Sabine Carpin

Acute metal stress in Populus tremula × P. alba (717-1B4 genotype): leaf and cambial proteome changes induced by cadmium2+.

The comprehension of metal homeostasis in plants requires the identification of molecular markers linked to stress tolerance. Proteomic changes in leaves and cambial zone of Populus tremula×P. alba (717‐1B4 genotype) were analyzed after 61 days of exposure to cadmium (Cd) 360 mg/kg soil dry weight in pot‐soil cultures. The treatment led to an acute Cd stress with a reduction of growth and photosynthesis. Cd stress induced changes in the display of 120 spots for leaf tissue and 153 spots for the cambial zone. It involved a reduced photosynthesis, resulting in a profound reorganisation of carbon and carbohydrate metabolisms in both tissues. Cambial cells underwent stress from the Cd actually present inside the tissue but also a deprivation of photosynthates caused by leaf stress. An important tissue specificity of the response was observed, according to the differences in cell structures and functions.

Insights into Populus XIP aquaporins: evolutionary expansion, protein functionality, and environmental regulation

A novel category of major intrinsic proteins which share weak similarities with previously identified aquaporin subfamilies was recently identified in land plants, and named X (for unrecognized) intrinsic proteins (XIPs). Because XIPs are still ranked as uncharacterized proteins, their further molecular characterization is required. Herein, a systematic fine-scale analysis of XIP sequences found in flowering plant databases revealed that XIPs are found in at least five groups. The phylogenetic relationship of these five groups with the phylogenetic organization of angiosperms revealed an original pattern of evolution for the XIP subfamily through distinct angiosperm taxon-specific clades. Of all flowering plant having XIPs, the genus Populus encompasses the broadest panel and the highest polymorphism of XIP isoforms, with nine PtXIP sequences distributed within three XIP groups. Comprehensive PtXIP gene expression patterns showed that only two isoforms (PtXIP2;1 and PtXIP3;2) were transcribed in vegetative tissues. However, their patterns are contrasted, PtXIP2;1 was ubiquitously accumulated whereas PtXIP3;2 was predominantly detected in wood and to a lesser extent in roots. Furthermore, only PtXIP2;1 exhibited a differential expression in leaves and stems of drought-, salicylic acid-, or wounding-challenged plants. Unexpectedly, the PtXIPs displayed different abilities to alter water transport upon expression in Xenopus laevis oocytes. PtXIP2;1 and PtXIP3;3 transported water while other PtXIPs did not.

Leaf proteome analysis of eight Populus × euramericana 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.

Poplar under drought: comparison of leaf and cambial proteomic responses.

The forest ecosystem is of particular importance from an economic and ecological perspective. However, the stress physiology of trees, perennial and woody plants, is far from being fully understood. For that purpose, poplar plants were exposed to drought; the plants exhibited commonly reported drought stress traits in the different plant tissues. Leafy rooted cuttings of poplar were investigated through a proteomic approach in order to compare the water constraint response of two plant tissues, namely leaf and cambium. Sampling was realized during the drought period at 2 time points with increased drought intensity and 7 days after rewatering. Our data show that there is a difference in the moment of response to the water constraint between the two tissues, cambium being affected later than leaves. In leaves, drought induced a decrease in rubisco content, and an increase in the abundance of light harvesting complex proteins as well as changes in membrane-related proteins. In the cambial tissue, the salient proteome pattern change was the decrease of multiple proteins identified as bark storage proteins. After rewatering, almost all changes in cambial proteome disappeared whereas a significant number of leaf proteins appeared to be differentially regulated only during the recovery from drought.

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

To 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.

Molecular characterization of a cytokinin-inducible periwinkle protein showing sequence homology with pathogenesis-related proteins and the Bet v 1 allergen family.

Cytokinin treatment of periwinkle callus cultures increased the accumulation of a protein, designated T1, in two-dimensional separated protein extracts. The first 30 NH2-terminal amino acids were determined by Edman degradation and showed significant sequence homology with intracellular pathogenesis-related (IPR) plant proteins and the Bet v 1 allergen family. The deduced amino acid sequence of cDNAs coding for T1, isolated by RT-PCR and 5′ RACE-PCR, exhibited an average sequence identity of 40% with both IPR and Bet v 1-related allergens. T1 and all related proteins contained a p-loop motif typically found in nucleotide-binding proteins as the most conserved sequence feature. Northern blot analysis showed that cytokinin treatment of periwinkle callus induced T1 transcripts, whereas addition of 2,4-dichlorophenoxyacetic acid inhibited this accumulation. Hybridization of genomic periwinkle DNA with the T1 cDNA suggested that the protein is encoded by a single-copy gene. Immunoblot studies with a panel of Bet v 1-specific antibodies and sera from Bet v 1 allergic individuals identified T1 as a protein that is immunologically distinct from the Bet v 1 allergen family and has no allergenic properties.

