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The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl.

Plant developmental processes are controlled by both endogenous programs and environmental stimuli. As a photomorphogenetic mutant, hy5 of Arabidopsis has been isolated and characterized. Our detailed characterization has revealed that the mutant is deficient in a variety of stimulus responses, including gravitropic response and waving growth of roots, as well as light-dependent hypocotyl elongation. In the roots and hypocotyl, the hy5 mutation also affects greening and specific cell proliferation such as lateral root formation and secondary thickening. Those phenotypes indicate that the HY5 gene is responsible for the regulation of fundamental developmental processes of the plant cell: cell elongation, cell proliferation, and chloroplast development. Molecular cloning of the HY5 gene using a T-DNA-tagged mutant has revealed that the gene encodes a protein with a bZIP motif, one of the motifs found in transcriptional regulators. Nuclear localization of the HY5 protein strongly suggests that the HY5 gene modulates the signal transduction pathways under the HY5-related development by controlling expression of genes downstream of these pathways.

Amino Acid Sequence↗

A possible cellular mechanism of neuronal loss in the dorsal root ganglia of Dystonia musculorum (dt) mice.

Dystonia musculorum (dt) is a mutant mouse with hereditary sensory neuropathy. A defective bullous pemphigoid antigen 1 (BPAG1) gene is responsible for this mutation. In the present study, we examined the distribution of neuronal intermediate filament proteins in the central and peripheral processes of the dorsal root ganglia (DRG) in adult dt mice using different approaches. We found that not only BPAG1, but also alpha-internexin was absent in the DRG neurons in adult dt mice. To study the relationship between the absence of alpha-internexin and the progressive neuronal loss in the DRG of dt mice, we further cultured DRG neurons from embryonic dt mutants. Immunocytochemical assay of cultured DRG neurons from dt embryos revealed that alpha-internexin was aggregated in the proximal region of axons and juxtanuclear region of the cytoplasma, yet the other intermediate filament proteins were widely distributed in all processes. The active caspase-3 activity was observed in the dt neuron with massive accumulation of alpha-internexin. From our observations, we suggest that the interaction between BPAG1 and alpha-internexin may be one of the key factors involved in neuronal degeneration, and abnormal accumulation of alpha-internexin may impair the axonal transport and subsequently turns on the cascade of neuronal apoptosis in dt mice.

Animals↗

Quorum sensing and phenazines are involved in biofilm formation by Pseudomonas chlororaphis (aureofaciens) strain 30-84.

The biological control bacterium Pseudomonas chlororaphis (aureofaciens) strain 30-84 employs two quorum sensing (QS) systems: PhzR/PhzI regulates the production of the antibiotics phenazine-1-carboxylic acid, 2-hydroxy-phenazine-1-carboxylic acid, and 2-hydroxy-phenazine, whereas CsaR/CsaI regulates currently unknown aspects of the cell surface. Previously characterized derivatives of strain 30-84 with mutations in each QS system and in the phenazine biosynthetic genes were screened for their ability to form surface-attached biofilm populations in vitro, using microtiter plate and flow cell biofilm assays, and on seeds and roots. Results from in vitro, seed, and root studies demonstrated that the PhzR/PhzI and the CsaR/CsaI QS regulatory systems contribute to the establishment of biofilms, with mutations in PhzR/PhzI having a significantly greater effect than mutations in CsaR/CsaI. Interestingly, phenazine antibiotic production was necessary for biofilm formation to the same extent as the PhzR/PhzI QS system, suggesting the loss of phenazines was responsible for the majority of the biofilm defect in these mutants. In vitro analysis indicated that genetic complementation or AHL addition to the growth medium restored the ability of the AHL synthase phzI mutant to form biofilms. However, only phenazine addition or genetic complementation of the phenazine biosynthetic mutation in trans restored biofilm formation by mutants defective in the transcriptional activator phzR or the phzB structural mutant. QS and phenazine production were also involved in the establishment of surface-attached populations on wheat seeds and plant roots, and, as observed in vitro, the addition of AHL extracts restored the ability of phzI mutants, but not phzR mutants, to form surface attached populations on seeds. Similarly, the presence of the wild type in mixtures with the mutants restored the ability of the mutants to colonize wheat roots, demonstrating that AHL and/or phenazine production by the wild-type population could complement the AHL- and phenazine-deficient mutants in situ. Together, these data demonstrate that both QS systems are involved in the formation of surface-attached populations required for biofilm formation by P. chlororaphis strain 30-84, and indicate a new role for phenazine antibiotics in rhizosphere community development beyond inhibition of other plant-associated microorganisms.

