Identification of ascorbic acid-deficient Arabidopsis thaliana mutants

Vitamin C (l-ascorbic acid) is a potent antioxidant and cellular reductant present at millimolar concentrations in plants. This small molecule has roles in the reduction of prosthetic metal ions, cell wall expansion, cell division, and in the detoxification of reactive oxygen generated by photosynthesis and adverse environmental conditions. However, unlike in animals, the biosynthesis of ascorbic acid (AsA) in plants is only beginning to be unraveled. The previously described AsA-deficient Arabidopsis mutant vtc1 (vitamin c-1) was recently shown to have a defect in GDP-mannose pyrophosphorylase, providing strong evidence for the recently proposed role of GDP-mannose in AsA biosynthesis. To genetically define other AsA biosynthetic loci, we have used a novel AsA assay to isolate four vtc mutants that define three additional VTC loci. We have also isolated a second mutant allele of VTC1. The four loci represented by the vtc mutant collection have been genetically characterized and mapped onto the Arabidopsis genome. The vtc mutants have differing ozone sensitivities. In addition, two of the mutants, vtc2-1 and vtc2-2, have unusually low levels of AsA in the leaf tissue of mature plants.     

Identification and characterization of victorin sensitivity in Arabidopsis thaliana

Cochliobolus victoriae is a necrotrophic fungus that produces a host-selective toxin called victorin. Victorin is considered to be host selective because it has been known to affect only certain allohexaploid oat cultivars containing the dominant Vb gene. Oat cultivars containing Vb are also the only genotypes susceptible to C. victoriae. Assays were developed to screen the "nonhost" plant of C. victoriae, Arabidopsis thaliana, for victorin sensitivity. Sensitivity to victorin was identified in six of 433 bulk populations of Arabidopsis. In crosses of Col-4 (victorin-insensitive) x victorin-sensitive Arabidopsis ecotypes, victorin sensitivity segregated as a single dominant locus, as it does in oats. This Arabidopsis locus was designated LOV, for locus orchestrating victorin effects. Allelism tests indicate that LOV loci are allelic or closely linked in all six victorin-sensitive ecotypes identified. LOV was localized to the north arm of Arabidopsis thaliana chromosome I. The victorin-sensitive Arabidopsis line LOV1 but not the victorin-insensitive line Col-4 was susceptible to C. victoriae infection. Consequently, the LOV gene appears to be a genetically dominant, disease susceptibility gene.     

Identification of new minisatellites loci in Arabidopsis thaliana

In order to characterize new CG-rich minisatellites present in the Arabidopsis thaliana genome, a genomic library was screened at low stringency with a probe containing nine repeated-units of a minisatellite (CMs1) previously identified. Both minisatellites and minisatellite-like elements were identified. The minisatellites, with a tandemly-repeated structure, all contain the Arabidopsis thaliana-core sequence previously defined (Tourmente et al., 1994). Both minisatellite and minisatellite-like sequences occur in the Arabidopsis genome in low copy and are weakly polymorphic between ecotypes. The genetic mapping of these markers has shown that they are dispersed on the genome. YACs clones of the CIC library carrying these minisatellites and minisatellite-like sequences were identified.Key words: Arabidopsis thaliana, minisatellites, polymorphism      

Identification of a copper transporter family in Arabidopsis thaliana

Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes. In this work, a five-member family (COPT1–5) of putative Arabidopsis copper transporters is described. We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant. The specific expression pattern of the Arabidopsis COPT1–5 mRNA in different tissues was analyzed by RT-PCR. Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed. Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S. cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells.     

Identification of Botrytis cinerea susceptibility loci in Arabidopsis thaliana

Botrytis cinerea is a major pathogen of fruit and vegetable crops causing both pre- and post-harvest grey mould. We have analysed 16 Arabidopsis thaliana ecotypes for natural variation in B. cinerea susceptibility. Susceptibility was associated with lower camalexin accumulation, and three ecotypes (Cape Verdi Islands (Cvi-0), Slavice (Sav-0) and Kindalville (Kin-0)) showed differential susceptibility to the two B. cinerea isolates used. Subsequently, to better understand the genetic control of grey mould disease, we assayed the Arabidopsis Landsberg erecta (Ler) x Columbia (Col-0) recombinant inbred population with the two isolates, and identified multiple small-to-medium-effect quantitative trait loci (QTL) governing susceptibility. Interestingly, the QTL for each isolate are distinct, suggesting that different mechanisms govern defence against these two isolates. Two QTL for each isolate exhibited epistatic interactions with specific allele combinations generating heightened B. cinerea susceptibility.     

Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and Saccharomyces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.     

Identification and structural analysis of SINE elements in the Arabidopsis thaliana genome

An insertion sequence was found in a Mu homologue in the genome of Arabidopsis thaliana. The insertion sequence had poly(A) at the 3' end, and promoter motifs (A- and B-boxes) recognized by RNA polymerase III. The sequence was flanked by direct repeats of a 15-bp sequence of the Mu homologue, which appears to be a target-site sequence duplicated upon insertion. These findings indicate that the insertion sequence is a retroposon SINE, and it was therefore named AtSN (A. thaliana SINE). Many members of the AtSN family were identified through a computer-aided homology search of databases and classified into two subfamilies, AtSN1 and AtSN2, having consensus sequences 159 and 149 bp in length, respectively. These had no homology to SINEs in other organisms. About half of AtSN members were truncated through loss of a region at either end of the element. Most of them were truncated at the 5' end, and had a duplication of the target-site sequence. This suggests that the ones with 5' truncation retroposed by the same mechanism as those without truncation. Members of the AtSN1 or AtSN2 subfamilies had many base substitutions when compared with the consensus sequence. All of the members examined were present in three different ecotypes of A. thaliana (Columbia, Landsberg erecta, and Wassilewskija). These findings suggest that AtSN members had proliferatedbefore the A. thaliana ecotype strains diverged.     

Primary Structure and Properties of Glutathione Reductase from Arabidopsis thaliana

A cDNA encoding glutathione reductase (EC 1.6.4.2 [EC] ) from Arabidopsisthaliana was cloned by immunoscreening. The amino acid sequencededuced from the nucleotide sequence agrees with the N-terminalamino acid sequence of the major isozyme (GR II) purified fromleaves of A. thaliana. The predicted polypeptide comprises anN-terminal leader sequence of 74 amino acids, which has featuresof chloroplast-targeting peptides, and a mature polypeptideof 491 residues with a molecular mass of 52.7 kDa, which showshomology with glutathione reductases from other species. TheK_m for GSSG was 44 µM and that for NADPH was 5.0 µMfor GR II at 25C. The pH optimum for GR II was 7.5 to 8.0.The native molecular mass of GR II was 110 kDa, indicating thatGR II is a homodimer. GR II had an isoelectric point of 4.8.The cDNA hybridizes with a 2.1-kb poly(A)⁺RNA from leaves ofA. thaliana. Genomic Southern analysis indicates that the genecorresponding to the cDNA is likely a single-copy gene. (Received July 1, 1993; Accepted September 8, 1993)     

Identification of novel subunits of the TOM complex from Arabidopsis thaliana

The preprotein translocase of the outer mitochondrial membrane (also called TOM complex) from Arabidopsis thaliana was characterized by Blue-native gel electrophoresis (BN-PAGE) and Electrospray Tandem Mass Spectrometry (ESI-MS/MS). BN-PAGE allows to prepare a very stable 390 kDa complex that includes six different protein types: the 34 kDa translocation pore TOM40, the 21/23 kDa preprotein receptor TOM20, the small TOM component TOM7 and three further subunits of 10, 6.3 and 6.0 kDa. Primary structures of all TOM subunits were elucidated. The 10 kDa subunit represents a truncated version of the TOM22 preprotein receptor and the two 6 kDa proteins represent subunits possibly homologous to fungal TOM6 and TOM5, although sequence conservation is at the borderline of significance. TOM40, TOM7 and one or both of the 6 kDa subunits form a subcomplex of about 100 kDa. The six TOM proteins from Arabidopsis are encoded by 12 genes, at least 11 of which are expressed. While the subunit composition of the TOM complex from fungi, animals and plants is remarkably conserved, the domain structure of individual TOM proteins differs, e.g. acidic domains in TOM22 and the 6 kDa TOM subunits from Arabidopsis are absent. The domain structure of the Arabidopsis TOM complex does not support the so-called ‘acid chain hypothesis’, which explains the translocation of proteins across the outer mitochondrial membrane of mitochondria by the binding of preproteins to acidic protein domains within the TOM complex. Functional implications are discussed.     

