Osmotic stress sensing in Populus: Components identification of a phosphorelay system

AbstractTo study the Populus response to an osmotic stress, we have isolated one cDNA encoding a histidine-aspartate kinase (HK1) and four cDNAs encoding histidine-containing phosphotransfer proteins (HPts), HPt1–4. The predicted HK1 protein shares a typical structure with ATHK1 and SLN1 osmosensors. The 4 HPTs are characterized by the histidine phosphotransfer domain. We have shown that HK1 is upregulated during an osmotic stress in hydroponic culture. We have detected an interaction between HK1 and HPt2, using the yeast two-hybrid system. These results suggest the existence of a multi-step phosphorelay pathway probably involved in osmotic stress sensing in Populus

Recherche d un phosphorelais multiple impliqué dans la perception et la transduction du signal stress hydrique chez le peuplier

A partir d une banque d ADNc de racines de peuplier Dorskamp , nous avons isolé un ADNc codant pour une histidine-aspartate kinase appelé HK1 et quatre ADNc codant pour trois protéines à domaine transmetteur de phosphate à histidine, HPt1, HPt2 et HPt3/4. Nous avons montré dans les racines de peuplier cultivé en hydroponie que le taux de transcrits HK1 augmente 5 min après l application d une contrainte hyper-osmotique et qu il existe une interaction entre HK1 et HPt2 chez la levure. L ensemble de ces résultats est un argument fort en faveur de l implication d un système de phosphorelais multiple dans la perception et la transduction du signal stress hydrique chez le peuplier.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF

Molecular cloning and tissue-specific expression of an anionic peroxidase in zucchini

A calcium-pectate-binding anionic isoperoxidase (APRX) from zucchini (Cucurbita pepo) was purified and subjected to N-terminal amino acid microsequencing. The cDNA encoding this enzyme was obtained by reverse transcriptase polymerase chain reaction from a cDNA library. It encoded a mature protein of 309 amino acids exhibiting all of the sequence characteristics of a plant peroxidase. Despite the presence of a C-terminal propeptide, APRX was found in the apoplast. APRX protein and mRNA were found in the root, hypocotyls, and cotyledons. In situ hybridization showed that the APRX-encoding gene was expressed in many different tissues. The strongest expression was observed in root epidermis and in some cells of the stele, in differentiating tracheary elements of hypocotyl, in the lower and upper epidermis, in the palisade parenchyma of cotyledons, and in lateral and adventitious root primordia. In the hypocotyl hook there was an asymmetric expression, with the inner part containing more transcripts than the outer part. Treatment with 2,3,5-triiodobenzoic acid reduced the expression of the APRX-encoding gene in the lower part of the hypocotyl. Our observations suggest that APRX could be involved in lignin formation and that the transcription of its gene was related to auxin level

Leaf proteome analysis of eight Populus × euramericana genotypes: genetic variation in drought response and in water-use efficiency involves photosynthesis-related proteins

Climatic events predicted for this century should involve drier and warmer summers, such as those that occurred in 2003 and 2005 in France. The higher temperatures could become a limiting factor for the regeneration of Populus nigra L. species. During establishment and development in summer period, seedlings must be able to resist high temperatures (until 57.8°C on sand on a Loire island in July 2009). Therefore, maintaining a high transpiration rate (E) for leaf cooling (and therefore a high stomatal conductance to water vapour (gs)), may be a prerequisite for seedlings to survive under high temperatures. To test this hypothesis, we used bulk leaf carbon isotope discrimination (Δ) as a time-integrated index of gas exchange activity (Farquhar et al. 1989). The objectives of this study were (1) to explore the genetic variability of ∆ among P. nigra seedlings from different female trees coming from contrasting sites and (2) the plasticity in response to two contrasting temperatures. Seeds from 16 open-pollinated females originating from the Loire river (France) and from the Paglia river (Italy) were first grown during 7 weeks in two growth chambers at 25°C. At this time (t0), one chamber remained at 25°C and in the second one, the temperature progressively increased until 43°C until the first symptoms of wilting appeared (t1). At t0, an important genetic variability for ∆ (∆t0 25°C) was measured ranged from 24.0‰ to 28.0‰ in both chambers, but no provenances differences were detected. At t1, an important genetic variability for ∆t1 25°C and ∆t1 43°C was observed ranged from 22.0‰ to 26.0‰ in each chamber and a significant provenance effect was detected. At t1, ∆t1 43°C values were significantly higher (0.5‰) than ∆t1 25°C. Moreover, a significant correlation was detected between the ∆t1 43°C values and the symptoms of wilting seedlings, seedlings totally healthy exhibiting higher ∆t1 43°C values. We conclude, under the hypothesis that ∆ is mainly controlled by gs, as already reported in poplar species (Monclus et al. 2006), maintaining high E and therefore high ∆ may enable seedlings to survive under high temperature

