Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K_D values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.     

Structural and Functional Studies of the Mitochondrial Cysteine Desulfurase from Arabidopsis thaliana

AtNfs1 is the Arabidopsis thaliana mitochondrial homolog of the bacterial cysteine desulfurases NifS and IscS, having an essential role in cellular Fe-S cluster assembly. Homology modeling of AtNfs1m predicts a high global similarity with E. coli IscS showing a full conservation of residues involved in the catalytic site, whereas the chloroplastic AtNfs2 is more similar to the Synechocystis sp. SufS. Pull-down assays showed that the recombinant mature form, AtNfs1m, specifically binds to Arabidopsis frataxin (AtFH). A hysteretic behavior, with a lag phase of several minutes, was observed and hysteretic parameters were affected by pre-incubation with AtFH. Moreover, AtFH modulates AtNfs1m kinetics, increasing V (max) and decreasing the S (0.5) value for cysteine. Results suggest that AtFH plays an important role in the early steps of Fe-S cluster formation by regulating AtNfs1 activity in plant mitochondria.     

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.     

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.     

The peroxisomal NAD+ carrier of Arabidopsis thaliana transports coenzyme A and its derivatives

The peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 has very recently been found to transport NAD+, NADH, AMP and ADP. In this work we have reinvestigated the substrate specificity and the transport properties of PXN by using a wide range of potential substrates. Heterologous expression in bacteria followed by purification, reconstitution in liposomes, and uptake and efflux experiments revealed that PNX transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3', 5'-phosphate (PAP), besides NAD+, NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport and was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline and tannic acid. Transport was saturable with a submillimolar affinity for NAD+, CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD+ and CoA.     

Molecular Cloning and Characterization of a Calmodulin Gene from Arabidopsis thaliana

The calcium binding protein, calmodulin Is involved in regulating various cellular and biochemical processes. A gene tor calmodulin (CaM) has been Isolated from a genomic library of Arabidopsis thaliana constructed in ; EMBL-4 using a heterologous cDNA probe from electric eel. One of the positive clones was characterized and the region containing the calmodulin gene sequences was Identified, excised using appropriate restriction enzymes and subcloned Into a plasmid vector. The genomic clone contains a complete copy of the calmodulin gene. A comparison of the nucleotide sequence of the part of the clone with those of the other plant and animal systems confirms that the clone In fact contains the calmodulin gene sequences. Southern hybridization ulling the calmodulin gene sequences as a probe reveals the presence of more than one copy of the calmodulin gene. The results of this investigation taken together with those Iff the other. indicate that the calmodulin gene belongs to a small mutigene family consisting of atieast four member. In the Arabidopsis genome.     

The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in Arabidopsis thaliana

Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD⁺ biosynthesis is exclusively cytosolic. Hence, NAD⁺ must be imported into organelles to support their metabolic functions. Three NAD⁺ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T‐DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD⁺ homeostasis for both metabolic and developmental processes in plants.     

拟南芥LecRK-ConA基因突变体的筛选鉴定及其功能初步研究

植物凝集素类受体激酶(lectin receptor-like kinases,LecRLKs)在植物抗逆性、激素信号转导及生长发育调节等方面扮演重要角色。LecRK-ConA基因是拟南芥Lec RLK基因家族中的一员。利用三引物法鉴定获得LecRK-ConA基因两个缺失突变体的纯合子。启动子顺式作用元件分析显示,LecRK-ConA具有多个与干旱、光、赤霉素(gibberellic acid,GA)等应答相关的元件。实时定量PCR结果表明:LecRK-ConA基因具有组织表达差异性,且在春化后4 d的种子中表达量最强,用NaCl和甘露醇处理均能抑制该基因的表达。用NaCl和甘露醇处理缺失突变体lecrk-cona和Col-0后,发现lecrk-cona的种子萌发率显著低于野生型。此外,突变体lecrkcona相对野生型Col-0来说,具有早花现象。上述实验结果为进一步深入研究LecRK-ConA基因在抗逆与开花调控途径中的生理功能奠定了基础。     

The Partial Characterization of the Major Seed Storage Proteins in Arabidopsis thaliana L.

