Functional Analysis Of Tomato NAC1Transcription Factor

Ripening of fleshy fruit is a subtle genetic regulatory process involving the combinedaction of plant hormones, developmental regulators, numerous of biotic and abioticenvironmental factors. Tomato (Solanum lycopersicum) is a model for biology and genetics,regulating specific ripening pathways including ethylene, carotenoids, and cell wallmetabolism in addition to upstream signaling and transcriptional regulators. Though manyefforts were taken to reveal the mechanism of tomato fruit ripening, the core of geneticcomponent that starts fruit ripening up is still uncertain.In the last decade, NAC (NAM，ATAF1/2，CUC2) transcription factors which are uniqueto plants have been the new type transcription regulatory factors that have multiple biologicalfunctions. And all NAC proteins constitute one of the largest families of plant specifictranscription factors. Members of NAC transcription factor family play important roles inplant growth and development. They regulate development of embryo and lateral roots,formation of apical meristem and secondary cell wall, senescence of plants, ripening of fruits,and so on. In addition, NAC transcription factors are involved in hormone regulation andplant responses to biotic and abiotic environmental stresses.In this study, we cloned a NAC transcription factor SlNAC1from tomato leaves.SlNAC1regulated tomato fruit ripening by regulating synthesis of ethylene and ABA.Moreover, overexpression of SlNAC1enhanced chilling tolerance of tomato plants.Consequently, study on SlNAC1is not only important to understanding molecular mechanismof fruit ripening, but also benefits to illuminating the mechanism of NAC transcriptionalfactors regulating plant defense to chilling stress. The major results are as follows:(1) We isolated the full length cDNA of SlNAC1by RT-PCR. The full length of SlNAC1is954bp and the ORF is906bp encoding302amino residues. And the molecular weight ofNAC1protein is about34.8KDa. SlNAC1has a conserved NAC domain in the N-terminaland the C-terminal acting as transcription activating domain. Transcripts of SlNAC1werehigher in flowers and fruits and accumulated gradually along with fruit ripening. The expression of SlNAC1was induced by chilling, heat, salt, drought, wounding, oxidativestresses, and several kinds of hormones.(2) SlNAC1::GFP fusion protein was transiently expressed in onion epidermal cells andwe found that it was located in the nucleus by using confocal laser scanning microscope.(3) Prokaryotic expression vector pET-SlNAC1was constructed and transformed intoE.Coli BL21. The proteins induced by IPTG was purified and used to immunize the mice toobtain antiserum. The titer of antibody was1:100,000. The results of western blot showedthat NAC1protein was induced by chilling, heat, drought, and high-salinity. The expressionlevels of NAC1protein were increased and decreased in over-expression lines and antisenseline, respectively.(4) The full-length ORF of SlNAC1was subcloned into the expression vector pBI121under the control of the35S-CaMV promoter to form sense and antisense constructs. TheAgrobacterium tumefaciens-mediated leaf disk method was used to generate transgenictomato plants. Kanamycin-resistant plants were detected by PCR, qRT-PCR and western blot.The results suggested that over-expression lines and antisense lines were obtained.Compared with wild-type plants, over-expression lines showed yellow/orange mature fruits,reduced carpel number and pericarp thickness, as well as increased seed number and seed size.By contrast, antisense transgenic fruits exhibited deeper red and thicker pericarps, reducedcarpels, and reduced seed number and seed size compared with the WT fruits. HPLC analysisof mature WT and transgenic fruits showed a dramatic reduction of lycopene in SlNAC1over-expression fruits, but the relative content of lutein and β-carotene in SlNAC1over-expression fruits was increased. Lycopene of antisense fruits was increased comparedwith that of WT fruits. Gas chromatograph analysis indicated that ethylene emission ofoverexpression lines was significantly lower than that of WT fruits, but overexpression fruitssoftened earlier than WT fruits. However, the respiration peaks of antisense fruits weredelayed and lower than that of WT fruits. And antisense fruits softened later than WT fruits.(5) The results of qRT-PCR showed that genes related to lycopene synthesis weredownregulated while genes decomposing lycopene into lutein and β-carotene were upregulated in overexpression fruits, compared to those in WT fruits. In overexpression fruits,expression levels of genes related to ethylene synthesis and signal transduction weredecreased whereas transcripts of genes involved in cell-wall metabolism were increased,compared with those in WT fruits. Nevertheless, the expression levels of these genes above inantisense fruits were opposite to the results in overexpression fruits. The results of microarrayanalysis were consistent with those of qRT-PCR analysis. And yeast one-hybrid assay showedthat SlNAC1directly regulated the expression levels of ACS2, ACS4, ACO1, and PSY1.(6) Compared with fruits of wild type, ABA content of SlNAC1over-expressing fruits wasincreased while that of antisense fruits was reduced. The firmness of MG fruits of theover-expressing fruits treated with NDGA increased and the softening of treated fruits wasdelayed compared with those without NDGA treatment at the same stage. And the firmnessof MG fruits of the antisense fruits treated with ABA decreased and the ripening of the fruitswas ahead. qRT-PCR analysis suggested that expression levels of NCED1and NCED2whichare two key genes involved in ABA synthesis were upregulated and downregulated inoverexpression and antisense fruits, respectively.(7) After chilling treatment, the content of H2O2and O2as well as REC and MDA wereincreased, but those of WT plants were increased more than those in overexpression lines.The rate of oxygen emission and Fv/Fm decreased both in overexpression lines and WTplants, but those of WT plants decreased more markedly. Expression level of CBF1inoverexpression line was higher than that in WT by qRT-PCR. These results suggested thatSlNAC1may enhance the chilling tolerance of tomato plants by upregulating expression ofCBF1which can activate expression of cold response genes downstream.