Plastidial NAD-Dependent Malate Dehydrogenase Is Critical for Embryo Development and Heterotrophic Metabolism in Arabidopsis

In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis.Reduction-oxidation (redox) reactions play pivotal roles for most metabolic processes and occur in all cellular compartments. The origin of all reducing power in plants is the chloroplast thylakoid membrane system, where light-driven photosynthetic electron transport leads to the coupled formation of ATP and the reducing equivalent NADPH (Dietz and Pfannschmidt, 2011). Sudden changes in light intensity and withdrawal of ATP and NADPH for biosynthetic processes in varying amounts can potentially disturb the ATP:NADPH ratio. Maintaining this ratio within certain limits, however, is crucial for plant metabolism, because it avoids the accumulation of excess electrons and the production of cytotoxic reactive oxygen species and allows for the continued production of ATP (Apel and Hirt, 2004; Logan, 2006; Scheibe and Dietz, 2012). Accordingly, plants have several mechanisms to dissipate excess electrons, avoid damage to cellular components, and maintain redox homeostasis. These mechanisms include nonphotochemical energy quenching, chlororespiration, cyclic electron transport, and the Mehler reaction (Scheibe et al., 2005).Reducing equivalents in the form of dedicated electron carriers or reduced cofactors (e.g. ferredoxin and NADH) are not generally transported directly across membranes; however, they can be shuttled indirectly as malate in exchange for oxaloacetic acid (OAA). This redox-poising mechanism is known as the malate valve in illuminated plastids or more generally, the malate-OAA shuttle (Heber, 1974; Scheibe, 2004; Taniguchi and Miyake, 2012). The key enzyme of the malate-OAA shuttle is malate dehydrogenase (MDH), which catalyses the reversible interconversion of malate and OAA. Isoforms of MDH are present in various cell compartments (Gietl, 1992), and each isoform is specific to either cosubstrate NAD (NAD-MDH; EC 1.1.1.37) or NADP (NADP-MDH; EC 1.1.1.82). The Arabidopsis genome encodes eight putative NAD-MDH isoforms: two isoforms are peroxisomal MDH (PMDH; PMDH1 and PMDH2; Pracharoenwattana et al., 2007; Eubel et al., 2008), two isoforms are mitochondrial MDH (MMDH; MMDH1 and MMDH2; Millar et al., 2001; Lee et al., 2008; Tomaz et al., 2010), and one isoform is plastidial MDH (plastid-localized NAD-dependent MDH [pdNAD-MDH]; Berkemeyer et al., 1998). The remaining three isoforms have no detectable target sequence and are thought to be cytosolic MDH (CMDH; CMDH1, CMDH2, and CMDH3). The Arabidopsis genome also encodes an additional NADP-dependent isoform of MDH, which is localized to the plastid (Hebbelmann et al., 2012).The physiological role of the different isoforms depends on the subcellular localization and the different metabolic pathways occurring there. For instance, MMDH was reported to be involved in two processes that are at least partly mitochondrial: leaf respiration and photorespiration (Tomaz et al., 2010). An MMDH null mutant (mmdh1 mmdh2) was slow growing and showed elevated leaf respiration in the dark and the light, although photosynthetic capacity was not affected. Tomaz et al. (2010) proposed that MMDH uses NADH to reduce OAA to malate, which is then shuttled to the cytosol, rather than generate NADH to fuel mitochondrial respiration (Tomaz et al., 2010). PMDH might serve at least two different functions. First, during fatty acid β-oxidation, which generates NADH, PMDH is proposed to regenerate the electron acceptor NAD by reducing OAA to malate, which is then shuttled to the cytosol in exchange for OAA (Pracharoenwattana et al., 2007). Second, PMDH is thought to generate NADH during photorespiration by