Plant Biology and UC Davis Genome Center, University of California, Davis, 451 Health Sciences Drive, 4409 GBSF, Davis, CA, USA.
Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, 68 HaMaccabim Road, P.O.B 15159, Rishon LeZion, 7505101, Israel.
Plant J. 2018 Nov;96(4):855-868. doi: 10.1111/tpj.14073. Epub 2018 Oct 8.
miR160 adjusts auxin-mediated development by post-transcriptional regulation of the auxin response factors ARF10/16/17. In tomato, knockdown of miR160 (sly-miR160) suggested that it is required for auxin-driven leaf blade outgrowth, but whether additional developmental events are adjusted by sly-miR160 is not clear. Here, the SlMIR160 genes and the genes of its SlARFs targets were edited by CRISPR/Cas9 resulting in the isolation of loss-of-function mutants. In addition, hypomorphic mutants that accumulate variable reduced levels of sly-miR160a were isolated. We found that the loss-of-function mutants in SlMIR160a (CR-slmir160a-6/7) produced only four wiry leaves, whereas the hypomorphic mutants developed leaves and flowers with graded developmental abnormalities. Phenotypic severity correlated with the upregulation of SlARF10A. Consistent with that, double mutants in SlMIR160a and SlARF10A restored leaf and flower development indicating that over-accumulation of SlARF10A underlay the developmental abnormalities exhibited in the CR-slmir160a mutants. Phenotype severity also correlated with the upregulation of the SHOOT MERISTEMLESS homolog Tomato Knotted 2, which in turn activated the transcription of the cytokinin biosynthesis genes SlIPT2 and SlIPT4. However, no change in Tomato Knotted 2 was detected in the absence of SlARF10A, suggesting that it is upregulated due to auxin signaling suppression by SlARF10A. Knockout of sly-miR160a-targeted SlARFs showed that whereas SlARF10A is indispensable for leaf blade outgrowth and floral organ patterning, the functions of SlARF16A and SlARF17 are redundant. Taken together our results suggest that sly-miR160a promotes blade outgrowth as well as leaf and leaflet initiation and floral organ development through the quantitative regulation of its major target SlARF10A.
miR160 通过对生长素响应因子 ARF10/16/17 的转录后调控来调节生长素介导的发育。在番茄中，miR160 的敲低（sly-miR160）表明它是生长素驱动的叶片生长所必需的，但 sly-miR160 是否调节其他发育事件尚不清楚。在这里，通过 CRISPR/Cas9 编辑 SlMIR160 基因及其 SlARFs 靶基因，分离得到功能丧失突变体。此外，还分离得到了积累可变低水平 sly-miR160a 的拟态突变体。我们发现，SlMIR160a 的功能丧失突变体（CR-slmir160a-6/7）仅产生四个丝状叶片，而拟态突变体发育的叶片和花朵具有分级发育异常。表型严重程度与 SlARF10A 的上调相关。一致的是，SlMIR160a 和 SlARF10A 的双突变体恢复了叶片和花朵的发育，表明 SlARF10A 的过度积累是 CR-slmir160a 突变体中表现出的发育异常的基础。表型严重程度也与 SHOOT MERISTEMLESS 同源物番茄 Knot2 的上调相关，番茄 Knot2 反过来激活细胞分裂素生物合成基因 SlIPT2 和 SlIPT4 的转录。然而，在没有 SlARF10A 的情况下，未检测到番茄 Knot2 的变化，表明由于 SlARF10A 对生长素信号的抑制，它被上调。sly-miR160a 靶向 SlARFs 的敲除表明，虽然 SlARF10A 对于叶片生长和花器官模式形成是必不可少的，但 SlARF16A 和 SlARF17 的功能是冗余的。总之，我们的结果表明，sly-miR160a 通过其主要靶标 SlARF10A 的定量调节来促进叶片生长以及叶片和小叶的起始和花器官的发育。
