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Histone deacetylation is one of the well characterized post-translational modifications related to transcriptional repression in eukaryotes. The process of histone deacetylation is achieved by histone deacetylases (HDACs). Over the last decade, substantial advances in our understanding of the mechanism of fruit ripening have been achieved, but the role of HDACs in this process has not been elucidated. In our study, an RNA interference (RNAi) expression vector targeting SlHDA1 was constructed and transformed into tomato plants. Shorter fruit ripening time and decreased storability were observed in SlHDA1 RNAi lines. The accumulation of carotenoid was increased through an alteration of the carotenoid pathway flux. Ethylene content, ethylene biosynthesis genes (ACS2, ACS4 and ACO1, ACO3) and ripening-associated genes (RIN, E4, E8, Cnr, TAGL1, PG, Pti4 and LOXB) were significantly up-regulated in SlHDA1 RNAi lines. In addition, the expression of fruit cell wall metabolism genes (HEX, MAN, TBG4, XTH5 and XYL) was enhanced compared with wild type. Furthermore, SlHDA1 RNAi seedlings displayed shorter hypocotyls and were more sensitive to ACC (1-aminocyclopropane-1-carboxylate) than the wild type. The results of our study indicate that SlHDA1 functions as a negative regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation.

The basic helix-loop-helix (bHLH) proteins are a large family of transcription factors that control various developmental processes in eukaryotes, but the biological roles of most bHLH proteins are not very clear, especially in tomato. In this study, a PRE-like atypical bHLH gene was isolated and designated as SlPRE2 in tomato. SlPRE2 was highly expressed in immature-green fruits, moderately in young leaves, flowers, and mature-green fruits. To further research the function of SlPRE2, a 35 S:PRE2 binary vector was constructed and transformed into wild type tomato. The transgenic plants showed increased leaf angle and stem internode length, rolling leaves with decreased chlorophyll content. The water loss rate of detached leaves was increased in young transgenic lines but depressed in mature leaves. Besides, overexpression of SlPRE2 promoted morphogenesis in seedling development, producing light-green unripening fruits and yellowing ripen fruits with reduced chlorophyll and carotenoid accumulation in pericarps, respectively. Quantitative RT-PCR analysis showed that expression of the chlorophyll related genes, such as GOLDEN 2-LIKE and RbcS, were decreased in unripening 35 S:PRE2 fruit, and carotenoid biosynthesis-related genes PHYTOENE SYNTHASE1A and ζ-CAROTENE DESATURASE in ripening fruit were also down-regulated. These results suggest that SlPRE2 affects plant morphology and is a negative regulator of fruit pigment accumulation.

upa20 induces cell enlargement and hypertrophy development. In our research, overexpression of SlUPA-like, orthologous to upa20, severely affected the growth of vegetative and reproductive tissues. Wilted leaves curled upwardly and sterile flowers were found in transgenic lines. Through anatomical analysis, palisade and spongy tissues showed fluffy and hypertrophic development in transgenic plants. Gene expression analysis showed that GA responsive, biosynthetic and signal transduction genes (e.g. GAST1, SlGA20OXs, SlGA3OXs, SlGID1s and SlPREs) were significantly upregulated, indicating that GA response is stimulated by overproduction of SlUPA-like. Furthermore, SlUPA-like was strongly induced by exogenous JA and wounding. Decreased expression of PI-I and induced expression of SlJAZs (including SlJAZ2, SlJAZ10 and SlJAZ11) were observed in transgenic plants, suggesting that JA response is repressed. In addition, SlUPA-like overexpressed plant exhibited more opened stoma and higher water loss than the control when treated with dehydration stress, which was related to decreased ABA biosynthesis, signal transduction and response. Particularly, abnormal developments of transgenic plants promote the plant susceptibility to Xanthomonas campestris pv. campestris. Therefore, it is deduced from these results that SlUPA-like plays vital role in regulation of plant development and stress tolerance through GA, JA and ABA pathways.

MADS-box transcription factors play important role in plant growth and development, especially floral organ identities. In our study, a MADS-box gene SlGLO1- tomato floral homeotic protein FBP1-like gene was isolated. Its tissue-specific expression profile analysis showed that SlGLO1 was highly expressed in petals and stamens. RNAi (RNA interference) repression of SlGLO1 resulted in floral organ abnormal phenotypes, including green petals with shorter size and aberrant carpelloid stamens. SlGLO1-silenced lines are male sterile. Total chlorophyll content was increased and chlorophyll biosynthetic genes were significantly up-regulated in SlGLO1-silenced petals and stamens. Furthermore, B-class genes expression analysis indicated that the repressed function of SlGLO1 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 SlGLO1-RNAi plants. Additionally, pollen grains of transgenic lines were aberrant and failed to germinate and tomato pollen-specific genes were down-regulated by more than 90% in SlGLO1-silenced lines. These results suggest that SlGLO1 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 our study, a tomato MADS-box gene, SlFYFL, was isolated. SlFYFL is expressed in all tissues of tomato and significantly higher in mature leave, fruit of different stages, AZ (abscission zone) and sepal. Delayed leaf senescence and fruit ripening, increased storability and longer sepals were observed in 35S:FYFL tomato. The accumulation of carotenoid was reduced, and ethylene content, ethylene biosynthetic and responsive genes were down-regulated in 35S:FYFL fruits. Abscission zone (AZ) did not form normally and abscission zone development related genes were declined in AZs of 35S:FYFL plants. Yeast two-hybrid assay revealed that SlFYFL protein could interact with SlMADS-RIN, SlMADS1 and SlJOINTLESS, respectively. These results suggest that overexpression of SlFYFL regulate fruit ripening and development of AZ via interactions with the ripening and abscission zone-related MADS box proteins.