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SUMMARY: Long non‐coding RNAs (lncRNAs) play crucial roles in various biological processes in plants. However, the functional mechanism of lncRNAs in fruit ripening, particularly the transition from unripe to ripe stages, remains elusive. One such lncRNA1840, reported by our group, was found to have important role in tomato fruit ripening. In the present study, we gain insight into its functional role in fruit ripening. CRISPR‐Cas9 mediated lncRNA1840 mutants caused the delayed tomato fruit ripening. Notably, loss function of lncRNA1840 did not directly impact ethylene signaling but rather delay ethylene synthesis. Transcriptomic analysis revealed differences in the expression of ripening related genes in lncRNA1840 mutants, suggesting that it is involved in gene regulation of fruit ripening. We used Chromatin Isolation by RNA Purification (ChIRP)‐Seq to identify lncRNA1840 binding sites on chromatin. ChIRP‐seq suggested that lncRNA1840 had occupancy on 40 genes, but none of them is differentially expressed genes in transcriptomic analysis, which indicated lncRNA1840 might indirectly modulate the gene expression. ChIRP‐mass spectrometry analysis identified potential protein interactors of lncRNA1840, Pre‐mRNA processing splicing factor 8, highlighting its involvement in post‐transcriptional regulatory pathways. In summary, lncRNA1840 is key player in tomato plant growth and fruit ripening, with multifaceted roles in gene expression and regulatory networks. Significance Statement: Long non‐coding RNAs (lncRNAs) play important roles in various biological processes in plants, but the mechanism of action of lncRNAs during fruit ripening remains unexplored. lncRNA1840 was identified in tomato, which confirmed that it affected the ripening process of tomato fruit and achieved post‐transcriptional regulation by participating in alternative splicing, which improved the regulatory network of tomato fruit ripening and filled the gap of lncRNA in the field of fruit ripening. [ABSTRACT FROM AUTHOR]

Summary: RIPENING INHIBITOR (RIN)‐deficient fruits generated by CRISPR/Cas9 initiated partial ripening at a similar time to wild‐type (WT) fruits but only 10% WT concentrations of carotenoids and ethylene (ET) were synthesized. RIN‐deficient fruit never ripened completely, even when supplied with exogenous ET. The low amount of endogenous ET that they did produce was sufficient to enable ripening initiation and this could be suppressed by the ET perception inhibitor 1‐MCP.The reduced ET production by RIN‐deficient tomatoes was due to an inability to induce autocatalytic system‐2 ET synthesis, a characteristic feature of climacteric ripening. Production of volatiles and transcripts of key volatile biosynthetic genes also were greatly reduced in the absence of RIN.By contrast, the initial extent and rates of softening in the absence of RIN were similar to WT fruits, although detailed analysis showed that the expression of some cell wall‐modifying enzymes was delayed and others increased in the absence of RIN.These results support a model where RIN and ET, via ERFs, are required for full expression of ripening genes. Ethylene initiates ripening of mature green fruit, upregulates RIN expression and other changes, including system‐2 ET production. RIN, ET and other factors are required for completion of the full fruit‐ripening programme. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. [ABSTRACT FROM AUTHOR]

The development and ripening of tomato fruit are complex processes involving many gene regulatory pathways at the transcriptional and post-transcriptional level. Ripening inhibitor ( RIN) is a vital transcription factor, which targets numerous ripening-related genes at the transcriptional level during tomato fruit ripening. Micro RNAs (mi RNAs) are a class of short noncoding RNAs that play important roles in post-transcriptional gene regulation. To elucidate the potential regulatory relationship between rin and mi RNAs during fruit development and ripening, we identified known mi RNAs and profiled their expression in wild-type tomato and rin mutant using a deep sequencing approach combined with quantitative RT- PCR. A total of 33 known mi RNA families were identified, of which 14 mi RNA families were differently accumulated. Subsequent promoter analysis showed that possible RIN-binding motifs ( CAr G-box) tended to occur frequently in the promoter regions of partial differently expressed mi RNAs. In addition, ethylene may participate in the regulation of mi RNAs accumulation during tomato fruit ripening. Chromatin immunoprecipitation ( Ch IP) and electrophoretic mobility shift assay confirmed the direct binding of RIN to the promoter of MIR172a. Collectively, these results showed a close correlation between mi RNA expression and RIN as well as ethylene, which further elucidated the regulatory roles of mi RNAs during fruit development and ripening and enriched the regulatory network of RIN in tomato fruit. [ABSTRACT FROM AUTHOR]

