AtHMA1 contributes to the detoxification of excess Zn(II) in Arabidopsis

AtHMA1 is a member of the heavy metal-transporting ATPase family. It exhibits amino acid sequence similarity to two other Zn(II) transporters, AtHMA2 and AtHMA4, and contains poly-His motifs that are commonly found in Zn(II)-binding proteins, but lacks some amino acids that are typical for this class of transporters. AtHMA1 localizes to the chloroplast envelope. In comparison with wild-type plants, we observed a more pronounced sensitivity in the presence of high Zn(II) concentrations, and increased accumulation of Zn in the chloroplast of T-DNA insertional mutants in AtHMA1 . The Zn(II)-sensitive phenotype of AtHMA1 knock-out plants was complemented by the expression of AtHMA1 under the control of its own promoter. The Zn(II)-transporting activity of AtHMA1 was confirmed in a heterologous expression system, Saccharomyces cerevisiae . The sensitivity of yeast to high concentrations of Zn(II) was altered by the expression of AtHMA1 lacking its N-terminal chloroplast-targeting signal. Taken together, these results suggest that under conditions of excess Zn(II), AtHMA1 contributes to Zn(II) detoxification by reducing the Zn content of Arabidopsis thaliana plastids.     

Alcohol and aldehyde dehydrogenase polymorphisms and risk for suicide: a preliminary observation in the Japanese male population

Epidemiological studies have shown that excessive alcohol consumption is a potent risk factor to develop suicidal behavior. Genetic factors for suicidal behavior have been observed in family, twin, and adoption studies. Because alcohol dehydrogenase (ADH1B) His47Arg and mitochondrial aldehyde dehydrogenase (ALDH2) Glu487Lys single nucleotide polymorphisms (SNPs), which affect alcohol metabolism, have been reported to exert significant impacts on alcohol consumption and on the risk for alcoholism in East Asia populations, we explored associations of the two functional SNPs with suicide using a case–control study of 283 completed suicides and 319 control subjects in the Japanese population. We found that the inactive ALDH2 allele (487Lys) was significantly less frequent in the completed suicides (19.3%) than in the controls (29.3%), especially in males, whereas this was not the case in females. The males bearing alcoholism‐susceptible homozygotes at both loci (inactive ADH1B Arg/Arg and active ALDH2 Glu/Glu genotypes) have a 10 times greater risk for suicide compared with the males bearing alcoholism‐protective homozygotes at both loci. Our data show the genetic impact of the two polymorphisms on suicidal behavior in the Japanese population, especially in males. Because we did not verify the daily alcohol consumption, the association of these SNPs with suicide might be due to alcoholism itself. Further studies using case–control subjects, which verifies the details of current and past alcohol consumption and diagnosis for alcoholism, are required to confirm these findings.     

Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase null mutants ofArabidopsis thaliana (L.) Heynh

Several mutants have been isolated at theArabidopsis thaliana (L.) Heynh. alcohol dehydrogenase (ADH) gene locus using allyl alcohol selection on ethyl methanesulfonate (EMS)-mutagenized seeds. Eleven mutants were isolated in theADH1-A electrophoretic allele, and 21 in theADH1-S allele. These null mutants are characterized by the absence of measurable ADH activity and genetic data showed that the mutations were confined to theADH1 gene locus ofArabidopsis. Eleven mutants in theADH1-A background were further characterized at the protein and mRNA level. These experiments revealed striking differences in the ADH protein and mRNA content. Some of the mutants did not synthesize any mRNA or ADH-like protein, whereas some of them had a nearly normal level of ADH protein and mRNA. Others had a very low level of both protein and mRNA. ADH null mutants differed physiologically from the wild type by their higher sensitivity to anaerobic treatment in plants and significantly reduced resistance to acetaldehyde in suspension cultures.This research was supported by the Geconcerteerde Onderzoeksactie, Grant 86/91–103, and the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL), Grant 4972A.     

光敏启动子控制的酵母Hem1基因在烟草中表达

拟南芥hemA1基因启动子是一种光响应型启动子,它控制着植物体内5-氨基乙酰丙酸(ALA)昼夜节律型合成.将该启动子与酿酒酵母Hem1基因构建的二价载体转入烟草中,获得转基因植株.以转二价基因的烟草T0代种子为材料,用不同浓度卡那霉素(Km)溶液浸种,结果显示,种子发芽率和子叶绿化率随着Km浓度升高而降低.将1 000 mg·L-1Km溶液中长出的162株抗性植株移植至盆钵中培养,GUS检测出的阳性比率为92%.用特异引物PCR法检测Km抗性-GUS阳性植株,发现含有拟南芥HemA1基因启动子的比率为92.5%,含有酿酒酵母Hem1基因的比率为88%,含有二价重组基因的比率为84.2%.RT-PCR检测表明,Hem1基因在黑暗中的表达量明显低于光照下,证明光敏启动子有效地控制了结构基因Hem1的表达.生理指标分析表明,与野生型相比,转基因植株ALA合成速率、叶绿素含量明显增加,叶绿素b/a比值提高.野生型植株基部叶片SPAD值显著降低,而转基因植株基部叶片SPAD值保持较高水平.以上结果说明,外源二价基因已经成功整合到烟草基因组中,并且能够在光照条件下过量表达,合成过量ALA.     

