Arabidopsis thaliana type I and II chaperonins

An examination of the Arabidopsis thaliana genome sequence led to the identification of 29 predicted genes with the potential to encode members of the chaperonin family of chaperones (CPN60 and CCT), their associated cochaperonins, and the cytoplasmic chaperonin cofactor prefoldin. These comprise the first complete set of plant chaperonin protein sequences and indicate that the CPN family is more diverse than previously described. In addition to surprising sequence diversity within CPN subclasses, the genomic data also suggest the existence of previously undescribed family members, including a 10-kDa chloroplast cochaperonin. Consideration of the sequence data described in this review prompts questions about the complexities of plant CPN systems and the evolutionary relationships and functions of the component proteins, most of which have not been studied experimentally.     

Integrated analysis of co-expressed MAP kinase substrates in Arabidopsis thaliana

MAP kinase (MAPK) signal transduction cascades are conserved eukaryotic pathways that modulate stress responses and developmental processes. In a recent report we have identified novel Arabidopsis MAPKK/MAPK/Substrate signaling pathways using microarrays containing 2,158 unique Arabidopsis proteins. Subsequently, several WRKY and TGA targets phosphorylated by MAPKs were verified in planta. We have also reported that specific MAPKK/MAPK modules expressed in Nicotiana benthamiana induced a cell death phenotype related to the immune response. We have generated a MAPK phosphorylation network based on our protein microarray experimental data. Here we further analyze our network by integrating phosphorylation and gene expression information to identify biologically relevant signaling modules. We have identified 108 phosphorylation events that occur among 96 annotated genes with highly similar pairwise expression profiles. Our analysis brings a new perspective on MAPK signaling by revealing new relationships between components of signaling pathways.Key words: MAPK, protein microarray, network, cell death, co-expression, signaling     

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.     

Two Arabidopsis metacaspases AtMCP1b and AtMCP2b are arginine/lysine-specific cysteine proteases and activate apoptosis-like cell death in yeast

Metacaspases in plants, fungi, and protozoa constitute new members of a conserved superfamily of caspase-related proteases. A yeast caspase-1 protein (Yca1p), which is the single metacaspase in Saccharomyces cerevisiae, was shown to mediate apoptosis triggered by oxidative stress or aging in yeast. To examine whether plant metacaspase genes are functionally related to YCA1, we carried out analyses of AtMCP1b and AtMCP2b, representing the two subtypes of the Arabidopsis metacaspase family, utilizing yeast strains with wild-type and the disrupted YCA1 gene (yca1Delta). Inducible expression of AtMCP1b and AtMCP2b significantly promoted yeast apoptosis-like cell death of both the wild-type and yca1Delta strains, relative to the vector controls, during oxidative stress and early aging process. Mutational analysis of the two AtMCPs revealed that their cell-death-inducing activities depend on their catalytic center cysteine residues as well as caspase-like processing. In addition, the phenotype induced by the expression of two AtMCPs was effectively prevented when the cells were pretreated with a broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone. These results suggest that the two subtypes of Arabidopsis metacaspases are functionally related to Yca1p with caspase-like characteristics. However, we found that bacterial and yeast extracts containing AtMCP1b, AtMCP2b, or Yca1p exhibit arginine/lysine-specific endopeptidase activities but cannot cleave caspase-specific substrates. Together, the results strongly implicate that expression of metacaspases could result in the activation of downstream protease(s) with caspase-like activities that are required to mediate cell death activation via oxidative stress in yeast. Metacaspases from higher plants may serve similar functions.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9: Regulation of lysine methyltransferases by physical interaction with their substrates

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 mark. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that at least these four KMTs require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.Key words: histone methylation, lysine methyltransferase, H3K4^me3, H3K9^me3, SETDB1, G9A, ING2     

拟南芥AtCBL9蛋白的原核表达纯化及其晶体生长研究

将拟南芥AtCBL9基因构建入原核表达载体pET-22b(+),在大肠杆菌中得到大量表达,用亲和层析和分子筛排阻层析2种方法对表达蛋白进行纯化。SDS-PAGE和动态光散射分析结果显示,AtCBL9蛋白在体外主要以单体和二体形式存在,单体和二体为同一蛋白,并具有较高的纯度。从单体蛋白中筛选出了微晶,为AtCBL9晶体生长优化及其最终结果解析和功能阐明奠定了基础。     

Response of Wild Type of Arabidopsis thaliana to Copper Stress

Wild type of Arabidopsis thaliana plants were cultivated hydroponically in Hoagland and Arnon nutrient solution and treated with copper (5 – 100 µM) for 2, 4, 7 and 14 d. A progressive decrease of the root length and biomass was observed at increasing Cu concentration in the nutrient solution. Roots accumulated higher amounts of Cu than shoots at all Cu treatments. Changes of cell and chloroplast ultrastructure of Cu-treated plants were also observed. Cu application did not induce formation of Cu-phytochelatin complexes. Changes in glutathione and glutathione disulfide content observed in roots and shoots of Cu-treated plants suggest their participation in amelioration of metal-induced oxidative stress.     