oxidation of malate imported from the cytosol (Reumann and Weber, 2006; Cousins et al., 2008). Arabidopsis mutants lacking PMDH (pmdh1 pmdh2) are severely impaired in β-oxidation, and seedling establishment is strongly impaired and dependent on the supply of exogenous sugar (Pracharoenwattana et al., 2007), a phenotype characteristic of β-oxidation mutants (Pinfield-Wells et al., 2005; Baker et al., 2006). However, after transfer of established pmdh1 pmdh2 seedlings to compost, they grew only slightly slower than wild-type plants (Pracharoenwattana et al., 2007).Until recently, genetic evidence for the roles of the plastidial MDH isoforms was scarce. In most C₄ plants, NADP-MDH is directly involved in CO₂ fixation, catalyzing the formation of the stable CO₂ carrier malate from the primary CO₂ fixation product OAA (Scheibe, 1987). However, in C₃ plants, NADP-MDH has long been proposed to have its major function in the malate valve, leading to shuttling of reducing power (as malate) from the chloroplast to the cytosol during the day and thereby regenerating the electron acceptor NADP inside the chloroplasts (Heber, 1974; Lance and Rustin, 1984; Scheibe, 1987). NADP-MDH is redox activated by thioredoxins in the light and essentially inactive in the dark (Scheibe, 1987; Buchanan and Balmer, 2005). The widely accepted belief that chloroplasts only possess this one strictly light-/redox-activated NADP-MDH temporarily led to the conclusion that the malate valve only works in illuminated chloroplasts (Berkemeyer et al., 1998; Scheibe, 2004). However, a recent study showed that Arabidopsis plants lacking NADP-MDH (nadp-mdh) were indistinguishable from wild-type plants, even under conditions that are supposed to provoke the accumulation of excess electrons and the production of cytotoxic reactive oxygen species (high light and short days; Hebbelmann et al., 2012). This finding indicates that NADP-MDH is not crucial for providing electron acceptors in chloroplasts, but it rather suggests that other mechanisms can counteract or prevent overreduction of the chloroplast.The existence of a second MDH isoform in plastids, which uses NAD as cofactor, has been questioned, because it could not be ruled out that NAD-MDH activity detected in isolated chloroplasts was caused by contamination from other organelles (Siebke et al., 1991; Backhausen et al., 1998). In 1998, Berkemeyer et al. (1998) reported the cloning, heterologous expression, and in vitro characterization of a pdNAD-MDH from Arabidopsis (At3g47520). In contrast to NADP-MDH, pdNAD-MDH is active under both light and dark conditions in isolated chloroplasts, and the activities of both enzymes are within the same range in the light (Backhausen et al., 1998; Berkemeyer et al., 1998). However, up to now, genetic evidence for the in vivo function of pdNAD-MDH is missing, and experimental data are scarce. Backhausen et al. (1998) showed that chloroplasts and heterotrophic chromoplasts isolated from different sources followed by incubation in the dark concomitantly produced 3-phosphoglycerate and malate on addition of dihydroxyacetone phosphate and OAA to the medium. It was proposed that 3-phosphoglycerate production was in a glycolytic step involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and that pdNAD-MDH regenerates the electron acceptor NAD required by GAPDH through reduction of OAA to malate, thus operating the malate-OAA shuttle in the dark and in nongreen tissues (Scheibe, 2004; Taniguchi and Miyake, 2012).Here, we aimed to evaluate the function of this MDH isoform in plastid metabolism by analyzing Arabidopsis plants with a transposon insertion in the pdNAD-MDH gene and Arabidopsis plants with reduced pdNAD-MDH by means of artificial microRNA silencing.     

