NAC transcription factors play an important role in ethylene biosynthesis, reception and signaling of tomato fruit ripening.

NAC proteins comprise a large family of transcription factors that play important roles in diverse physiological processes during development. To explore the role of NAC transcription factors in the ripening of fruits, we predicted the secondary and tertiary structure as well as regulative function of the SNAC4 (SlNAC48, Accession number: NM 001279348.2) and SNAC9 (SlNAC19, Accession number: XM 004236996.2) transcription factors in tomato. We found that the tertiary structure of SNAC9 was similar to that of ATNAP, which played an important role in the fruit senescence and was required for ethylene stimulation. Likewise, the bio-function prediction results indicated that SNAC4 and SNAC9 participated in various plant hormone signaling and senescence processes. More information about SNACs was obtained by the application of VIGS (virus-induced gene silencing). The silencing of SNAC4 and SNAC9 dramatically repressed the LeACS2, LeACS4 and LeACO1 expression, which consequently led to the inhibition of the ripening process. The silencing of SNACs down-regulated the mRNA levels of the ethylene perception genes and, at the same time, suppressed the expression of ethylene signaling-related genes except for LeERF2 which was induced by the silencing of SNAC4. The expressions of LeRIN were different in two silenced fruits. In addition, the silencing of SNAC4 reduced its mRNA level, while the silencing of SNAC9 induced its expression. Furthermore, the silencing of LeACS4, LeACO1 and LeERF2 reduced the expression of SNAC4 and SNAC9, while the silencing of NR induced the expression of all of them. In particular, these results indicate that SNAC transcription factors bind to the promoter of the ethylene synthesis genes in vitro. This experimental evidence demonstrates that SNAC4 and SNAC9 could positively regulate the tomato fruit ripening process by functioning upstream of ethylene synthesis genes. These outcomes will be helpful to provide a theoretical foundation for further exploring the tomato fruit ripening and senescence mechanism.