The specificity of two proteinases that cleave adjacent to arginine, C1 esterase and acrosin, for peptide p-nitroanilide substrates

Relative values of Vmax/Km for hydrolysis of 40 peptide p-nitroanilides catalyzed by human Cl-s and human acrosin are reported. For Cl-s, Ac-Lys(gamma Cbz)-Gly-Arg is the optimum sequence, but 25% of the substrates have (Vmax/Km)rel greater than 0.25 compared to this sequence. The best acrosin substrate tested has the sequence Tos-Gly-Pro-Arg, although (Vmax/Km)rel greater than 0.15 for more than half of the substrates. Proline at P2 is preferred by acrosin. Both enzymes prefer arginine at P1 greater than or equal to 3-fold over lysine and will not accept citrulline. In addition, occupancy of site S3 may yield an increase in Vmax/Km of greater than or equal to 10-fold with either enzyme, but many residues are accepted at S2, S3 and S4. Thus, an acrosin assay using Tos-Gly-Pro-Arg p-nitroanilide as a substrate is more than 20-times as sensitive as existing assays with blocked arginine derivatives.     

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene -glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase.The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and -amylase inhibitors.     

Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.     

Mutations in the Type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis

Human PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) encodes a type II protein arginine (Arg) methyltransferase and its homologs in animals and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) are known to regulate RNA processing, signal transduction, and gene expression. However, PRMT5 homologs in higher plants have not yet been reported and the biological roles of these proteins in plant development remain elusive. Here, using conventional biochemical approaches, we purified a plant histone Arg methyltransferase from cauliflower (Brassica oleracea) that was nearly identical to AtPRMT5, an Arabidopsis (Arabidopsis thaliana) homolog of human PRMT5. AtPRMT5 methylated histone H4, H2A, and myelin basic protein in vitro. Western blot using symmetric dimethyl histone H4 Arg 3-specific antibody and thin-layer chromatography analysis demonstrated that AtPRMT5 is a type II enzyme. Mutations in AtPRMT5 caused pleiotropic developmental defects, including growth retardation, dark green and curled leaves, and FlOWERING LOCUS C (FLC)-dependent delayed flowering. Therefore, the type II protein Arg methyltransferase AtPRMT5 is involved in promotion of vegetative growth and FLC-dependent flowering time regulation in Arabidopsis.     

Co-action between phytochrome B and HY4 in Arabidopsis thaliana

A combination of physiological and genetic approaches was used to investigate whether phytochromes and blue light (BL) photoreceptors act in a fully independent manner during photomorphogenesis of Arabidopsis thaliana (L.) Heynh. Wild-type seedlings and phyA, phyBand hy4 mutants were daily exposed to 3 h BL terminated with either a red light (R) or a far-red light (FR) pulse. In wild-type and phyA-mutant seedlings, BL followed by an R pulse inhibited hypocotyl growth and promoted cotyledon unfolding. The effects of BL were reduced if exposure to BL was followed by an FR pulse driving phytochrome to the R-absorbing form (Pr). In the wild type, the effects of R versus FR pulses were small in seedlings not exposed to BL. Thus, maximal responses depended on the presence of both BL and the FR-absorbing form of phytochrome (Pfr) in the subsequent dark period. Impaired responses to BL and to R versus FR pulses were observed in phyB and hy4 mutants. Simultaneous irradiation with orange light indicated that BL, perceived by specific BL photoreceptors (i.e. not by phytochromes), required phytochrome B to display a full effect. These results indicate interdependent co-action between phytochrome B and BL photoreceptors, particularly the HY4 gene product. No synergism between phytochrome A (activated by continuous or pulsed FR) and BL photoreceptors was observed.Abbreviations BL blue light - D darkness - FR far-redlight - FRc continuous FR - Pfr FR-absorbing form of phytochrome - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - WT wild type We thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands), Professor J. Chory (Salk Institute, Calif., USA) and the Arabidopsis Biological Resource Center (Ohio State University, Ohio, USA) for their kind provision of the original seed batches. This work was financially supported by CONICET, Universidad de Buenos Aires (AG 040) and Fundación Antorchas (A-12830/1 0000/9)     

Photosynthetic Properties of Photosystem Ⅱ in Arabidopsis thaliana Ipa1 Mutant

In a previous study, we characterized a high chlorophyll fluorescence Ipal mutant of Arabidopsis thallana, in which approximately 20% photosystem （PS） Ⅱ protein is accumulated. In the present study, analysis of fluorescence decay kinetics and thermoluminescence profiles demonstrated that the electron transfer reaction on either the donor or acceptor side of PSII remained largely unaffected in the Ipa1 mutant. In the mutant, maximal photochemical efficiency （Fv/Fm, where Fm is the maximum fluorescence yield and Fv is variable fluorescence） decreased with increasing light intensity and remained almost unchanged in wildtype plants under different light conditions. The Fv/Fm values also increased when mutant plants were transferred from standard growth light to low light conditions. Analysis of PSll protein accumulation further confirmed that the amount of PSll reaction center protein is correlated with changes in Fv/Fm in Ipal plants. Thus, the assembled PSll in the mutant was functional and also showed increased photosensitivity compared with wild-type plants.