摘要
Chloroplast genes are transcribed by the plastidencoded RNA polymerase（PEP） or nucleus-encoded RNA polymerase. FRUCTOKINASE-LIKE PROTEINS（FLNs） are phosphofructokinase-B（Pfk B）-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（hsa_1） mutant in rice. Map-based cloning revealed that HSA_1 encodes a putative OsFLN_2. Here, we further demonstrated that knockdown or knockout of the OsFLN_1, a close homolog of HSA_1/OsFLN_2, considerably inhibits chloroplast biogenesis and the fln_1 knockout mutants, created by clustered regularly interspaced short palindromic repeats（CRISPR） and CRISPR-associate protein_9, exhibit severe albino phenotype and seedling lethality. Moreover, OsFLN_1 localizes to the chloroplast.Yeast two-hybrid, pull-down and bimolecular fluorescencecomplementation experiments revealed that OsFLN_1 and HSA_1/OsFLN_2 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 fln_1 mutants. Quantitative reverse transcription polymerase chain reaction and immunoblot analysis revealed that the transcription and translation of PEP-dependent genes were strongly inhibited in fln_1 and trxz mutants, indicating that loss of OsFLN_1, HSA_1/OsFLN_2, or OsTRXz function perturbs the stability of the transcriptionally active chromosome complex and PEP activity. These results show that OsFLN_1 and HSA_1/OsFLN_2 contribute to chloroplast biogenesis and plant growth.
Chloroplast genes are transcribed by the plastidencoded RNA polymerase（PEP） or nucleus-encoded RNA polymerase. FRUCTOKINASE-LIKE PROTEINS（FLNs） are phosphofructokinase-B（Pfk B）-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（hsa_1） mutant in rice. Map-based cloning revealed that HSA_1 encodes a putative OsFLN_2. Here, we further demonstrated that knockdown or knockout of the OsFLN_1, a close homolog of HSA_1/OsFLN_2, considerably inhibits chloroplast biogenesis and the fln_1 knockout mutants, created by clustered regularly interspaced short palindromic repeats（CRISPR） and CRISPR-associate protein_9, exhibit severe albino phenotype and seedling lethality. Moreover, OsFLN_1 localizes to the chloroplast.Yeast two-hybrid, pull-down and bimolecular fluorescencecomplementation experiments revealed that OsFLN_1 and HSA_1/OsFLN_2 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 fln_1 mutants. Quantitative reverse transcription polymerase chain reaction and immunoblot analysis revealed that the transcription and translation of PEP-dependent genes were strongly inhibited in fln_1 and trxz mutants, indicating that loss of OsFLN_1, HSA_1/OsFLN_2, or OsTRXz function perturbs the stability of the transcriptionally active chromosome complex and PEP activity. These results show that OsFLN_1 and HSA_1/OsFLN_2 contribute to chloroplast biogenesis and plant growth.
基金
supported by National Natural Science Foundation of China (31371606, 31601284, 31661143006)
The Transgenic Plant Research and Commercialization Project of the Ministry of Agriculture of China (2016ZX08001003-002)
Zhejiang Province Outstanding Youth Fund (LR16C130001)
The Collaborative Innovation Project of the Chinese Academy of Agricultural Sciences (Y2016XT05)
State Key Laboratory of Rice Biology Research Project (2017ZZKT10103)