Disruption of ptLPD1 or ptLPD2, Genes That Encode Isoforms of the Plastidial Lipoamide Dehydrogenase,Confers Arsenate Hypersensitivity in Arabidopsis

Arsenic is a ubiquitous environmental poison that inhibits root elongation and seed germination to a variable extent depending on the plant species. To understand the molecular mechanisms of arsenic resistance, a genetic screen was developed to isolate arsenate overly sensitive (aos) mutants from an activation-tagged Arabidopsis (Arabidopsis thaliana) population. Three aos mutants were isolated, and the phenotype of each was demonstrated to be due to an identical disruption of plastidial LIPOAMIDE DEHYDROGENASE1 (ptLPD1), a gene that encodes one of the two E3 isoforms found in the plastidial pyruvate dehydrogenase complex. In the presence of arsenate, ptlpd1-1 plants exhibited reduced root and shoot growth and enhanced anthocyanin accumulation compared with wild-type plants. The ptlpd1-1 plants accumulated the same amount of arsenic as wild-type plants, indicating that the aos phenotype was not due to increased arsenate in the tissues but to an increase in the innate sensitivity to the poison. Interestingly, a ptlpd1-4 knockdown allele produced a partial aos phenotype. Two loss-of-function alleles of ptLPD2 in Arabidopsis also caused elevated arsenate sensitivity, but the sensitivity was less pronounced than for the ptlpd1 mutants. Moreover, both the ptlpd1 and ptlpd2 mutants were more sensitive to arsenite than wild-type plants, and the LPD activity in isolated chloroplasts from wild-type plants was sensitive to arsenite but not arsenate. These findings show that the ptLPD isoforms are critical in vivo determinants of arsenite-mediated arsenic sensitivity in Arabidopsis and possible strategic targets for increasing arsenic tolerance.Arsenic (As) is a naturally occurring metalloid found in soil, water, and air, but anthropogenic activities, including smelting and fossil fuel combustion, have led to increased environmental exposure (Mandal and Suzuki, 2002). In the environment, As exists in both organic and inorganic forms. Arsenate [As(V)] is the principal inorganic form of As in aerobic soils, while arsenite [As(III)] is the main form found under anaerobic conditions (Marin et al., 1993; Onken and Hossner, 1995, 1996; Mandal and Suzuki, 2002; Masscheleyn et al., 2002).Both As(V) and As(III) are toxic to plants, inducing symptoms ranging from poor seed germination and inhibited root growth to death (Meharg and Hartley-Whitaker, 2002; Lee et al., 2003; Ahsan et al., 2008; Smith et al., 2010). The modes of action of As(V) and As(III) differ, owing to their distinct chemical properties. As(V), with its structural similarity to phosphate, can compete with phosphate in oxidative phosphorylation, leading to the production of ADP-As(V) (Gresser, 1981). However, half-maximal stimulation of ADP-As(V) formation requires physiologically unlikely concentrations of approximately 0.8 mm As(V) (Moore et al., 1983). As(V) has been recently shown to enhance membrane fluidity, and thus membrane permeability, by binding and replacing phosphate or choline head groups (Tuan et al., 2008). The resulting damage to the membrane would disrupt the transport of mineral nutrients and water (Smith et al., 2010). As(V) can be promptly reduced in plants, including Arabidopsis (Arabidopsis thaliana), to As(III) by endogenous As(V) reductases, so that often more than 90% of As in plant cells is in the form of As(III) (Zhao et al., 2009). As(III) readily forms covalent bonds with sulfhydryl groups, especially vicinal dithiols. Binding to the free thiols of proteins is believed to be the basis of As(III) toxicity, either by inhibiting activity directly or by disrupting protein structure. Many enzymes have been proposed to be targets leading to As(III) toxicity, and the As(III) sensitivity of some of these enzymes has been investigated in nonplant systems (Adamson and Stevenson, 1981; Cavigelli et al., 1996; Lynn et al., 1997; Hu et al., 1998; Kitchin and Wallace, 2008). Of the many potential protein targets, only the pyruvate dehydrogenase complex (PDC) has been shown to be inactivated by physiologically relevant micromolar concentrations of As(III) (Hu et al., 1998), suggesting that PDC may be the primary target for As(III)-mediated cytotoxicity. However, little is known about the mechanism of As toxicity in vivo, especially in plants.Although As is phytotoxic, some plants species are resistant to high levels of As through avoidance mechanisms, while species of the Pteridaceae family of ferns hyperaccumulate As without toxic effects (Verbruggen et al., 2009; Zhao et al., 2009). As an analog of phosphate, As(V) is readily taken up by plants through high-affinity phosphate transporters encoded by the PHOSPHATE TRANSPORTER1 (PHT1) gene family (Shin et al., 2004; González et al., 2005; Catarecha et al., 2007). Except for the hyperaccumulating ferns, avoidance of As toxicity by resistant species is often accomplished by a decrease in phosphate uptake activity (Meharg and Hartley-Whitaker, 2002). Unlike As(V), the transport of As(III) is facilitated by aquaporin nodulin 26-like intrinsic proteins (Bienert et al., 2008; Isayenkov and Maathuis, 2008; Ma et al., 2008; Kamiya et al., 2009). In roots and fronds of hyperaccumulating ferns, As(III) is sequestered in the vacuole (Lombi et al., 2002; Pickering et al., 2006). Much of the As(III) taken up by nonaccumulating resistant species may be released back to the rhizosphere through an undefined efflux pathway (Zhao et al., 2009). As(III) that remains in tissues reacts with thiol-containing molecules, such as glutathione or phytochelatins, both of which are usually produced in greater abundance in response to As (Grill et al., 1987; Sneller et al., 1999; Schmöger et al., 2000; Schulz et al., 2008). As(III)-glutathione adducts can be sequestered in the vacuole (Dhankher et al., 2002; Bleeker et al., 2006). However, increased synthesis of glutathione or phytochelatins alone is unlikely to confer a very high level of tolerance (Zhao et al., 2009).To identify genes essential for As resistance in plants, we used a genetic screen to identify mutants of Arabidopsis that were hypersensitive to As(V). The screen was analogous to that used to isolate the salt overly sensitive (sos) mutants of Arabidopsis (Wu et al., 1996) that led to the identification of the SOS pathway for salt tolerance (Zhu, 2000, 2003). Our hypothesis was that arsenate overly sensitive (aos) mutants would reveal a different set of genes from those identified in mutants showing increased resistance to As(V).     

