Identification and characterization of silicon efflux transporters in horsetail (Equisetum arvense).

Silicon (Si) is a beneficial element to plants, and its absorption via transporters leads to protective effects against biotic and abiotic stresses. In higher plants, two groups of root transporters for Si have been identified: influx transporters (Lsi1) and efflux transporters (Lsi2). Lsi1 transporters belong to the NIPIII aquaporins, and functional Lsi1s have been found in many plants species. Much less is known about Lsi2s that have been characterized in only a few species. Horsetail (Equisetum arvense), known among the highest Si accumulators in the plant kingdom, is a valuable model to study Si absorption and deposition. In this study, we first analyzed discrete Si deposition patterns in horsetail shoots, where ubiquitous silicification differs markedly from that of higher plants. Then, using the sequenced horsetail root transcriptome, two putative Si efflux transporter genes, EaLsi2-1 and EaLsi2-2, were identified. These genes share low sequence similarity with their homologues in higher plants. Further characterisation of EaLsi2-1 in transient expression assay using Nicotiana benthamiana epidermal cells confirmed transmembrane localization. In order to determine their functionality, the EaLsi2-1 was expressed in Xenopus oocytes, confirming that the translated protein was efficient for Si efflux. Both genes were equally expressed in roots and shoots, but interestingly, showed a much higher expression in the shoots than in the roots in contrast to Lsi2s found in other plants, a result consistent with the specific anatomy of horsetail and its rank as one of the highest Si accumulators among plant species.

Cloning, functional characterization and heterologous expression of TaLsi1, a wheat silicon transporter gene.

Silicon (Si) is known to be beneficial to plants, namely in alleviating biotic and abiotic stresses. The magnitude of such positive effects is associated with a plant's natural ability to absorb Si. Many grasses can accumulate as much as 10% on a dry weight basis while most dicots, including Arabidopsis, will accumulate less than 0.1%. In this report, we describe the cloning and functional characterization of TaLsi1, a wheat Si transporter gene. In addition, we developed a heterologous system for the study of Si uptake in plants by introducing TaLsi1 and OsLsi1, its ortholog in rice, into Arabidopsis, a species with a very low innate Si uptake capacity. When expressed constitutively under the control of the CaMV 35S promoter, both TaLsi1 and OsLsi1 were expressed in cells of roots and shoots. Such constitutive expression of TaLsi1 or OsLsi1 resulted in a fourfold to fivefold increase in Si accumulation in transformed plants compared to WT. However, this Si absorption caused deleterious symptoms. When the wheat transporter was expressed under the control of a root-specific promoter (a boron transporter gene (AtNIP5;1) promoter), a similar increase in Si absorption was noted but the plants did not exhibit symptoms and grew normally. These results demonstrate that TaLsi1 is indeed a functional Si transporter as its expression in Arabidopsis leads to increased Si uptake, but that this expression must be confined to root cells for healthy plant development. The availability of this heterologous expression system will facilitate further studies into the mechanisms and benefits of Si uptake.

Dehydrin variants associated with superior freezing tolerance in alfalfa (Medicago sativa L.).

A cDNA (msaCIG) encoding a cold-inducible Y(2)K(4) dehydrin in alfalfa (Medicago sativa spp. sativa) was shown to share extensive homology with sequences from other species and subspecies of Medicago. Differences were mainly the result of the occurrence of large indels, amino acids substitutions/deletions and sequence duplications. Using a combination of a bulk segregant analysis and RFLP hybridization, we uncovered an msaCIG polymorphism that increases in frequency in response to recurrent selection for superior freezing tolerance. Progenies from crosses between genotypes with (D+) or without (D-) the polymorphic dehydrin significantly differed in their tolerance to subfreezing temperatures. Based on the msaCIG sequence, we looked for intragenic variations that could be associated to the polymorphism detected on Southern blots. Amplifications with primers targeting the 3' half side of msaCIG revealed fragment size variations between pools of genotypes with (+) or without (-) the polymorphism. Three major groups of amplicons of approximately 370 nt (G1), 330 nt (G2), and 290 nt (G3) were distinguished. The G2 group was more intensively amplified in pools of genotypes with the polymorphic dehydrin and was associated to a superior freezing tolerance phenotype. Sequences analysis revealed that size variation in the 3' half was attributable to the variable occurrence of large indels. Single amino acid substitutions and/or deletions caused major differences in the prediction of the secondary structure of the polypeptides. The identification of dehydrin variants associated to superior freezing tolerance paves the way to the development of functional markers and the fixation of favorable alleles in various genetic backgrounds.