Bacterial Proteins↗

Early symbiotic responses induced by Sinorhizobium meliloti iIvC mutants in alfalfa.

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype. ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC- background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC- strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.

Alcohol Oxidoreductases↗

Influence of metham sodium on suppression of collar rot disease of peanut, in vitro antibiosis, siderophore production and root colonization by a fluorescent pseudomonad strain FPO4.

A hydroxamate type siderophore producing fluorescent Pseudomonas strain, isolated from the rhizoplane of paddy root showing plant growth promoting activity, exhibited a decreased in vitro antibiosis, production of siderophore and suppression of collar rot in presence of metham sodium. Use of herbicide had a detrimental effect on the plant growth promoting activity of this organism. The multiple drug resistant mutant strain derived from this rhizobacteria colonized the roots, but the herbicide application had a negative effect on their population. HPLC analysis of the siderophore showed five peaks of which the peak number three confirmed the antifungal activity.

Anti-Bacterial Agents↗

Molecular and physiological analysis of Arabidopsis mutants defective in cytosolic or chloroplastic aspartate aminotransferase.

Arabidopsis mutants deficient in cytosolic (AAT2) or chloroplastic (AAT3) aspartate (Asp) aminotransferase were characterized at the molecular and physiological levels. All of the ethyl methane sulfonate- or nitrosomethylurea-generated mutants are missense mutations, as determined by sequencing of the ASP2 gene from the cytosolic aat2 mutants (aat2-1, aat2-2, aat2-4, and aat2-5) and the ASP5 gene from the chloroplastic aat3 mutants (aat3-1, aat3-2, and aat3-4). A T-DNA insertion mutant in cytosolic AAT2 (aat2-T) was also identified. All the cytosolic aat2 and chloroplastic aat3 mutants have less than 6% AAT2 and less than 3% AAT3 activity, respectively, as determined by the native gel assay; however, none are nulls. The metabolic and physiological affect of these mutations in AAT isoenzymes was determined by measuring growth and amino acid levels in the aat mutants. Two aat2 mutants (aat2-2 and aat2-T) show reduced root length on Murashige and Skoog medium. For aat2-2, this growth defect is exaggerated by Asp supplementation, suggesting a defect in Asp metabolism. Amino acid analysis of the aat mutants showed alterations in levels of Asp and/or Asp-derived amino acids in several aat2 alleles. Two aat2 mutants show dramatic decreases in Asp and asparagine levels in leaves and/or siliques. As such, the cytosolic AAT2 isoenzyme appears to serve a nonredundant function in plant nitrogen metabolism of Asp and Asp-derived amino acids.

Amino Acid Substitution↗

Rhizobium meliloti exopolysaccharide Mutants Elicit Feedback Regulation of Nodule Formation in Alfalfa.

Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells.

Journal Article↗

Protein isolation and second-dimension electrophoretic separation.

Protocols for the proteome analysis of Arabidopsis tissues based on 2 D gel electrophoresis are given for leaves and roots as well as for seeds. The protocols contain a detailed description of the sample preparation step and of the solubilization of proteins. We then describe our protocols for the isoelectric focusing, the transfer of the strips from the first to the second dimension, SDS-PAGE as the second dimension of electrophoretic separation, staining of the gels using colloidal Coomassie blue, and finally drying of the gels between cellophane sheets. Notes contain additional background information on critical steps. Our protocols allow the analysis of leaves, roots, or seeds from different Arabidopsis ecotypes, from mutants or from plants subjected to various environmental conditions.

Arabidopsis↗

Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum.

In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans. One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it. On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only. All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads. All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed. We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics.

Cellulose↗

AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells.

In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.

Adenine↗

Developmental abnormalities and reduced fruit softening in tomato plants expressing an antisense Rab11 GTPase gene.

A cDNA clone from tomato fruit encodes a protein with strong homology with the rab11/YPT3 class of small GTPases that is thought to be involved in the control of protein trafficking within cells. The gene, LeRab11a, showed a pattern consistent with a single copy in DNA gel blots. The corresponding mRNA was developmentally regulated during fruit ripening, and its expression was inhibited in several ripening mutants. Its reduced expression in the Never-ripe mutant indicates that it may be induced by ethylene in fruit. The ripening-induced expression in tissues that are undergoing cell wall loosening immediately suggests a possible role in trafficking of cell wall-modifying enzymes. The message also was produced in leaves and flowers but not in roots. Antisense transformation was used to generate a "mutant phenotype." Antisense fruit changed color as expected but failed to soften normally. This was accompanied by reduced levels of two cell wall hydrolases, pectinesterase and polygalacturonase. There were other phenotypic effects in the plants, including determinate growth, reduced apical dominance, branched inflorescences, abnormal floral structure, and ectopic shoots on the leaves. In some plants, ethylene production was reduced. These data suggest an alternative or additional role in exocytosis or endocytosis of homeotic proteins, hormone carriers, or receptors.