Discovery of Novel Allosteric Effectors Based on the Predicted Allosteric Sites for Escherichia coli D-3-Phosphoglycerate Dehydrogenase

D-3-phosphoglycerate dehydrogenase (PGDH) from Escherichia coli catalyzes the first critical step in serine biosynthesis, and can be allosterically inhibited by serine. In a previous study, we developed a computational method for allosteric site prediction using a coarse-grained two-state Gō Model and perturbation. Two potential allosteric sites were predicted for E. coli PGDH, one close to the active site and the nucleotide binding site (Site I) and the other near the regulatory domain (Site II). In the present study, we discovered allosteric inhibitors and activators based on site I, using a high-throughput virtual screen, and followed by using surface plasmon resonance (SPR) to eliminate false positives. Compounds 1 and 2 demonstrated a low-concentration activation and high-concentration inhibition phenomenon, with IC₅₀ values of 34.8 and 58.0 µM in enzymatic bioassays, respectively, comparable to that of the endogenous allosteric effector, L-serine. For its activation activity, compound 2 exhibited an AC₅₀ value of 34.7 nM. The novel allosteric site discovered in PGDH was L-serine- and substrate-independent. Enzyme kinetics studies showed that these compounds influenced K_m, k_cat, and k_cat/K_m. We have also performed structure-activity relationship studies to discover high potency allosteric effectors. Compound 2-2, an analog of compound 2, showed the best in vitro activity with an IC₅₀ of 22.3 µM. Compounds targeting this site can be used as new chemical probes to study metabolic regulation in E. coli. Our study not only identified a novel allosteric site and effectors for PGDH, but also provided a general strategy for designing new regulators for metabolic enzymes.     