Abstract: The fruit ripening of Green-ripe (Gr) mutant tomato was inhibited dramatically. To determine the expression patterns of Gr in tomato, we first produced the polyclonal antibody of Gr protein. RT-PCR was used to amplify the Gr gene from green ripe tomato fruit. And the PCR product was subcloned into prokaryotic protein expression vectors pET-30a to generate recombinant plasmid. The Gr protein was induced by IPTG in BL21 (DE3) and purified by Ni-NTA agarose column. The anti-Gr serum was produced by immunizing rabbits, and the titer of the anti-Gr serum was above 5000 by ELISA analysis. Purified by the DEAE-52 ion-column, the high purification level of anti-Gr polyclonal antibody was obtained. Furthermore, RT-CPR was used in the RNA level to demonstrate that the expression of Gr gene was specialized in some cultures of tomato. For example, the expressions of Gr were higher in seed, flower and green ripe fruit than others, and the expression level were reduced by exogenous ethylene treatment in the flower and green ripe fruit. Moreover, Polyclonal antibody of Gr was used to investigate the expression pattern of Gr in protein level by the Western blotting. Our results show that the expression level of Gr in protein level was complied with the expressions in RNA. So, we suggested that the regulation of Gr was transcriptional. [Copyright &y& Elsevier]

For elucidating the regulatory mechanism of ethylene on carotenoid-related volatiles (open chain) compounds and the relationship between lycopene and carotenoid-related volatiles, transgenic tomato fruits in which ACC synthase was suppressed were used. The transgenic tomato fruit showed a significant reduction of lycopene and aroma volatiles with low ethylene production. 6-methyl-5-hepten-2-one, 6-methyl-5-hepten-2-ol and geranylacetone, which were suspected to be lycopene degradation products, were lower than those in wild type tomato fruits. In order to identify whether lycopene accumulation effects the biosynthesis of some carotenoid-related volatiles independent of ethylene in tomato or not, the capability of both wild type and transgenic tomato fruits discs to convert lycopene into carotenoid-related volatiles was evaluated. The data showed that external lycopene could convert into 6-methyl-5-hepten-2-one and 6-methyl-5-hepten-2-ol in vivo, indicating that the strong inhibition of ethylene production had no effect on enzymes in the biosynthesis pathway of some carotenoid-related volatiles. Therefore, in ACS-suppression transgenic tomato fruits, the low levels of 6-methyl-5-hepten-2-one, 6-methyl-5-hepten-2-ol was due to decreased lycopene accumulation, not ethylene production. Ethylene only affected the accumulation of lycopene, and then indirectly influenceed the level of lycopene-related volatiles. [ABSTRACT FROM AUTHOR]

Two new genes,LeERF1andLeERF2, were isolated from a tomato (Lycopersicon esculentumcv. Lichun) cDNA library. Phylogenetic analysis indicated that they encoded Ethylene Responsive Element Binding Proteins (EREBPs), characterized by a conserved ERF (ethylene response factor) domain of specific binding plant cis-acting elements GCC box. Both LeERF1 and LeERF2 proteins were obtained via prokaryotic expression and purification. Electrophoretic mobility shift assay showed that LeERF1 and LeERF2 protein could bind to the promoter of theNP24gene coding for pathogenesis-related protein osmotin precursor but not the mutant promoter where its GCC box was deleted. Polyclonal antibodies of LeERF1 and LeERF2 blocked their bindingin vitro. [ABSTRACT FROM AUTHOR]

Long-Non-Coding RNAs (LncRNAs) are a class of non-coding endogenous RNAs contributing to numerous biological processes. LeERF1 is a tomato ethylene response factor (ERF) near the end of the ethylene signal transduction pathway. To identify lncRNAs in tomato and elucidate their roles in ethylene signaling, deep sequencing was deployed in over-expression and repression LeERF1 transgenic and control tomato fruits. A total of 397 lncRNAs were identified, including 169 tomato lncRNAs that had not previously been identified. Among these, 12 were differentially expressed between the transgenic and control tomato fruits. Numerous lncRNA target genes were identified including many associated with ethylene signaling including auxin response factors and auxin-induced proteins, F-box proteins, ERFs and MADS-box proteins. In addition, two lncRNAs were found to be the precursor of three miRNAs and four lncRNAs could be targeted by five miRNAs. We propose a regulatory model highlighting the relationships between lncRNAs and their targets involved in ethylene signal transduction which establishes a foundation for addressing the role of LncRNAs in ethylene response. [ABSTRACT FROM AUTHOR]