Interference between arsenic-induced toxicity and hypoxia.

Plants often face combinatorial stresses in their natural environment. Here, arsenic (As) toxicity was combined with hypoxia (Hpx) in the roots of Arabidopsis thaliana as it often occurs in nature. Arsenic inhibited growth of both roots and leaves, whereas root growth almost entirely ceased in Hpx. Growth efficiently resumed, and Hpx marker transcripts decreased upon reaeration. Compromised recovery from HpxAs treatment following reaeration indicated some persistent effects of combined stresses despite lower As accumulation. Root glutathione redox potential turned more oxidized in Hpx and most strongly in HpxAs. The more oxidizing root cell redox potential and the lowered glutathione amounts may be conducive to the growth arrest of plants exposed to HpxAs. The stresses elicited changes in elemental and transcriptomic composition. Thus, calcium, magnesium, and phosphorous amounts decreased in rosettes, but the strongest decline was seen for potassium. The reorganized potassium-related transcriptome supports the conclusion that disturbed potassium homeostasis contributes to the growth phenotype. In a converse manner, photosynthesis-related parameters were hardly affected, whereas accumulated carbohydrates under all stresses and anthocyanins under Hpx exclude carbohydrate limitation. The study demonstrates the existence of both synergistic since mutually aggravating effects and antagonistic effects of single and combined stresses.

Vacuolar compartmentalization as indispensable component of heavy metal detoxification in plants.

Plant cells orchestrate an array of molecular mechanisms for maintaining plasmatic concentrations of essential heavy metal (HM) ions, for example, iron, zinc and copper, within the optimal functional range. In parallel, concentrations of non-essential HMs and metalloids, for example, cadmium, mercury and arsenic, should be kept below their toxicity threshold levels. Vacuolar compartmentalization is central to HM homeostasis. It depends on two vacuolar pumps (V-ATPase and V-PPase) and a set of tonoplast transporters, which are directly driven by proton motive force, and primary ATP-dependent pumps. While HM non-hyperaccumulator plants largely sequester toxic HMs in root vacuoles, HM hyperaccumulators usually sequester them in leaf cell vacuoles following efficient long-distance translocation. The distinct strategies evolved as a consequence of organ-specific differences particularly in vacuolar transporters and in addition to distinct features in long-distance transport. Recent molecular and functional characterization of tonoplast HM transporters has advanced our understanding of their contribution to HM homeostasis, tolerance and hyperaccumulation. Another important part of the dynamic vacuolar sequestration syndrome involves enhanced vacuolation. It involves vesicular trafficking in HM detoxification. The present review provides an updated account of molecular aspects that contribute to the vacuolar compartmentalization of HMs.

The relationship between metal toxicity and cellular redox imbalance.

The relationship between cellular redox imbalances leading to oxidative stress and metal toxicity in plants has been studied intensely over the past decades. This interdependency was often considered to reflect a rather indirect metal effect of cellular disregulation and progressive secondary damage development. By contrast, recent experiments revealed a clear relationship between metal stress and redox homeostasis and antioxidant capacity. Analysis of plants expressing targeted modifications of components of the antioxidant system, the comparison of closely related plant species with different degrees of toxic metal sensitivity and effector studies with, for instance, salicylic acid have established a link between the degree of plant tolerance to metals and the level of antioxidants.

Emerging Trends in Metalloid-Dependent Signaling in Plants.

Metalloids are semiconducting elements that constitute a small group in the periodic table of elements. Their occurrence in nature either poses an environmental threat or benefit to plants. The precise mechanisms or manner of crosstalk of metalloid interference and sensing remain open questions. Standard plant nutrient solutions contain the metalloid boron (B) as a micronutrient, while silicon (Si) is considered a beneficial element routinely supplied only to some plants such as grasses. By contrast, arsenic (As) is a severe environmental hazard to most organisms, including plants, while the less abundant metalloids germanium (Ge), antimony (Sb), and tellurium (Te) display variable degrees of toxicity. Here we review the molecular events and mechanisms that could explain the contrasting (or overlapping) action of metalloids on the cell and cell signaling.

Remodeling of Root Growth Under Combined Arsenic and Hypoxia Stress Is Linked to Nutrient Deprivation.

Root architecture responds to environmental stress. Stress-induced metabolic and nutritional changes affect the endogenous root development program. Transcriptional and translational changes realize the switch between stem cell proliferation and cell differentiation, lateral root or root hair formation and root functionality for stress acclimation. The current work explores the effects of stress combination of arsenic toxicity (As) and hypoxia (Hpx) on root development in Arabidopsis thaliana. As revealed previously, combined As and Hpx treatment leads to severe nutritional disorder evident from deregulation of root transcriptome and plant mineral contents. Both As and Hpx were identified to pose stress-specific constraints on root development that lead to unique root growth phenotype under their combination. Besides inhibition of root apical meristem (RAM) activity under all stresses, As induced lateral root growth while root hair density and lengths were strongly increased by Hpx and HpxAs-treatments. A dual stimulation of phosphate (Pi)-starvation response was observed for HpxAs-treated plant roots; however, the response under HpxAs aligned more with Hpx than As. Transcriptional evidence along with biochemical data suggests involvement of PHOSPHATE STARVATION RESPONSE 1; PHR1-dependent systemic signaling. Pi metabolism-related transcripts in close association with cellular iron homeostasis modulate root development under HpxAs. Early redox potential changes in meristematic cells, differential ROS accumulation in root hair zone cell layers and strong deregulation of NADPH oxidases, NADPH-dependent oxidoreductases and peroxidases signify a role of redox and ROS signaling in root architecture remodeling under HpxAs. Differential aquaporin expression suggests transmembrane ROS transport to regulate root hair induction and growth. Reorganization of energy metabolism through NO-dependent alternate oxidase, lactate fermentation, and phosphofructokinase seems crucial under HpxAs. TOR and SnRK-signaling network components were potentially involved in control of sustainable utilization of available energy reserves for root hair growth under combined stress as well as recovery on reaeration. Findings are discussed in context of combined stress-induced signaling in regulation of root development in contrast to As and Hpx alone.

