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 distribution of arsenate and arsenite in shoots and roots of Holcus lanatus is influenced by arsenic tolerance and arsenate and phosphate supply

The recent discovery that phytochelatins are important for arsenic (As) detoxification in terrestrial plants results in the necessity to understand As speciation and metabolism in plant material. A hydroponic study was therefore conducted to examine the effects of different levels of phosphate and arsenate [As(V)] on As speciation and distribution in tolerant and non-tolerant clones of Holcus lanatus. Speciation of As in tissue (using high-performance liquid chromatography-inductively coupled plasma mass spectrometry) revealed that the predominant species present were the inorganic As species (As(V) and arsenite [As(III)]), although small levels (<1%) of organic As species (dimethylarsinic acid and monomethylarsonic acid) were detected in shoot material. In roots, the proportion of total As present as As(III) generally increased with increasing levels of As(V) in the nutrient solution, whereas in shoots, the proportion of total As present as As(III) generally decreased with increasing levels of As(V). H. lanatus plants growing in the high-phosphorus (P) (100 micro M) solution contained a higher proportion of As(V) (with regard to total As) in both roots and shoots than plants supplied with low P (10 micro M); in addition, tolerant clones generally contained a higher proportion of As(V) with regard to total As than non-tolerant clones. The study further revealed that As(V) can be reduced to As(III) in both roots and shoots. Although the reduction capacity was limited, the reduction was closely regulated by As influx for all treatments. The results therefore provide a new understanding about As metabolism in H. lanatus.     

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.     

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.     

The nature of arsenic-phytochelatin complexes in Holcus lanatus and Pteris cretica

We have developed a method to extract and separate phytochelatins (PCs)-metal(loid) complexes using parallel metal(loid)-specific (inductively coupled plasma-mass spectrometry) and organic-specific (electrospray ionization-mass spectrometry) detection systems-and use it here to ascertain the nature of arsenic (As)-PC complexes in plant extracts. This study is the first unequivocal report, to our knowledge, of PC complex coordination chemistry in plant extracts for any metal or metalloid ion. The As-tolerant grass Holcus lanatus and the As hyperaccumulator Pteris cretica were used as model plants. In an in vitro experiment using a mixture of reduced glutathione (GS), PC(2), and PC(3), As preferred the formation of the arsenite [As((III))]-PC(3) complex over GS-As((III))-PC(2), As((III))-(GS)(3), As((III))-PC(2), or As((III))-(PC(2))(2) (GS: glutathione bound to arsenic via sulphur of cysteine). In H. lanatus, the As((III))-PC(3) complex was the dominant complex, although reduced glutathione, PC(2), and PC(3) were found in the extract. P. cretica only synthesizes PC(2) and forms dominantly the GS-As((III))-PC(2) complex. This is the first evidence, to our knowledge, for the existence of mixed glutathione-PC-metal(loid) complexes in plant tissues or in vitro. In both plant species, As is dominantly in non-bound inorganic forms, with 13% being present in PC complexes for H. lanatus and 1% in P. cretica.     

Multifactorial Growth Responses in Holcus lanatus: Optima and Limiting Factors

A factorial, controlled-environment experiment employing fourlevels each of photosynthetically-active radiation (PAR), nutrientsupply and ambient temperature was conducted on the grass Holcuslanatus L. (Yorkshire fog). The back-transformed means of log total dry weight per plantat 28 d for the highest- and lowest-yielding treatments were533 mg and 1·8 mg respectively. An analysis of varianceshowed that all three environmental variables have strong andcomplex interaction with one another. Multivariable, polynomialresponse surfaces were fitted to answered questions such as:Where does maximal yield lie? How curved is the response? Howsteep are the gradients of yield against the three environmentalvariables? How sensitive are individual response curves to backgroundlevels of other factors? The direction of change in the logarithms of total dry weightand total leaf area was positive throughout with changes inPAR, nutrient supply and temperature, except at certain extremecombinations of factor values. For leaf area ratio, the directionof change with nutrient supply and temperature was also consistentlypositive, but with PAR it was consistently negative, creatingsaddle-point in the response surface. For all three yields variables, the response to all three factorsconsidered individually was relatively independent of variationsin the background levels of the remaining two. The ranking ofpercentage yields was particularly stable to such variationsin background, even under markedly sub-optimal combinationsof conditions. General conclusions regarding the significance of multivariatetests were drawn, with particular reference to the conduct ofinter-specific screening operations and the interpretation ofmulti-factor environment scenarios such as those involved inclimate change research.Copyright 1993, 1999 Academic Press Multifactorial experiment, temperature, photosynthetically-active radiation, mineral nutrients, response surfaces, optima, limiting factors, screening, climate change     

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.     

