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.     

Polymorphism and physiology of arsenate tolerance in Holcus lanatus L. from an uncontaminated site

The polymorphism of arsenate tolerance in a Holcus lanatus L. population from an uncontaminated soil was investigated and a high percentage of tolerant individuals (65%) was found in the population studied. Influx of arsenate was highly correlated to arsenate tolerance within the population, with the most tolerant individuals having the lowest rates of arsenate influx. Isotherms for the high affinity arsenate uptake systems were determined in six tolerant and six non-tolerant genotypes. Tolerant plants had the lowest rates of arsenate influx. This was achieved by adaptation of the V_max of arsenate influx with the V_max of the high affinity uptake system saturating at lower substrate concentrations in the tolerant plants. The polymorphism is discussed with relation to adaptation to the extreme environments to which the plants are subjected on mine-spoil soils.     

The Growth Response of Ecotypes of Holcus lanatus L. from Different Soil Types in Northwestern Europe to Phenolic Acids

Abstract: Phenolics are often discussed in relation to either allelopathy or to herbivory. This work, however, was undertaken to determine if phenolic acids benefit the growth of plants in very acid soils. We here report racial differences in the phenolic acid concentrations of the important plant species found in five sites within Central Scotland which covered a wide range in soil acidity from very acid (organic peats) to mildly acid (calcareous), and describe the racial differences in the growth response of Holcus lanatus L. to phenolic acids with increasing acidity. The total concentrations of phenolic acids in the ecotypes of important species were correlated with the total concentrations found in their respective soils. In general, the most phenolic-rich ecotypes of the five came from the organic acid soils (Flanders Moss [FM] and Sheriffmuir [SMB]). However, with the exception of ferulic acid which was a major component of both acid soils and their associated vegetation, individual simple phenolic acids extracted from either plants or soils were not consistently correlated. The addition of dry plant material collected from the five sites (0.5 g plant material 100 g^-1 soil) to the acid-organic Flanders Moss (FM) soil stimulated the growth of two ecotypes of Holcus (acid-mineral Sheriffmuir [SMM], calcareous Kinloch Rannoch [KR]) but the same litter addition in non-organic, less acidic or calcareous soil inhibited growth of these ecotypes. In hydroponic solutions, growth response of Flanders Moss (FM) and Kinloch Rannoch (KR) to pH and phenolic acid mixtures was interdependent: in acid solutions (pH 4.0) but not at pH 6.5 root elongation rates (RER) of both ecotypes, and shoot elongation rates (SER) of Flanders Moss (FM), increased after treatment with a mixture of seven commonly occurring phenolic acids.     

A new hypothesis for the origin of pentaploid Holcus from diploid Holcus lanatus L. and tetraploid Holcus mollis L. in France

The Holcus complex in France consists of two species, Holcus lanatus L. (Yorkshire fog; 2n= 2x= 14) and Holcus mollis L. (Creeping soft-grass; 2n= 4x= 28) and an interspecific hybrid H.m.×H.l. (2n= 5x= 35), which is morphologically similar to Holcus mollis. A heterologous rDNA probe from wheat was used to detect the corresponding region in Holcus (s. l.) genomic DNA'fragments, for six to eight plants from 13 populations located south-west of Paris. A restriction enzyme map of the ribosomal RNA genes (rDNA) in Holcus (s. l.) was also constructed. The length polymorphism detected in the IGS region was used as a DNA fingerprint for the identification of different cytotypes and species of the Holcus complex and for the typing and delimitation of individuals in populations. In the light of the results we reconsider the assumption that the pentaploid hybrid H.m.×H.l. is purely clonal. New hypotheses concerning the origin of the pentaploid hybrid and its reproduction are proposed, and the consequences for genetic diversity in natural populations discussed.     

Copper- and arsenate-induced oxidative stress in Holcus lanatus L. clones with differential sensitivity

The biochemical responses of Holcus lanatus L. to copper and arsenate exposure were investigated in arsenate‐tolerant and ‐non‐tolerant plants from uncontaminated and arsenic/copper‐contaminated sites. Increases in lipid peroxidation, superoxide dismutase (SOD) activity and phytochelatin (PC) production were correlated with increasing copper and arsenate exposure. In addition, significant differences in biochemical responses were observed between arsenate‐tolerant and ‐non‐tolerant plants. Copper and arsenate exposure led to the production of reactive oxygen species, resulting in significant lipid peroxidation in non‐tolerant plants. However, SOD activity was suppressed upon metal exposure, possibly due to interference with metallo‐enzymes. It was concluded that in non‐tolerant plants, rapid arsenate influx resulted in PC production, glutathione depletion and lipid peroxidation. This process would also occur in tolerant plants, but by decreasing the rate of influx, they were able to maintain their constitutive functions, detoxify the metals though PC production and quench reactive oxygen species by SOD activity.     

