SUMMARY The shoot apical meristem (SAM) comprises a group of undifferentiated cells that divide to maintain the meristem and also give rise to all plant shoot organs. SAM fate is specified by HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) transcription factors, which are targets of miR166/165. In Arabidopsis, AGO10 is a critical regulator of SAM maintenance, but the mechanism of regulation remains unknown. Here we demonstrate that AGO10 specifically recruits miR166/165. The AGO10-miR166/165 association is determined by the distinct structure of the miR166/165 duplex. Deficient loading of miR166 into AGO10 results in a defective SAM. AGO10 has a higher binding affinity for miR166 than does AGO1, a master repressor for miRNA targets. Notably, the miR166/165-binding ability of AGO10, but not its catalytic activity, is required for SAM development. We propose that AGO10 functions as a decoy for miR166/165 to maintain the SAM, preventing their incorporation into AGO1 complexes and the subsequent repression of HD-ZIP gene expression.

SummaryVirus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants. Here we report that either by syringe-infiltrating the tobacco rattle virus (TRV)-vector into the surface, stem or carpopodium of a tomato fruit attached to the plant or by vacuum-infiltrating into a tomato fruit detached from the plant, TRV can efficiently spread and replicate in the tomato fruit. Although VIGS can be performed in tomato fruit by all of the means mentioned above, the most effective method is to inject the TRV-vector into the carpopodium of young fruit attached to the plant about 10 days after pollination. Several reporter genes related to ethylene responses and fruit ripening, including LeCTR1 and LeEILs genes, were also successfully silenced by this method during fruit development. In addition, we found that the silencing of the LeEIN2 gene results in the suppression of tomato fruit ripening. The results of our study indicate that the application of VIGS techniques by the described methods can be successfully applied to tomato fruit and is a valuable tool for studying functions of the relevant genes during fruit developing.

HighlightA relatively reliable list of tomato lncRNAs was provided. Silencing of novel lncRNAs greatly delayed the ripening of tomato fruits, implying that lncRNA might be an essential factor for fruit ripening.

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.

miRNAs originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures. Accurate processing of pri-miRNAs is required for functional miRNAs. Here, using pri-miR166 family as a paradigm, we report the decisive role of pri-miRNA terminal loops in miRNA biogenesis. We found that multi-branched terminal loops in pri-miR166s substantially suppressed miR166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal-loop-branched (TLBed) pri-miRNAs can be processed by Dicer-like1 (DCL1) complexes bi-directionally: from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In either case, DCL1 complexes canonically cut pri-miRNAs at a distance of 16-17 base pairs (bp) from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. Thus, these results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complicated secondary structures.