Josefa M. Alamillo

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.

Local inhibition of nitrogen fixation and nodule metabolism in drought-stressed soybean

Drought stress is a major factor limiting symbiotic nitrogen fixation (NF) in soybean crop production. However, the regulatory mechanisms involved in this inhibition are still controversial. Soybean plants were symbiotically grown in a split-root system (SRS), which allowed for half of the root system to be irrigated at field capacity while the other half remained water deprived. NF declined in the water-deprived root system while nitrogenase activity was maintained at control values in the well-watered half. Concomitantly, amino acids and ureides accumulated in the water-deprived belowground organs regardless of transpiration rates. Ureide accumulation was found to be related to the decline in their degradation activities rather than increased biosynthesis. Finally, proteomic analysis suggests that plant carbon metabolism, protein synthesis, amino acid metabolism, and cell growth are among the processes most altered in soybean nodules under drought stress. Results presented here support the hypothesis of a local regulation of NF taking place in soybean and downplay the role of ureides in the inhibition of NF.

Molecular analysis of ureide accumulation under drought stress in Phaseolus vulgaris L.

Under water deficit, ureidic legumes accumulate ureides in plant tissues, and this accumulation has been correlated with the inhibition of nitrogen fixation. In this work we used a molecular approach to characterize ureide accumulation under drought stress in Phaseolus vulgaris. Accumulation of ureides, mainly allantoate, was found in roots, shoots and leaves, but only a limited transient increase was observed in nodules from drought-stressed plants. We show that ureide accumulation is regulated at the transcriptional level mainly through induction of allantoinase (ALN), whereas allantoate amidohydrolase (AAH), involved in allantoate degradation, was slightly reduced, indicating that inhibition of this enzyme, key in ureide breakdown in aerial tissues, is not the main cause of allantoate accumulation. Expression of the ureide metabolism genes analysed in this study was induced by abscisic acid (ABA), suggesting the involvement of this plant hormone in ureide accumulation. Moreover, we observed that increases of ureide levels in P. vulgaris drought-stressed tissues were similar in non-nodulated, nitrate-fed plants, and in plants cultured under nitrogen-fixation conditions. Our results indicate that ureide accumulation in response to water deficit is independent from de novo synthesis of ureides in nodules, and therefore uncoupled from nitrogen fixation.

An antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts

Summary To investigate the potential of antibody derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed a small immune protein (SIP) in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The ɛSIP molecule consisted of a single‐chain antibody (scFv) specific for the porcine coronavirus transmissible gastroenteritis virus (TGEV) linked to the ɛ‐CH4 domain from human immunoglobulin E (IgE). In some constructs, the sequence encoding the ɛSIP molecule was flanked by the leader peptide from the original murine antibody at its N‐terminus and an endoplasmic reticulum retention signal (HDEL) at its C‐terminus to allow the expressed protein to be directed to, and retained within, the endoplasmic reticulum. Western blot analysis of samples from Nicotiana clevelandii or cowpea tissue infected with constructs revealed the presence of SIP molecules which retained their ability to dimerize. The analysis of crude plant extracts revealed that the plant‐expressed ɛSIP molecules could bind to and neutralize TGEV in tissue culture, the levels of binding and neutralization reflecting the level of expression. Oral administration of crude extracts from SIP‐expressing plant tissue to 2‐day‐old piglets demonstrated that the extracts which showed the highest levels of in vitro neutralization could also provide in vivo protection against challenge with TGEV.

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 Stokess 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.

Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L.

The ureides allantoin and allantoate are key molecules in the transport and storage of nitrogen in ureide legumes. In shoots and leaves from Phaseolus vulgaris plants using symbiotically fixed nitrogen as the sole nitrogen source, ureide levels were roughly equivalent to those of nitrate-supported plants during the whole vegetative stage, but they exhibited a sudden increase at the onset of flowering. This rise in the level of ureides, mainly in the form of allantoate, was accompanied by increases in allantoinase gene expression and enzyme activity, consistent with developmental regulation of ureide levels mainly through the tissue-specific induction of allantoate synthesis catalysed by allantoinase. Moreover, surprisingly high levels of ureides were also found in non-nodulated plants fertilized with nitrate, at both early and late developmental stages. The results suggest that remobilized N from lower leaves is probably involved in the sharp rise in ureides in shoots and leaves during early pod filling in N(2)-fixing plants and in the significant amounts of ureides observed in non-nodulated plants.

Use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies

Abstract To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full‐length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The agr;SIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the α‐CH3 domain from human IgA. To express the full‐length IgA, the individual light and heavy chains from the TGEV‐specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co‐infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant‐expressed αSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody‐expressing plant tissue to 2‐day‐old piglets showed that both the αSIP and full‐length IgA molecules can provide in vivo protection against TGEV.

Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonasreinhardtii

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.

Kinetic and catalytic characterization of urate oxidase from Chlamydomonas reinhardtii

Abstract Urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from Chlamydomonas reinhardtii is a tetramer of M r =124 000, composed of similar-sized subunits and containing four atoms of copper per enzyme molecule (J. M. Alamillo, J. Cardenas and M. Pineda, Biochim Biophys. Acta, 1076 (1991) 203-208). The enzyme exhibits a strict specificity toward its substrates, oxygen and urate, and has optimal activity in the pH 8.5–9.5 range, as determined by the V max / K m ratio. Purified uricase obeys hyperbolic kinetics for urate but shows a sigmoidal response to oxygen (Hills coefficient = 3.7), indicative of a positive homotropic cooperatively. Under steady-state conditions, sets of intersecting lines were found in the double-reciprocal plots, which indicates that uricase reaction proceeds through a ternary complex involving enzyme, urate and oxygen. Since each substrate bonded to the enzyme in the absence of the other, a random mechanism is proposed for the complex formation. Uricase activity was competitively inhibited by hypoxanthine, xanthine and their 8-aza derivatives, as well as by oxonate, allantoin and CO 2 . Hydrogen peroxide acted as an uncompetitive inhibitor, and 1- and 9-methylurate showed mixed inhibition. Dissociation constants calculated from the effect of pH on K m and V max indicate that two dissociable groups with p K values of about 7.6 and 10.0 are probably involved in both substrate binding and catalytic oxidation. Inhibition by the group-specific reagents diethylpyrocarbonate and phenylglyoxal strongly suggests that those dissociable groups are histidyl and arginyl residues. From these data a picture emerges in which copper and dissociable groups on the enzyme interact with CO and NH groups on urate.

Molecular and functional characterization of allantoate amidohydrolase from Phaseolus vulgaris

Allantoate degradation is an essential step for recycling purine-ring nitrogen in all plants, but especially in tropical legumes where the ureides allantoate and allantoin are the main compounds used to store and transport the nitrogen fixed in nodules. Two enzymes, allantoate amidohydrolase (AAH) and allantoate amidinohydrolase (allantoicase), could catalyze allantoate breakdown, although only AAH-coding sequences have been found in plant genomes, whereas allantoicase-related sequences are restricted to animals and some microorganisms. A cDNA for AAH was cloned from Phaseolus vulgaris leaves. PvAAH is a single-copy gene encoding a polypeptide of 483 amino acids that conserves all putative AAH active-site domains. Expression and purification of the cDNA in Nicotiana benthamiana showed that the cloned sequence is a true AAH protein that yields ureidoglycine and ammonia, with a Km of 0.46 mM for allantoate. Optimized in vitro assay, quantitative RT-PCR and antibodies raised to the PvAAH protein were used to study AAH under physiological conditions. PvAAH is ubiquitously expressed in common bean tissues, although the highest transcript levels were found in leaves. In accordance with the mRNA expression levels, the highest PvAAH activity and allantoate concentration also occurred in the leaves. Comparison of transcript levels, protein amounts and enzymatic activity in plants grown with different nitrogen sources and upon drought stress conditions showed that PvAAH is regulated at posttranscriptional level. Moreover, RNAi silencing of AAH expression increases allantoate levels in the transgenic hairy roots, indicating that AAH should be the main enzyme involved in allantoate degradation in common bean.