Luc Richard

Calcium Signaling via Phospholipase C Is Essential for Proline Accumulation upon Ionic But Not Nonionic Hyperosmotic Stresses in Arabidopsis

Proline (Pro) accumulation occurs in various plant organisms in response to environmental stresses. To identify the signaling components involved in the regulation of Pro metabolism upon water stress in Arabidopsis (Arabidopsis thaliana), a pharmacological approach was developed. The role of phosphoinositide-specific phospholipases C (PLCs) in Pro accumulation was assessed by the use of the aminosteroid U73122, a commonly employed specific inhibitor of receptor-mediated PLCs. We found that U73122 reduced pyrroline-5-carboxylate synthetase transcript and protein as well as Pro levels in salt-treated seedlings. Inhibition of PLC activity by U73122 was quantified by measuring the decrease of inositol 1,4,5-trisphosphate (InsP3) levels. Moreover, the utilization of diacylglycerol kinase and InsP3-gated calcium release receptor inhibitors suggested that InsP3 or its derivatives are essential for Pro accumulation upon salt stress, involving calcium as a second messenger in ionic stress signaling. This observation was further supported by a partial restoration of Pro accumulation in salt- and U73122-treated seedlings after addition of extracellular calcium, or when calcium homeostasis was perturbed by cyclopiazonic acid, a blocker of plant type IIA calcium pumps. Taken together, our data indicate that PLC-based signaling is a committed step in Pro biosynthesis upon salinity but not in the case of mannitol stress. Calcium acts as a molecular switch to trigger downstream signaling events. These results also demonstrated the specific involvement of lipid signaling pathway to discriminate between ionic and nonionic stresses.

Regulation of phosphatidylcholine biosynthesis under salt stress involves choline kinases in Arabidopsis thaliana

Increasing evidence suggests a major role for phosphatidylcholine (PC) in plant stress adaptation. The present work investigated the regulation of choline, PC and interconnected phosphatidylethanolamine biosynthesis in Arabidopsis thaliana L. as a function of cold‐ and salt‐ or mannitol‐mediated hyperosmotic stresses. While PC synthesis is accelerated in both salt‐ and cold‐treated plants, the choline kinase (CK) and phosphocholine cytidylyltransferase genes are oppositely regulated with respect to these abiotic treatments. Salt stress also stimulates CK activity in vitro. A possible regulatory role of CK in stimulating PC biosynthesis rate in salt‐stressed plants is discussed.

Characterization of the pectin methylesterase-like gene AtPME3: a new member of a gene family comprising at least 12 genes in Arabidopsis thaliana

Pectin demethylesterification appears to be catalysed by a number of pectin methylesterase (PME) isoenzymes in higher plant species. In order to better define the biological role of these isoenzymes in plant cell growth and differentiation, we undertook molecular studies on the PME-encoding genes in Arabidopsis thaliana. In this paper, we report the characterization of AtPME3, a new PME-related gene of 4kb in length that we have mapped on Chromosome III. AtPME3 encodes a putative mature PME-related isoenzyme of 34kDa with a basic isoelectric point. Since the extent of the gene family encoding PME in higher plant species is still unknown, we resorted to the use of degenerate primers designed from several well-known consensus regions to identify new PME-related genes in the genome of Arabidopsis. Our results, in combination with several known expressed sequences tags (ESTs), indicate that the Arabidopsis genome contains at least 12 PME-related genes. Consequently, a method of systematic gene expression analysis has been applied in order to discern the expression pattern of these 12 genes throughout the plant at the floral stage. Whereas most of these genes appeared to be more or less ubiquitously expressed throughout the plant, several genes are distinguishable by their strikingly specific expression in certain organs. The present data bring a new insight into the role of specific PME-related genes in flower and root development.

Proline dehydrogenase: a key enzyme in controlling cellular homeostasis.