Base Sequence↗

CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana.

Cullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.

Arabidopsis↗

Phosphatase under-producer mutants have altered phosphorus relations.

Phosphorus (P) acquisition and partitioning are essential for plant homeostasis. P is available for plant uptake when in its inorganic form (H2PO4-, or Pi), but Pi is often limiting in soils. Plants secrete acid phosphatases (APases) into the apoplastic space, which may be important for obtaining Pi from organic P sources; however, the relative importance of these enzymes for plant P nutrition has yet to be determined. We demonstrate that the root-associated APase pool is increased in Arabidopsis when Pi is limiting and document five APase isoforms secreted from Arabidopsis roots. Previously, we presented the identification of the phosphatase under-producer (pup) mutants, which have decreased in vivo root APase staining when grown under low P conditions. Here, we present the characterization of one of these, pup3, and further studies with pup1. pup3 has 49%, 38%, and 37% less specific APase activity in exudates, roots, and shoots, respectively. Root-associated APase activity is decreased by 16% in pup1 and 25% in pup3, regardless of P treatment. Two APase activity isoforms are reduced in pup3 exudates, and root and shoot isoforms are also affected. One of the two exudate isoforms is recognized by a polyclonal antibody raised to an Arabidopsis purple APase recombinant protein (AtPAP12); however, AtPAP12 transcript levels are unaffected in the mutant. The pup3 mutation was mapped to 68.4 +/- 6.0 centimorgans on chromosome 5. Although P concentrations were not altered in pup1 and pup3 tissues when grown in nutrient solution in which Pi was the sole source of P, the mutants had 10% (pup1) and 17% (pup3) lower shoot P concentrations when grown in a peat-vermiculite mix in which the majority of the total P was present as organic P. Therefore, the pup defects, which include secreted APases, are functionally important for plant P nutrition.

Acid Phosphatase↗

Plasma membrane-associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis.

Abscisic acid (ABA) is an important plant hormone that modulates seed germination and plant growth and stress responses, but its signaling remains poorly understood. We investigated the role of ROP10, a member of the Arabidopsis Rop subfamily of Rho GTPases, in ABA signaling. A null rop10 mutant exhibits enhanced responses to ABA in seed germination, root elongation, and stomatal closure assays and in the induction of expression of the transcription factor MYB2, but it shows wild-type levels of ABA and normal responses to other hormones. Consistently, transgenic expression of a constitutively active form of ROP10 reduces ABA inhibition of seed germination, whereas dominant-negative mutants of ROP10 enhance ABA response and partially suppress abi2. Furthermore, ABA specifically downregulates ROP10 transcription in root tips. ROP10 is localized to the plasma membrane (PM), and PM localization is crucial for its function. These results suggest that ROP10 is a PM-localized signaling molecule that is involved specifically in the negative regulation of ABA signaling.

Abscisic Acid↗

Isolation of a premycorrhizal infection (pmi2) mutant of tomato, resistant to arbuscular mycorrhizal fungal colonization.

Arbuscular mycorrhizae (AM) represent an ancient symbiosis between mycorrhizal fungi and plant roots which co-evolved to exhibit a finely tuned, multistage interaction that assists plant growth. Direct screening efforts for Myc- plant mutants resulted in the identification of a tomato (Lycopersicon esculentum L. cv. Micro-Tom) mutant, M20, which was impaired in its ability to support the premycorrhizal infection (pmi) stages. The Myc- phenotype of the M20 mutant was a single Mendelian recessive trait, stable for nine generations, and nonallelic to a previously identified M161 pmi mutant. The M20 mutant was resistant to infection by isolated AM spores and colonized roots. Formation of Glomus intraradices appressoria on M20 roots was normal, as on wild-type (WT) plants, but in significantly reduced numbers. A significant reduction in spore germination was observed in vitro in the presence of M20 exudates relative to WT. Our results indicate that this new mutant shares similar physiological characteristics with the M161 pmi mutant, but has a more suppressive Myc- phenotype response.

