Functional characterization of pathogen-responsive protein AtDabb1 with an antifungal activity from Arabidopsis thaliana

A plant antifungal protein was purified from Arabidopsis thaliana leaves by using a typical procedure consisting of anion exchange chromatography and high-performance liquid chromatography. We determined the amino acid sequence of the purified protein using MALDI-TOF/MS analysis, and found that the sequence matched that of a hypothetical Arabidopsis protein in GenBank (accession number NP_175547). We designated the protein as AtDabb1. After the cDNA encoding the AtDabb1 gene was cloned from an Arabidopsis leaf cDNA library, the recombinant protein was expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the AtDabb1 gene was induced by pathogen-related signaling molecules including salicylic acid and jasmonic acid. These results suggest that AtDabb1 may contribute to the induced plant defense mechanism against diverse pathogenic fungi.     

The prohibitin genes in Arabidopsis thaliana: expression in seeds, hormonal regulation and possible role in cell cycle control during seed germination

A fragment encoding a partial sequence of a prohibitin (Phb) gene was isolated. The expression of Phb mRNA and protein in seeds of wild type and mutant Arabidopsis thaliana is presented. Phb mRNA is abundant in wild-type seeds; thus, it may have sequence or structural characteristics responsible for this stability. The 3' untranslated region sequence of a Phb gene has interesting features. We found that Arabidopsis Phb does not interact with a retinoblastoma-related protein or E2F in a yeast two-hybrid system, thus suggesting that the plant protein may have not conserved such interaction, described for mammalian Phb. The possible role of Phb in cell cycle regulation during germination is discussed.     

Characterization of an Arabidopsis lipoxygenase gene responsive to methyl jasmonate and wounding.

A cDNA corresponding to the gene AtLox2 was isolated from an Arabidopsis thaliana library using a lipoxygenase (LOX) probe from soybean. AtLox2 encodes a 102-kD protein, AtLOX2, which has 42 to 45% amino acid sequence identity with other plant LOX sequences. The AtLOX2 sequence is more than 30 amino acids longer at the amino terminus than other plant LOX sequences, and this extension has features reminiscent of chloroplast transit peptides, suggesting that AtLOX2 may be chloroplast localized. AtLox2 mRNA levels are high in leaves and inflorescences but very low in seeds, roots, and stems. AtLox2 mRNA accumulation is rapidly induced in leaves in response to methyl jasmonate. Leaves that have been wounded and adjacent leaves on the same plant also accumulate AtLox2 mRNA.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein

Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. We isolated a complementary DNA (ATLP-3) encoding a PR5-like protein from Arabidopsis which is distinct from two other previously reported PR5 cDNAs from the same plant species. The predicted ATLP-3 protein with its amino-terminal signal sequence is 245 amino acids in length and is acidic with a pI of 4.8. The deduced amino acid sequence of ATLP-3 shows significant sequence similarity with PR5 and thaumatin-like proteins from Arabidopsis and other plants and contains a putative signal sequence at the amino-terminus. The expression of ATLP-3 and a related gene (ATLP-1) that we previously isolated from Arabidopsis was induced by pathogen infection and salicylic acid, a known inducer of pathogenesis-related genes. Southern blot analysis indicates that the ATLP-1 and ATLP-3 are coded by single-copy genes. To study the effect of ATLP-1 and ATLP-3 proteins on fungal growth, the cDNA regions corresponding to putative mature protein were expressed in Escherichia coli and the cDNA encoded proteins were purified. ATLP-1 and ATLP-3 proteins cross-reacted with anti-osmotin and anti-zeamatin antibodies. ATLP-3 protein showed antifungal activity against several fungal pathogens suggesting that ATLP-3 may be involved in plant defense against fungal pathogens.     

Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana.

A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene. A fragment of the predicted size was amplified from genomic DNA from Arabidopsis thaliana. Overlapping cDNA clones, some with homology to the PCR amplified fragment, were identified and sequenced. The assembled nucleic acid sequence is 4720 bp and encodes a protein of 1534 amino acids which has significant homology to prokaryote and mammalian cytosine methyltransferases. Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain. The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytosine-5 methyltransferases and shows 50% homology to the murine enzyme in the methyltransferase domain. The amino terminal domain is only 24% homologous to the murine enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man. In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana.     

Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.     

The IgM antigen receptor of B lymphocytes is associated with prohibitin and a prohibitin-related protein.