Metabolite Profiling Reveals a Role for Atypical Cinnamyl Alcohol Dehydrogenase CAD1 in the Synthesis of Coniferyl Alcohol in Tobacco Xylem

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes. Sequences of NtCAD1-1 and NtCAD1-7 were deposited in GenBank under accession numbers AY911854 and AY911855, respectively.     

Evolution of the Catalytic Activity of Arabidopsis thaliana Glutathione Transferase Zeta Class-1 by Saturation Mutagenesis

Saturation mutagenesis was performed on three non-catalytic residues, Asn71, Leu108, and Gly177, in and near the active site of Arabidopsis thaliana GSTZ-1 (AtGSTZ-1). Forty unique mutants with more than 10% activity increases, were obtained. Of these, 12 resulted in large activity improvement and were purified for further characterization. Remarkably, 11 of them contained mutations at Leu108, suggesting that Leu108 plays an important role in dichloroacetic acid-dechlorinating (DCA-DC) activity. Kinetic analysis revealed that multiple mutations at these residues increased k _cat/K _m toward DCA and GSH by as much as 2.2- and 5.8-fold, respectively. Since the catalytic residues were not involved in mutagenesis, the activity enhancements were presumably due to structural change in the active site rather than to a change in catalysis. Our results also suggest that the specific shape of the active site in AtGSTZ-1 is essential to its unique DCA-DC activity.     

Regulator and substrate: Dual roles for the ATG1-ATG13 kinase complex during autophagic recycling in Arabidopsis

Like other organisms, plants rely on autophagy to recycle intracellular components needed for development, new growth and survival during nutrient stress. This 'self eating' is a catabolic process by which unwanted cytoplasmic materials and dysfunctional organelles are sequestered into vesicles and subsequently delivered to the vacuole for breakdown. The process is tightly regulated by the autophagy-related 1(ATG1)-ATG13 kinase complex which is controlled by multiple nutrient-responsive upstream regulators that integrate nutrient demand with availability. To further appreciate how autophagy is controlled in plants, we recently examined the functions of the ATG1-ATG13 complex in Arabidopsis thaliana. Our data revealed a dual role for the ATG1-ATG13 complex, first as a regulator of plant autophagy, and second as a substrate of this recycling process.     

Examination of the Conditions for Protoplast Isolation from Tobacco Cells Cultured in vitro

Protoplasts were isolated from cultured tobacco cells by removing the cell wall enzymatically. We examined the time courses of treatment with some different concentrations of enzymes (cellulase and macerozyme) and with some different kinds and concentrations of sugars (sucrose, sorbitol and mannitol) which produce good conditions for protoplast isolation. The best conditions for protoplast preparation from tobacco cells cultured in vitro were: use of actively growing cells and isolation of protoplasts with 5% cellulase and 0.1% macerozyme in 0.5% sorbitol.     

Genetics and ontogeny of alcohol dehydrogenase isozymes in the mouse: Evidence for a cis-acting regulator gene (Adt-1) controlling C2 isozyme expression in reproductive tissues and close linkage of Adh-3 and Adt-1 on chromsome 3

An electrophoretic variant previously reported for the stomach isozyme of alcohol dehydrogenase (ADH-C2) in inbred strains of Mus musculus (Holmes, 1977) has been used to localize the gene encoding this enzyme (Adh-3) on chromosome 3 near Va (varitint) (9.6 ± 3.6% recombinants). Genetic variation of ADH-C2 activity in male and female reproductive tissues among inbred strains and Harwell linkage testing stocks was also observed. Reproductive tissue ADH-C2 phenotypes were inherited in a normal Mendelian fashion among F₂ progeny of an F₁ (LII × C57BL/Go) × C57BL/Go backcross as though controlled by a single cis-acting regulator locus (designated Adt-1) with two alleles: Adt-1 ^a (presence of ADH-C2) and Adt-1 ^b (absence or low activity of ADH-C2). No recombinants were observed among 73 progeny or among 13 inbred strains and six Harwell linkage testing stocks of mice, indicating that Adh-3 and Adt-1 are closely linked or identical genes. A single recombinant phenotype was observed in Peru-Coppock mice, suggesting that they are separate genes. Ontogenetic analyses demonstrated that ADH-B₂ is present throughout development from late fetal stages in stomach, liver, and kidney; similar results were found for ADH-C2 in developing kidney and stomach extracts, whereas ADH-A2 exhibited high activity in liver extracts after 3 weeks of age in both sexes and in male kidney extracts after 6 weeks.     

Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis

The completion of the Arabidopsis thaliana genome has revealed that there are nine members of the Pht1 family of phosphate transporters in this species. As a step towards identifying the role of this gene family in phosphorus nutrition, we have isolated the promoter regions from each of these genes, and fused them to the reporter genes beta-glucuronidase and/or green fluorescent protein. These chimeric genes have been introduced into A. thaliana, and reporter gene expression has been assayed in plants grown in soil containing high and low concentrations of inorganic phosphate (Pi). Four of these promoters were found to direct reporter gene expression in the root epidermis, and were induced under conditions of phosphate deprivation in a manner similar to previously characterised Pht1 genes. Other members of this family, however, showed expression in a range of shoot tissues and in pollen grains, which was confirmed by RT-PCR. We also provide evidence that the root epidermally expressed genes are expressed most strongly in trichoblasts, the primary sites for uptake of Pi. These results suggest that this gene family plays a wider role in phosphate uptake and remobilisation throughout the plant than was previously believed.     

Interallelic complementation at the Arabidopsis CRE1 locus uncovers independent pathways for the proliferation of vascular initials and canonical cytokinin signalling

The differentiation of vascular tissue plays a central role in root architecture and its functionality. Regardless of its importance, the molecular mechanisms involved in the inception of vascular morphogenesis and their interaction with hormones are only now beginning to be understood. The characterisation of the WOODEN LEG (wol/cre1 mutant), impaired in procambial cell proliferation and the identification of WOL/CRE1 as a cytokinin receptor, provided the first genetic evidence pointing to a role of cytokinins in the formation of vascular initials. However, the striking wol phenotype in vascular differentiation is unique among all the available cre1 alleles collection. In this work, we identified a mutant with identical deficiencies in vascular differentiation as wol. Complementation analysis revealed that this mutant rescued the wol short-root phenotype. However, genetic characterisation of the mutant showed that the mutation was located at the CRE1 locus, indicating that both alleles displayed interallelic complementation. Trans-heterozygotes characterisation showed that these plants fully restored the deficiency in vascular differentiation but not the canonical cytokinin signalling. Furthermore, we show that, as measured in root growth inhibition, calli regeneration assays and northern analysis, the original wol allele is in fact more sensitive to cytokinins than the trans-heterozygous plants, or some cre1 alleles showing wild-type vascular morphogenesis. Thus, there is no strict correlation between the phenotype in vascular differentiation displayed by the cre1/wol alleles and canonical cytokinin signalling. These results indicate that at least partially independent regulatory circuits may operate in procambial cell proliferation and in cytokinin responsiveness exerted through the CRE1 receptor.     

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.     

Reduced activity of Arabidopsis chromosome-cohesion regulator gene CTF7/ECO1 alters cytosine methylation status and retrotransposon expression

Multicellular organisms such as higher plants require timely regulation of DNA replication and cell division to grow and develop. Recent work in Arabidopsis has shown that chromosome segregation during meiosis and mitosis depends on the activity of several genes that in yeast are involved in the establishment of chromosomal cohesion. In this process, proteins of the STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) family tether chromosomes and establish inter- and intrachromosomal connections. In Arabidopsis, recruitment of SMC proteins and establishment of cohesion during key stages of the cell cycle depend on the activity of CHROMOSOME TRANSMISSION FIDELITY 7/ESTABLISHMENT OF COHESION 1 (CTF7/ECO1). Here we show that loss of CTF7/ECO1 activity alters the status of cytosine methylation in both intergenic regions and transposon loci. An increase in expression was also observed for transposon copia28, which suggests a link between CTF7/ECO1 activity, DNA methylation and gene silencing. More work is needed to determine the mechanistic relationships that intervene in this process.     

Requirement of KNAT1/BP for the Development of Abscission Zones in Arabidopsis thaliana

The KNAT1 gene is a member of the Class I KNOXhomeobox gene family and is thought to play an important role in meristem development and leaf morphogenesis. Recent studies have demonstrated that KNAT1/BP regulates the architecture of the inflorescence by affecting pedicle development in Arabidopsis thaliana. Herein, we report the characterization of an Arabidopsis T-DNA insertion mutant that shares considerable phenotypic similarity to the previously identified mutant brevipedicle （bp）. Molecular and genetic analyses showed that the mutant is allelic to bp and that the T-DNA is located within the first helix of the KNAT1 homeodomain （HD）. Although the mutation causes a typical abnormality of short pedicles, propendent siliques, and semidwarfism, no obvious defects are observed in the vegetative stage. A study on cell morphology showed that asymmetrical division and inhibition of cell elongation contribute to the downward-pointing and shorter pedicle phenotype. Loss of KNAT/BPfunction results in the abnormal development of abscission zones. Mlcroarray analysis of gene expression profiling suggests that KNAT1/BP may regulate abscission zone development through hormone signaling and hormone metabolism in Arabidopsis.