Proline dehydrogenase (ProDH), also called proline oxidase (POX), is a universal enzyme in living organisms. It catalyzes the oxidation of L-proline to delta1-pyrroline-5-carboxylate leading to the release of electrons, which can be transferred to either electron transfer systems or to molecular oxygen. ProDH is not only essential for proline catabolism but also plays key roles in providing energy, shuttling redox potential between cellular compartments and reactive oxygen species production. Structural analysis of prokaryotic ProDHs already gives some insights into the biochemical activity and biological functions of this enzyme, which can be extended to eukaryotic ProDHs based on sequence similarities. Here we report the most recent investigations on the biochemical and regulation of ProDH at transcriptional, post-transcriptional and translational levels. The biological roles of ProDH in cell homeostasis and adaptation through energetic, developmental, adaptive, physiological and pathological processes in eukaryotes are presented and discussed to create a framework for future research direction.

Molecular cloning and characterisation of a putative pectin methylesterase cDNA in Arabidopsis thaliana (L.)

Pectin methylesterase (PME) is a cell wall enzyme that catalyses the de‐esterification of pectins leading to fundamental changes which confer new properties to the micro‐environment of each cell. In order to elucidate the meaning of PME‐mediated changes of pectin in the time course of cell differentiation, we attempted to study the regulation of PME genes in Arabidopsis thaliana. In this report, the first full cDNA sequence showing sequence similarities with other PME genes characterised so far in other plant species has been isolated from an Arabidopsis shoot cDNA library. This ATPME1 cDNA is 1,970 bp long and contains an open reading frame encoding a protein of 64,1 kDa and a basic pI of 8.7 as predicted from the nucleotide sequence. Northern blot analyses denoted changes in the expression level of the ATPME1 mRNA according to plant organs. High mRNA levels were found in young developing organs such as cauline leaves while they were significantly lower in rosette leaves, stems and inflorescences, and almost undetectable in roots. Beside this molecular approach, isoelectrofocusing analyses revealed the occurrence of three PME isoforms in Arabidopsis. Two PME isoforms with pI values of 4.9 and 9.1 were found throughout the plant, but at a higher level in the root, while an other PME isoform with a pI of 5.7 was essentially detected in the inflorescence. The relationship between our observations and the data reported for other plant species is discussed.

Clustered genes within the genome of Arabidopsis thaliana encoding pectin methylesterase-like enzymes

We focused our attention on the isolation and regulation of genes encoding for pectin methylesterase (PME) isoenzymes in Arabidopsis thaliana (At). The present data reports the identification of two PME-like genes, named AtPME1 and AtPME2, that are closely linked in the At genome. These genes possess different structural organisations. While all higher plant PME characterised so far possess an intron at a similar location relative to their coding sequence, AtPME2 shows an additional intron whose presence within other higher plant PME-like genes has not been previously reported. Sequence comparison of the N-terminal region suggests that the secretory pathways of the putative AtPME1 and AtPME2 isoforms are different, and that this region may contribute to specify a biological function to each isoform. Moreover, phylogenetic analysis reflects the possible existence of functional groups of PME isoforms in higher plant species that seem to have been conserved during evolution.

PCR cloning and expression analysis of a cDNA encoding a pectinacetylesterase from Vigna radiata L.

A cDNA clone encoding a pectinacetylesterase (PAE) was isolated from 3‐day‐old mung bean seedlings using PCR‐based techniques. Degenerate oligonucleotide primers were designed according to the N‐terminus and internal peptides from the purified PAE. The full‐length clone of 1453 bp codes for a signal peptide of 24 amino acids and a mature protein of 375 amino acids. The M r and the pI of the cDNA‐deduced amino acid sequence agree with the values estimated for the purified enzyme. No significant sequence identity between the PAE and any known protein could be found in the databases. Northern analysis revealed developmentally regulated expression of the mRNA in mung been seedlings.

