| The reproductive development of higher plants is composed of several successively developmental processes including inflorescence meristem formation,flower meristem differentiation,flower bud generation,flower organ formation,fruit development and ripening.All these biological processes result in the seed formation and dispersal,to ensure the survival of progeny and the transmission of genetic material.Multiple transcription factor families are involved in the transcriptional regulation of reproductive development.MADS-box protein family is one of the most important transcription factor family.MADS-box proteins is a large family of transcription factors that are widely distributed in eukaryotes.MADS-box transcription factors are mainly involved in the regulation of flower organ identity,flower development,inflorescence development,fruit development and ripening.Up to now,great progress has been made in the investigation of MADS-box transcription factor family and numerous studies on this gene family have been reported in multiple plants such as rice,arabidopsis,and tomato,and so on.Tomato is generally used as an excellent model plant for growth and developmental study,especially for floral development,fruit development and ripening,because of several desirable attributes,such as short life cycle,small genome size,efficient stable transformation,high-density genetic maps and the completion of tomato genome sequence.Moreover,the existence of lots of well-characterized mutants in tomato also facilitates the study of plant growth and development regulation.Among these mutants,the formation of mc(macrocalyx),j(jointless),and rin(ripening inhibitor)mutants was caused by the mutation of MADS-box gene MC,JOINTLESS,and Sl MADS-RIN gene,respectively.This prompted us to further study other new MADS-box transcription factors that may be involved in the regulation of plant reproductive development.In this study,two MADS-box genes,SlCMB1 and SlMBP3,were selected from the tomato SGN database.No functional investigations of these two genes in tomato till now were found.Their funtions in tomato are still unclear and need to be studied deeply.We studied the biological functions of SlCMB1 and SlMBP3 in tomato growth and development by transgenic technology,morphological study,physiological and biochemical analysis,transcriptome sequencing analysis,and yeast two-hybrid assay.The main results of this study are following:The MADS-box genes,SlCMB1 and SlMBP3,were cloned from tomato and bioinformatics analysis of these two genes were performed in our study.The results indicated that the amino acid sequences encoded respectively by SlCMB1 and SlMBP3 have high homology with other known MADS-box proteins,especially the MADS-box domain.Gene structure analysis indicated that SlCMB1 and SlMBP3 gene have the conserved intron-exon structure.The results of a homologous protein alignment showed that SlCMB1 and SlMBP3 have the typical MADS-box domains(i.e.the MADS domain,the K domain and the I domain)and belonged to the MADS-box transcription factor family.SlCMB1 shares higher similarity with Sl MADS1,Sl MBP21 and MADS-RIN of tomato,and SlMBP3 shares higher similarity with FBP7 and FBP11 of Petunia,STK of Arabidopsis and Sl-AGL11 of tomato,compared with other functional MADS-box proteins.Phylogenetic analysis displayed that SlCMB1 was a SEPALLATA(SEP)MADS-box protein,and SlMBP3 was a AGAMOUS(AG)MADS-box protein.Promoter analysis of SlCMB1 and SlMBP3 exhibited that two gibberellin-responsive elements and an ethylene-responsive element were found in the promoter region of SlCMB1 gene,and three ethylene-responsive elements,nine seed development responsive elements,a meristem responsive element,a auxin responsive element,and three abscisic acid responsive elements.SlCMB1 is a SEP MADS-box gene,and expression pattern analysis showed that SlCMB1 was prominently expressed in stem,flowers,sepals,inflorescence and IMG,MG fruits of the wild-type(WT)tomato(Mill.cv.Ailsa Craig AC++),and was faintly expressed in vegetative organs.SlCMB1 transcripts rapidly declined with fruit ripening in WT,Nr and rin fruits,but were much lower in IMG,MG and B