Genetic variation and drought response in two Populus × euramericana genotypes through 2-DE proteomic analysis of leaves from field and glasshouse cultivated plants

Abstract

Genotype and water deficit effects on leaf 2-DE protein profiles of two Populus deltoides × Populus nigra, cv. ‘Agathe_F’ and ‘Cima’, were analysed over a short-term period of 18 days in glasshouse using 4-month-old rooted cuttings and over a long-lasting period of 86 days in open field using 4-year-old rooted cuttings. Leaf proteomes were analyzed using two-dimensional gel electrophoresis, and proteins were identified after database searching from MS peptide spectra.

A reliable genotype effect was observed in the leaf proteome over experiment locations, water regimes and sampling dates. Quantitative differences between genotypes were found. Most of them corresponded to proteins matching isoforms or post-translational modification variants. However, ‘Cima’ displayed the highest abundance of antioxidant enzymes.

In response to water deficit, about 10% of the reproducible spots significantly varied regardless of the experiment location, among which about 25% also displayed genotype-dependent variations. As a whole, while ‘Cima’ differed from ‘Agathe_F’ by increased abundance of enzymes involved in photorespiration and in oxidative stress, ‘Agathe_F’ was mainly differentiated by increased abundance of enzymes involved in photosynthesis.

Introduction

Significant genetic variation in productivity and in leaf-ecophysiological variables has been reported for Populus deltoides Bartr. ex Marsh. × Populus nigra L. (Populus × euramericana) hybrids (Marron et al., 2005, Marron et al., 2007, Monclus et al., 2005, Al Afas et al., 2006, Al afas et al., 2007, Voltas et al., 2006, Bonhomme et al., 2008). Leaf-ecophysiological variables encompassed: (i) structural variables such as specific leaf area (SLA), stomatal density, leaf nitrogen and carbon contents, and (ii) functional variables such as water-use efficiency (WUE) directly estimated from gas exchange (intrinsic water-use efficiency, W_i) or indirectly from leaf carbon isotope discrimination (Δ). In addition, although poplars are among the woody species the most susceptible to drought stress (Tschaplinski et al., 1994, Dreyer et al., 2004), P. × euramericana genotypes also displayed significant genetic variations in their drought tolerance level and in their pattern of responses to water deficit (Brignolas et al., 2000, Marron et al., 2002, Marron et al., 2003, Monclus et al., 2006).

Proteomic studies have already proven their value to assess genetic variation in various plant species (Thiellement et al., 1999, Thiellement et al., 2002, Cánovas et al., 2004, Agrawal et al., 2005, Vincent et al., 2007). However, although the sequenced genome of Populus trichocarpa had provided considerable key genomic resources for protein identification (Tuskan et al., 2006), little information was reported in poplars. Poplar proteomics have been carried out to study the molecular plasticity of a particular genotype in response to drought (Pelah et al., 1995, Plomion et al., 2006, Bogeat-Triboulot et al., 2007, He et al., 2008) or to other abiotic stresses such as heat, cold or heavy metals (Renaut et al., 2004, Ferreira et al., 2006, Bohler et al., 2007, He et al., 2008, Kieffer et al., 2008). To our knowledge, no study has dealt neither with the genotypic variations of poplar leaf proteomes, nor with the genotypic variations of their plasticity in response to drought. The occurrence of genetic variations in expressed protein content could be of considerable advantage for marker-assisted selection. Indeed, the identification of molecular markers related to important traits such as WUE or drought tolerance could be useful for the selection of poplar genotypes in breeding programs.

In this study, a proteomic approach was conducted using two hybrid poplar genotypes (P. × euramericana cv. ‘Agathe_F’ and cv. ‘Cima’) contrasted for some leaf-ecophysiological variables such as Δ or specific leaf area (Monclus et al., 2005, Monclus et al., 2006). The main objectives were (i) to evaluate the extent of genetic variation in leaf proteome regardless of environmental factors such as growth condition, water regime or sampling date, and (ii) to evaluate the leaf proteome changes induced by a moderate drought in open field and a simulated moderate water deficit in glasshouse. Therefore, genotype differences and drought responses were evaluated in an open field and in a glasshouse experiments where 4-year-old or 4-month-old rooted cuttings were held irrigated or submitted to a moderate water deficit during 86 days or 18 days, respectively.