Functional Study Of Tomato SlNAC4 And SlDEAD31 Genes In Fruit Ripening And Abiotic Stress Responses

Both NAC(NAM/ATAF1/2/CUC2) transcription factors and DEAD-box helicases play crucial roles in plant growth and development, biotic and abiotic stress responses. Among which, NAC transcription factors comprise a large plant-specific gene family. Currently, considerable progress has been made to unravel the diverse roles of NAC transcription factors in plants, whereas their functional studies are mainly limited in the model plants Arabidopsis and rice. In addition, although a few NAC proteins have been characterized so far, the actually molecular mechanisms of most plant NAC proteins are still remain to be further elucidated. Tomato(Solanum lycopersicum) is one of the most common fruits and vegetables, and the best model species for the study of fleshy fruit development and ripening. However, to date, only little information regarding development- and/or stress-related NAC transcription factors is available in tomato. Tomato nor(non-ripening) fruit mutant is caused by the mutation in the NOR of the N AC gene family, which inspires us to continue to explore the new NAC members which may also be involved in the regulation of fruit ripening. The recent completion of the tomato genome sequence also provided excellent information for genome-wide analysis of the genes belonging to specific gene families. In this study, seven tomato NAC genes, designated as Sl NAC4-Sl NAC10, were characterized to encode NAC proteins that share high similarity with other known plant NAC proteins, especially in the NAC domains. These Sl NAC genes, except Sl NAC10, contain conserved intron-exon structure. Phylogenetic analysis suggested that Sl NAC4 protein may belong to the stress-related NACs, while Sl NAC5, Sl NAC7, and Sl N AC10 proteins may belong to the development-related NACs. Q uantitative RT-PCR analysis revealed that these seven Sl NAC genes exhibited different tissue-specific(such as roots, leaves, flowers and fruits) expression profiles, suggesting that they may have diverse and crucial roles during tomato growth and development. Varying degrees of induction were detected in the expression of these Sl NAC genes except Sl NAC6 when treated with various hormones, including ABA, ACC, GA3, Me JA, and IAA. Furthermore, the expression of Sl NAC4-Sl NAC10 genes was also checked under various abiotic stresses, including Na Cl, high/low temperature, dehydration, and wounding, we found that these seven Sl NAC genes were induced by these stresses with differential induction levels. These results provide valuable information for further exploring the functional roles of NAC genes during tomato development and in response to environmental stresses.Then we screened out the Sl NAC4 gene by gene expression pattern analysis whose transcripts show high accumulation in ripening fruits, indicating that Sl NAC4 may be involved in the regulation of fruit ripening. Fruit ripening in tomato is a complicated development process affected by both endogenous hormonal and genetic regulators, and external variety of signals. Although the role of NOR was proved to act upstream of ethylene and determine the competency of tomato fruit ripening, its underlying molecular mechanisms remain to be further elucidated. To explore further the role of Sl NAC4 in fruit ripening, reduced expression of Sl NAC4 gene by RNAi in tomato resulted in inhibited fruit ripening with suppressed chlorophyll breakdown, delayed fruit ripening, decreased ethylene synthesis mediated mainly through inhibited expression of ethylene biosynthesis genes, and reduced carotenoid by altering carotenoid pathway flux. Besides, transgenic tomato fruits also displayed obvious reduction of multiple ripening-associated genes, indicating that Sl NAC4 functions as a positive regulator in fruit ripening via affecting the ethylene synthesis and carotenoid accumulation. Furthermore, Sl NAC4 expression was not induced by ethylene and may function upstream of the ripening regulator RIN protein, and positively regulate its expression. Yeast two-hybrid assay further showed that Sl N AC4 could interact with NOR and RIN proteins, respectively. These results suggested that ethylene-dependent and ethylene- independent processes that are mediated by Sl NAC4 protein in fruit ripening regulatory network.Besides, taking into account the expression of Sl NAC4 was significantly induced by salt and dehydration stress, and decreased ethylene, which has been confirmed to play important roles in stress response was detected in Sl NAC4-RNAi fruits, indicating the function of Sl NAC4 in response to abiotic stress. To confirm this, we investigated the effects of salt and drought stress on Sl NAC4-RNAi transgenic plants. The results demonstrated that the shoot and root growth of Sl NAC4-RNAi plants was more inhibited by salt stress than that of WT at post-germination stage. The leaf salt assay also showed less tolerance in RNAi plants by retaining lower chlorophyll content. Moreover, Sl NAC4-RNAi plants became less tolerant to salt and drought stress in soil, which were demonstrated by lower levels of chlorophyll and water contents, and higher water loss rate of leaves as compared to the WT plants. In addition, the expressions of various stress-related genes were reduced in RNAi plants under both control and salt-stressed conditions. Collectively, these results highlight that the crucial role of Sl NAC4 protein functions as a stress-responsive transcription factor in positive modulation of abiotic stress tolerance, and may hold promising applications in the engineering of drought- and salt-tolerant tomato. In summary, Sl NAC4 gene not only plays important roles in tomato fruit ripening as a positive regulator by modulating the ethylene biosynthesis and carotenoid accumulation, our results demonstrate that Sl NAC4 also participates in the response to abiotic stress.To date, DEAD-box helicases are shown to participate in the development of flower, seed, and root, while their importance in response to biotic and abiotic stress is only beginning to emerge. Presently, the functional studies of DEAD-box RNA helicase genes in plants are mainly limited in the model plant Arabidopsis, and the specific biological functions of RNA helicase in most plant still remain to be elucidated. So far, none of DEAD-box RNA helicase genes were well characterized in tomato. In this study, we reported o n the identification and characterization of two DEAD-box RNA helicase genes from tomato, here named as Sl DEAD30 and Sl DEAD31. Phylogenetic analysis suggested that both proteins may belong to the stress-related DEAD-box RNA helicases. Expression analysis indicated that Sl DEAD30 was mainly expressed in mature levees and roots, whereas Sl DEAD31 was constantly expressed in various tissues. Different induction levels of Sl DEAD30 and Sl DEAD31 expression were detected in response to multiple abiotic stresses, including Na Cl, dehydration, high temperature, and low temperature. Besides, the expression of Sl DEAD30 was not induced by the hormones used, while Sl DEAD31 expression was increased by Me JA. The functions of Sl DEAD31 gene were further investigated by constructing its overexpression and RNAi vectors. We found that the silencing of Sl DEAD31 may be deleterious, while Sl DEAD31 overexpression did not significantly affect tomato normal growth and development. Then we further explored the possible functions of Sl DEAD31 in salt and drought stress. The results showed that transgenic tomato plants exhibited enhanced salt resistance at postgermination stage. Furthermore, transgenic plants also exhibited dramatically enhanced tolerance to salt stress and slightly improved drought resistance, which were demonstrated simultaneously by significantly enhanced expression of multiple stress-related genes, and higher survival rate, chlorophyll content, relative water content, and lower water loss rate and malondialdehyde(MDA) production compared to WT plants. Collectively, these results provided a preliminary characterization of Sl DEAD30 and Sl DEAD31 genes in tomato, and suggested that Sl DEAD31 functions as a stress-responsive DEAD-box RNA helicase in positive modulation of abiotic stress tolerance, and was potentially useful for engineering drought- and salt-tolerant crops.In summary, seven N AC transcription factor genes and two DEAD-box RNA helicase genes were identified in this study, and we focus on analyzing the potential functions of Sl NAC4 and Sl DEAD31 genes in fruit ripening and abiotic stress response, which provided a set of significant data and important clue s for elucidation of the functions of Sl NAC4 and Sl DEAD31 in tomato and laid a foundation for potential applications in genetic engineering.