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.     

Ecotypes of Holcus lanatus Tolerant to Zinc Toxicity also Tolerate Zinc Deficiency

Genotypes tolerant to zinc (Zn) toxicity, if they accumulateZn in their roots, may grow better than Zn-sensitive genotypes,even in Zn-deficient soil. In the present study, Holcus lanatusL. ecotypes differing in tolerance to Zn toxicity were grownin Zn-deficient Laffer soil which was amended with Zn to createa range of conditions from Zn deficiency to Zn toxicity. IncreasingZn additions to the soil, up to the sufficiency level, improvedgrowth of all ecotypes. At toxic levels of added Zn, the Zn-sensitiveecotype suffered a greater decrease in growth than the Zn-tolerantecotypes. All ecotypes accumulated more Zn in roots than inshoots, with root concentrations exceeding 8 g Zn kg^-1dry weightin extreme cases. When grown in Zn-deficient or Zn-sufficientsoil (up to 0.5 mg Zn kg^-1soil added), ecotypes tolerant toZn toxicity took up more Zn, grew better and had greater rootand shoot Zn concentration than the control (Zn-sensitive ecotype).Zn-tolerant ecotypes transported more Zn, copper (Cu) and iron(Fe) from roots to shoots in comparison with the Zn-sensitiveecotype. The average Zn uptake rate from Zn-deficient soil (noZn added) was greater in the Zn-tolerant ecotypes than in theZn-sensitive ecotype. In conclusion, ecotypes of H. lanatusthat are tolerant to Zn toxicity also tolerate Zn deficiencybetter than the Zn-sensitive ecotype because of their greatercapacity for taking up Zn from Zn-deficient soil. This is thefirst report of the coexistence of traits for tolerance to Zntoxicity and Zn deficiency in a single plant genotype. Copyright2000 Annals of Botany Company Copper, heavy metal, Holcus lanatus, iron, zinc deficiency, zinc toxicity     

Leaf growth in the fast-growing Holcus lanatus and the slow-growing Deschampsia flexuosa: tissue maturation

Inherent variation in the relative growth rate of grasses is negatively correlated with that in leaf mass per unit leaf area (LMA). To scrutinize this correlation, the LMA of two grass species was analysed. Changes in LMA and cell wall synthesis in leaf blades of the fast-growing grass Holcus lanatus and the slow-growing grass Deschampsia flexuosa were investigated above the elongation zone of the leaf blade. After the leaf had obtained its final length, in H. lantus final LMA values of 40-44 gm^-2 were obtained at full leaf length, whereas in D. flexuosa LMA values continued to rise to 110 gm^-2. During this period of tissue maturation the LMA value doubled in H. lanatus, whereas in D. flexuosa an increase of 30% was measured. Most of the cell walls could be hydrolysed with driselase, the residue was hydrolysed with sulphuric acid. Driselase hydrolysates were identical in sugar composition, whereas the sugars released by sulphuric acid treatment changed gradually in composition as the tissue matured. The major sites of cell wall deposition during cell maturation were the outer walls of epidermal cells, fibres adjacent to the epidermis and the mestome ring around the vascular bundles. Lignin deposition was restricted to the vascular bundles and lignin levels of the leaf blade did not exceed 0.9% of the total amount of cell wall polysaccharides. Lignin accumulation occurred mainly after the increase in LMA and is unlikely to affect measurably the growth of these leaves.     

Holcus lanatus invasion slows decomposition through its interaction with a macroinvertebrate detritivore, Porcellio scaber

Detritus based food webs may mediate the impacts of invasive species on ecosystem processes. Holcus lanatus (L.) is an invasive perennial grass that is rapidly spreading in the coastal prairie of California. We used litterbags to determine if H. lanatus altered the rate of litter decomposition through its interaction with the dominant macroinvertebrate detritivore at our study site, the isopod Porcellio scaber (Latreille). Over the course of a year, H. lanatus litter loss was 15% less than annual grasses, a difference that was directly attributable to P. scaber. Although there was no effect of isopods on litter loss during the winter, when most decomposition occurred, isopods had substantial effects on litter loss of annual grasses during the summer. P. scaber had no effect on litter loss of H. lanatus litter. Our findings suggest that H. lanatus invasion slows decomposition in the coastal prairie because it is unusually refractory to an important detritivore. The decreased decomposition of H. lanatus litter, along with increased production during the growing season, lead to a doubling of the end-of-season litter biomass where H. lanatus had invaded.