Basia Vinocur

Gradual Soil Water Depletion Results in Reversible Changes of Gene Expression, Protein Profiles, Ecophysiology, and Growth Performance in Populus euphratica , a Poplar Growing in Arid Regions

The responses of Populus euphratica Oliv. plants to soil water deficit were assessed by analyzing gene expression, protein profiles, and several plant performance criteria to understand the acclimation of plants to soil water deficit. Young, vegetatively propagated plants originating from an arid, saline field site were submitted to a gradually increasing water deficit for 4 weeks in a greenhouse and were allowed to recover for 10 d after full reirrigation. Time-dependent changes and intensity of the perturbations induced in shoot and root growth, xylem anatomy, gas exchange, and water status were recorded. The expression profiles of approximately 6,340 genes and of proteins and metabolites (pigments, soluble carbohydrates, and oxidative compounds) were also recorded in mature leaves and in roots (gene expression only) at four stress levels and after recovery. Drought successively induced shoot growth cessation, stomatal closure, moderate increases in oxidative stress-related compounds, loss of CO2 assimilation, and root growth reduction. These effects were almost fully reversible, indicating that acclimation was dominant over injury. The physiological responses were paralleled by fully reversible transcriptional changes, including only 1.5% of the genes on the array. Protein profiles displayed greater changes than transcript levels. Among the identified proteins for which expressed sequence tags were present on the array, no correlation was found between transcript and protein abundance. Acclimation to water deficit involves the regulation of different networks of genes in roots and shoots. Such diverse requirements for protecting and maintaining the function of different plant organs may render plant engineering or breeding toward improved drought tolerance more complex than previously anticipated.

Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert

BackgroundPlants growing in their natural habitat represent a valuable resource for elucidating mechanisms of acclimation to environmental constraints. Populus euphratica is a salt-tolerant tree species growing in saline semi-arid areas. To identify genes involved in abiotic stress responses under natural conditions we constructed several normalized and subtracted cDNA libraries from control, stress-exposed and desert-grown P. euphratica trees. In addition, we identified several metabolites in desert-grown P. euphratica trees.ResultsAbout 14,000 expressed sequence tag (EST) sequences were obtained with a good representation of genes putatively involved in resistance and tolerance to salt and other abiotic stresses. A P. euphratica DNA microarray with a uni-gene set of ESTs representing approximately 6,340 different genes was constructed. The microarray was used to study gene expression in adult P. euphratica trees growing in the desert canyon of Ein Avdat in Israel. In parallel, 22 selected metabolites were profiled in the same trees.ConclusionOf the obtained ESTs, 98% were found in the sequenced P. trichocarpa genome and 74% in other Populus EST collections. This implies that the P. euphratica genome does not contain different genes per se, but that regulation of gene expression might be different and that P. euphratica expresses a different set of genes that contribute to adaptation to saline growth conditions. Also, all of the five measured amino acids show increased levels in trees growing in the more saline soil.

Transgenic Populus tremula: a step-by-step protocol for its Agrobacterium-mediated transformation

In recent years, Populus species have acquired an important place in basic and applied research of woody plants. The practical role of Populus species in world forestry and their importance to research as a woody-plant model have led to increasing interest in tissue-culture and molecular techniques, as well as the development of transformation procedures for this genus. A simple technical procedure is described here step-by-step, for the first time, as a routine method for transforming Populus tremula using a disarmed Agrobacterium tumefaciens hypervirulent strain. The procedure begins with the inoculation of stem explants with bacterial suspension, followed by a short period of co-cultivation on a highly regenerative medium. Transformed shoots are selected on regeneration medium containing antibiotics and the presence of the inserted target genes is checked using a rapid and efficient PCR test. Selected shoots are transferred to a rooting medium, under the same selection pressure, and propagated via stem cuttings. Selected plants can be hardened and transferred to the green-house within 4 months of inoculation. The method has proven efficient for several gene constructs, selection on Kan or Hyg, and three different Agrobacterium strains.

Linking the Salt Transcriptome with Physiological Responses of a Salt-Resistant Populus Species as a Strategy to Identify Genes Important for Stress Acclimation

