Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

The Roots of Plant Frost Hardiness and Tolerance.

Frost stress severely affects agriculture and agroforestry worldwide. Although many studies about frost hardening and resistance have been published, most of them focused on the aboveground organs and only a minority specifically targets the roots. However, roots and aboveground tissues have different physiologies and stress response mechanisms. Climate models predict an increase in the magnitude and frequency of late-frost events, which, together with an observed loss of soil insulation, will greatly decrease plant primary production due to damage at the root level. Molecular and metabolic responses inducing root cold hardiness are complex. They involve a variety of processes related to modifications in cell wall composition, maintenance of the cellular homeostasis and the synthesis of primary and secondary metabolites. After a summary of the current climatic models, this review details the specificity of freezing stress at the root level and explores the strategies roots developed to cope with freezing stress. We then describe the level to which roots can be frost hardy, depending on their age, size category and species. After that, we compare the environmental signals inducing cold acclimation and frost hardening in the roots and aboveground organs. Subsequently, we discuss how roots sense cold at a cellular level and briefly describe the following signal transduction pathway, which leads to molecular and metabolic responses associated with frost hardening. Finally, the current options available to increase root frost tolerance are explored and promising lines of future research are discussed.

Selection of Appropriate Reference Genes for Gene Expression Analysis under Abiotic Stresses in Salix viminalis.

Salix viminalis is a fast growing willow species with potential as a plant used for biomass feedstock or for phytoremediation. However, few reference genes (RGs) for quantitative real-time polymerase chain reaction (qPCR) are available in S. viminalis, thereby limiting gene expression studies. Here, we investigated the expression stability of 14 candidate reference genes (RGs) across various organs exposed to five abiotic stresses (cold, heat, drought, salt, and poly-metals). Four RGs ranking algorithms, namely geNormPLUS, BestKeeper, NormFinder, and GrayNorm were applied to analyze the qPCR data and the outputs were merged into consensus lists with RankAggreg, a rank aggregation algorithm. In addition, the optimal RG combinations were determined with geNormPLUS and GrayNorm. The genes that were the most stable in the roots were TIP41 and CDC2. In the leaves, TIP41 was the most stable, followed by EF1b and ARI8, depending on the condition tested. Conversely, GAPDH and ß-TUB, two genes commonly used for qPCR data normalization were the least stable across all organs. Nevertheless, both geNormPLUS and GrayNorm recommended the use of a combination of genes rather than a single one. These results are valuable for research of transcriptomic responses in different S. viminalis organs.