Homolog of Human placenta-specific gene 8, PcPLAC8-10, enhances cadmium uptake by Populus roots.

Cadmium (Cd) pollution of soil occurs worldwide. Phytoremediation is an effective approach for cleaning up Cd polluted soil. Fast growing Populus species with high Cd uptake capacities are desirable for phytoremediation. Thus, it is important to elucidate the molecular functions of genes involved in Cd uptake by poplars. In this study, PcPLAC8-10, a homolog of Human placenta-specific gene 8 (PLAC8) implicated in Cd transport was functionally characterized in Populus × canescens. PcPLAC8-10 was transcriptionally induced in Cd-treated roots and it encoded a plasma membrane-localized transporter. PcPLAC8-10 exhibited Cd uptake activity when expressed in yeast cells. No difference in growth was observed between wild type (WT) and PcPLAC8-10-overexpressing poplars. PcPLAC8-10-overexpressing poplars exhibited increases in net Cd2+ influxes by 192% and Cd accumulation by 57% in the roots. However, similar reductions in biomass were found in WT and transgenic poplars when exposed to Cd. The complete motif of CCXXXXCPC in PcPLAC8-10 was essential for its Cd transport activity. These results suggest that PcPLAC8-10 is a plasma membrane-localized transporter responsible for Cd uptake in the roots and the complete CCXXXXCPC motif of PcPLAC8-10 plays a key role in its Cd transport activity in poplars.

PcNRAMP1 Enhances Cadmium Uptake and Accumulation in Populus × canescens.

Poplars are proposed for the phytoremediation of heavy metal (HM) polluted soil. Characterization of genes involved in HM uptake and accumulation in poplars is crucial for improving the phytoremediation efficiency. Here, Natural Resistance-Associated Macrophage Protein 1 (NRAMP1) encoding a transporter involved in cadmium (Cd) uptake and transport was functionally characterized in Populus × canescens. Eight putative PcNRAMPs were identified in the poplar genome and most of them were primarily expressed in the roots. The expression of PcNRAMP1 was induced in Cd-exposed roots and it encoded a plasma membrane-localized protein. PcNRAMP1 showed transport activity for Cd2+ when expressed in yeast. The PcNRAMP1-overexpressed poplars enhanced net Cd2+ influxes by 39-52% in the roots and Cd accumulation by 25-29% in aerial parts compared to the wildtype (WT). However, Cd-induced biomass decreases were similar between the transgenics and WT. Further analysis displayed that the two amino acid residues of PcNRAMP1, i.e., M236 and P405, play pivotal roles in regulating its transport activity for Cd2+. These results suggest that PcNRAMP1 is a plasma membrane-localized transporter involved in Cd uptake and transporting Cd from the roots to aerial tissues, and that the conserved residues in PcNRAMP1 are essential for its Cd transport activity in poplars.

Sulfur metabolism, organic acid accumulation and phytohormone regulation are crucial physiological processes modulating the different tolerance to Pb stress of two contrasting poplars.

To investigate the pivotal physiological processes modulating lead (Pb) tolerance capacities of poplars, the saplings of two contrasting poplar species, Populus × canescens with high Pb sensitivity and Populus nigra with relatively low Pb sensitivity, were treated with either 0 or 8 mM Pb for 6 weeks. Lead was absorbed by the roots and accumulated massively in the roots and leaves, leading to overproduction of reactive oxygen species, reduced photosynthesis and biomass in both poplar species. Particularly, the tolerance index of P. × canescens was significantly lower than that of P. nigra. Moreover, the physiological responses including the concentrations of nutrient elements, thiols, organic acids, phytohormones and nonenzymatic antioxidants, and the activities of antioxidative enzymes in the roots and leaves were different between the two poplar species. Notably, the differences in concentrations of nutrient elements, organic acids and phytohormones were remarkable between the two poplar species. A further evaluation of the Pb tolerance-related physiological processes showed that the change of 'sulfur (S) metabolism' in the roots was greater, and that of 'organic acid accumulation' in the roots and 'phytohormone regulation' in the leaves were markedly smaller in P. × canescens than those in P. nigra. These results suggest that there are differences in Pb tolerance capacities between P. × canescens and P. nigra, which is probably associated with their contrasting physiological responses to Pb stress, and that S metabolism, organic acid accumulation and phytohormone regulation are probably the key physiological processes modulating the different Pb tolerance capacities between the two poplar species.

