【作者】 张斌；

【导师】 栾升； 兰文智；

【作者基本信息】 南京大学 ， 生物学， 2017， 博士

【摘要】 锰(Mn)是植物生长发育必需的微量元素之一。作为多种酶的辅因子和激活剂,Mn不仅参与抗氧化作用、DNA的合成、糖代谢与蛋白修饰等多种过程,而且为光系统Ⅱ(PSⅡ)中的放氧复合物的核心原子参与催化水的光解。因此,叶绿体缺锰将直接降低光合效率,必然导致农作物产量和品质的大幅下降。叶绿体起源于内共生的蓝藻,是具有内、外双层被膜系统的半自主细胞器。由于外膜通透性较高,而内膜具有高度选择性,因此,叶绿体的物质交换需要借助于内膜上相应的转运体蛋白。叶绿体中Mn2+稳态的维持对植物的生长发育有着至关重要的影响,然而介导Mn2+进入叶绿体的分子基础并未见报道。本论文利用反向遗传学方法筛选到模式植物拟南芥中一个新型 Mn2+转运体 CMT1(Chloroplastic Manganese Transporter1),并结合分子生物学、细胞生物学、叶绿体蛋白质组学和离子组学等多种研究手段,分析该转运体在植物体内的功能及其作用机理。主要研究结果如下:(1)CMT1影响了植物的生长发育为发掘定位于叶绿体内膜的离子转运体,本论文利用蛋白质组学分析可能定位于拟南芥叶绿体内膜的多跨膜蛋白,并构建了相应T-DNA插入突变体库。在筛选该突变体库过程中,发现At4g13590基因的T-DNA插入突变体表现出叶片黄化、植株矮小和生长周期延长等表型。利用基因组DNA成功回补该突变体的表型,证明该表型的出现确由At4g13590基因的突变造成。通过生物信息学预测发现,At4g13590编码的蛋白具有359个氨基酸,5个预测跨膜区,其N端具有叶绿体信号肽。通过序列保守性分析,该基因编码的蛋白属于一个进化保守且具有阳离子转运功能的蛋白家族UPF0016(Uncharacterized protein family 0016)。我们根据本论文的结果将其命名为CMT1。(2)CMT1定位于叶绿体内膜为探究CMT1的亚细胞定位,我们首先利用CMT1与绿色荧光蛋白GFP构建融合载体CMT1-GFP并瞬时转染拟南芥叶肉细胞的原生质体,发现CMT1定位于细胞中的叶绿体,表现出与叶绿体内膜蛋白PIC1相同的定位模式。进一步将CMT1与叶PIC1共表达,发现CMT1的绿色荧光信号与PIC1的红色荧光信号几乎完全重合,证明CMT1定位于叶绿体内膜。通过GUS染色及qRT-PCR验证,发现该基因主要在地上部分表达,与其叶绿体定位相符。(3)CMT1影响了光合作用为分析CMT1在光合作用中的功能,首先我们利用透射电镜对叶绿体超微结构进行分析。与野生型相比,cmt1突变体植株的叶片基本结构没有变化,而叶绿体中基质类囊体减少,基粒类囊体垛叠增厚。我们进一步利用BN-PAGE与2D电泳对类囊体膜蛋白分析,发现突变体中的PSⅡ超级复合物以及其他复合物含量降低,而LHCⅡ三聚体和单体的含量明显增加;为进一步定量分析类囊体膜蛋白变化,我们通过免疫印迹实验发现突变体cmt1放氧复合物中围绕锰簇的外周蛋白PsbO的含量基本不变,但是PsbP与PsbQ的含量却仅有野生型的25%左右。利用叶绿素a荧光动力学参数和氧电极分析,分别发现突变体的最大光化学效率(Fv/Fm)和实际光化学效率(ΦPSⅡ)明显降低,类囊体膜蛋白放氧速率仅为野生型的32%。这些结果说明CMT1通过维持放氧复合物的功能和结构,从而影响PSⅡ所介导的光合效率。(4)CMT1具有锰离子转运活性基于UPF0016家族的离子转运活性,我们推测CMT1可能与叶绿体离子稳态相关。通过Percoll梯度离心法制备完整叶绿体,利用电感耦合等离子体质谱法(ICP-MS)对野生型与突变体cmt1叶绿体的离子组进行分析,发现突变体叶绿体的Mn含量仅为野生型的38%。利用锰离子吸收缺陷型酵母突变株Δsmf1进行生长回补,发现切除叶绿体信号肽的CMT1表达在质膜,而且能有效地恢复Δsmf1在低Mn2+环境下的生长。这些结果表明CMT1具有Mn2+转运能力。综上所述,本论文鉴定到一个控制叶绿体Mn2+吸收的转运体CMT1,其缺失降低叶绿体内Mn2+含量,影响PSⅡ中放氧复合物的组装和功能,降低了光合效率,从而导致植物的生长发育受到显著阻遏,本论文的研究内容可为分子培育高效利用Mn2+的作物提供基因储备和理论指导。更多还原