Relationship between plant phosphorus status and the kinetics of arsenate influx in clones ofdeschampsia cespitosa (L.) beauv. that differ in their tolerance to arsenate

Uptake kinetics of arsenate were determined in arsenate tolerant and non-tolerant clones of the grassDeschampsia cespitosa under differing root phosphorus status to investigate the mechanism controlling the suppression of arsenate influx observed in tolerant clones. Influx was always lower in tolerants compared to non-tolerants. Short term influx of arsenate by the high affinity uptake system in both tolerant clones was relatively insensitive to root phosphorus status. This was in contrast to the literature where the regulation of the phosphate (arsenate) uptake system is normally much more responsive to plant phosphorus status. The low affinity uptake system in both tolerant and non-tolerant clones, unlike the high affinity uptake system, was more closely regulated by root phosphate status and was repressed to a much greater degree under increasing root phosphorus levels than the high affinity system.     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms     

Effect of silicate on the growth and arsenate uptake by rice (Oryza sativa L.) seedlings in solution culture

A solution culture experiment was conducted to investigate the effect of silicate on the yield and arsenate uptake by rice. Rice seedlings (Oryza sativaL. cv. Weiyou 77) were cultured in modified Hoagland nutrient solution containing three arsenate levels (0, 0.5 and 1.0 mg L ^–1 As) and four silicate levels (0, 14, 28 and 56 mg L ^–1 Si). Addition of Si significantly increased shoot dry weight (P=0.001) but had little effect on root dry weight (P=0.43). Addition of As had no significant effect on shoot dry weight (P=0.43) but significantly increased root dry weight (P=0.01). Silicon concentrations in shoots and roots increased proportionally to increasing amounts of externally supplied Si (P < 0.001). The presence of As in the nutrient solution had little effect on shoot Si concentration (P=0.16) but significantly decreased root Si concentration (P=0.005). Increasing external Si concentration significantly decreased shoot and root As concentrations and total As uptake by rice seedlings (P <0.001). In addition, Si significantly decreased shoot P concentration and shoot P uptake (P <0.001). The data clearly demonstrate a beneficial effect of Si on the growth of rice seedlings. Addition of Si to the growth medium also inhibited the uptake of arsenate and phosphate by the rice seedlings.     

Effects of arsenate and phosphate on their accumulation by an arsenic-hyperaccumulator Pteris vittata L.

Arsenate and phosphate interactions are important for better understanding their uptake and accumulation by plant due to their similarities in chemical behaviors. The present study examined the effects of arsenate and phosphate on plant biomass and uptake of arsenate and phosphate by Chinese brake (Pteris vittata L.), a newly-discovered arsenic hyperaccumulator. The plants were grown for 20 weeks in a soil, which received the combinations of 670, 2670, or 5340 mol kg^–1 arsenate and 800, 1600, or 3200 mol kg^–1 phosphate, respectively. Interactions between arsenate and phosphate influenced their availability in the soil, and thus plant growth and uptake of arsenate and phosphate. At low and medium arsenate levels (670 and 2670 mol kg^–1), phosphate had slight effects on arsenate uptake by and growth of Chinese brake. However, phosphate substantially increased plant biomass and arsenate accumulation by alleviating arsenate phytotoxicity at high arsenate levels (5340 mol kg^–1). Moderate doses of arsenate increased plant phosphate uptake, but decreased phosphate concentrations at high doses because of its phytotoxicity. Based on our results, the minimum P/As molar ratios should be at least 1.2 in soil solution or 1.0 in fern fronds for the growth of Chinese brake. Our findings suggest that phosphate application may be an important strategy for efficient use of Chinese brake to phytoremediate arsenic contaminated soils. Further study is needed on the mechanisms of interactive effects of arsenate and phosphate on Chinese brake in hydroponic systems.