A Plastidial Localization and Origin of l-Glutamate Dehydrogenase in a Soybean Cell Culture

The subcellular distribution of l-glutamate dehydrogenase (GDH, EC 1.4.1.3.) was studied in SB3 soybean (Glycine max) cells using subcellular fractionation techniques. Compounds that inhibit protein synthesis either on 80s or 70s ribosomes were also used to give a preliminary idea of which subcellular fraction is involved in GDH synthesis. It was found that whereas cycloheximide and puromycin considerably reduced the total amount of protein synthesized by the cells, they did not appear to inhibit the synthesis of GDH. In the presence of chloramphenicol, both GDH activity and protein level in the cells were considerably reduced, suggesting that this enzyme was synthesized in organelles and not in the cytosol. Streptomycin, which inhibits plastid protein synthesis, also inhibited synthesis of GDH, indicating that a fraction of GDH activity was plastidial in origin. This is supported by the data on subcellular distribution of the enzyme, which showed that a major fraction of GDH is found in the plastidial fraction, although some activity is found associated with the mitochondrial fraction also. Since a major fraction of GDH activity was found in the plastidial fraction, we studied protein synthesis using isolated plastids and ³⁵S-methionine. Using antibodies raised against purified GDH, we identified a ³⁵S-labeled 41-kilodalton polypeptide synthesized by plastids as GDH.     

F-box蛋白家族的功能研究进展

F-box蛋白是一类含有F-box基序(motif),在泛素介导的蛋白质水解过程中具有底物识别特性的蛋白质家族.这类蛋白质在细胞时相转换、信号传导、发育等多种生理过程中都具有重要功能.     

Characterization of an HSP70 Cognate Gene Family in Arabidopsis

Analysis of the polypeptide composition of extracts from heat-shocked leaves of Arabidopsis indicated the presence of at least 12 HSP70-related polypeptides, most of which were constitutively expressed. In vitro translation of mRNA from heat-shocked and control leaves indicated that the amount of mRNA encoding four HSP70 polypeptides was increased strongly by heat-shock. Three Arabidopsis genes which exhibit homology to a Drosophila HSP70 gene were cloned. Two of the three genes are arranged in direct orientation approximately 1.5 kilobases apart. The third gene is not closely linked to the other two. Nucleotide sequence analysis of the 5′ regions of the two linked genes revealed that both contain a TATA box, the CAAT motif, and several short sequences which are homologous to the Drosophila heat-shock consensus sequence. The deduced partial amino acid sequence of the open reading frames were 79 and 72% homologous to the corresponding regions of the Drosophila HSP70-cognate and HSP70 sequences, respectively. As with the two maize HSP70 genes which have been characterized, and the Drosophila HSP70-cognate genes, the Arabidopsis genes contained a putative intron in the codon specifying amino acid 72. Analysis of mRNA levels with gene-specific oligonucleotide probes indicated that two of the genes were not expressed or were expressed at very low levels in leaves during normal growth or after heat-shock, whereas the other gene was constitutively expressed. By analogy with the results of similar studies of other organisms, it appears that the three cloned genes are members of a small family which are most closely related to the HSP70-cognate genes found in other species.     

Functional Characterization of cis-Elements Conferring Vascular Vein Expression of At4g34880 Amidase Family Protein Gene in Arabidopsis

The expression of At4g34880 gene encoding amidase in Arabidopsis was characterized in this study. A promoter region of 1.5 kb on the upstream of the start codon of the gene (referred as AmidP) was fused with uidA (GUS) reporter gene, and transformed into Arabidopsis plant for determining its spatial expression. The results indicated that AmidP drived GUS expression in vascular system, predominately in leaves. Truncation analysis of AmidP demonstrated that VASCULAR VEIN ELEMENT (VVE) motif with a region of 176 bp sequence (−1500 to −1324) was necessary and sufficient to direct the vascular vein specific GUS expression in the transgenic plant. Tandem copy of VVE increased vascular system expression, and 5′- and 3′- deletions of VVE motif in combination with a truncated −65 CaMV 35S minimal promoter showed that 11bp cis-acting element, naming DOF2 domain, played an essential role for the vascular vein specific expression. Meanwhile, it was also observed that the other cis-acting elements among the VVE region are also associated with specificity or strength of GUS activities in vascular system.     