Identification of Arabidopsis thaliana variants with differential glyphosate responses

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.     

Identification and functional characterization of the BAG protein family in Arabidopsis thaliana

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.     

Major changes in the cell wall during silique development in Arabidopsis thaliana

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.     

Identification and characterization of nuclear pore complex components in Arabidopsis thaliana

The nuclear pore complex (NPC) facilitates nucleocytoplasmic transport, a crucial process for various cellular activities. The NPC comprises ~30 nucleoporins and is well characterized in vertebrates and yeast. However, only eight plant nucleoporins have been identified, and little information is available about the complete molecular structure of plant NPCs. In this study, an interactive proteomic approach was used to identify Arabidopsis thaliana nucleoporins. A series of five cycles of interactive proteomic analysis was performed using green fluorescent protein (GFP)-tagged nucleoporins. The identified nucleoporins were then cloned and subcellular localization analyses were performed. We found that the plant NPC contains at least 30 nucleoporins, 22 of which had not been previously annotated. Surprisingly, plant nucleoporins shared a similar domain organization to their vertebrate (human) and yeast (Saccharomyces cerevisiae) counterparts. Moreover, the plant nucleoporins exhibited higher sequence homology to vertebrate nucleoporins than to yeast nucleoporins. Plant NPCs lacked seven components (NUCLEOPORIN358 [Nup358], Nup188, Nup153, Nup45, Nup37, NUCLEAR DIVISION CYCLE1, and PORE MEMBRANE PROTEIN OF 121 kD) that were present in vertebrate NPCs. However, plants possessed a nucleoporin, Nup136/Nup1, that contained Phe-Gly repeats, and sequence analysis failed to identify a vertebrate homolog for this protein. Interestingly, Nup136-GFP showed greater mobility on the nuclear envelope than did other nucleoporins, and a Nup136/Nup1 deficiency caused various defects in plant development. These findings provide valuable new information about plant NPC structure and function.     

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.     

Identification of functionally important residues of Arabidopsis thaliana S-adenosylmethionine decarboxylase

The Arabidopsis thaliana S-adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank(TM) U63633) was cloned, and the AdoMetDC protein was expressed, purified, and characterized. The K(m) value for S-adenosylmethionine (AdoMet) is 23.1 microM and the K(i) value for methylglyoxal bis-(guanylhydrazone) (MGBG) is 0.15 microM. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations at conserved cysteine (Cys(50), Cys(83), and Cys(230)) and lysine(81) residues, chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. The AdoMetDC mutants K81A and C83A retained up to 60 and 10% of wild type activity, respectively, demonstrating that lysyl and sulfhydryl groups are required for full catalytic activity. However, changing Cys(50) and Cys(230) to alanine had minimal effects on the catalytic activity. Changing Lys(81) to alanine produced an altered substrate specificity. When lysine was used as a substrate instead of AdoMet, the substrate specificity for lysine increased 6-fold. The K(m) value for AdoMet is 11-fold higher than that of the wild type, but the V(max) value is more than 60%. Taken together, the results suggest that the lysine(81) residue is critical for substrate binding.     

拟南芥中一种AtBAG4蛋白的抗体制备和特异性检测

以拟南芥AtBAG4的全长cDNA,构建pET-51780重组质粒,转化大肠杆菌BL21(DE3),IPTG可诱导融合蛋白高效表达,表达产物以可溶形式存在;经Ni-NTA层析柱分离纯化,可得到分子质量约为49kDa的PAGE纯蛋白。用纯化的融合蛋白pET-51780免疫家兔,可得到抗AtBAG4抗体。Western blot结果显示该抗体能特异识别原核系统内表达的抗原以及拟南芥自身的抗原。     

Identification and characterization of a plastidial phosphatidylglycerophosphate phosphatase in Arabidopsis thaliana

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1‐2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1‐1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1‐1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation.