植物离子组学及其研究方法与应用进展

植物离子组学是一门研究植物体内元素组成、分布与累积以及这些元素随植物生理状况、生物与非生物刺激、发育阶段、生境和遗传等因素的变化及其机制的新兴学科。离子组学在数量遗传性状定位、生理状况判别以及植物体内调控元素吸收、运输和贮藏的潜在基因鉴别等方面至关重要。该文综述了离子组学的基本概念、研究方法和主要研究进展,并就离子组学的研究热点、面临的挑战和未来发展趋势作了简要评述。     

植物抗旱和耐重金属基因工程研究进展

干旱和重金属污染严重影响植物的生长发育.植物耐逆相关基因的克隆和功能鉴定研究,为通过基因工程途径提高植物的抗逆性奠定了理论基础.水分亏缺、高盐、低温和重金属胁迫都能诱导LEA(late embryogenesis abundant protein)基因的表达.转基因研究表明,LEA蛋白具有抗旱保护作用、离子结合特性以及抗氧化活性;水孔蛋白存在于细胞膜和液泡膜上,在细胞乃至整个植物体水分吸收和运输过程中发挥重要作用.干旱和盐胁迫促进水孔蛋白基因转录物的积累.过量表达水孔蛋白可增强水分吸收和运输,提高植物的抗旱能力.金属转运蛋白参与重金属离子的吸收、运输和累积等过程.这些蛋白基因在改良草坪草植物的抗旱节水和耐重金属能力等方面具有潜在的应用价值.     

铅胁迫下植物抗性机制的研究进展

综述了近10多年来国内外研究重金属铅对植物的伤害机理,以及植物对铅的耐性机制的研究进展.首先从细胞分裂、细胞膜透性、光合作用和抗氧化酶系统等生理生化方面讨论了铅对植物的伤害机理,其次分析了铅离子跨膜运输、根系分泌物、金属配位体、铅离子区域化分布与植物抗性的关系.     

植物对重金属的吸收和分布

植物修复是利用植物来清除污染土壤中重金属的一项技术。该技术成功与否取决于植 物从土壤中吸取金属以及向地上部运输金属的能力。植物对金属的吸收主要取决于自由态离子活度。许多螯合剂能诱导植物对重金属的吸收。金属离子在液泡中的区域化分布是植物耐 重金属的主要原因。同时,细胞内的金属硫蛋白、植物螯合肽等蛋白质以及有机酸、氨基酸等在金属贮存和解毒方面也起重要作用。本文还论述了重金属在植物体内运输的生理及分子 方面的研究进展。     

植物对重金属的吸收和分布

植物修复是利用植物来清除污染土壤中重金属的一项技术。该技术成功与否取决于植物从土壤中吸取金属以及向地上部运输金属的能力。植物对金属的吸收主要取决于自由态离子活度。许多螯合剂能诱导植物对重金属的吸收。金属离子在液泡中的区域化分布是植物耐重金属的主要原因。同时,细胞内的金属硫蛋白、植物螯合脓等蛋白质以及有机酸、氨基酸等在金属贮存和解毒方面也起重要作用。本文还论述了重金属在植物体内运输的生理及分子方面的研究进展。     

植物细胞无机离子的区域化及通量分析

目前,区域化通量分析方法广泛应用于植物组织的离子吸收研究。在本文中,考虑植株离子吸收处于稳态和非稳态的两种情况,着重介绍区域化通量分析的数学模型、实际应用及局限性。特别考虑到离子向地上部运输对区域化通量分析的影响,使模型更符合植物组织的实际情况。     

HKT1在植物耐盐机制中的研究进展

土地盐碱化是导致作物产量降低的重要影响因素之一.在盐胁迫下,植物进化出一系列应对盐胁迫的策略,其中,离子转运蛋白在植物应对盐胁迫的过程中发挥着举足轻重的作用.HKT1转运蛋白是一类具有转运Na+功能的离子转运蛋白,主要定位在维管束组织附近,广泛存在于单子叶植物和双子叶植物中,参与植物体内Na+的长距离运输,调节植物中N...     

超富集植物遏蓝菜对重金属吸收、运输和累积的机制

遏蓝菜Thlaspi caerulescens可以在其地上部累积大量重金属如锌、镉等,是公认的超富集植物。由于该植物生物量小,不宜直接用于重金属污染的土壤植物修复,而被广泛作为一种模式植物来进行重金属富集机制研究。遏蓝菜对重金属离子的累积大致经过螯合剂解毒、地上部长距离运输以及在液泡中的储存等生理过程。已经发现的植物体内的金属螯合剂——有机酸、氨基酸、植物络合素(PCs)、金属硫蛋白(MT)和尼克烟酰胺NA等,区室化以及长距离运输相关的转运蛋白——ZIP(ZRT/IRTlike protein)、CDF(Cation diffusion facilitator)、Nramp(Natural resistance and macrophage protein)和HMA(Heavy metal ATPase)等家族,以上各种基因、多肽与蛋白等共同参与了植物对金属累积与耐受过程并发挥各自重要的作用。以下主要介绍了遏蓝菜重金属超富集相关的基因、多肽和蛋白,以及它们在重金属螯合作用和运输过程中的功能。     

钙依赖蛋白激酶(CDPKs)在植物钙信号转导中的作用

CDPKs在植物钙信号转导中起重要作用。本文介绍了植物钙信号转导及CDPKs的结构与生化性质,在此基础上,重点总结了CDPKs在植物钙信号转导中的潜在调节作用,包括基因表达、代谢、离子和水分的跨膜运输、细胞骨架的动态变化、气孔运动和生长发育等,并提出了在CDPKs研究中已达成的共识和需要解决的问题。     

钙依赖蛋白激酶（CDPKs）在植物钙信号转导中的作用

CDPKs在植物钙信号转导中起重要作用。本文介绍了植物钙信号转导及CDPKs的结构与生化性质,在此基础上,重点总结了CDPKs在植物钙信号转导中的潜在调节作用,包括基因表达、代谢、离子和水分的跨膜运输、细胞骨架的动态变化、气孔运动和生长发育等,并提出了在CDPKs研究中已达成的共识和需要解决的问题。     

Quantitation of plasma membrane calcium pump phosphorylated intermediates by electrophoresis

P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates (EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [gamma-(32)P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and (32)P-labeled EP by digital analysis of both the stained gel and its autoradiogram, respectively. The principal advantages of this method over typical procedures (filtration and centrifugation) are the low amount of enzyme required and the substantial decrease in the blank values and data scattering produced by unspecific phosphorylation and nonquantitative recovering of the enzyme. Application of this new method to a purified preparation of the plasma membrane calcium ATPase (PMCA) results in overcoming the difficulties of measuring EP at high ATP concentrations. A biphasic behavior of the substrate curve for EP was observed when the study was extended to ATP levels within the physiological range. Since, in principle, the method does not require the use of highly purified preparations, it could be helpful for the study of phosphorylated intermediates especially under conditions in which small amounts of protein are available, e.g., mutated variants of P-ATPases.     

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.     

CHO cells expressing the human P₅-ATPase ATP13A2 are more sensitive to the toxic effects of herbicide paraquat

P-ATPases are membrane transporters energized by ATP. The subfamily of P₅-ATPases is the least studied P-ATPases and the ion substrate specificity of the P₅ subfamily is not known. Mutations of the human P₅ATPase gene ATP13A2 has been shown to underlie a form of Parkinson disease (PD). We investigated the link between ATP13A2 and environmental factors related to PD development. Increasing concentrations of the synthetic polyamine analog paraquat induced a greater cytotoxic effect over CHO cells expressing ATP13A2. Paraquat-toxicity was associated with increased production of cellular reactive oxygen species and this increment was reversed by the natural polyamine spermidine. Acridine orange fluorescence intensity suggested that ATP13A2 induced the expansion of acidic vesicles that become more alkaline upon external addition of spermidine. Polyamine uptake is proposed to be initiated by a plasma membrane carrier followed by sequestration into acidic vesicles of the late endocytic compartment through an unidentified active mechanism; because ATP13A2 is located in lysosomes and late endosomes, our results open the possibility that ATP13A2 could be one of those active transporters capable of transporting polyamines like spermidine as well as its toxic analog paraquat.     

植物重金属转运蛋白P_(1B)-ATPase结构和功能研究进展

植物调节体内重金属的累积量以维持自身生存,其中,金属阳离子转运蛋白发挥了关键作用。P1B-ATPase是在生物中广泛存在的P-ATPase中的一个亚族,也是P-ATPase多个亚族中唯一参与重金属稳态的转运蛋白。拟南芥中共发现8个P1B-ATPase。研究表明,P1B-ATPase在植物体内具有维持金属的稳态、转运以及金属解毒的功能;与金属离子在根部区域的活化、吸收、地上部分的运输、贮存,以及植物对重金属的耐受性均相关。以下综述了P1B-ATPase的进化分类、结构特征以及功能方面的最新研究进展,并展望了其在植物修复领域的应用前景。     

Structure,molecular genetics,and evolution of vacuolar H+-ATPases

Proton-ATPases can be divided into three classes denoted as P-, F-, and V-ATPases. The P-ATPases are evolutionarily distinct from the F- and V-type ATPases which have been shown to be related, probably evolved from a common ancestral enzyme. Like F-ATPases, V-ATPases are composed of two distinct structures: a catalytic sector that is hydrophilic in nature and a hydrophobic membrane sector which functions in proton conduction. Recent studies on the molecular biology of vacuolar H⁺-ATPases revealed surprising findings about the evolution of pronon pumps as well as important clues for the evolution of eukaryotic cells.     

Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase

As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H(+)-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His). To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles. Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport. Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport. Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.     

Immunocytological localization of an epitope-tagged plasma membrane proton pump (H(+)-ATPase) in phloem companion cells.

In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.     

Turgor pressure, membrane tension and the control of exocytosis in higher plants

Both turgor pressure and differences in membrane tension are capable of providing an energy input into exocytosis, the process of fusion of Golgi vesicles with the cell membrane in plants. It is shown that the contribution of turgor pressure is much larger than that of membrane tension, so that the exocytotic process is not likely on thermodynamic grounds to be reversible unless another source of energy is made available. However, recycling of membrane material as flattened, empty vesicles is energetically possible and is likely to be favoured when the magnitude of membrane tension in the cell membrane is low. Thus the outward flows of membrane and cell wall material are in principle linked to turgor, whereas membrane tension influences the inward flow of membrane material.     

稻瘟菌Magnaporthe oryzae P-ATPases基因家族分析

利用TCDB(Transporter Classification Database)网站数据库中的P-ATPases氨基酸序列对稻瘟菌全基因组表达序列(Coding Sequence,CDS)数据库进行搜索和分析,共发现23个P-ATPases基因,进化树分析表明这23个基因分属于4个家族和7个亚家族。构建了本地ESTs数据库,通过P-ATPases基因CDS序列与EST序列比对分析发现,在这些基因中有20个存在EST同源体,另外3个基因没有发现EST同源体,因此这20个基因是真实P-ATPases基因的可能性更高。运用MEME程序分析了这些P-ATPases蛋白结构域的基序,有6种基序在90%以上的基因氨基酸序列中出现,属保守基序。对这23个P-ATPases基因GC含量的分析表明,它们的平均GC含量在0.519-0.628之间,稍高于稻瘟菌整个基因组GC平均含量(0.516),同时这些基因内各区段GC含量变化不大,没有明显的梯度变化。本文结果为下一步深入研究稻瘟菌中P-ATPases基因家族的功能奠定了基础。