José M. Bellés

Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance

The yeast trehalose-6-phosphate synthase gene (TPS1) was engineered under the control of the cauliflower mosaic virus regulatory sequences (CaMV35S) for expression in plants. Using Agrobacterium-mediated transfer, the gene was incorporated into the genomic DNA and constitutively expressed in Nicotiana tabacum L. plants. Trehalose was determined in the transformants, by anion-exchange chromatography coupled to pulsed amperometric detection. The non-reducing disaccharide accumulated up to 0.17 mg per g fresh weight in leaf extracts of transgenic plants. Trehaloseaccumulating plants exhibited multiple phenotypic alterations, including stunted growth, lancet-shaped leaves, reduced sucrose content and improved drought tolerance. These pleiotropic effects, and the fact that water loss from detached leaves was not significantly affected by trehalose accumulation, suggest that synthesis of this sugar, rather than leading to an osmoprotectant effect, had altered sugar metabolism and regulatory pathways affecting plant development and stress tolerance.

The Short-Chain Alcohol Dehydrogenase ABA2 Catalyzes the Conversion of Xanthoxin to Abscisic Aldehyde

Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a Km value for xanthoxin of 19 μM and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC–mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin→abscisic aldehyde→ABA transition as the last steps of the major ABA biosynthetic pathway.

The Yeast HAL2 Nucleotidase Is an in Vivo Target of Salt Toxicity

The yeast halotolerance gene HAL2 encodes a nucleotidase that dephosphorylates 3′-phosphoadenosine 5′-phosphate (PAP) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS), intermediates of the sulfate assimilation pathway. This nucleotidase is inhibited by Na+ and Li+ but not by K+. Incubation of wild-type yeast cells with NaCl and LiCl, but not with KCl, increased intracellular PAP to millimolar concentrations. No depletion of the pool of adenine nucleotides (AMP, ADP, ATP) was observed. Other stresses such as heat shock or oxidative stress did not result in PAP accumulation. PAPS concentrations also increased during salt stress but remained lower than 0.5 μM. S-Adenosylmethionine concentrations decreased by 50%, reflecting inhibition of sulfate assimilation during salt stress. Salt-induced PAP accumulation was attenuated in a yeast strain overexpressing HAL2. This strain grew better than the wild type under salt stress. These results suggest that the cation sensitivity of the HAL2 nucleotidase is an important determinant of the inhibition of yeast growth by sodium and lithium salts. In addition to blocking sulfate assimilation by product inhibition of PAPS reductase, PAP accumulation may have other unidentified toxic effects.

Metabolic response of tomato leaves upon different plant-pathogen interactions.

INTRODUCTION Plants utilise various defence mechanisms against their potential biotic stressing agents such as viroids, viruses, bacteria or fungi and abiotic environmental challenges. Among them metabolic alteration is a common response in both compatible and incompatible plant-pathogen interactions. However, the identification of metabolic changes associated with defence response is not an easy task due to the complexity of the metabolome and the plant response. To address the problem of metabolic complexity, a metabolomics approach was employed in this study. OBJECTIVE To identify a wide range of pathogen (citrus exocortis viroid, CEVd, or Pseudomonas syringae pv. tomato)-induced metabolites of tomato using metabolomics. METHODOLOGY Nuclear magnetic resonance (NMR) spectroscopy in combination with multivariate data analysis were performed to analyse the metabolic changes implicated in plant-pathogen interaction. RESULTS NMR-based metabolomics of crude extracts allowed the identification of different metabolites implicated in the systemic (viroid) and hypersensitive response (bacteria) in plant-pathogen interactions. While glycosylated gentisic acid was the most important induced metabolite in the viroid infection, phenylpropanoids and a flavonoid (rutin) were found to be associated with bacterial infection. CONCLUSIONS NMR metabolomics is a potent platform to analyse the compounds involved in different plant infections. A broad response to different pathogenic infections was revealed at metabolomic levels in the plant. Also, metabolic specificity against each pathogen was observed.

A Novel Mammalian Lithium-sensitive Enzyme with a Dual Enzymatic Activity, 3′-Phosphoadenosine 5′-Phosphate Phosphatase and Inositol-polyphosphate 1-Phosphatase

We report the molecular cloning in Rattus norvegicus of a novel mammalian enzyme (RnPIP), which shows both 3′-phosphoadenosine 5′-phosphate (PAP) phosphatase and inositol-polyphosphate 1-phosphatase activities. This enzyme is the first PAP phosphatase characterized at the molecular level in mammals, and it represents the first member of a novel family of dual specificity enzymes. The phosphatase activity is strictly dependent on Mg2+, and it is inhibited by Ca2+ and Li+ ions. Lithium chloride inhibits the hydrolysis of both PAP and inositol-1,4-bisphosphate at submillimolar concentration; therefore, it is possible that the inhibition of the human homologue of RnPIP by lithium ions is related to the pharmacological action of lithium. We propose that the PAP phosphatase activity of RnPIP is crucial for the function of enzymes sensitive to inhibition by PAP, such as sulfotransferase and RNA processing enzymes. Finally, an unexpected connection between PAP and inositol-1,4-bisphosphate metabolism emerges from this work.