To investigate early salt acclimation mechanisms in a salt-tolerant poplar species (Populus euphratica), the kinetics of molecular, metabolic, and physiological changes during a 24-h salt exposure were measured. Three distinct phases of salt stress were identified by analyses of the osmotic pressure and the shoot water potential: dehydration, salt accumulation, and osmotic restoration associated with ionic stress. The duration and intensity of these phases differed between leaves and roots. Transcriptome analysis using P. euphratica-specific microarrays revealed clusters of coexpressed genes in these phases, with only 3% overlapping salt-responsive genes in leaves and roots. Acclimation of cellular metabolism to high salt concentrations involved remodeling of amino acid and protein biosynthesis and increased expression of molecular chaperones (dehydrins, osmotin). Leaves suffered initially from dehydration, which resulted in changes in transcript levels of mitochondrial and photosynthetic genes, indicating adjustment of energy metabolism. Initially, decreases in stress-related genes were found, whereas increases occurred only when leaves had restored the osmotic balance by salt accumulation. Comparative in silico analysis of the poplar stress regulon with Arabidopsis (Arabidopsis thaliana) orthologs was used as a strategy to reduce the number of candidate genes for functional analysis. Analysis of Arabidopsis knockout lines identified a lipocalin-like gene (AtTIL) and a gene encoding a protein with previously unknown functions (AtSIS) to play roles in salt tolerance. In conclusion, by dissecting the stress transcriptome of tolerant species, novel genes important for salt endurance can be identified.

Abiotic Resistance and Chaperones: Possible Physiological Role of SP1, a Stable and Stabilizing Protein from Populus

The molecular basis of abiotic stress tolerance in plants, especially drought, salinity and extreme temperatures, is rather limited. In view, the worldwide devastating problems of salinization and desertification, efforts are continuing to unravel some of the molecular controls and tolerance mechanisms. Discovery of new genes for abiotic stress tolerance, combined with controlled molecular breeding will have an important role in shaping agricultural plants in the post-genomic era (Wang et al., 200la).

rol-Transgenic Populus tremula: root development, root-borne bud regeneration and in vitro propagation efficiency

Abstract The potential use of the rol genes from Agrobacterium rhizogenes to improve the root system horticultural characteristics was evaluated in transgenic aspen (Populus tremula) plants, harboring the rol genes under their native promoters. Southern blot and RT-PCR analyses confirmed the presence and expression of A. rhizogenesrolC and rolB genes in four different phenotypically selected transgenic clones. Several of the observed phenotypic modifications were related to rol-gene expression and included, in particular, modified root systems. All in vitro-cultured rol-transgenic plants exhibited extensive root formation in a hormone-free medium, as well as a larger root surface area and mass, as compared to a uidA (β-glucuronidase-encoding)-transgenic aspen line and control (non-transformed) plants. Adventitious root formation in stem segments of rol-transgenic plants exhibited very rapid kinetics, resulting in a much shorter rooting time for rol-transgenic stem segments (e.g. 10 days for 80% rooting in rol-transgenic lines T-26 and T-27, as compared to more than 18 days for control non-transformed or uidA-transgenic aspen plants). rol-Transgenic plants maintained the capacity for 100% rooting throughout the year, versus 70–80% rooting in non-transformed plants during the winter. The four rol-transgenic lines exhibited differences in root development; in two of them enhanced root development was accompanied by increased shoot fresh weight. The root:shoot fresh weight ratio was always higher in rol-transgenic lines than in non-transformed plants. In the T-27 rol-transgenic line, the propagation coefficient of shoot-bud regeneration in liquid root culture was almost three times higher than in non-transformed plants. To the best of our knowledge this is the first report on quantitative phenotypic alterations in rol-transgenic woody plants.

Cellulose binding domain increases cellulose synthase activity in Acetobacter xylinum, and biomass of transgenic plants.

Recombinant cellulose-binding domain (CBD) was found to modulate the elongation of different plant cells in-vitro. At low concentrations (0.01–1 µg ml−1), CBD enhanced elongation of Arabidopsis thaliana L. roots. At high concentrations (100–500 µg ml−1), CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 µg ml−1, whereas root elongation was inhibited at that concentration. Using Acetobacter xylinum L. as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to fivefold as compared with the control. Electron microscopy examination of the cellulose ribbons produced by A.xylinum, showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits as compared with a thin and uniform ribbon in the control. Expression of cbd modulated the growth of transgenic plants. Biomass production was significantly higher in selected clones as compared with the control.

Bud Regeneration and Growth from Transgenic and Non-Transgenic Aspen (Populus tremula) Root Explants

Poplars, including aspen, (Populus tremula), are fast growing trees employed in the wood industry and have a potential value for biomass and energy production. Vegetative propagation is carried out by cuttings, root suckers and grafting, but an increase of the propagation efficiency is highly desired. In vitro procedures are becoming available for efficient large-scale clonal propagation of poplar genotypes. These procedures can benefit from large-scale root culture in liquid media, based on the natural ability of aspen for shoot bud formation on roots (Carmi, 1994). The effect of different types of root expiants upon bud regeneration of aspen was studied using root segments from adventitious roots that formed on stem sections of two transgenic lines that harbored the rolB and rolC genes, and one non-transgeme line.