Comparison of inhibition of N2 fixation and ureide accumulation under water deficit in four common bean genotypes of contrasting drought tolerance.

BACKGROUND AND AIMS: Drought is the principal constraint on world production of legume crops. There is considerable variability among genotypes in sensitivity of nitrogen fixation to drought, which has been related to accumulation of ureides in soybean. The aim of this study was to search for genotypic differences in drought sensitivity and ureide accumulation in common bean (Phaseolus vulgaris) germplasm that may be useful in the improvement of tolerance to water deficit in common bean. METHODS: Changes in response to water deficit of nitrogen fixation rates, ureide content and the expression and activity of key enzymes for ureide metabolism were measured in four P. vulgaris genotypes differing in drought tolerance. KEY RESULTS: A variable degree of drought-induced nitrogen fixation inhibition was found among the bean genotypes. In addition to inhibition of nitrogen fixation, there was accumulation of ureides in stems and leaves of sensitive and tolerant genotypes, although this was higher in the leaves of the most sensitive ones. In contrast, there was no accumulation of ureides in the nodules or roots of stressed plants. In addition, the level of ureides in the most sensitive genotype increased after inhibition of nitrogen fixation, suggesting that ureides originate in vegetative tissues as a response to water stress, probably mediated by the induction of allantoinase. CONCLUSIONS: Variability of drought-induced inhibition of nitrogen fixation among the P. vulgaris genotypes was accompanied by subsequent accumulation of ureides in stems and leaves, but not in nodules. The results indicate that shoot ureide accumulation after prolonged exposure to drought could not be the cause of inhibition of nitrogen fixation, as has been suggested in soybean. Instead, ureides seem to be produced as part of a general response to stress, and therefore higher accumulation might correspond to higher sensitivity to the stressful conditions.

Identification of a plum pox virus CI-interacting protein from chloroplast that has a negative effect in virus infection.

The cylindrical inclusion (CI) protein of potyviruses is involved in virus replication and cell-to-cell movement. These two processes should rely on multiple plant-virus interactions; however, little is known about the host factors that are involved in, or that may interfere with, CI functions. By using a yeast two-hybrid system, the CI protein from Plum pox virus (PPV) was found to interact with the photosystem I PSI-K protein, the product of the gene psaK, of Nicotiana benthamiana. Coexpression of PPV CI was shown to cause a decrease in the accumulation level of PSI-K transiently expressed in N. benthamiana leaves. To test the biological relevance of this interaction, we have analyzed the infection of PPV in N. benthamiana plants in which psaK gene expression has been silenced by RNA interference, as well as in Arabidopsis thaliana psaK knockout plants. Our results show that downregulation of the psaK gene leads to higher PPV accumulation, suggesting a role for the CI-PSI-K interaction in PPV infection.

Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana.

Twelve Arabidopsis accessions were challenged with Plum pox potyvirus (PPV) isolates representative of the four PPV strains. Each accession supported local and systemic infection by at least some of the PPV isolates, but high variability was observed in the behavior of the five PPV isolates or the 12 Arabidopsis accessions. Resistance to local infection or long-distance movement occurred in about 40% of all the accession-isolate combinations analyzed. Except for Nd-1, all accessions showed resistance to local infection by PPV-SoC; in the Landsberg erecta (Ler) accession, this resistance was compromised by sgt1 and rar1 mutations, suggesting that it could be controlled by an R gene-mediated resistance pathway. While most of the susceptible accessions were symptomless, PPV induced severe symptoms on inflorescences in C24, Ler, and Bay-0 as early as 15 days after inoculation. Genetic analyses indicated that these interaction phenotypes are controlled by different genetic systems. The restriction of long-distance movement of PPV-El Amar and of another member of genus Potyvirus, Lettuce mosaic virus, in Col-0 requires the RTM genes, indicating for the first time that the RTM system may provide a broad range, potyvirus-specific protection against systemic infection. The restriction to PPV-PS long-distance movement in Cvi-1 is controlled by a single recessive gene, designated rpv1, which was mapped to chromosome 1. The nuclear inclusion polymerase b-capsid protein region of the viral genome appears to be responsible for the ability of PPV-R to overcome rpv1-mediated resistance.

Purification and molecular properties of urate oxidase from Chlamydomonas reinhardtii.

Urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic and immunological homogeneity by a procedure which includes as main steps ammonium sulfate fractionation, gel filtration, ion exchange and xanthine-agarose affinity chromatography. The native enzyme has a relative molecular mass (Mr) of 124,000 and consists of four identical or similar-sized subunits of Mr 31,000 each. The enzyme has a Stokes's radius of 3.87 nm, a sedimentation coefficient of 6.8 S and an f/f0 of 1.23, and exhibits its maximal absorption at 276 nm. Optimum pH was 8.5 and maximum activity was shown at 40 degrees C, with an activation energy of 53 kJ.mol-1 and a Q10 of 1.96. Absorption spectrum of native reduced enzyme showed two transient maxima at 392 and 570 nm, very similar to those of metal-urate complexes, which disappeared in the presence of cyanide. Inhibition by cyanide and neocuproin, but not by salicylhydroxamic acid, strongly suggests that copper is the metal involved in enzymatic urate oxidation. By using a sensitive photokinetic method for copper determination, a content of 4 mol of copper per mol of enzyme has been found.

Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii.

Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.

Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence.

Genes encoding plant antibiotic peptides show expression patterns that are consistent with a defence role. Transgenic over-expression of defence peptide genes is potentially useful to engineer resistance of plants to relevant pathogens. Pathogen mutants that are sensitive to plant peptides in vitro have been obtained and a decrease of their virulence in planta has been observed, which is consistent with their hypothetical defence role. A similar approach has been followed to elucidate the potential direct anti-microbial role of hydrogen peroxide. Additionally, a scavenger of peroxynitrite has been used to investigate its involvement in plant defence.

Identification of secret agent as the O-GlcNAc transferase that participates in Plum pox virus infection.

Serine and threonine of many nuclear and cytoplasmic proteins are posttranslationally modified with O-linked N-acetylglucosamine (O-GlcNAc). This modification is made by O-linked N-acetylglucosamine transferases (OGTs). Genetic and biochemical data have demonstrated the existence of two OGTs of Arabidopsis thaliana, SECRET AGENT (SEC) and SPINDLY (SPY), with at least partly overlapping functions, but there is little information on their target proteins. The N terminus of the capsid protein (CP) of Plum pox virus (PPV) isolated from Nicotiana clevelandii is O-GlcNAc modified. We show here that O-GlcNAc modification of PPV CP also takes place in other plant hosts, N. benthamiana and Arabidopsis. PPV was able to infect the Arabidopsis OGT mutants sec-1, sec-2, and spy-3, but at early times of the infection, both rate of virus spread and accumulation were reduced in sec-1 and sec-2 relative to spy-3 and wild-type plants. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, we determined that a 39-residue tryptic peptide from the N terminus of CP of PPV purified from the spy-3 mutant, but not sec-1 or sec-2, was O-GlcNAc modified, suggesting that SEC but not SPY modifies the capsid. While our results indicate that O-GlcNAc modification of PPV CP by SEC is not essential for infection, they show that the modification has a role(s) in the process.

Effects of desiccation on photosynthesis pigments and the ELIP-like dsp 22 protein complexes in the resurrection plant Craterostigma plantagineum.

Desiccation and abscisic acid treatment lead to major changes in thylakoid membranes of the desiccation-tolerant plant Craterostigma plantagineum. The chlorophyll contents and proteins of the light harvesting complexes decrease during desiccation, although some chlorophyll is retained in the dehydrated state. The xanthophyll cycle pigment zeaxanthin, however, increased. Under these conditions, a 22 kDa ELIP-like desiccation-induced protein (dsp 22) accumulated in the thylakoid membranes. Fractionation of pigment-protein complexes of stressed plants revealed that the dsp 22 protein co-localized with the carotenoid zeaxanthin. Inhibition of zeaxanthin production had a negative effect on the accumulation of the dsp 22 protein. It is suggested that dsp 22 contributes to the protection against photoinhibition caused by dehydration.

Effects of urate, a natural inhibitor of peroxynitrite-mediated toxicity, in the response of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae.

Urate, a natural peroxynitrite scavenger, has been used to investigate the possible role of peroxynitrite during plant-pathogen interactions. Urate greatly reduced lesion formation in Arabidopsis leaves treated with an abiotic peroxynitrite-generating system or with a peroxynitrite solution, indicating that it can act as an effective scavenger in planta. In the interaction with the avirulent Pseudomonas syringae pv. phaseolicola (avrRPM1+), cell death in the inoculated area was strongly reduced by urate, without compromising disease resistance. In contrast, urate promoted discrete cell death in response to an isogenic Pseudomonas syringae (avrRPM1-), which did not trigger an HR when inoculated alone, and it induced resistance and arrest of pathogen growth. Scavenging of peroxynitrite did not modify the response of Arabidopsis to an avirulent strain of Xanthomonas campestris pv campestris, that showed a high resistance to NO and peroxynitrite. Our data indicate that peroxynitrite plays a significant role in the responses of plants to Pseudomonas syringae.

Processing of thionin precursors in barley leaves by a vacuolar proteinase.

Thionins are synthesized as precursors with a signal peptide and a long C-terminal acidic peptide that is post-translationally processed. A fusion protein including the maltose-binding protein from Escherichia coli (MalE), thionin DG3 from barley leaves, and its acidic C-terminal peptide has been used to obtain antibodies that recognize both domains of the precursor. In barley leaf sections, mature thionins accumulated in the vacuolar content, while the acidic peptide was not detected in any cell fraction. Brefeldin A and monensin inhibited processing of the precursor but its export from the microsomal fraction was not inhibited. Both purified vacuoles and an acid (pH 5.5) extract from leaves processed the fusion protein into a MalE-thionin and an acidic peptide fragment. A 70-kDa proteinase that effected this cleavage was purified from the acid extract. Processing of the fusion protein by both lysed vacuoles and the purified proteinase was inhibited by Zn2+ and by Cu2+, but not by inhibitors of the previously described vacuolar processing thiol or aspartic proteinases. In vivo processing of the thionin precursor in leaf sections was also inhibited by Zn2+ and Cu2+. Variants of the fusion protein with altered processing sites that represented those of thionin precursors from different taxa were readily processed by the proteinase, whereas changing the polarity of either the C-terminal or N-terminal residues of the processing site prevented cleavage by the proteinase.

A novel embryo-specific barley cDNA clone encodes a protein with homologies to bacterial glucose and ribitol dehydrogenase.

In order to analyze the genetic programme expressed during the early stages of embryogenesis a cDNA clone bank was constructed from desiccation-tolerant excised barley embryos 18 d after pollination (D. Bartels et al., 1988, Planta 175, 485-492). One of the selected cDNA clones pG31 encodes a transcript of 1300 nucleotides and a protein of 31 kDa, both are specifically expressed in developing embryos and are not detected in other tissues. The expression of the pG31 mRNA is not modulated by the plant hormone cis-abscisic acid but it ceases to be expressed in germinating embryos. The protein sequence deduced from the pG31 transcript shows substantial sequence homologies to bacterial glucose dehydrogenase and ribitol dehydrogenase. Biochemical analysis indicates that glucose dehydrogenase activity is present in protein extracts from embryos 18 d after pollination. This glucose dehydrogenase activity is inhibited by antiserum raised against the recombinant pG31 protein. These findings provide evidence for the discovery of a novel pathway in carbohydrate metabolism acting specifically during embryogenesis.

Plant defense peptides.

Eight families of antimicrobial peptides, ranging in size from 2 to 9 kD, have been identified in plants. These are thionins, defensins, so-called lipid transfer proteins, hevein- and knottin-like peptides, MBP1, IbAMP, and the recently reported snakins. All of them have compact structures that are stabilized by 2-6 disulfide bridges. They are part of both permanent and inducible defense barriers. Transgenic overexpression of the corresponding genes leads to enhanced tolerance to pathogens, and peptide-sensitive pathogen mutants have reduced virulence.