Effect of cytokinin on alkaloid accumulation in periwinkle callus cultures transformed with a light-inducible ipt gene

The effect of cytokinins on accumulation of indole alkaloids in periwinkle callus cultures was investigated. Firstly, it was found that exogenously-applied cytokinin increased the ajmalicine and serpentine content of untransformed callus culture obtained from cotyledons. Secondly, periwinkle cotyledons were transformed with the isopentenyl transferase (ipt) gene under the control of a light-inducible promoter and two transformed callus lines were used in order to investigate whether endogenously-produced cytokinin could also increase the alkaloid production. We found that the ipt-transgenic tissues accumulated higher levels of isopentenyl transferase transcripts as well as zeatin riboside, even under non-inductive condition, but lower concentration of alkaloids compared to that of untransformed tissues. A 28 kDa polypeptide whose accumulation was previously found to be associated with alkaloid production in a periwinkle cell suspension was also present in the non-transformed tissue and its level was increased in parallel to the CK-enhanced alkaloid production. Neither light induction condition, nor exogenous cytokinin treatment led to the increase of the 28 kDa polypeptide accumulation in the transformed tissues. All these data show that endogenously-produced cytokinin does not mimic the effect of exogenously-applied cytokinin on the alkaloid production in periwinkle calli.

Cadmium and Zinc are differentially distributed in Populus tremula x P. alba exposed to metal excess

Abstract Poplar plants were exposed during 61 days to a soil added with heavy metals so as to contain 300 mg Zn2+.kg−1 soil dry weight (SDW) (Zinc) or 50 mg Cd2+.kg−1 SDW (Cadmium). The Cd treatment induced a delayed growth of poplar, whereas Zn induced no change in physiological parameters. Both treatments resulted in a significant metal accumulation in plants. Zn2+ and Cd2+ exhibited contrasting distribution within tissues, indicating dissimilar handling by the plant. The main difference was the efficient compartmentalisation of Zn2+ in specific organ parts: old leaves and bark, while Cd2+ did not exhibit such a compartmentalisation. Results were also compared with a previous work where plants were exposed to 360 mg Cd2+.kg−1 SDW.

The relationship between the accumulation of a 28 kd polypeptide and that of indole alkaloids in Catharanthus roseus cell suspension cultures

Summary We previously found that the accumulation of the indole alkaloid ajmalicine and that of a polypeptide of 28kD were induced by 2,4-dichlorophenoxyacetic acid removal in periwinkle ( Catharanthus roseus ) cell cultures and further increased by zeatin addition to the culture medium. In this work, we compared the effect of various plant growth regulators and stresses on the accumulation patterns of both compounds. In a first series of experiments, the periwinkle cells were subcultured in auxin-free medium for five passages and their contents in ajmalicine and 28 kD polypeptide analysed. The maximal biomass progressively decreased along subcultures; the 28 kD polypeptide and ajmalicine accumulated concomitantly in the cells (treated with or without zeatin) up to the third passage but fully disappeared afterwards. In a second series of experiments, treatment with gibberellic acid deeply inhibited, whereas NaCl treatment (given together with or without zeatin) increased the accumulation of both ajmalicine and the 28 kD polypeptide. Moreover, growing the cells in hypoxia for 2 days inhibited the accumulation of both compounds. These concomitant changes in the accumulation pattern of ajmalicine and the 28 kD polypeptide suggest that the latter can play a (direct or indirect) regulatory role in the indole alkaloid pathway of periwinkle cells.

Screening for changes in leaf and cambial proteome of Populus tremula × P. alba under different heat constraints.

Young poplar plants were exposed to different heat regimes, a rapid heat constraint at 42°C (heat shock HS) alone or preceded by a stepwise increase in temperature (heat gradient HG). Proteomics analyses were carried out on both leaf and cambial tissues. The responses of both tissues were compared and linked to morphological and physiological observations. Both heat treatments negatively affected the photosynthetic rate while increasing the stomatal conductance. In the leaf, the HS impacted some photosynthetic proteins, and particularly induced an increase in abundance of proteins of the oxygen evolving complexes. On the other hand, the HG reduced carbohydrate metabolism and induced mainly an increase in germin-like proteins. In the cambial zone, the HS caused a decrease in sucrose synthase content and in enzymes related to protein synthesis. The main effect of HG was the accumulation of thaumatin-like proteins as well as an increase in the abundance of proteins involved in carbohydrate metabolism. Further, both tissues underwent changes in the content of heat shock proteins, but more importantly, of peroxiredoxins. The results show more sustainable changes in leaf and cambial proteomes in response to HS compared to HG.