Cadmium-induced changes in vacuolar aspects of Arabidopsis thaliana.

We have examined the changes due to Cd treatment in the vacuolar form in root tip cortical cells in Arabidopsis thaliana employing a transformant with GFP fused to a tonoplast protein. A Cd-induced enhancement in complexity with general expansion of vacuolar system within 24 h was evident. The changes in the vacuolar form were dependent on the applied Cd concentrations. Concomitantly, as revealed through dithizone staining, Cd accumulated in the seedling roots exhibiting abundance of Cd-dithizone complexes in root tip, root hairs and vasculature. To get insight into the involvement of SNARE protein-mediated vesicle fusion in Cd detoxification, the magnitude of Cd toxicity in a couple of knock out mutants of the vacuolar Qa-SNARE protein VAM3/SYP22 was compared with that in the wild type. The Cd toxicity appeared to be comparable in the mutants and the wild type. In order to analyze the Cd effects at cellular level, we treated the Arabidopsis suspension-cultured cells with Cd. Cd, however, did not induce a change in the vacuolar form in suspension-cultured cells although Cd measured with ICP-MS was obviously taken up into the cell. The V-ATPase activity in the microsomal fractions from vacuoles isolated from A. thaliana suspension cultured cells remained unaffected by Cd. Changes in the levels of certain metabolites of Cd-treated cells were also not so distinct except for those of glutathione. The significance of findings is discussed.

Characterization of seed germination, seedling growth, and associated metabolic responses of Brassica juncea L. cultivars to elevated nickel concentrations.

Seed germination and seedling growth responses of three Brassica juncea L. cultivars, namely Varuna, Kranti, and Pusa Jai Kisan, to nickel have been characterized. Ni suppressed the seed germination differentially, suppression being greater in cv. Kranti than in others. On the basis of seedling growth performance, cv. Varuna proved most tolerant to Ni. The Ni accumulation in seedlings differed strongly among the three cultivars. The lowest and highest Ni contents were observed in the seedlings of cvs. Kranti and Pusa Jai Kisan, respectively. Despite substantial Ni accumulation, cv. Varuna was most tolerant to Ni. Ni accumulation in seedlings was accompanied by differentially altered Fe and K contents and increased levels of non-protein thiols and free proline. The O2- and H2O2 contents and their respective scavenging enzymes in the seedlings responded differentially to the Ni treatment suggesting the involvement of redox imbalance in the development of Ni toxicity. Interestingly, the greater Ni tolerance of cv. Varuna coincided with the elevated constitutive activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). The data have implications for seedling establishment under Ni-rich conditions and in turn for phytoremediation.

Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex.

BACKGROUND: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. RESULTS: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. CONCLUSIONS: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

Modulation by phenolic compounds of ABA-induced inhibition of mustard (Brassica juncea L. cv. RLM 198) seed germination.

Exogenous abscisic acid (ABA) inhibited the germination of B. juncea seeds in a concentration dependent manner. As revealed in a time-course study, the ABA-induced inhibition got progressively alleviated with the lapse of time following ABA treatment possibly due to metabolic conversion of applied ABA in the seed tissue. A simultaneous application of certain phenolic compounds namely, p-coumaric-, vanillic-, gallic-, and chlorogenic acid (but not caffeic acid) also caused an alleviation of ABA effect. Of the above two patterns of recovery, the phenolic-dependent alleviation of ABA effect was apparent much earlier (24-48 hr treatment) than the time-dependent one (72 hr). It is likely that phenolics could accelerate ABA metabolism in the seed tissue leading to an early recovery from ABA-induced inhibition.

The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress.

Plants exposed to heavy metals accumulate an array of metabolites, some to high millimolar concentrations. This review deals with N-containing metabolites frequently preferentially synthesized under heavy metal stress such as Cd, Cu, Ni, and Zn. Special focus is given to proline, but certain other amino acids and oligopeptides, as well as betaine, polyamines, and nicotianamine are also addressed. Particularly for proline a large body of data suggests significant beneficial functions under metal stress. In general, the molecules have three major functions, namely metal binding, antioxidant defence, and signalling. Strong correlative and mechanistic experimental evidence, including work with transgenic plants and algae, has been provided that indicates the involvement of metal-induced proline in metal stress defence. Histidine, other amino acids and particularly phytochelatins and glutathione play a role in metal binding, while polyamines function as signalling molecules and antioxidants. Their accumulation needs to be considered as active response and not as consequence of metabolic dys-regulation.

In vitro alleviation of heavy metal-induced enzyme inhibition by proline.

Accumulation of proline in response to toxic heavy metal exposure seems to be wide-spread among plants. To elucidate the role for proline in plant responses to heavy metal stress, we studied the effect of proline on Cd-induced and Zn-induced inhibition of glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) and nitrate reductase (NR; EC 1.6.6.2) in vitro. Proline appeared to protect both enzymes against Zn and, though less effectively, against Cd. Measurements with a Cd(2+)-specific electrode strongly suggested that this protection was based on a reduction of the free metal ion activity in the assay buffer, due to the formation of metal-proline complexes. There were no indications of any significant role for proline-water or proline-protein interactions. The significance of these findings with regard to heavy metal-induced proline accumulation in vivo is discussed.