The two major classes of antigen receptors on murine B lymphocytes, mIgM and mIgD, are both contained in a complex with two additional molecules, Ig-alpha and Ig-beta, which permit signal transduction. Accordingly, early biochemical events after antigen binding to either receptor are similar; biological effects, however, are different. Here, we describe three newly discovered intracellular proteins of 32, 37 and 41 kDa molecular mass, that are non-covalently associated with mIgM, but not with mIgD. These proteins coprecipitate with mIgM in Triton X-100 and Nonidet P-40, but not in digitonin lysates. In addition, mIgM is to some extent associated with 29 and 31 kDa proteins that are predominantly associated with mIgD (see accompanying paper). Amino acid sequencing of p32 and p37 identified p32 as mouse prohibitin; this was corroborated by Western blot analysis with antibodies specific for rat prohibitin. p37 is a newly discovered protein. cDNA clones for both proteins were isolated and sequenced. The deduced amino acid sequence of p32 is identical to that of rat prohibitin. p37 is highly homologous to p32. Since prohibitin was identified as an inhibitor of cell proliferation, its association with mIgM, but not mIgD, could explain the different biological events elicited after engagement of each receptor.     

甘蓝型油菜Toc33基因编码区的分离及蛋白序列的比较

以甘蓝型油菜新鲜嫩叶为实验材料提取其总DNA,以其为模板,根据拟南芥Toc33基因编码区序列设计引物,PCR扩增甘蓝型油菜叶绿体外膜蛋白转运机器的构件蛋白基因Toc33,得到两条扩增带,测序结果显示克隆到的两个片段分别长1370bp、1490bp,将这两个片段分别命名为Bn Tpc33-1,Bn Toc33-2,序列比较发现它们之间的同源性为78％,其中外显子的同源性为96％,而内含子的同源性仅为60％。为研究Toc33与同一基因家族的Toc34基因功能间的关系,对拟南芥、油菜、诸葛菜等植物的Toc33、Toc34蛋白序列进行比较分析并构建了分子系统进化树。     

Application of a high-throughput HPLC-MS/MS assay to Arabidopsis mutant screening; evidence that threonine aldolase plays a role in seed nutritional quality

Beyond their essential function as the building blocks of proteins, amino acids contribute to many aspects of plant biochemistry and physiology. Despite this, there are relatively large gaps in our understanding of the biochemical pathways and regulation of amino acid synthesis in plants. A rapid (1.5 min versus 20-90 min for standard methods) HPLC-MS/MS assay for separating 19 amino acids was developed for quantifying levels of free amino acids in plant tissue. This assay was used to determine the free amino acid content in the seeds of 10,000 randomly mutagenized Arabidopsis lines, and 322 Arabidopsis lines with increased levels of one or more amino acids were identified. The heritability of the mutant phenotype was confirmed for 43 lines with increased seed levels of the aspartate-derived amino acids Ile, Lys, Thr, or Met. Genetic mapping and DNA sequencing identified a mutation in an Arabidopsis threonine aldolase (AT1G08630, EC 4.1.2.5) as the cause of increased seed Thr levels in one mutant. The assay that was developed for this project has broad applicability to Arabidopsis and other plant species.     

Abscisic acid and desiccation-dependent expression of a novel putative SNF5-type chromatin-remodeling gene in Pisum sativum.

Snf5-like proteins are components of multiprotein chromatin remodeling complexes involved in the ATP-dependent alteration of DNA-histone contacts. Mostly described in yeast and animals, the only plant SNF5-like gene characterized so far has been BSH from Arabidopsis thaliana (L.) Heynh. We report the cloning and characterization of expression of a SNF5-like gene from pea (Pisum sativum L. cv. Lincoln), which has been designated PsSNF5. Southern analysis showed a single copy of the gene in the pea genome. The cDNA contained a 723bp open reading frame encoding a 240 amino acid protein of 27.4kDa with a potential nuclear localization signal. PsSNF5 protein sequence closely resembled BSH, with which it showed an overall amino acid identity of 78.5%. Two-hybrid experiments showed that PsSNF5 is functionally interchangeable with Arabidopsis BSH in the interactions with other components of the remodeling complex. Phylogenetic analysis demonstrated that PsSNF5 clustered with translated expressed sequence tags from other Leguminosae, hypothetically coding for new Snf5-like proteins. RT-PCR expression analysis demonstrated that the PsSNF5 gene is constitutively expressed in all the tissues examined, with minor differences in expression level in different tissues. Nevertheless, expression analysis revealed that PsSNF5 was up-regulated in the last stages of embryo development, when water content decreases. Moreover, abscisic acid and drought stress induced PsSNF5 accumulation in germinating embryos and vegetative tissues, suggesting that chromatin remodeling induced by PsSNF5-containing complexes might contribute to the response to that phytohormone.