Gentisic Acid As a Pathogen-Inducible Signal, Additional to Salicylic Acid for Activation of Plant Defenses in Tomato

Citrus exocortis viroid (CEVd) and tomato mosaic virus (ToMV), which produce a systemic non-necrotizing infection in tomato (Lycopersicon esculentum cv. Rutgers), strongly induced the accumulation of a phenolic compound that we have characterized as 2,5-dihydroxybenzoic acid (gentisic acid, GA) by nuclear magnetic resonance, following purification by high-performance liquid chromatography. Levels of free and total GA increased more than 150-fold in response to CEVd and ToMV infections. Unlike these non-necrotizing infections, the necrotizing reaction elicited by Pseudomonas syringae pv. syringae in this host did not produce any accumulation of GA. It is also shown that, in healthy leaf tissues, benzoic acid (BA) and salicylic acid (SA) were rapidly converted to GA, SA being the immediate precursor of GA, according to radiolabeling studies. Interestingly, exogenous GA elicited accumulation of the previously described CEVd-induced antifungal pathogenesis-related (PR) proteins P23, P32, and P34. These protei...

Comparative physiology of salt tolerance in Candida tropicalis and Saccharomyces cerevisiae

The salt tolerance of the respiratory yeast Candida tropicalis and the fermentative yeast Saccharomyces cerevisiae have been compared in glucose media. C. tropicalis showed a better adaptation to Na+ and Li+ and maintained higher intracellular K+:Na+ and K+:Li+ ratios than S. cerevisiae. However, C. tropicalis showed a poorer adaptation to osmotic stress (produced by KCl and sorbitol) and exhibited reduced glycerol production as compared to S. cerevisiae. In media with the non-repressing sugar galactose as carbon source, S. cerevisiae exhibited reduced glycerol production and increased sensitivity to osmotic stress. Under these conditions, S. cerevisiae, but not C. tropicalis, utilized trehalose as a more important osmolyte than glycerol. These results suggest that the relative tolerance of yeast to the osmotic and cation toxicities of NaCl, and the underlying relative capabilities for osmolyte synthesis and cation transport, are modulated by the general catabolite control exerted by glucose.

Induction of p-Coumaroyldopamine and Feruloyldopamine, Two Novel Metabolites, in Tomato by the Bacterial Pathogen Pseudomonas syringae

Inoculation of tomato plants (Solanum lycopersicum cv. Rutgers) with Pseudomonas syringae pv. tomato led to the production of a hypersensitive-like response in this pathovar of tomato. Accumulation of hydroxycinnamic acid amides (HCAA) of tyramine (p-coumaroyltyramine and feruloyltyramine) and dopamine (p-coumaroyldopamine and feruloyldopamine) was detected after bacterial infection. Two of them, p-coumaroyldopamine and feruloyldopamine, are described for the first time. The accumulation of HCAA was preceded by an increment of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase (THT) gene expression. HCAA also accumulated in transgenic NahG tomato plants overexpressing a bacterial salicylic hydroxylase. However, treatment of plants with the ethylene biosynthesis inhibitor, aminoethoxyvinilglycine, led to a reduction in the accumulation of THT transcripts and HCAA. Together, the results suggest that pathogen-induced induction of ethylene is essential for HCAA synthesis, whereas salicylic acid is not required for this response. In addition, notable antibacterial and antioxidant activities were found for the new HCAA, thus indicating that they could play a role in the defense of tomato plants against bacterial infection.

Shared and novel molecular responses of mandarin to drought

Drought is the most important stress experienced by citrus crops. A citrus cDNA microarray of about 6.000 genes has been utilized to identify transcriptomic responses of mandarin to water stress. As observed in other plant species challenged with drought stress, key genes for lysine catabolism, proline and raffinose synthesis, hydrogen peroxide reduction, vacuolar malate transport, RCI2 proteolipids and defence proteins such as osmotin, dehydrins and heat-shock proteins are induced in mandarin. Also, some aquaporin genes are repressed. The osmolyte raffinose could be detected in stressed roots while the dehydrin COR15 protein only accumulated in stressed leaves but not in roots. Novel drought responses in mandarin include the induction of genes encoding a new miraculin isoform, chloroplast β-carotene hydroxylase, oleoyl desaturase, ribosomal protein RPS13A and protein kinase CTR1. These results suggest that drought tolerance in citrus may benefit from inhibition of proteolysis, activation of zeaxanthin and linolenoyl synthesis, reinforcement of ribosomal structure and down-regulation of the ethylene response.

A novel target of lithium therapy

Phosphatases converting 3′‐phosphoadenosine 5′‐phosphate (PAP) into adenosine 5′‐phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems. These enzymes are lithium‐sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy. A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity. The enzyme exhibits high affinity for PAP (K m<1 μM) and is sensitive to subtherapeutic concentrations of lithium (IC50=0.3 mM). The human enzyme also hydrolyzes inositol‐1,4‐bisphosphate with high affinity (K m=0.4 μM), therefore it can be considered as a dual specificity enzyme with high affinity (μM range) for both PAP and inositol‐1,4‐bisphosphate. Hydrolysis of inositol‐1,4‐bisphosphate was also inhibited by lithium (IC50=0.6 mM). Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy.