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Ripening of the model fruit tomato (Solanum lycopersicum) is controlled by a transcription factor network including NAC (NAM, ATAF1/2, and CUC2) domain proteins such as No-ripening (NOR), SlNAC1, and SlNAC4, but very little is known about the NAC targets or how they regulate ripening. Here, we conducted a systematic search of fruit-expressed NAC genes and showed that silencing NOR-like1 (Solyc07g063420) using virus-induced gene silencing (VIGS) inhibited specific aspects of ripening. Ripening initiation was delayed by 14 days when NOR-like1 function was inactivated by CRISPR/Cas9 and fruits showed obviously reduced ethylene production, retarded softening and chlorophyll loss, and reduced lycopene accumulation. RNA-sequencing profiling and gene promoter analysis suggested that genes involved in ethylene biosynthesis (SlACS2, SlACS4), color formation (SlGgpps2, SlSGR1), and cell wall metabolism (SlPG2a, SlPL, SlCEL2, and SlEXP1) are direct targets of NOR-like1. Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assay (DLR) confirmed that NOR-like1 bound to the promoters of these genes both in vitro and in vivo, and activated their expression. Our findings demonstrate that NOR-like1 is a new positive regulator of tomato fruit ripening, with an important role in the transcriptional regulatory network.

Proanthocyanidins (PAs) and anthocyanins are involved in the response of plants to various environmental stresses. However, the mechanism behind defense-induced PA biosynthetic regulation is still not completely elucidated, also in grapevine. This study performed a transcriptome sequencing analysis of grape berries infected with Colletotrichum gloeosporioides to highlight the induction of the VabHLH137 factor from the basic helix–loop–helix (bHLH) XII subfamily by the fungus, which appeared to be significantly co-expressed with PA-related genes. The functional analysis of VabHLH137 overexpression and knockdown in transgenic grape calli showed that it positively regulated PA and anthocyanin biosynthesis. Moreover, VabHLH137 overexpression in the grape calli significantly increased resistance to C. gloeosporioides. A yeast one-hybrid and electrophoretic mobility shift assay revealed that VabHLH137 directly bound to the VaLAR2 promoter, enhancing its activity and interacting with VaMYBPAR, a transcriptional activator of PA biosynthesis. Furthermore, transient experiments showed that although the VabHLH137 + VaMYBPAR complex activated VaLAR2 expression, it failed to further enhance VaLAR2 expression compared to VaMYBPAR alone. The findings indicated that VabHLH137 enhanced PA biosynthesis by activating of VaLAR2 expression, providing new insight into the transcriptional regulation of defense-induced PA biosynthesis in grapevine. [ABSTRACT FROM AUTHOR]