stage fruits of the Nr and rin mutant fruits than in WT.Furthermore,SlCMB1 m RNA was preferentially accumulated in sepals and declined with with sepal development.These results indicated that SlCMB1 may play important roles in tomato flower development and fruit ripening.RNAi suppression of SlCMB1 in tomato resulted in branched and exceptionally long inflorescences with 4-5 times as many flowers as WT and longer internodes.The SlCMB1-RNAi lines exhibited longer and stouter peduncles and a loss of inflorescence determinacy that a transition from reproductive to vegetative growth(i.e.,the formation of new leaves and apical meristem)in inflorescences was observed.Significantly changed inflorescence architecture with more branches in SlCMB1-RNAi lines were observed.In addition,the SlCMB1-RNAi lines displayed enlarged and longer sepals,and the overwhelming majority of flowers had fused sepals from anthesis to the young fruit period.The content of GA3,one kind of gibberellin,,was dramatically increased in the peduncles of the SlCMB1-RNAi lines.Anatomical investigation of paraffin sections suggested that suppression of SlCMB1 facilitates the growth of tomato peduncles through increasing cell size but not cell number,and sepals of the transgenic lines were linked to each other with several layers of cells.Real-time quantification PCR(q RT-PCR)analysis showed that the expression levels of inflorescences-related genes(BL,LS,SFT,TMF,UF and SP)were notably increased,whereas S was dramatically downregulated and no significant differences in MC and J transcript levels were observed in inflorescences of SlCMB1-RNAi lines.Four putative tomato PRE genes(PRE1,PRE2,PRE3 and PRE4)related to cell elongation and three gibberellin biosynthesis genes(GA20ox1,G3ox1 and G3ox2)were markedly upregulated,while GA2ox1,which is responsible for the deactivation of gibberellin,was significantly reduced in peduncle of SlCMB1-RNAi lines.Three sepal development related genes,AP2 a,MC and GOBLET,were dramatically decreased in sepals of SlCMB1-RNAi lines.These results indicated that SlCMB1 plays a key role in the regulation of inflorescence and sepal development.In addition,suppression of SlCMB1 also resulted in delayed ripening time for 3 to 5 days.The ethylene production,the carotenoid and lycopene contents were markedly reduced in SlCMB1-RNAi fruits compared with WT.Expression profile analysis showed that three lycopene cyclase genes(CYCB,LCYB and LCYE)were significantly upregulated,while lycopene synthesis genes(PSY1 and PDS),ethylene biosynthesis and response genes(ACO1,ACO3,ACS2,ACS4,E4,E8 and ERF1)and ripening related genes(RIN,TAGL1,FUL1,FUL2,Lox C and PE)were notably downregulated in SlCMB1-RNAi tomato fruits.These results indicated that SlCMB1 is also involved in the regulation of tomato fruit ripening.Yeast two-hybrid assay SlCMB1 could interact with inflorescence and sepal development and fruit ripening related regulators(MC,JOINTLESS,Sl MBP21,Sl AP2 a,Sl MADS-RIN,Sl MADS1 and TAGL1).In conclusion,these results demonstrate that SlCMB1 plays essential roles in inflorescence and sepal development and fruit ripening by impacting the expression of other related genes or by interacting with related regulators.These results provided a set of significant data for exploring the regulatory mechanisms of SlCMB1 gene in reproductive development and provided a new method to improve the productivity and ripening time of tomato or even other crops.SlMBP3 is a AG MADS-box gene in tomato.Expression profile analysis showed that SlMBP3 was preferentially expressed in flowers and early stages of WT,Nr and rin mutant fruits,and faintly expressed in vegetative organs.Its transcripts declined with fruit ripening in WT,Nr and rin fruits,and were much higher in IMG,MG and B stage fruits of the rin mutant than in WT and Nr fruits.Moreover,SlMBP3 was prominently expressed in the pistils of floral organs,then showed increased abundance with flower development and decreased abundance during placenta and seed development.To further study SlMBP3 function in tomato development,we generated several transgenic lines harboring constructed SlMBP3-RNAi and