Adaptation to acidic soil is achieved by increased numbers of cis-acting elements regulating ALMT1 expression in Holcus lanatus.

Yorkshire fog (Holcus lanatus), which belongs to the Poaceae family and is a close relative of the agronomic crop oat (Avena sativa), is a widely adaptable grass species that is able to grow on highly acidic soils with high levels of Al, but the mechanism underlying the high Al tolerance is unknown. Here, we characterized two accessions of H. lanatus collected from an acid plot (soil pH 3.6, HL-A) and a neutral plot (pH 7.1, HL-N) in terms of Al tolerance, organic acid anion secretion and related gene expression. In response to Al (pH 4.5), the HL-A roots secreted approximately twice as much malate as the HL-N roots, but there was no difference in citrate secretion. Cloning of the gene HlALMT1 responsible for malate secretion showed that the encoded amino acid sequence did not differ between two accessions, but the expression level in the outer cell layers of the HL-A roots was twice as high as in the HL-N roots. This difference was not due to the genomic copy number, but was due to the number of cis-acting elements for an Al-responsive transcription factor (HlART1) in the promoter region of HlALMT1, as demonstrated by both a yeast one-hybrid assay and a transient assay in tobacco protoplasts. Furthermore, introduction of HlALMT1 driven by the HL-A promoter into rice resulted in significantly more Al-induced malate secretion than introduction of HlALMT1 driven by the HL-N promoter. These findings indicate that the adaptation of H. lanatus to acidic soils may be achieved by increasing number of cis-acting elements for ART1 in the promoter region of the HlALMT1 gene, enhancing the expression of HlALMT1 and the secretion of malate.

Trait-directed de novo population transcriptome dissects genetic regulation of a balanced polymorphism in phosphorus nutrition/arsenate tolerance in a wild grass, Holcus lanatus.

The aim of this study was to characterize the transcriptome of a balanced polymorphism, under the regulation of a single gene, for phosphate fertilizer responsiveness/arsenate tolerance in wild grass Holcus lanatus genotypes screened from the same habitat. De novo transcriptome sequencing, RNAseq (RNA sequencing) and single nucleotide polymorphism (SNP) calling were conducted on RNA extracted from H. lanatus. Roche 454 sequencing data were assembled into c. 22,000 isotigs, and paired-end Illumina reads for phosphorus-starved (P-) and phosphorus-treated (P+) genovars of tolerant (T) and nontolerant (N) phenotypes were mapped to this reference transcriptome. Heatmaps of the gene expression data showed strong clustering of each P+/P- treated genovar, as well as clustering by N/T phenotype. Statistical analysis identified 87 isotigs to be significantly differentially expressed between N and T phenotypes and 258 between P+ and P- treated plants. SNPs and transcript expression that systematically differed between N and T phenotypes had regulatory function, namely proteases, kinases and ribonuclear RNA-binding protein and transposable elements. A single gene for arsenate tolerance led to distinct phenotype transcriptomes and SNP profiles, with large differences in upstream post-translational and post-transcriptional regulatory genes rather than in genes directly involved in P nutrition transport and metabolism per se.

Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus.

Decreased arsenate [As(V)] uptake is the major mechanism of naturally selected As(V) hypertolerance in plants. However, As(V)-hypertolerant ecotypes also show enhanced rates of phytochelatin (PC) accumulation, suggesting that improved sequestration might additionally contribute to the hypertolerance phenotype. Here, we show that enhanced PC-based sequestration in As(V)-hypertolerant Holcus lanatus is not due to an enhanced capacity for PC synthesis as such, but to increased As(V) reductase activity. Vacuolar transport of arsenite-thiol complexes was equal in both ecotypes. Based on homology with the yeast As(V) reductase, Acr2p, we identified a Cdc25-like plant candidate, HlAsr, and confirmed the As(V) reductase activity of both HlAsr and the homologous protein from Arabidopsis thaliana. The gene appeared to be As(V)-inducible and its expression was enhanced in the As(V)-hypertolerant H. lanatus ecotype, compared with the non-tolerant ecotype. Homologous ectopic overexpression of the AtASR cDNA in A. thaliana produced a dual phenotype. It improved tolerance to mildly toxic levels of As(V) exposure, but caused hypersensitivity to more toxic levels. Arabidopsis asr T-DNA mutants showed increased As(V) sensitivity at low exposure levels and enhanced arsenic retention in the root. It is argued that, next to decreased uptake, enhanced expression of HlASR might act as an additional determinant of As(V) hypertolerance and As transport in H. lanatus.