Functional Study Of Two MIKC~C-type MADS-box Genes In Tomato

MADS-box genes encode a highly conserved gene family of transcription factors that mainly regulate reproductive processes in plants,such as floral organ identity,flowering and fruit ripening.Although it has been reported in many higher plant species,such as Arabidopsis thaliana,Oryza sativa,Brassica rapa and Raphanus sativus,the systematic identification and characterization of MADS-box transcription factors family is still limited in tomato(Solanum lycopersicon L.).The recent completion of the tomato genome project also provided excellent information for genome-wide analysis of the genes belonging to specific gene families.In this study,131 tomato MADS-box genes were identified to encode MADS-box proteins from Sol Genomics Network(SGN)database.Through the phylogenetic analysis between tomato and Arabidopsis thaliana,all tomato MADS-box genes were classified into two groups including,81 type I and 50 type II.We further analyzed their molecular characteristics and named systematically MADS-box genes having unknown function.Genome wide screening analysis and naming of MADS-box gene in tomato provides convenient and valuable information for sequentially exploring the function of MADS-box genes in tomato.Microarray expression data of MIKC-type MADS-box genes showed their transcripts accumulated mostly in reproductive organs,indicating that these genes may play an important role during tomato reproductive process.In addition,this study screened 15 floral organ identity genes in tomato,including 2 A-class genes,4 B-class genes,2 C-class genes,2 D-class genes and 5 E-class genes.Expression analysis of four whorl flower organs in tomato showed that these genes have obvious tissue specific expression characteristics,suggesting that these genes play different functions in regulating floral organ development.Then we screened out two MADS-box gene(Sl GLO1 and Sl MBP11)by gene expression pattern analysis whose transcripts show high accumulation in reproductive organs such as flower and fruits,indicating that they may be involved in the regulation of reproductive growth process.These two MIKC~C-type proteins were selected and studied by transgenic technology.MADS-box transcription factors play important role in plant growth and development,especially floral organ identities.In this study,a MADS-box gene Sl GLO1-tomato floral homeotic protein FBP1-like gene was isolated.Its tissue-specific expression profile analysis showed that Sl GLO1 was highly expressed in petals and stamens.RNAi(RNA interference)repression of Sl GLO1 resulted in floral organ abnormal phenotypes,including green petals with shorter size and aberrant carpelloid stamens.Total chlorophyll content was increased and chlorophyll biosynthetic genes were significantly up-regulated in Sl GLO1-silenced petals and stamens.Furthermore,B-class genes expression analysis indicated that the repressed function of Sl GLO1 led to the enhanced expression of TAP3 and the down-regulation of TPI in the petals and stamens,while the expression of TM6 was reduced in petals and increased in stamens and carpels of Sl GLO1-RNAi plants.Additionally,Sl GLO1-silenced lines are male sterile.Pollen grains of transgenic lines were aberrant and failed to germinate and tomato pollen-specific genes were down-regulated by more than 90 % in Sl GLO1-silenced lines.These results suggest that Sl GLO1 plays important role in regulating plant floral organ and pollen development in tomato.MADS-domain proteins are important transcription factors involved in many biological processes of plants.In the present study,Sl MBP11,a member of AGL15 subfamily,was cloned in tomato.Sl MBP11 is ubiquitously expressed in all tissues we examined,whereas Sl MBP11 transcript levels were significantly higher in reproductive tissues than in vegetative tissues.Plants exhibiting increased Sl MBP11 levels displayed reduced plant height,leaf size,internode length,loss of dominance in young seedling,highly branched growth from each leaf axil,increased number of nodes and leaves.Moreover,overexpression lines also exhibited reproductive phenotypes such as shorter style and split ovary led to polycarpous fruits,while the wild type showed normal floral organization.In addition,delayed perianth senescence was observed in transgenic tomato.These phenotypes were further confirmed by analyzing morphological,anatomical and molecular features of overexpressing lines.These results suggest that Sl MBP11 plays an important role in regulating plant architecture and reproductive development in tomato.These findings add a new class of transcription factors to the group of genes controlling axillary bud growth and illuminate a previously uncharacterized function of MADS-box genes in tomato.The MADS-box family transcription factors are the key regulators of plants involved in many biological processes.However,to date only little information regarding stress-related MADS-box genes is available in tomato.To further unravel the function of Sl MBP11 in response to abiotic stress,we generated transgenic tomato plants with knockdown Sl MBP11 by RNA-interfered(RNAi)and overexpressing Sl MBP11,and investigated the effects of salt stress on wild-type(WT),RNAi and overexpressing plants.In this study,seedling growth of Sl MBP11-RNAi was more inhibited by salt than that of WT at post-germination stage,and RNAi plants became less tolerant to salt stress than WT plants in soil,which were demonstrated by lower relative water and chlorophyll contents,and higher relative electrolyte leakage and malondialdehyde(MDA).By contrast,overexpression plants had no obvious difference compared with WT seedlings when challenged by Na Cl at post-germination stage,and overexpression of Sl MBP11 in tomato enhanced tolerance to salt stress,which were confirmed by lower relative electrolyte leakage,MDA content,higher water and chlorophyll content transgenic plants.In addition,the expression of chlorophyll biosynthesis,photosystem and stress related genes were changed in the opposite direction in Sl MBP11-RNAi and overexpressing plants under both control and salt-stressed conditions.Together,these results highlighted the important role of Sl MBP11 as a stress-responsive transcription factor in positive modulation of salt stress tolerance possibly through an ABA-independent signaling network,and may hold promising applications in the engineering of salt tolerant tomato.In summary,131 MADS-box transcription factor genes were identified in this study,and we focus on analyzing the potential function of Sl GLO1 and Sl MBP11 genes in floral organ development,plant architecture and abiotic stress response,which provided a set of significant data and important clues to elucidate the function of Sl GLO1 and Sl MBP11 in tomato.