拟南芥RALF多肽家族的功能多样性初步分析

绿色植物中的快速碱化因子(Rapid alkalinization factor,RALF)为一类进化保守的多肽信号分子,以基因家族形式存在。模式植物拟南芥中至少存在35个RALF基因成员,前期研究显示拟南芥RALF家族的部分成员,比如RALF1/23,RALF4/19可分别作为Cr RLK1L类蛋白受体激酶家族成员FERONIA及BUPS1/2的配体,调控细胞伸长、植物免疫应答及双受精等过程,但是RALF家族其他成员是否具有生物学活性,以及不同成员之间是否具有功能性差异均尚不清楚。因此,本研究异源表达了19个代表性的RALF,并对其生物学活性和功能性差异进行了分析。实验结果表明,19个RALF均对根的生长起到不同程度的抑制作用,进一步挑选了部分代表性RALF成员进行了活性氧(Reactive oxygen species,ROS)迸发及丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)磷酸化实验分析,实验结果表明,我们确证了11个RALF蛋白参与了MAPK信号的响应,同时,证实16个RALF蛋白抑制了由flg22引起的ROS的释放。此外,不同RALF成员在下胚轴细胞伸长上的作用也存在明显差异,比如RALF10促进下胚轴的伸长。以上研究结果表明不同RALF之间既存在功能冗余性,又存在功能性差异。本研究丰富了对RALF功能复杂性和多样性的认识。     

拟南芥AtMGT3基因转运功能研究

对拟南芥AtMGT3基因的M矿转运功能进行了初步研究．AtMG73转录本的半定量分析表明。AtMG73在根、茎、叶、花、角果中均有表达,但在花中表达量最高,根中最少．MM281功能互补和液体生长曲线结果表明：At-MGT3功能互补细菌Mg^2＋转运突变株,具有Mg^2＋转运能力;AtMGT3介导Mg^2＋的低亲和性吸收,是一个低亲和性Mg^2＋转运蛋白;AtMGT3可能还能转运Fe^2＋,但转运Fe^2＋浓度超出了正常的生理浓度．在正常生理条件下．AtMGT3的主要生理功能是作为Mg2＋转运蛋白起作用．     

Functional Characterization of Phospholipid N-Methyltransferases from Arabidopsis and Soybean

