A xyloglucan endotransglucosylase/hydrolase involves in growth of primary root and alters the deposition of cellulose in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of enzymes that mediate the construction and restructure of the cellulose/xyloglucan framework by splitting and reconnecting xyloglucan molecule cross-linking among cellulose microfibrils. Remodification of cellulose microfibrils within cell-wall matrices is realized to be one of the most critical steps in the regulation of cells expansion in plants. Thirty-three XTH genes have been found in Arabidopsis thaliana but their roles remain unclear. AtXTH21 (At2g18800), an Arabidopsis XTH gene that mainly expresses in root and flower, exhibits different expression profiles from other XTH members under hormone treatment. We examined loss-of-function mutants using T-DNA insertion lines and overexpression lines and found that the AtXTH21 gene played a principal role in the growth of the primary roots by altering the deposition of cellulose and the elongation of cell wall.     

Loss of function of COBRA, a determinant of oriented cell expansion, invokes cellular defence responses in Arabidopsis thaliana

An Arabidopsis T-DNA insertion mutant that results in complete loss-of-function of the COBRA gene has been identified. The COBRA gene encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that modulates cellulose deposition and oriented cell expansion in roots. The loss-of-function mutant allele (named "cob-5") exhibits abnormal cell growth throughout the entire plant body and accumulates massive amounts of stress response chemicals such as anthocyanins and callose. To gain further insight into the mechanism by which COBRA affects cell growth and physiology, the whole-genome gene expression profile of cob-5 plants was compared with that of wild-type plants. Consistent with the mutant phenotype, many genes involved in anthocyanin biosynthesis were up-regulated in the cob-5 plants, whereas genes involved in cell elongation were down-regulated. The most striking feature of the gene expression profile of cob-5 was the massive and co-ordinate induction of defence- and stress-related genes, many of which are regulated by the plant stress signal jasmonic acid (JA). Indeed, the cob-5 plants over-accumulated JA by nearly 8-fold compared with wild-type plants. Furthermore, induction of cell elongation defects in conditional allele cob-3 plants triggers the expression of a defence-responsive gene. These results provide potential clues to the mechanisms by which plant cells initially perceive biotic stress at the cell surface.     

Isolation and characterization of a novel semi-lethal Arabidopsis thaliana mutant of gene for pentatricopeptide (PPR) repeat-containing protein

A novel Arabidopsis thaliana mutant of one member of the pentatricopeptide repeat (PPR) gene family has been identified among T-DNA insertion lines. Tagging of the At1g53330 gene caused the appearance of a semi-lethal mutation with a complex phenotypic expression from embryo lethality associated with the abnormal pattern of cell division during globular to heart transition to fertile plants with just subtle phenotypic changes. The PPR protein At1g53330.1 was predicted to be targeted to mitochondria by TargetP and MitoProt programs. Complementation analysis confirmed that the phenotype is a result of a single T-DNA integration. A thorough functional analysis of this mutant aimed at finding a particular organelle target of At1g53330.1 protein will follow.     

The GH3 acyl adenylase family member PBS3 regulates salicylic acid-dependent defense responses in Arabidopsis

The pbs3-1 mutant, identified in a screen for Arabidopsis (Arabidopsis thaliana) mutants exhibiting enhanced susceptibility to the avirulent Pseudomonas syringae pathogen DC3000 (avrPphB), also exhibits enhanced susceptibility to virulent P. syringae strains, suggesting it may impact basal disease resistance. Because induced salicylic acid (SA) is a critical mediator of basal resistance responses, free and glucose-conjugated SA levels were measured and expression of the SA-dependent pathogenesis-related (PR) marker, PR1, was assessed. Surprisingly, whereas accumulation of the SA glucoside and expression of PR1 were dramatically reduced in the pbs3-1 mutant in response to P. syringae (avrRpt2) infection, free SA was elevated. However, in response to exogenous SA, the conversion of free SA to SA glucoside and the induced expression of PR1 were similar in pbs3-1 and wild-type plants. Through positional cloning, complementation, and sequencing, we determined that the pbs3-1 mutant contains two point mutations in the C-terminal region of the protein encoded by At5g13320, resulting in nonconserved amino acid changes in highly conserved residues. Additional analyses with Arabidopsis containing T-DNA insertion (pbs3-2) and transposon insertion (pbs3-3) mutations in At5g13320 confirmed our findings with pbs3-1. PBS3 (also referred to as GH3.12) is a member of the GH3 family of acyl-adenylate/thioester-forming enzymes. Characterized GH3 family members, such as JAR1, act as phytohormone-amino acid synthetases. Thus, our results suggest that amino acid conjugation plays a critical role in SA metabolism and induced defense responses, with PBS3 acting upstream of SA, directly on SA, or on a competitive inhibitor of SA.     

