National Institute of Agrobiological Sciences, Tsukuba, Japan.
National Agriculture and Food Research Organization (NARO), Tsukuba, Japan.
Ann Bot. 2018 Aug 1;122(2):251-265. doi: 10.1093/aob/mcy058.
Floret opening in barley is induced by the swelling of the lodicule, a trait under the control of the cleistogamy1 (cly1) gene. The product of cly1 is a member of the APETALA2 (AP2) transcription factor family, which inhibits lodicule development. A sequence polymorphism at the miR172 target site within cly1 has been associated with variation in lodicule development and hence with the cleistogamous phenotype. It was unclear whether miR172 actually functions in cly1 regulation and, if it does, which miR172 gene contributes to cleistogamy. It was also interesting to explore whether miR172-mediated cly1 regulation occurs at transcriptional level or at translational level.
Deep sequencing of small RNA identified the miR172 sequences expressed in barley immature spikes. miR172 genes were confirmed by computational and expression analysis. miR172 and cly1 expression profiles were determined by in situ hybridization and quantitative expression analysis. Immunoblot analysis provided the CLY1 protein quantifications. Definitive evidence of the role of miR172 in cleistogamy was provided by a transposon Ds-induced mutant of Hv-miR172a.
A small RNA analysis of the immature barley spike revealed three isomers, miR172a, b and c, of which miR172a was the most abundant. In situ hybridization analysis showed that miR172 and cly1 co-localize in the lodicule primordium, suggesting that these two molecules potentially interact with one another. Immunoblot analysis showed that the sequence polymorphism at the miR172 target site within cly1 reduced the abundance of the CLY1 protein, but not that of its transcript. In a Ds-induced mutant of Hv-miR172a, which generates no mature miR172a, the lodicules fail to grow, resulting in a very small lodicule.
Direct evidence is presented to show that miR172a acts to reduce the abundance of the CLY1 protein, which enables open flowering in barley.
大麦小花蕾的绽放是由内稃的膨胀引起的，这一特性受闭花受精 1（cly1）基因的控制。cly1 的产物是 APETALA2（AP2）转录因子家族的一员，它抑制内稃的发育。cly1 内 miR172 靶位点的序列多态性与内稃发育的变化有关，从而与闭花受精表型有关。尚不清楚 miR172 是否真的参与了 cly1 的调控，如果是，哪个 miR172 基因对闭花受精有贡献。同样有趣的是，探索 miR172 介导的 cly1 调控是否发生在转录水平或翻译水平。
通过对大麦幼穗的小 RNA 进行深度测序，确定了在大麦幼穗中表达的 miR172 序列。通过计算和表达分析证实了 miR172 基因的存在。通过原位杂交和定量表达分析确定了 miR172 和 cly1 的表达谱。免疫印迹分析提供了 CLY1 蛋白的定量。通过 Hv-miR172a 的转座子 Ds 诱导突变体提供了 miR172 在闭花受精中作用的明确证据。
对大麦幼穗的小 RNA 分析显示，有三个 miR172 的异构体，miR172a、b 和 c，其中 miR172a 最为丰富。原位杂交分析显示，miR172 和 cly1 在内稃原基中共定位，表明这两种分子可能相互作用。免疫印迹分析表明，cly1 内 miR172 靶位点的序列多态性降低了 CLY1 蛋白的丰度，但不影响其转录本的丰度。在 Hv-miR172a 的 Ds 诱导突变体中，由于没有成熟的 miR172a，内稃无法生长，导致内稃非常小。
本文提供了直接证据表明，miR172a 可降低 CLY1 蛋白的丰度，从而使大麦开放开花。
