Transgenic Medicago truncatula Plants That Accumulate Proline Display Enhanced Tolerance to Cadmium Stress.

Cadmium (Cd) accumulation in agricultural soils constitutes a serious problem for crop yields and food safety. It is known that proline (Pro) can rapidly accumulate in plant tissues in response to abiotic stress. To analyze the potential protective effect of Pro accumulation against Cd toxicity, we compared the response to Cd stress of wild-type (WT) Medicago truncatula and a transgenic line that we had previously obtained and characterized (p18), which expressed the Δ 1-pyrroline-5-carboxylate synthetase gene from Vigna aconitifolia (VaP5CS), and accumulated high Pro levels. Cadmium significantly reduced germination of WT seeds compared to p18 seeds, and seedling relative root growth, a valid indicator of metal tolerance, was significantly higher for p18 than WT seedlings. We analyzed the relative expression of genes related to Pro metabolism, phytochelatin biosynthesis. antioxidant machinery, and NADPH recycling, which are relevant mechanisms in the response to Cd stress. They presented differential expression in the seedlings of both genotypes both under control conditions and under Cd stress, suggesting that the Cd response mechanisms might be constitutively activated in the transgenic line. Pro accumulation promoted higher survival, enhanced growth performance, and minor nutrient imbalance in transgenic p18 plants compared to WT plants. These facts, together with the recorded gluthatione levels, lipid peroxidation and antioxidant enzyme activities strongly suggested that VaP5CS expression and Pro accumulation conferred enhanced Cd tolerance to M. truncatula p18 plants, which was likely mediated by changes in Pro metabolism, increased phytochelatin biosynthesis and a more efficient antioxidant response. Moreover, p18 roots accumulated significantly higher Cd amounts than WT roots, while Cd translocation to the aerial part was similar to WT plants, thus suggesting that high Pro levels increased not only Cd tolerance, but also Cd phytostabilization by rhizosequestration.

Cadmium-Tolerant and -Sensitive Cultivars Identified by Screening of Medicago truncatula Germplasm Display Contrasting Responses to Cadmium Stress.

Cadmium (Cd) pollution in soils is an increasing problem worldwide, and it affects crop production and safety. We identified Cd-tolerant and -sensitive cultivars by testing 258 accessions of Medicago truncatula at seedling stage, using the relative root growth (RRG) as an indicator of Cd tolerance. The factorial analysis (principal component analysis method) of the different growth parameters analyzed revealed a clear differentiation between accessions depending on the trait (tolerant or sensitive). We obtained a normalized index of Cd tolerance, which further supported the suitability of RRG to assess Cd tolerance at seedling stage. Cd and elements contents were analyzed, but no correlations with the tolerance trait were found. The responses to Cd stress of two accessions which had similar growth in the absence of Cd, different sensitivity to the metal but similar Cd accumulation capacity, were analyzed during germination, seedling stage, and in mature plants. The results showed that the Cd-tolerant accession (CdT) displayed a higher tolerance than the sensitive cultivar (CdS) in all the studied stages. The increased gene expression of the three main NADPH recycling enzymes in CdT might be key for this tolerance. In CdS, Cd stress produced strong expression of most of the genes that encode enzymes involved in glutathione and phytochelatin biosynthesis (MtCYS, MtγECS, and MtGSHS), as well as GR, but it was not enough to avoid a redox status imbalance and oxidative damages. Our results on gene expression, enzyme activity, antioxidant content, and lipid peroxidation indicate different strategies to cope with Cd stress between CdS and CdT, and provide new insights on Cd tolerance and Cd toxicity mechanisms in M. truncatula.

Wide cross-species RNA-Seq comparison reveals convergent molecular mechanisms involved in nickel hyperaccumulation across dicotyledons.

The Anthropocene epoch is associated with the spreading of metals in the environment increasing oxidative and genotoxic stress on organisms. Interestingly, c. 520 plant species growing on metalliferous soils acquired the capacity to accumulate and tolerate a tremendous amount of nickel in their shoots. The wide phylogenetic distribution of these species suggests that nickel hyperaccumulation evolved multiple times independently. However, the exact nature of these mechanisms and whether they have been recruited convergently in distant species is not known. To address these questions, we have developed a cross-species RNA-Seq approach combining differential gene expression analysis and cluster of orthologous group annotation to identify genes linked to nickel hyperaccumulation in distant plant families. Our analysis reveals candidate orthologous genes encoding convergent function involved in nickel hyperaccumulation, including the biosynthesis of specialized metabolites and cell wall organization. Our data also point out that the high expression of IREG/Ferroportin transporters recurrently emerged as a mechanism involved in nickel hyperaccumulation in plants. We further provide genetic evidence in the hyperaccumulator Noccaea caerulescens for the role of the NcIREG2 transporter in nickel sequestration in vacuoles. Our results provide molecular tools to better understand the mechanisms of nickel hyperaccumulation and study their evolution in plants.

