State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China.
Hebei Key Laboratory of Molecular and Cellular Biology, Hebei Normal University, Shijiazhuang 050024, China.
J Integr Plant Biol. 2018 Feb;60(2):94-111. doi: 10.1111/jipb.12631.
Chloroplast genes are transcribed by the plastid-encoded RNA polymerase (PEP) or nucleus-encoded RNA polymerase. FRUCTOKINASE-LIKE PROTEINS (FLNs) are phosphofructokinase-B (PfkB)-type carbohydrate kinases that act as part of the PEP complex; however, the molecular mechanisms underlying FLN activity in rice remain elusive. Previously, we identified and characterized a heat-stress sensitive albino (hsa1) mutant in rice. Map-based cloning revealed that HSA1 encodes a putative OsFLN2. Here, we further demonstrated that knockdown or knockout of the OsFLN1, a close homolog of HSA1/OsFLN2, considerably inhibits chloroplast biogenesis and the fln1 knockout mutants, created by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associate protein 9, exhibit severe albino phenotype and seedling lethality. Moreover, OsFLN1 localizes to the chloroplast. Yeast two-hybrid, pull-down and bimolecular fluorescence complementation experiments revealed that OsFLN1 and HSA1/OsFLN2 interact with THIOREDOXINZ (OsTRXz) to regulate chloroplast development. In agreement with this, knockout of OsTRXz resulted in a similar albino and seedling lethality phenotype to that of the fln1 mutants. Quantitative reverse transcription polymerase chain reaction and immunoblot analysis revealed that the transcription and translation of PEP-dependent genes were strongly inhibited in fln1 and trxz mutants, indicating that loss of OsFLN1, HSA1/OsFLN2, or OsTRXz function perturbs the stability of the transcriptionally active chromosome complex and PEP activity. These results show that OsFLN1 and HSA1/OsFLN2 contribute to chloroplast biogenesis and plant growth.
叶绿体基因由质体编码的 RNA 聚合酶 (PEP) 或核编码的 RNA 聚合酶转录。果糖激酶样蛋白 (FLNs) 是磷酸果糖激酶-B (PfkB) 型碳水化合物激酶，作为 PEP 复合物的一部分发挥作用；然而，水稻中 FLN 活性的分子机制仍不清楚。以前，我们在水稻中鉴定并表征了一个热应激敏感白化突变体 (hsa1)。基于图谱的克隆表明，HSA1 编码一个假定的 OsFLN2。在这里，我们进一步证明，OsFLN1（HSA1/OsFLN2 的密切同源物）的敲低或敲除，极大地抑制了叶绿体生物发生，而由成簇规律间隔短回文重复 (CRISPR) 和 CRISPR 相关蛋白 9 产生的 fln1 敲除突变体表现出严重的白化表型和幼苗致死性。此外，OsFLN1 定位于叶绿体。酵母双杂交、下拉和双分子荧光互补实验表明，OsFLN1 和 HSA1/OsFLN2 与硫氧还蛋白 Z (OsTRXz) 相互作用，以调节叶绿体发育。与此一致，OsTRXz 的敲除导致与 fln1 突变体相似的白化和幼苗致死表型。定量逆转录聚合酶链反应和免疫印迹分析显示，在 fln1 和 trxz 突变体中，PEP 依赖性基因的转录和翻译受到强烈抑制，表明 OsFLN1、HSA1/OsFLN2 或 OsTRXz 功能的丧失扰乱了转录活性染色体复合物和 PEP 活性的稳定性。这些结果表明，OsFLN1 和 HSA1/OsFLN2 有助于叶绿体生物发生和植物生长。