Physiological Characteristics and Transcriptomic Dissection in Two Root Segments with Contrasting Net Fluxes of Ammonium and Nitrate of Poplar Under Low Nitrogen Availability.

To investigate physiological and transcriptomic regulation mechanisms underlying the distinct net fluxes of NH4+ and NO3- in different root segments of Populus species under low nitrogen (N) conditions, we used saplings of Populus × canescens supplied with either 500 (normal N) or 50 (low N) µM NH4NO3. The net fluxes of NH4+ and NO3-, the concentrations of NH4+, amino acids and organic acids and the enzymatic activities of nitrite reductase (NiR) and glutamine synthetase (GS) in root segment II (SII, 35-70 mm to the apex) were lower than those in root segment I (SI, 0-35 mm to the apex). The net NH4+ influxes and the concentrations of organic acids were elevated, whereas the concentrations of NH4+ and NO3- and the activities of NiR and GS were reduced in SI and SII in response to low N. A number of genes were significantly differentially expressed in SII vs SI and in both segments grown under low vs normal N conditions, and these genes were mainly involved in the transport of NH4+ and NO3-, N metabolism and adenosine triphosphate synthesis. Moreover, the hub gene coexpression networks were dissected and correlated with N physiological processes in SI and SII under normal and low N conditions. These results suggest that the hub gene coexpression networks play pivotal roles in regulating N uptake and assimilation, amino acid metabolism and the levels of organic acids from the tricarboxylic acid cycle in the two root segments of poplars in acclimation to low N availability.

Temperature-induced lipocalin (TIL) is translocated under salt stress and protects chloroplasts from ion toxicity.

Temperature-induced lipocalins (TIL) have been invoked in the defense from heat, cold and oxidative stress. Here we document a function of TIL for basal protection from salinity stress. Heterologous expression of TIL from the salt resistant poplar Populus euphratica did not rescue growth but prevented chlorophyll b destruction in salt-exposed Arabidopsis thaliana. The protein was localized to the plasma membrane but was re-translocated to the symplast under salt stress. The A. thaliana knock out and knock down lines Attil1-1 and Attil1-2 showed stronger stress symptoms and stronger chlorophyll b degradation than the wildtype (WT) under excess salinity. They accumulated more chloride and sodium in chloroplasts than the WT. Chloroplast chloride accumulation was found even in the absence of salt stress. Since lipocalins are known to bind regulatory fatty acids of channel proteins as well as iron, we suggest that the salt-induced trafficking of TIL may be required for protection of chloroplasts by affecting ion homeostasis.

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.

Salt stress affects xylem differentiation of grey poplar (Populus x canescens).

In this study the impact of salt stress on the physiology and wood structure of the salt-sensitive Populus x canescens was investigated. Two weeks of salt stress altered wood anatomy significantly. The xylem differentiation zone was reduced and the resulting vessels exhibited reduced lumina. To understand this phenomenon, ion composition, levels of corresponding transcripts and of the stress hormone ABA were analysed. With increasing sodium and chloride concentrations, a general reduction of potassium was found in roots and shoots, but not in leaves. Consequently, the corresponding K+ channel transcripts in roots favoured K+ release. The overall osmolarity in leaves was up to fourfold higher than in roots or shoots. Therefore, adjustment of the K+/Na+ balance seemed not to be required in leaves. Sodium increased gradually from roots to shoots and then to leaves indicating that sodium storage took place first in roots, then in shoots, and finally in leaves to protect photosynthesis from salt effects as long as possible. Since leaf abscisic acid levels markedly increased, stomatal closure seemed to limit CO2 uptake. As a consequence, diminished nutrient supply to the cambium in combination with lowered shoot K+ content led to decreased vessel lumina, and a reduction of the radial cambium was observed. Thus, xylem differentiation was curtailed and the development of full size vessels was impaired.

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.