Biomolecular approaches to understanding metal tolerance and hyperaccumulation in plants.

Trace metal elements are essential for plant growth but become toxic at high concentrations, while some non-essential elements, such as Cd and As, show toxicity even in traces. Thus, metal homeostasis is tightly regulated in plants. Plant species colonising metalliferous soils have evolved mechanisms to hypertolerate metals and, in rare cases, can hyperaccumulate them in excess amounts in their shoots. The molecular mechanisms of metal hypertolerance and hyperaccumulation are likely derived from alterations in the basic mechanisms involved in general metal homeostasis. Genes involved in metal transport, synthesis of metal chelators and oxidative stress responses are constitutively and highly expressed in metal hypertolerant and hyperaccumulator species. Plant specialized metabolites and cell wall components have been proposed as major players in these mechanisms. In addition, the high intra-specific natural variation of metal hypertolerance and hyperaccumulation suggests that various molecular mechanisms might be involved in the evolution of these traits. To date, the potential of wild plant populations as systems to study metal tolerance and hyperaccumulation has not been fully exploited. The advent of next-generation sequencing (NGS) has enabled the study of non-model species, providing an opportunity to study natural populations and new tolerant and/or hyperaccumulating species, and will provide new insights into metal tolerance and hyperaccumulation. In this review we highlight background knowledge about metal tolerance and hyperaccumulation in plants and the current state-of-the-art techniques to study and identify the underlying mechanisms of metal hypertolerance and hyperaccumulation. We also outline for the reader the importance of the multidisciplinarity of this research field and how the integration of multiomic approaches will benefit facing the future scientific challenges.

Vacuolar Iron Stores Gated by NRAMP3 and NRAMP4 Are the Primary Source of Iron in Germinating Seeds.

During seed germination, iron (Fe) stored in vacuoles is exported by the redundant NRAMP3 and NRAMP4 transporter proteins. A double nramp3 nramp4 mutant is unable to mobilize Fe stores and does not develop in the absence of external Fe. We used RNA sequencing to compare gene expression in nramp3 nramp4 and wild type during germination and early seedling development. Even though sufficient Fe was supplied, the Fe-responsive transcription factors bHLH38, 39, 100, and 101 and their downstream targets FRO2 and IRT1 mediating Fe uptake were strongly upregulated in the nramp3 nramp4 mutant. Activation of the Fe deficiency response was confirmed by increased ferric chelate reductase activity in the mutant. At early stages, genes important for chloroplast redox control (FSD1 and SAPX), Fe homeostasis (FER1 and SUFB), and chlorophyll metabolism (HEMA1 and NYC1) were downregulated, indicating limited Fe availability in plastids. In contrast, expression of FRO3, encoding a ferric reductase involved in Fe import into the mitochondria, was maintained, and Fe-dependent enzymes in the mitochondria were unaffected in nramp3 nramp4 Together, these data show that a failure to mobilize Fe stores during germination triggered Fe deficiency responses and strongly affected plastids, but not mitochondria.

Genotypic variations in the dynamics of metal concentrations in poplar leaves: a field study with a perspective on phytoremediation.

Poplar is commonly used for phytoremediation of metal polluted soils. However, the high concentrations of trace elements present in leaves may return to soil upon leaf abscission. To investigate the mechanisms controlling leaf metal content, metal concentrations and expression levels of genes involved in metal transport were monitored at different developmental stages on leaves from different poplar genotypes growing on a contaminated field. Large differences in leaf metal concentrations were observed among genotypes. Whereas Mg was remobilized during senescence, Zn and Cd accumulation continued until leaf abscission in all genotypes. A positive correlation between Natural Resistance Associated Macrophage Protein 1 (NRAMP1) expression levels and Zn bio-concentration factors was observed. Principal component analyses of metal concentrations and gene expression levels clearly discriminated poplar genotypes. This study highlights a general absence of trace element remobilization from poplar leaves despite genotype specificities in the control of leaf metal homeostasis.