Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls

Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant development. The analyses of reporter gene lines indicate gene expression of UDP-glucose dehydrogenases in growing tissues. The biochemical characterization of the different isoforms shows equal affinities for the cofactor NAD+ (~40 µM) but variable affinities for the substrate UDP-glucose (120–335 µM) and different catalytic constants, suggesting a regulatory role for the different isoforms in carbon partitioning between cell wall formation and sucrose synthesis as the second major UDP-glucose-consuming pathway. UDP-glucose dehydrogenase is feedback inhibited by UDP-xylose. The relatively (compared with a soybean UDP-glucose dehydrogenase) low affinity of the enzymes for the substrate UDP-glucose is paralleled by the weak inhibition of the enzymes by UDP-xylose. The four Arabidopsis UDP-glucose dehydrogenase isoforms oxidize only UDP-glucose as a substrate. Nucleotide-sugars, which are converted by similar enzymes in bacteria, are not accepted as substrates for the Arabidopsis enzymes

Cell wall remodeling under abiotic stress

Plants exposed to abiotic stress respond to the unfavorable conditions at multiple levels. One challenge under drought stress is to reduce shoot growth while maintaining root growth, a process requiring differential cell wall synthesis and remodeling. Key players in this process are the formation of reactive oxygen species (ROS) and peroxidases, which initially cross-link phenolic compounds and glycoproteins of the cell walls causing stiffening. The function of ROS shifts after having converted all of the peroxidase substrates in the cell wall. If ROS-levels remain high during prolonged stress, OH°-radicals are formed which lead to polymer cleavage. In concert with xyloglucan modifying enzymes and expansins, the resulting cell wall loosening allows further growth of stressed organs

DCD – a novel plant specific domain in proteins involved in development and programmed cell death

BACKGROUND: Recognition of microbial pathogens by plants triggers the hypersensitive reaction, a common form of programmed cell death in plants. These dying cells generate signals that activate the plant immune system and alarm the neighboring cells as well as the whole plant to activate defense responses to limit the spread of the pathogen. The molecular mechanisms behind the hypersensitive reaction are largely unknown except for the recognition process of pathogens. We delineate the NRP-gene in soybean, which is specifically induced during this programmed cell death and contains a novel protein domain, which is commonly found in different plant proteins. RESULTS: The sequence analysis of the protein, encoded by the NRP-gene from soybean, led to the identification of a novel domain, which we named DCD, because it is found in plant proteins involved in development and cell death. The domain is shared by several proteins in the Arabidopsis and the rice genomes, which otherwise show a different protein architecture. Biological studies indicate a role of these proteins in phytohormone response, embryo development and programmed cell by pathogens or ozone. CONCLUSION: It is tempting to speculate, that the DCD domain mediates signaling in plant development and programmed cell death and could thus be used to identify interacting proteins to gain further molecular insights into these processes

Ascorbate oxidation activates systemic defence against root-knot nematode Meloidogyne graminicola in rice

Ascorbic acid (AA) is the major antioxidant buffer produced in the shoot tissue of plants. Previous studies on root-knot nematode (RKN; Meloidogyne graminicola)-infected rice (Oryza sativa) plants showed differential expression of AA-recycling genes, although their functional role was unknown. Our results confirmed increased dehydroascorbate (DHA) levels in nematode-induced root galls, while AA mutants were significantly more susceptible to nematode infection. External applications of ascorbate oxidase (AO), DHA, or reduced AA, revealed systemic effects of ascorbate oxidation on rice defence versus RKN, associated with a primed accumulation of H2O2 upon nematode infection. To confirm and further investigate these systemic effects, a transcriptome analysis was done on roots of foliar AO-treated plants, revealing activation of the ethylene (ET) response and jasmonic acid (JA) biosynthesis pathways in roots, which was confirmed by hormone measurements. Activation of these pathways by methyl-JA, or ethephon treatment can complement the susceptibility phenotype of the rice Vitamin C (vtc1) mutant. Experiments on the jasmonate signalling (jar1) mutant or using chemical JA/ET inhibitors confirm that the effects of ascorbate oxidation are dependent on both the JA and ET pathways. Collectively, our data reveal a novel pathway in which ascorbate oxidation induces systemic defence against RKNs

Down-regulation of the myo-inositol oxygenase gene family has no effect on cell wall composition in Arabidopsis

The enzyme myo-inositol oxygenase (MIOX; E.C. 1.13.99.1) catalyzes the ring-opening four-electron oxidation of myo-inositol into glucuronic acid, which is subsequently activated to UDP-glucuronic acid (UDP-GlcA) and serves as a precursor for plant cell wall polysaccharides. Starting from single T-DNA insertion lines in different MIOX-genes a quadruple knockdown (miox1/2/4/5-mutant) was obtained by crossing, which exhibits greater than 90% down-regulation of all four functional MIOX genes. Miox1/2/4/5-mutant shows no visible phenotype and produces viable pollen. The alternative pathway to UDP-glucuronic acid via UDP-glucose is upregulated in the miox1/2/4/5-mutant as a compensatory mechanism. Miox1/2/4/5-mutant is impaired in the utilization of myo-inositol for seedling growth. The incorporation of myo-inositol derived sugars into cell walls is strongly (>90%) inhibited. Instead, myo-inositol and metabolites produced from myo-inositol such as galactinol accumulate in the miox1/2/4/5-mutant. The increase in galactinol and raffinose family oligosaccharides does not enhance stress tolerance. The ascorbic acid levels are the same in mutant and wild type plants

Myoinositol Oxygenase Controls the Level of Myoinositol in Arabidopsis, But Does Not Increase Ascorbic Acid1[OA]

Ascorbic acid (AsA) is a major plant antioxidant. Mutants like vtc1 show a reduced AsA concentration, which confirmed by genetic evidence the previously proposed AsA pathway via GDP-Man. Here we investigate the role of an animal-like alternative biosynthesis route to AsA, starting from the metabolite d-GlcUA, which is produced in plants by myoinositol oxygenase (Miox). Miox-overexpressing lines have a more than 30-fold up-regulated transcript level and higher enzymatic activity as shown by increased incorporation of Miox-derived sugars into cell wall polymers. In addition, Miox overexpressors exhibit a lower steady-state level of myoinositol and accumulate less myoinositol in feeding experiments due to an enhanced turnover rate. The AsA concentration remains the same in wild-type and Miox overexpressor lines. Even challenging plants with stress, which increases AsA concentration 4-fold, reveals no difference in AsA biosynthesis between wild-type and Miox-overexpressing lines. We conclude that d-GlcUA derived from the Miox reaction plays a negligible role for AsA biosynthesis. However, Miox controls the metabolite level of myoinositol in plants

Raffinose family oligosaccharides act as galactose stores in seeds and are required for rapid germination of Arabidopsis in the dark

Raffinose synthase 5 (AtRS5, At5g40390) was characterized from Arabidopsis as a recombinant enzyme. It has a far higher affinity for the substrates galactinol and sucrose than any other raffinose synthase previously reported. In addition raffinose synthase 5 is also working as a galactosylhydrolase, degrading galactinol, and raffinose under certain conditions. Together with raffinose synthase 4, which is predominantly a stachyose synthase, both enzymes contribute to the raffinose family oligosaccharide (RFO) accumulation in seeds. A double knockout in raffinose synthase 4 and raffinose synthase 5 (AtRS4,5) was generated, which is devoid of RFOs in seeds. Unstressed leaves of 4 week old AtRS4,5 plants showed drastically 23.8-fold increased concentrations of galactinol. Unexpectedly, raffinose appeared again in drought stressed AtRS4,5 plants, but not under other abiotic stress conditions. Drought stress leads to novel transcripts of raffinose synthase 6 suggesting that this isoform is a further stress inducible raffinose synthase in Arabidopsis. AtRS4,5 seeds showed a 5 days delayed germination phenotype in darkness and an elevated expression of the transcription factor phytochrome interacting factor 1 (AtPIF1) target gene AtPIF6, being a repressor of germination. This prolonged dormancy is not seen during germination in the light. Exogenous galactose partially promotes germination of AtRS4,5 seeds in the dark suggesting that RFOs act as a galactose store and repress AtPIF6 transcripts.P27323-B2