SlMBP3-overexpressing vector via Agrobacterium tumefaciens-mediated T-DNA transfer.The results showed that fruit development was affected in SlMBP3-RNAi plants,with reduced fruit size and weight.The SlMBP3-RNAi placenta differentiated into fleshy tissues with no jelly,grew infinitely to entirely fill the fruit locules and never liquefied in the whole life of tomato plants.Furthermore,the fleshy placenta in SlMBP3-RNAi fruits underwent a ripening-like process and noticeably pigment accumulation was also observed in SlMBP3-RNAi placentas with fruit ripening.The fruit ripening time was advanced for 3 to 4 days,and the ethylene production was markedly increased in SlMBP3-RNAi fruits.By contrast,overexpression of SlMBP3 in tomato resulted in accelerated placenta liquefaction in green fruits and no macroscopic differences of pigment accumulation between WT and SlMBP3-overexpressing lines were observed.Simultaneously,exceptionally malformed seeds having no seed coat and seed hairs and growing on the surface of SlMBP3-RNAi placenta were observed which was a significant difference from that in WT where the seeds can grow normally.Conversely,tomato seeds can grow normally in SlMBP3-overexpressing fruits and larger seeds with more seed hairs were observed.The results of physiological analysis showed that the placenta firmness,the contents of two pigments(lycopene and total carotenoid)and three cell wall components(protopectin,cellulose and hemicellulose)were significantly increased in SlMBP3-RNAi placentas.While the contents of total chlorophyll and water-soluble pectin(WSP)and four key enzymatic activities(polygalacturonase(PG),?-Galactosidase(TBG),endo-1,4-?-glucanohydrolase(CEL)and ?-xylosidase(XYL))related to cell wall modification,were dramatically reduced in SlMBP3-RNAi placentas.The lignin content was markedly reduced in SlMBP3-RNAi seeds and the SlMBP3-RNAi seeds lines never germinated compared with WT.However,the opposite changes of pigment and cell wall component contents,and enzymatic activities related to cell wall modification were observed in SlMBP3-overexpressing lines.And the seed lignin content and average fresh weight of single mature seed was remarkably increased in SlMBP3-overexpressing lines.The results of paraffin sections diplayed that jelly and extra space were generated to allow seeds to grow after placenta liquefaction in wild-type tomato fruits,but there was no space in fruit locules in SlMBP3-RNAi lines due to the filled fleshy placenta.In contrast,larger space of fruit locules was observed in SlMBP3-overexpressing lines due to the advanced placenta liquefaction compared with WT.The seed paraffin section analysis revealed that extremely malformed seeds with no seed hairs and seed coats were observed in SlMBP3-RNAi lines,however,the SlMBP3-overexpressing seeds had complete seed structure,more and longer seed hairs,and incrassate seed coats.The SEM(scanning electron microscopy)analysis further confirmed that the wrinkles on the seed surface and malformed seeds with no seed coats and seed hairs in SlMBP3-RNAi lines,and larger seeds with more and longer seed hairs in SlMBP3-overexpressing lines were observed.Transcriptomic analysis and q RT-PCR in placenta and seeds of WT and SlMBP3-RNAi lines showed that cell wall metabolism-,plant hormone signal transduction-,ethylene biosynthesis and response-,fruit ripeningand seed/placenta development-related genes were dramatically changed in SlMBP3-RNAi placenta or seeds.Moreover,the yeast two-hybrid assay revealed that SlMBP3 can interact with flower development regulator,TM5,and fruit ripening regulator,RIN.These results indicated that SlMBP3 plays a key role in placenta and seed,and fruit and fruit ripening,which provided a set of significant data to completely elucidate this gene's functions in tomato reproductive development.In summary,this study focus on analyzing the functions of two MADS-box proteins(SlCMB1 and SlMBP3)in tomato,which provided a set of significant data and important clue for elucidation of the potential functions of SlCMB1 and SlMBP3 and other MADS-box proteins in plants. |
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