Phospholipid N-methyltransferase (PLMT) enzymes catalyze the S-adenosylmethionine-dependent methylation of ethanolamine-containing phospholipids to produce the abundant membrane lipid phosphatidylcholine (PtdCho). In mammals and yeast, PLMT activities are required for the de novo synthesis of the choline headgroup found in PtdCho. PLMT enzyme activities have also been reported in plants, yet their roles in PtdCho biosynthesis are less clear because most plants can produce the choline headgroup entirely via soluble substrates, initiated by the methylation of free ethanolamine-phosphate. To gain further insights into the function of PLMT enzymes in plants, we isolated PLMT cDNAs from Arabidopsis and soybean (Glycine max) based upon primary amino acid sequence homology to the rat PLMT, phosphatidylethanolamine N-methyltransferase. Using a heterologous yeast expression system, it was shown that plant PLMTs methylate phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine but cannot utilize phosphatidylethanolamine as a substrate. Identification of an Arabidopsis line containing a knock-out dissociator transposon insertion within the single copy AtPLMT gene allowed us to investigate the consequences of loss of PLMT function. Although the accumulation of the PLMT substrates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine was considerably elevated in the atplmt knock-out line, PtdCho levels remained normal, and no obvious differences were observed in plant morphology or development under standard growth conditions. However, because the metabolic routes through which PtdCho is synthesized in plants vary greatly among differing species, it is predicted that the degree with which PtdCho synthesis is dependent upon PLMT activities will also vary widely throughout the plant kingdom.Phosphatidylcholine (PtdCho)2 is the most abundant phospholipid in most non-plastid membranes of eukaryotes. PtdCho biosynthesis has been studied intensively in plants not only because of its importance as a structural membrane lipid, but also because of its role as a precursor to important lipid-based signaling molecules, such as phosphatidic acid, and phospholipase A₂-derived free fatty acids (1). The choline headgroup of PtdCho serves multiple functions as well. In addition to being an essential human nutrient (2), in many plant species choline can be oxidized to produce the potent osmoprotectant glycine betaine (3, 4).For over 2 decades it has been apparent that there are fundamental differences between the manner in which PtdCho is produced in plants versus how it is synthesized in mammals and fungi. In the latter two systems, PtdCho can be formed through two distinct pathways as follows: (a) the “nucleotide pathway” in which free choline is incorporated in PtdCho using CDP-choline as an intermediate, and (b) the “methylation pathway” whereby PtdCho is produced directly from phosphatidylethanolamine (PtdEtn) via three sequential methylation reactions using S-adenosylmethionine (AdoMet) as the methyl donor (5, 6). In contrast, PtdCho biosynthesis in plants occurs through a branched pathway that utilizes components of both the nucleotide and methylation pathways (7). The greatest distinction between the contrasting mechanisms of PtdCho biosynthesis can be attributed to the presence of plant enzymes that are capable of converting ethanolamine headgroups to choline at the phospho-base level, activities that are absent in mammals and yeast. Conversely, mammals and fungi possess methylation enzymes that act directly on PtdEtn, a reaction that cannot be detected in most plant systems investigated (reviewed in Ref. 7).A diagram of the most widely accepted model of phosphoamino alcohol biosynthesis in plants is shown in Fig. 1. Similar to animals and yeast, free choline can be directly incorporated into PtdCho via nucleotide pathway enzymes in plants. In the absence of choline, however, the methylation of Etn-phosphate represents the first committed step in PtdCho biosynthesis. The resulting monomethylethanolamine-phosphate (MMEtn-P) metabolite can be further methylated at the phospho-base level to produce Cho-P. Alternatively, MMEtn-P can be incorporated into phosphatidylmonomethylethanolamine (PtdMMEtn) via the cytidylyltransferase and amino alcohol phosphotransferase activities of the nucleotide pathway and then methylated at the phosphatidyl-base level to complete the synthesis of PtdCho (Fig. 1). The extent with which PtdCho is formed by the flow of metabolites through phospho-bases as opposed to phosphatidyl-bases varies greatly among different plant species. In most higher plants, it is likely that the methylation of the phosphoamino alcohol headgroups involves the flow of metabolites through both branches of the pathway, as has been shown in species such as barley, carrot, and tobacco (3, 8, 9). Nevertheless, examples have also been reported where only one of the branches appears to be utilized. In Lemna paucicostata, for example, the methylation steps in PtdCho biosynthesis were shown to occur almost exclusively at the phospho-base level (10). At the other end of the spectrum is soybean, where all methylations beyond the initial formation of MMEtn-P were reported to occur on phosphatidyl-bases (8, 11). The tremendous variability observed among plants with regard to PtdCho formation is also exemplified by a study conducted by Williams and Harwood (12) where it was shown that the predominant route of PtdCho synthesis in olive culture cells involved the first two methylation reactions taking place at the phospho-base level (producing dimethylethanolamine phosphate) and the final methylation occurring on a phosphatidyldimethylethanolamine (PtdDMEtn) substrate.Open in a separate windowFIGURE 1.Phosphatidylcholine biosynthetic pathways. Steps common to plants, mammals, and yeast are indicated by black arrows. Dashed arrows indicate pathways specific to plants. Methylation of PtdEtn, which occurs in mammals and yeast, is indicated by on open arrow. Enzymes catalyzing phosphoamino alcohol methylation reactions in plants, mammals, and yeast are indicated.Our understanding on the mechanisms by which plants synthesize PtdCho and regulate its accumulation has been further enhanced as the genes encoding the various steps of the phosphoamino alcohol pathway have been isolated and characterized. For example, molecular characterizations led to the conclusion that all of the amino alcohol phosphotransferase reactions depicted in Fig. 1 can be mediated by the product of a single gene (designated AAPT1) that displays a broad substrate specificity (13, 14). Similarly, it was the isolation of the phosphoethanolamine methyltransferase (PEAMT) genes from Arabidopsis and spinach that led to the discovery that all three phospho-base methylation reactions could be catalyzed by a single enzyme (15, 16). Inhibition of PEAMT gene function in Arabidopsis through T-DNA insertion or co-suppression revealed unexpected associations between the phosphoamino alcohol pathway and root development, salt hypersensitivity, and male sterility (17, 18).Although most of the reactions depicted in Fig. 1 have been characterized at the molecular genetic level, conspicuously absent is information on the plant genes/enzymes responsible for the methylation reactions conducted at the phosphatidyl-base level. In contrast, these reactions are among the most well characterized in animals and yeast, catalyzed by enzymes commonly referred to as phospholipid N-methyltransferases (PLMTs). In mammals, the 18-kDa integral membrane protein phosphatidylethanolamine N-methyltransferase (PEMT) is a PLMT that is expressed primarily in the liver (19). PEMT catalyzes all three of the methylation reactions needed to convert PtdEtn to PtdCho. Yeast uses two distinct PLMT enzymes to catalyze the three methylation reactions as follows: Cho2p/Pem1p that mediates the direct methylation of PtdEtn to produce PtdMMEtn (20, 21), and Opi3p/Pem2p, an enzyme homologous to the mammalian PEMT, that primarily catalyzes the methylation of PtdMMEtn to PtdDMEtn and PtdDMEtn to PtdCho, the final two steps of the methylation pathway (20, 22). PLMT activities are critical in both of these systems. Mice possessing pemt knock-out mutations are completely dependent on dietary choline for survival, and they display abnormal levels of choline metabolites within the liver and develop hepatic steatosis even when fed diets supplemented with choline (23). Yeast lacking PLMT activities (cho2/opi3 double mutants) are obligate choline auxotrophs, unable to synthesize PtdCho de novo in the absence of exogenous choline.To gain a greater understanding of the specific function of PLMT reactions in higher plants, and their contribution toward PtdCho biosynthesis, we cloned and characterized PLMT homologs from Arabidopsis and soybean. By expressing the candidate cDNAs in yeast, we were able to confirm that they encoded functional PLMT activities as well as to establish their substrate specificities. We also identified a mutant Arabidopsis line containing a knock-out allele in the single copy PLMT gene found in the Arabidopsis genome, allowing us to characterize the consequences of loss of gene function in this model species.     

拟南芥Rab1家族基因的克隆和功能研究

Yptl蛋白是酵母唯一的Rab1 GTP酶,调控囊泡从内质网到高尔基体的运输.酵母温敏突变株 ASY01是一个Ypt1基因功能部分缺失菌株,在26℃可以正常生长,但在37℃不能生长.拟南芥有4个Rab1基因,分别是AtRab1A1、AtRab1B1、AtRab1B2、AtRab1C1.克隆了所有4个AtRab1基因,构建酵母表达载体,转化温敏突变型酵母ASY01.温度敏感性实验结果表明,所有转基因菌株在37℃都恢复正常生长.说明拟南芥4个Rab1基因都与酵母Ypt1基因功能互补,都具有调节囊泡从内质网到高尔基体运输的功能.