Leaf proteome analysis of eight Populus × euramericana genotypes: genetic variation in drought response and in water-use efficiency involves photosynthesis-related proteins

Climatic events predicted for this century should involve drier and warmer summers, such as those that occurred in 2003 and 2005 in France. The higher temperatures could become a limiting factor for the regeneration of Populus nigra L. species. During establishment and development in summer period, seedlings must be able to resist high temperatures (until 57.8°C on sand on a Loire island in July 2009). Therefore, maintaining a high transpiration rate (E) for leaf cooling (and therefore a high stomatal conductance to water vapour (gs)), may be a prerequisite for seedlings to survive under high temperatures. To test this hypothesis, we used bulk leaf carbon isotope discrimination (Δ) as a time-integrated index of gas exchange activity (Farquhar et al. 1989). The objectives of this study were (1) to explore the genetic variability of ∆ among P. nigra seedlings from different female trees coming from contrasting sites and (2) the plasticity in response to two contrasting temperatures. Seeds from 16 open-pollinated females originating from the Loire river (France) and from the Paglia river (Italy) were first grown during 7 weeks in two growth chambers at 25°C. At this time (t0), one chamber remained at 25°C and in the second one, the temperature progressively increased until 43°C until the first symptoms of wilting appeared (t1). At t0, an important genetic variability for ∆ (∆t0 25°C) was measured ranged from 24.0‰ to 28.0‰ in both chambers, but no provenances differences were detected. At t1, an important genetic variability for ∆t1 25°C and ∆t1 43°C was observed ranged from 22.0‰ to 26.0‰ in each chamber and a significant provenance effect was detected. At t1, ∆t1 43°C values were significantly higher (0.5‰) than ∆t1 25°C. Moreover, a significant correlation was detected between the ∆t1 43°C values and the symptoms of wilting seedlings, seedlings totally healthy exhibiting higher ∆t1 43°C values. We conclude, under the hypothesis that ∆ is mainly controlled by gs, as already reported in poplar species (Monclus et al. 2006), maintaining high E and therefore high ∆ may enable seedlings to survive under high temperature

Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold

Leaf proteome analysis of eight Populus xeuramericana genotypes : genetic variation in drought response and in water-use efficiency involves photosynthesis-related proteins

Genetic variation of leaf proteome in drought response was investigated among eight Populus ×euramericana genotypes contrasting for their leaf carbon isotope discrimination (Δ), an estimate of intrinsic water-use efficiency. Plants were grown in open field on two similar plots. Drought was induced by an 86-day irrigation cessation on one plot, whereas a second plot remained regularly irrigated. Using 2-DE, 863 reproducible spots were detected; about 60% presented at least one significant effect i.e. treatment, genotype and/or genotype by treatment interaction effect. A significant genotype by treatment interaction was detected for 62 reliably identified proteins among which, about 65% consisted in chloroplast-associated proteins either involved in the Calvin cycle or in the electron-transport chains. The other proteins were involved in oxidative stress, amino acid or protein metabolisms. Correlations between protein abundance and Δ variations were found for 45 reliably identified proteins. The abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms scaled negatively with Δ regardless of the treatment, suggesting that a large intrinsic water-use efficiency could be due to higher abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase. Under control condition, abundance of enzymes involved in carbon fixation was also negatively correlated with Δ, whereas abundance of enzymes involved in photorespiration or respiration was positively correlated with Δ