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.

Stearoyl-acyl carrier protein-desaturase (SACPD)-catalyzed synthesis of oleic acid (18:1) is an essential step in fatty acid biosynthesis. Arabidopsis mutants (ssi2) with reduced SACPD activity accumulate salicylic acid (SA) and exhibit enhanced resistance to multiple pathogens. We show that reduced levels of 18:1 induce similar defense-related phenotypes in soybean. A Bean pod mottle virus (BPMV)-based vector was employed to effectively silence soybean SACPDs. The silenced plants contained reduced 18:1 and increased stearic acid, developed spontaneous cell death lesions, increased SA accumulation, and constitutively expressed pathogenesis-related genes. These plants also expressed elevated levels of resistance-like genes and showed resistance to bacterial and oomycete pathogens. Exogenous application of glycerol induced similar phenotypes, mimicking the effect of silencing SACPDs in healthy soybean plants. Overexpression of a soybean SACPD increased 18:1 levels in ssi2 but not in wild-type Arabidopsis plants, suggesting that the soybean enzyme was under feedback regulation similar to that of the Arabidopsis isozymes. These results suggest that soybean and Arabidopsis respond similarly to 18:1-derived cues by inducing a novel broad-spectrum resistance-conferring pathway, even though they differ significantly in their lipid biosynthetic pathways. We also demonstrate the efficacy of BPMV-induced gene silencing as a tool for functional studies in soybean.

Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL) or the isochorismate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We investigated the relative contributions of PAL and ICS to defense-related SA accumulation in soybean (Glycine max). Soybean plants silenced for five PAL isoforms or two ICS isoforms were analyzed for SA concentrations and SA-derived defense responses to the hemibiotrophic pathogens Pseudomonas syringae and Phytophthora sojae. We show that, unlike in Arabidopsis, PAL and ICS pathways are equally important for pathogen-induced SA biosynthesis in soybean. Knock-down of either pathway shuts down SA biosynthesis and abrogates pathogen resistance. Moreover, unlike in Arabidopsis, pathogen infection is associated with the suppression of ICS gene expression. Pathogen-induced biosynthesis of SA via the PAL pathway correlates inversely with phenylalanine concentrations. Although infections with either virulent or avirulent strains of the pathogens increase SA concentrations, resistance protein-mediated response to avirulent P. sojae strains may function in an SA-independent manner. These results show that PAL- and ICS-catalyzed reactions function cooperatively in soybean defense and highlight the importance of PAL in pathogen-induced SA biosynthesis.

With the development of high-throughput sequencing, many long non-coding RNAs (lncRNAs) have been found to play important roles in diverse biological processes. However, the biological functions of most plant lncRNAs are still unknown. We have previously discovered a tomato ripening-related lncRNA, lncRNA1459. Here, we cloned the full-length lncRNA1459, giving two transcript isoforms. In addition, lncRNA1459 exhibited a specific location in the nucleus. Furthermore, in order to fully identify the function of lncRNA1459 in tomato ripening, loss-of-function mutants of lncRNA1459 were developed using clustered regularly interspaced short palindromic repeats (CRISPR)/-associated protein 9 (Cas9)-induced genome editing technology. Compared with wild-type fruits, the tomato ripening process was significantly repressed in lncRNA1459 mutants. Ethylene production and lycopene accumulation were largely repressed in lncRNA1459 mutants. Additionally, genes related to ethylene and carotenoid biosynthesis were distinctly downregulated in lncRNA1459 mutants compared with wild-type fruits. Moreover, expression of numerous ripening-related genes was changed significantly when lncRNA1459 was knocked out. Expression of potential tomato ripening-related lncRNAs was also specifically changed after knocking out lncRNA1459. Taken together, these results provide insight into the role of lncRNA1459 in tomato fruit ripening.

SummaryIn recent years, the type II CRISPR system has become a widely used and robust technique to implement site‐directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single‐site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited. Furthermore, we manipulated the γ‐aminobutyric acid (GABA) shunt in tomatoes using a multiplex pYLCRISPR/Cas9 system that targeted five key genes. Fifty‐three genome‐edited plants were obtained following single plant transformation, and these samples represented single to quadruple mutants. The GABA accumulation in both the leaves and fruits of genomically edited lines was significantly enhanced, and the GABA content in the leaves of quadruple mutants was 19‐fold higher than that in wild‐type plants. Our data demonstrate that the multiplex CRISPR/Cas9 system can be exploited to precisely edit tomato genomic sequences and effectively create multisite knockout mutations, which could shed new light on plant metabolic engineering regulations.