 and SlCTR4svs expressions were significantly up-regulated by 2,4-D treatment,while expression of Sly-miR1917 showed no alternation.There was no obvious phenotypic modulation in SlIAA29 RNAi transgenic lines,SlIAA15 RNAi transgenic lines reduced plant height and weaker apical dominance,and axillary shoots developed from the first leaf node,indicated that SlIAA15 may be involved in the crosstalk between auxin and ethylene signalling pathways.Overexpression of SlIAA15 leads to 2,4-D damage phenotypes.Further study showed that there was no alternation in the resistant to 2,4-D in the 35S::Sly-miR1917,35S::SlCTR4sv3 and 35S::mSlCTR4sv3 lines.The above results suggested that ethylene may mediate in 2,4-D herbicide mechanism via various aspects.