Analysis of two L-Galactono-1,4-lactone-responsive genes with complementary expression during the development of Arabidopsis thaliana

Unraveling the role of genes annotated as protein of unknown function is of importance in progression of plant science. l-Galactono-1,4-lactone (l-GalL) is the terminal precursor for ascorbic acid (AsA) biosynthesis in Arabidopsis thaliana, and a previous study showed two DUF (domains of unknown function) 642 family genes (At1g80240 and At5g25460, designated as DGR1 and DGR2, respectively) to be sensitive to it. In this work, leaves from wild-type Arabidopsis were fed with d-glucose, l-galactose, l-GalL and AsA, and the expression level of the At1g80240 and At5g25460 genes showed a specific response to l-GalL, but not to the other supplements despite the increases of the tissue AsA contents. Analysis of promoter-β-glucuronidase (GUS) transgenic plants showed the two genes to be complementarily expressed at the root apex and in the rest of the root excluding the apex, respectively, in both young and old seedlings, and to be expressed at the leaf primordia. The GUS activity under the control of the At5g25460 promoter was high in the cotyledon and leaf veins of young seedlings. These findings were consistent with the results of quantitative real-time PCR. Interestingly, the T-DNA insertion mutant of At5g25460 (SALK_125079) displayed shorter roots and smaller rosettes than Col-0; however, no phenotypic difference was observed between the T-DNA insertion mutant of At1g80240 and the wild type. This is the first report on the expression and functional analysis of these two DUF642 family genes, with the results revealing the contribution of DGR genes to the development of Arabidopsis.     

Identification of an Arabidopsis Nodulin-related protein in heat stress

We identified a Nodulin-related protein 1 (NRP1) encoded by At2g03440, which was previously reported to be RPS2 interacting protein in yeast-two-hybrid assay. Northern blotting showed that AtNRP1 expression was suppressed by heat stress (42°C) and induced by low temperature (4°C) treatment. Strong GUS staining was observed in the sites of meristematic tissues of pAtNRP1:: GUS transgenic plants, such as shoot apex and root tips, young leaf veins, stamens and stigmas of flowers, and abscission layers of young siliques. To study AtNRP1 biological functions, we have characterized both loss-of-function T-DNA insertion and transgenic overexpression plants for AtNRP1 in Arabidopsis. The T-DNA insertion mutants displayed no obvious difference as compared to wild-type Arabidopsis under heat stress, but the significant enhanced susceptibility to heat stress was revealed in two independent AtNRP1-overexpressing transgenic lines. Further study found that the decreased thermtolerance in AtNRP1-overexpressing lines accompanied significantly decreased accumulation of ABA after heat treatment, which was probably due to AtNRP1 playing a role in negative-feedback regulation of the ABA synthesis pathway. These results support the viewpoint that the application of ABA inhibits nodulation and nodulin-related gene expression and threaten adverse ambient temperature can impact the nodulin-related gene expression.     

Spermine is not essential for survival of Arabidopsis

Spermine is the final product of the polyamine biosynthetic pathway and is ubiquitously present in most organisms. The genome of Arabidopsis thaliana has two genes encoding spermine synthase: ACAULIS5 (ACL5), whose loss-of-function mutants show a severe defect in stem elongation, and SPMS. In order to elucidate the function of spermine in plants, we isolated a T-DNA insertion mutant of the SPMS gene. Free and conjugated spermine levels in the mutant, designated spms-1, were significantly decreased compared with those in the wild-type, but no obvious morphological phenotype was observed in spms-1 plants. We further confirmed that acl5-1 spms-1 double mutants contained no spermine. Surprisingly, acl5-1 spms-1 was fully as viable as the wild-type and showed no phenotype except for the reduced stem growth due to acl5-1. These results indicate that spermine is not essential for survival of Arabidopsis, at least under normal growth conditions.     

Creation of a collection of morphological insertional mutants of Arabidopsis thaliana

A collection of transgenic Arabidopsis thaliana plants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12-18% of the total number of A. thaliana genes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.     

Collection of Arabidopsis thalianaMorphological Insertion Mutants

A collection of transgenic Arabidopsis thalianaplants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12–18% of the total number of A. thalianagenes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.