【Abstract】 Manganese(Mn)is one of the essential microelements in the life cycle of plants.As the cofactor or activator of numerous enzymes,Mn plays the key role in diverse metabolic pathways,including the antioxidant defense,DNA synthesis,sugar metabolism and protein modification.In photosyntesis,Mn is the central atom of oxygen evolving complex that catalyzes water-splitting in PSII.Therefore,Mn deficiency is detrimental to photosynthesis thus leading to the decrease of crop yield and quality.Chloroplast is a semi-autonomous organelle with a dual-membrane system.The transport of nutrient ions through the outer envelope membrane is generally viewed as non-selective.In contrast,specific transporters are required for ions transport through the inner envelope membrane.Mn homeostasis in chloroplast is vital for plant growth and development.However,little is known about the mechanism of Mn uptake into chloroplast.In this study,we identified a new type of Mn2+transporter CMT1(Chloroplastic Manganese Transporter 1)by the reverse genetics.The molecular mechanism of CMT1 in vivo was analyzed by various methods,including molecular genetics,cell biology,proteomics and ionomics.The main conclusions are as follows:(1)CMT1 is essential for plant growth and developmentTo explore chloroplastic transporters,we collected putative multi-transmembrane proteins localized in the inner envelope membrane based on proteomics analysis and established a T-DNA mutants’ library corresponding to these candidate proteins.In screening the library,we found a mutant with a T-DNA insertion in the intron of At4g13590 showed a chlorosis,dwarf and stunted growth.To demonstrate that the phenotype was attributable to the mutation of At4g13590,complementation experiment was conducted with the cmt1 mutant as background by its genomic fragment,and the phenotypic defects in cmt1 could be complemented.At4g13590 ecncoded a protein of 359 amino acids with 5 putative transmembrane helices and a chloroplast transit peptide in its N-terminal.At4g13590 belonged to a well conserved family,UPF0016(Uncharacterized protein family 0016),and was named as CMT1 based on its properties according to our research described in this thesis.(2)CMT1 is localized in the inner envelope membrane of chloroplastTo investigate the subcellular localization of CMT1,the CMT1-GFP fusion construct was introduced into Arabidopsis mesophyll protoplasts by transient expression.The GFP signals were specifically located at the periphery of chloroplasts only,which was similar to PIC1,a well known inner envelope membrane protein.To further verify the inner envelope membrane localization of CMT1,PIC1-RFP and CMT1-GFP were co-transformed into Arabidopsis mesophyll protoplasts.GFP signals produced by CMT1-GFP were well overlapped with RFP signals generated by PIC1-RFP.Taken together,these results confirmed that CMT1 was a inner envelope protein.To analyze the expression pattern of CMT1,we performed the histochemical staining and qRT-PCR assay.The results revealed that CMT1 was expressed in various tissues,predominantly in aerial part.(3)CMT1 is crucial for photosynthesisThe chlorosis and stunted growth suggested a possible defect in photosynthesis of cmt1 mutant.Firstly,we analyzed the the ultrastructure of chloroplast by transmission electron microscopy.The results showed that few stroma lamellas were observed in cmt1,whereas grana stacks appeared to be more thick than wild type.Then we conducted the BN-PAGE followed by SDS-PAGE to investigate the thylakoid membrane protein complexes.The results exhibited that the PSⅡ supercomplexes(PSⅡ SC)and other supercomplexes were significantly reduced in the mutant.By contrast,the LHCⅡ trimer and LHCⅡ monomer showed a relative increase in abundance.To assess the levels of thylakoid membrane protein quantitatively,the immunoblotting was performed.In analysis of the nuclear-encoded subunits of the oxygen evolving complex(OEC)in PSⅡ SC,we noticed that level of PsbO was not altered in cmt1,however,contents of PsbP and PsbQ were reduced to only 25%compared with wild type.The chlorophyll a fluorescence parameters and oxygen evolution rate were analyzed respectively.Fv/Fm and ΦPSⅡ in cmt1 were significantly decreased compared with wild type,and the oxygen evolution activity in cmt1 is only 32%of that in wild type.These results indicated that the defect of PSⅡ is probably due to the loss function of OEC.(4)CMT1 functions in Mn2+ transport in the chloroplastMembers of UPF0016 family have been linked to the Ca2+/H+ homeostasis in yeast and human.So we speculated that CMT1 might alter the ion levels in the chloroplast.To assess the effect of the cmt1 mutation on metal homeostasis,we measured the ion contents of leaves and intact chloroplasts by inductively coupled plasma-mass spectrometry(ICP-MS).Even though total Mn levels in leaves were comparable between wild type and mutant,the content of Mn was significantly reduced in chloroplast of cmtl compared with wild type.To examine the transport property in vitro,CMT1 was introduced into the Δsmf1 yeast strain which is defective in Mn uptake.The plasma membrane localized ΔN65CMT1(without chloroplast transit peptide)could complement the phenotype of Δsmf1 in Mn limited condition.These results demonstrated that CMT1 was directly involved in Mn2+ transport.In conclusion,we identified a new Mn2+ transporter localized in the inner envelope membrane of chloroplast,which mediating Mn2+ uptake into stroma from cytoplasm.Mutation of CMT1 results in Mn deficiency in chloroplasts,affecting the OEC activity,thus leading to the suppression of plant growth and development.The thesis provides useful theoretical guidances and experimental evideces for molecular breeding to improve Mn2+utilization efficiency of crops.更多还原