Domenico Morabito

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.

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.

Carbon Metabolism Enzyme Activities and Carbon Partitioning in Pinus halepensis Mill, exposed to Mild Drought and Ozone

Summary Since several years, accelerated decline of Aleppo pine ( Pinus halepensis ) forests has been observed in mediterranean areas. In fact, the combination of various environmental factors (photochemical oxidants, drought, high light, ...) was suspected to cause this decline. In this study, three year-old Aleppo pines were exposed during 3 months to ozone fumigation (100 ppb) combined or not with mild drought to study the effects of these combined factors on some sequences linked to carbon partitioning and primary carbon metabolism within the tree. After a cumulative ozone exposure of 132ppm·h, ozone induced a significant decrease in specific activity of the whole-plant (−38%) combined with a disequilibrium of the carbon transfer between root and shoot in favour of the shoots (non significant). Moreover, while the same cumulative dose of ozone had no effect on total Rubisco activity in one year-old needles, mitochondfial NAD malic enzyme activity increased significantly (+32%). By combining ozone with mild drought, the ozone-induced responses of all the parameters were significantly amplified and Rubisco activity was significantly decreased (by 44%). These results allowed us to conclude that at 132ppm·h, ozone alone led to an increase in dark respiration. Moreover, by the combination of ozone and mild drought, a decrease carbon fixation capacity was associated to a decrease of the carbon transfered to the toots, leading to a reduced root growth. Thus, there are indications that high levels of ozone during the summer months may impair the ability of Pinus halepensis to withstand severe water stress in its natural environment.

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.

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.

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.

Differences in the physiological responses of two clones of Eucalyptus microtheca selected for their salt tolerance

Abstract Two Eucalyptus microtheca clones (clones 42 and 43) suspected to be salt tolerant were rooted and grown in a greenhouse. Five-month old cuttings were watered for 2 months with and without 200 mM NaCl. During the salt stress period, mineral (Na, Ca, K and Cl) and organic (soluble amino acids and proline) compounds were determined in roots, stems and leaves. Changes in protein profiles induced by salt stress were investigated after 2 weeks of salt treatment. Clone 42 demonstrated a delayed growth during salt stress while clone 43 showed a complete inhibition of shoot length. Salinity had a significant effect on mineral compounds: whatever the duration of the salt treatment, the uptake of sodium in roots was 2.5 times higher in the more tolerant clone 42 than in clone 43. Regarding potassium and calcium contents in roots, clone 42 demonstrated an increase in its amount whereas clone 43 showed a decrease. The increase in soluble amino acids induced by the stress in the different organs of the two clones was not significantly different whereas a higher content of proline was determined in clone 42 relative to clone 43. Salinity had significant effects on the content of one predominat polypeptide with an apparent molecular weight of 18 kDa which was specifically induced under salt stress in roots of clone 43. In clone 42, this polypeptide was present in low amounts in control conditions and salt treatment increased its synthesis.

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.

Genetic variation in productivity, leaf traits and carbon isotope discrimination in hybrid poplars cultivated on contrasting sites

Abstract• We examined the relationships between productivity, leaf traits and carbon isotope discrimination in bulk leaf matter (Δ1) and in phloem sap (Δs) from more than 5-year-old trees belonging to Populus deltoides × P. nigra and Populus trichocarpa × P. deltoides; trees were grown in alluvial and non alluvial sites in a commercial poplar plantation.• On both sites, a large genetic variability was evidenced for all variables. The genotypic ranking remained stable between years for all variables, while it differed between sites. Δ1 scaled positively with Δs and neither Δ1 nor Δs were correlated with productivity. A significant genotype by site interaction was evident for all variables. The non alluvial site resulted in lower productivity, and in thicker/denser leaves with lower nitrogen and carbon contents. Noteworthy, the genotypic ranking for Δ1 measured at the alluvial site was similar to that previously established in a glasshouse.• As observed in previous studies from younger trees, there is a potential to select genotypes, combining high productivity and high water-use efficiency, for growth in moderately drought-prone areas.Résumé• La productivité, des caractères foliaires et la discrimination isotopique vis-à-vis du carbone de la matière organique des feuilles (Δ1) et de la sève élaborée (Δs) ont été étudiés chez Populus deltoides × P. nigra et Populus trichocarpa × P. deltoides à partir d’arbres âgés de plus de cinq ans, cultivés en peupleraie sur des sites alluviaux et non alluviaux.• Des différences génotypiques ont été observées pour toutes les variables. Le classement des génotypes était conservé entre deux années, alors qu’il variait entre sites une année donnée. Δ1 était corrélé positivement avec Δs et aucun lien n’a été détecté entre Δ1 ou Δs et productivité. Une interaction significative entre génotype et site a été observée pour toutes les variables. Les arbres du site non alluvial se caractérisaient par une plus faible productivité et des feuilles plus épaisses/denses avec des teneurs en azote et en carbone plus faibles. De façon intéressante, le classement des génotypes pour Δ1 était maintenu entre les expériences réalisées sur site alluvial et celles précédemment menées en serre.• Toutes expériences confondues, il semble possible de sélectionner des génotypes productifs et efficients afin d’étendre les plantations à des terrains enclins à des sécheresses modérées.

The free oxygen radical scavenging enzymes and redox status in roots and leaves of Populus x Euramericana in response to osmotic stress, desiccation and rehydration

Summary The redox status (ascorbate, glutathione under reduced and oxidized forms) and the activities of antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase) were measured in leaves and roots of Populus euramericana cv. Luisa Avanzo cuttings exposed to wilting or to osmotic stress. Osmotic stress was applied for 12 h with 100 mmol/L mannitol; the wilting state (85% of the original fresh weight), which was reached in 15 min in roots, 75 min in old leaves and 145 min in young leaves, was maintained for 12 h. Superoxide synthesis was drastically enhanced in wilted organs vs. osmotically stressed ones. Wilted and osmotically stressed roots exhibited a lack of stress-inducible antioxidative enzymes and a decrease in glutathione and ascorbate contents with the duration of treatments. An enhanced capability to dismutate superoxide was observed in wilted leaves and in osmotically stressed old leaves; ascorbate peroxidase activity was solely enhanced in wilted leaves, but ascorbate content remained stable both in the case of desiccated and osmotically stressed leaves. Although glutathione reductase activity decreased in wilted and mannitol-treated leaves, glutathione content remained also stable in stressed leaves. After rehydration of 12-h wilted leaves and roots, no recovery of the initial rate of each enzyme activity was observed. The adaptive response of roots and leaves of poplar to oxidative stress generated by wilting and osmotic stress was discussed.