This study was aimed at the characterization of the major storage proteins in Arabidopsis thaliana. Two major protein fractions, i.e., the fraction Ⅰ and Ⅱ proteins, were isolated from the extract of mature seeds of this plant by molecular seive gel filtration chromatography. Various polyacrylarnide gel electrophoretic techniques were used to study the properties and polypeptide compositions of these two protein fractions. In was shown that during the SDS gel electrophoresis, fraction Ⅰ protein was separated into 6 major bands with the mol. was. of 34, 31, 29, 28 and 19-20 kD, respectively, whereas Fraction Ⅱ protein migrated as 3 low mol. wt. bands (10-12 kD) on the same gel. Non-denaturing native gel electrophoresis revealed that fraction Ⅰ was a neutral protein and Fraction Ⅱ was a positively charged basic protein with an isoelectric point (pI) higher than 8.8. Fraction I protein was further separated into at least 16 polypeptides in isoelectric focusing/SDS two-dimensional gel electrophoresis, i.e. each SDS band contained 3-4 polypeptides with the same mol. wt. but different pis. This suggested a more complex polypeptide composition of this protein. The properties of fraction Ⅰ and Ⅱ proteins were in good accordance with that of the 12s and 1.7s storage globulins in seeds of many other dicotyledonous plants, and therefore had been characterized as the two major seed storage proteins in this species. These two storage globulins were shown to be accumulated within a defined period during the late stage of seed development (12-14 DAF) and became predominant protein components in mature seeds. In the mean time, a few points in relation to the polypeptide composition and subunit molecular configuration of the 12s globulin were noted.     

Mitochondrial localization of Arabidopsis thaliana Isu Fe-S scaffold proteins

Isu are scaffold proteins involved in iron-sulfur cluster biogenesis and playing a key role in yeast mitochondria and Escherichia coli. In this work, we have characterized the Arabidopsis thaliana Isu gene family. AtIsu1,2,3 genes encode polypeptides closely related to their bacterial and eukaryotic counterparts. AtIsu expression in a Saccharomyces cerevisiae Deltaisu1Deltanfu1 thermosensitive mutant led to the growth restoration of this strain at 37 degrees C. Using Isu-GFP fusions expressed in leaf protoplasts and immunodetection in organelle extracts, we have shown that Arabidopsis Isu proteins are located only into mitochondria, supporting the existence of an Isu-independent Fe-S assembly machinery in plant plastids.     

Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection

The cell''s endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system.Membrane compartmentalization is an essential mechanism for eukaryotic life, by which cells separate and control biological processes. Plant growth, development, and adaptation to biotic and abiotic stress all rely on the highly dynamic endomembrane system, yet we know comparatively little about the proteins regulating these dynamic trafficking events. The plasma membrane (PM) provides the main interface between the cell and its environment, mediating the transfer of material to and from the cell and is a primary site for perception of external signals. Transmembrane proteins are synthesized in the endoplasmic reticulum (ER) and trafficked to the PM via the Golgi, although there are other secretory routes for soluble cargo (discussed in (1–4)). Post-Golgi trafficking is the main route by which newly synthesized transmembrane proteins and cell wall glycans are delivered to the PM. In plants, secretory and endocytic traffic converge at the trans-Golgi network (TGN), which also functions as an early endosome (EE). Multivesicular bodies (MVBs) are the other main endosomal compartment in plants and serve as prevacuolar compartments (PVCs) or late endosomes (LE) destined for vacuolar degradation (reviewed (1, 5, 6)).Recycling and sorting of plasma membrane proteins is essential for generating the polar localization of auxin efflux transporters (discussed in (7)), formation of the cell plate during cell division (8–11), and in defense such as localized deposition of papilla reviewed in (12, 13). Furthermore, the subcellular localization of transporters and receptors is dynamically regulated. For example, the boron transporter (BOR1) exhibits polar localization and is internalized and degraded under conditions of high boron to reduce toxicity (14, 15). Similarly the receptor-like kinases (RLKs) flagellin-sensing 2 (FLS2) and brassinosteroid insensitive 1 (BRI1), important transmembrane receptors in antibacterial immunity and plant development, respectively, are constitutively endocytosed and recycled to the PM (16–18). Both receptors and transporters are also cargoes of the LE/MVB trafficking route (16) and are probably sorted to the vacuole for degradation (19, 20). Importantly, FLS2 trafficking via the recycling endocytic or the late endocytic route depends on its activation status; inactive receptors are recycled while ligand-activated receptors are sorted to the late endosomal pathway (16). Similarly, the polar sorting of auxin efflux transporters depends on their phosphorylation status (21). These observations illustrate that membrane compartmentalization underpins important aspects of plant cell biology and has initiated a quest toward a better understanding of the endomembrane compartments and the routes and mechanisms by which cargo is trafficked and sorted within the cell.Membrane trafficking within the cell requires complex machinery consisting of a plethora of coat and adaptor proteins, small GTPases, targeting, tethering, and scission factors (reviewed in (22, 23)). Homologues of some animal and yeast and endomembrane regulators have been identified in plants, but the localization and function of many of these remain to be characterized. For example, members of the RAB GTPase family have been shown to have markedly different roles and localizations in plants compared with their animal and yeast homologs (24). Therefore, acquiring localization data for tethering complexes and other regulators in plant systems is essential. In Arabidopsis thaliana, some of these proteins have been developed as useful probes to visualize the different endomembrane compartments by fusion with fluorescent reporters (9, 25–27). These include regulators of trafficking events such as RAB GTPases that are molecular switches responsible for the assembly of tethering and docking complexes and compartment identity. RAB proteins are widely used markers of endomembrane compartments, for example RABD2a/ARA5 labels the Golgi and TGN/EE as well as post-Golgi vesicles (4, 24, 26, 28), RABF2b/ARA7 localizes to TGN/EE and LE (25), RABF1/ARA6 is a marker of the LE/MVB vesicles (25, 29), and RABG3f localizes to MVBs and the tonoplast (26, 30).Fluorescent-tagged marker lines for the live-cell imaging of plant cells have been invaluable in defining the location of proteins within and between organelles and endomembrane compartments (26). However, microscopic investigation of membrane trafficking is limited by throughput, as only few proteins can be studied simultaneously. A powerful approach to large-scale identification of proteins in endomembrane compartments is through subcellular fractionation based on physical properties to directly isolate or enrich for the subcellular compartment of interest. Subcellular fractionation-based proteomics have been successfully used to decipher the steady state and cargo proteomes of, including but not limited to, the ER, the vacuole, PM, mitochondria and chloroplasts, and smaller vesicle-like compartments such as peroxisomes and Golgi (31–41). However, the smaller, transitory vesicles of the secretory and endocytic pathways have proved challenging to purify for reliable proteomic analysis. To overcome this, affinity purification of vesicles was established in animal cells (42, 43) and recently successfully applied in plants in combination with subcellular fractionation. Affinity purification and mass spectrometry (MS) of syntaxin of plants 61 (SYP61)-positive TGN/EE compartments identified 145 proteins specifically enriched in (44), while affinity isolation of VHA-a1-GFP (vacuolar H⁺ ATPase A1) identified 105 proteins associated with the TGN/EE (45). The VHA-A1 affinity purification data were then further refined using density gradient centrifugation to differentiate cargo and steady-state proteins (45).We have further explored affinity purification of fluorescent-tagged markers localizing to defined compartments to identify proteins associated with trafficking. Our motivation was to dissect the trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with small GTPases in the RAB family. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for Golgi/TGN/EE/secretory vesicles, LE/MVB compartments, LE/MVB compartments and LE/MVB/tonoplast, respectively. Additionally, we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles (CCVs) and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast (26, 27, 29, 46, 47) as comparisons. Our objective was to identify transient cargo proteins, tethers, and docking factors associated with dynamic subdomains of the endomembrane system, to supplement better-characterized “steady-state” components, and to identify components of recycling and vacuolar trafficking pathways.     

Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.     

Characterization of the genome of Arabidopsis thaliana

The small crucifer Arabidopsis thaliana has many useful features as an experimental organism for the study of plant molecular biology. It has a four-week life-cycle, only five chromosomes and a genome size less than half that of Drosophila. To characterize the DNA sequence organization of this plant, we have randomly selected 50 recombinant lambda clones containing inserts with an average length of 12,800 base-pairs and analyzed their content of repetitive and unique DNA by various genome blot, restriction digestion and RNA blot procedures. The following conclusions can be drawn. The DNA represented in this random sample is composed predominantly of single-copy sequences. This presumably reflects the organization of the Arabidopsis genome as a whole and supports prior conclusions reached on the basis of kinetics of DNA reassociation. The DNA that encodes the ribosomal RNAs constitutes the only major class of cloned nuclear repetitive DNA. It consists of approximately 570 tandem copies of a heterogeneous 9900-base-pair repeat unit. There is an average of approximately 660 copies of the chloroplast genome per cell. Therefore, the chloroplast genome constitutes the major component of the repetitive sequences found in A. thaliana DNA made from whole plants. The inner cytosine residue in the sequence C-C-G-G is methylated more often than the outer in the tandem ribosomal DNA units, whereas very few differences in the methylation state of these two cytosine residues are detected in unique sequences.     

Loss of chloroplast-localized NAD kinase causes ROS stress in Arabidopsis thaliana

Journal of Plant Research - Chloroplast-localized NAD kinase (NADK2) is responsible for the production of NADP+, which is an electron acceptor in the linear electron flow of photosynthesis. The...