Fungal Structures↗

Identification of bacterial groups preferentially associated with mycorrhizal roots of Medicago truncatula.

The genetic structures of bacterial communities associated with Medicago truncatula Gaertn. cv. Jemalong line J5 (Myc+ Nod+) and its symbiosis-defective mutants TRV48 (Myc+ Nod-) and TRV25 (Myc- Nod-) were compared. Plants were cultivated in a fertile soil (Châteaurenard, France) and in soil from the Mediterranean basin showing a low fertility (Mas d'Imbert, France). Plant growth, root architecture, and the efficiency of root symbiosis of the three plant genotypes were characterized in the two soils. Structures of the bacterial communities were assessed by automated-ribosomal intergenic spacer analysis (A-RISA) fingerprinting from DNA extracted from the rhizosphere soil and root tissues. As expected, the TRV25 mutant did not develop endomycorrhizal symbiosis in any of the soils, whereas mycorrhization of line J5 and the TRV48 mutant occurred in both soils but at a higher intensity in the Mas d'Imbert (low fertility) than in the Châteaurenard soil. However, modifications of plant growth and root architecture, between mycorrhizal (J5 and TRV48) and nonmycorrhizal (TRV25) plants, were recorded only when cultivated in the Mas d'Imbert soil. Similarly, the genetic structures of bacterial communities associated with mycorrhizal and nonmycorrhizal plants differed significantly in the Mas d'Imbert soil but not in the Châteaurenard soil. Multivariate analysis of the patterns allowed the identification of molecular markers, explaining these differences, and markers were further sequenced. Molecular marker analysis allowed the delineation of 211 operational taxonomic units. Some of those belonging to the Comamonadaceae and Oxalobacteraceae (beta-Proteobacteria) families were found to be significantly more represented within bacterial communities associated with the J5 line and the TRV48 mutant than within those associated with the TRV25 mutant, indicating that these bacterial genera were preferentially associated with mycorrhizal roots in the Mas d'Imbert soil.

Betaproteobacteria↗

Rhizobium etli mutant modulates carbon and nitrogen metabolism in Phaseolus vulgaris nodules.

The aim of this study was to evaluate the biochemical events in root nodules which lead to increased yield when bean is inoculated with a Rhizobium etli mutant (CFN037) having increased respiratory capacity. CFN037-inoculated plants had 22% more nitrogen (N) than did wild-type (CE3)-inoculated plants. Root nodule enzymes involved in nodule carbon and nitrogen assimilation as well as in ureides and amides synthesis were assessed in plants inoculated with CFN037 and the CE3. Our results show that the xylem ureides content was lower while that of amino acids was higher in CFN037- compared with CE3-inoculated plants. Supporting these results, enzymes involved in ureide synthesis were reduced while activity of aspartate aminotransferase, glutamate synthase, sucrose synthase, and glucose-6-P dehydrogenase were increased in CFN037-induced nodules. Glutamate synthase and phosphoenolpyruvate carboxylase transcripts were detected early in the development of nodules induced by CFN037 compared with CE3. However, plants inoculated with strain CE3-vhb, which express the Vitreoscilla sp. hemoglobin and also displays increased respiratory capacity, did not have altered ureide transport in N2-fixing plants. The data suggest that inoculation with special selected mutant strains of R. etli can modulate nodule N assimilation and N transport compounds.

Amino Acids↗

Deletion of the herpes simplex virus type 1 ribonucleotide reductase gene alters virulence and latency in vivo.

The role of herpes simplex virus type 1 (HSV-1)-encoded ribonucleotide reductase (RR) has been investigated in mice and guinea pigs using a mutant from which 90% of the large subunit of the enzyme was deleted. The RR mutant was extremely impaired in its ability to induce external vaginal lesions or to cause death in mice following intracerebral, intraperitoneal, or intravaginal inoculation, or in guinea pigs following intraperitoneal or intravaginal inoculation. The RR mutant replicated poorly in the vagina of mice and guinea pigs when compared with the parental virus. Neither infectious nor latent virus was recovered from the trigeminal ganglia of mice or from the dorsal root ganglia of mice and guinea pigs after inoculation with the RR mutant. Using the polymerase chain reaction, RR mutant DNA was, nevertheless, detected in the dorsal root ganglia of guinea pigs. These studies suggest that HSV-1 RR is essential for virulence and may also play a role in the recovery of reactivatable latent virus from ganglia in both mice and guinea pigs.

Animals↗