Mutations in the Hyperosmotic Stress-Responsive Mitochondrial BASIC AMINO ACID CARRIER2 Enhance Proline Accumulation in Arabidopsis

Mitochondrial carrier family proteins are diverse in their substrate specificity, organellar location, and gene expression. In Arabidopsis (Arabidopsis thaliana), 58 genes encode these six-transmembrane-domain proteins. We investigated the biological role of the basic amino acid carrier Basic Amino Acid Carrier2 (BAC2) from Arabidopsis that is structurally and functionally similar to ARG11, a yeast ornithine and arginine carrier, and to Arabidopsis BAC1. By studying the expression of BAC2 and the consequences of its mutation in Arabidopsis, we showed that BAC2 is a genuine mitochondrial protein and that Arabidopsis requires expression of the BAC2 gene in order to use arginine. The BAC2 gene is induced by hyperosmotic stress (with either 0.2 m NaCl or 0.4 m mannitol) and dark-induced senescence. The BAC2 promoter contains numerous stress-related cis-regulatory elements, and the transcriptional activity of BAC2:β-glucuronidase is up-regulated by stress and senescence. Under hyperosmotic stress, bac2 mutants express the P5CS1 proline biosynthetic gene more strongly than the wild type, and this correlates with a greater accumulation of Pro. Our data suggest that BAC2 is a hyperosmotic stress-inducible transporter of basic amino acids that contributes to proline accumulation in response to hyperosmotic stress in Arabidopsis.

A New Method for Accurately Measuring Δ 1 -Pyrroline-5-Carboxylate Synthetase Activity

Proline is a key factor in plant adaptation to environmental stresses. The Delta(1)-pyrroline-5-carboxylate synthetase catalyzes the first committed step and the rate-limiting step for proline biosynthesis in both plants and mammals. This enzyme catalyzes the reduction of glutamate to pyrroline-5-carboxylate in two sequential steps including the phosphorylation and the reduction of its precursor. Several methods were established to assay P5CS activity but however none of them are fully reliable. Therefore, we developed a new simple and reliable assay which is based on the quantification of Pi. This assay allowed us to determine the optimal pH, the apparent K(m) and V(m) of P5CS with regard to ATP and glutamate.

Opposite lipid signaling pathways tightly control proline accumulation in Arabidopsis thaliana and Thellungiella halophila

Throughout evolution, plants have developed various strategies to tolerate water stress. Among them, the accumulation of proline has been reported in a wide range of species. The metabolic pathway of this compatible solute is relatively well characterized in Arabidopsis thaliana. However, the signaling cascades involved in its regulation remain largely unknown. Thellungiella halophila, which is a close relative of Arabidopsis, tolerates extreme salinity up to 500 mM NaCl. In this work, the involvement of lipid signaling pathways in the regulation of proline accumulation was investigated in these two species upon water stress. A pharmacological approach has been performed using specific inhibitors of key signaling elements. The effects of these inhibitors have been investigated on proline accumulation. The present data show that phospholipases D (PLDs) are negative regulators of proline anabolism under normal conditions inA. thaliana. When such PLD-mediated regulation is abolished by 1-butanol, plants show a higher proline responsiveness to osmotic stress. In contrast to Arabidopsis, 1-butanol does not have any effect on proline accumulation in T. halophila under non-stress conditions. However, upon water constraints, 1-butanol reduces rather than increase proline accumulation. Our data suggest the involvement of a PLD-mediated signaling pathway in the tight regulation of proline metabolism that acts in a opposite way in A. thaliana and T. halophila. On the other hand, phospholipases C exert a positive control on proline accumulation in A. thaliana upon salt stress and a negative control in T. halophila upon water stress and non-stress conditions. In conclusion, we provide experimental evidence that positive and negative regulators are involved in the fine regulation of proline metabolism upon moderate water stress. Our study has defined a critical role of lipid signaling pathways in proline accumulation in A. thaliana and in T. halophila. Thus, in Arabidopsis, our data indicate that PLC-based signaling is a committed step in proline biosynthesis upon salinity but not upon hyperosmotic stress.