Reactive green dye remediation by Alternanthera philoxeroides in association with plant growth promoting Klebsiella sp. VITAJ23: A pot culture study.

Contamination of soil by textile effluent is a major threat found worldwide. These pollutants have diverse range of negative effects on the ecosystem, therefore restoration through cost effective biological strategy is the need of the hour. The aim of the current study was to enhance the decolourization of reactive green dye (RGD) using phytoremediation coupled with augmentation of effective bacteria to the rhizosphere. The isolate Klebsiella sp. VITAJ23 was isolated from textile effluent polluted soil and was assessed for its plant growth promoting traits (PGP) and the PGP functional genes were amplified. The soil was artificially polluted with RGD concentration ranging from 1000 to 3000 mg kg-1 and Alternanthera philoxeroides plantlets were planted in phyto and rhizoremediation treatments, the setup was maintained upto 60 d. The isolate VITAJ23 was augmented in the rhizoremediation setup and the morphological parameters were assessed at regular interval. There was a significant increase in the chlorophyll content as well as root and shoot length of the plant when treated with the bacterial suspension. Decolourization study revealed 79% removal of reactive green dye with an enhanced oxido-reductase enzyme activity in the setup bioaugmented with bacteria. The biodegraded metabolites were identified as 2-allylnapthalene, l-alanine, n-acetyl-and propenoic acid by GC-MS analysis and a plant-bacteria degradation pathway was predicted using computational tools. Inoculation of PGP-Klebsiella sp. VITAJ23 enhanced the rate of plant growth and dye degradation.

Selenium and silicon reduce cadmium uptake and mitigate cadmium toxicity in Pfaffia glomerata (Spreng.) Pedersen plants by activation antioxidant enzyme system.

Cadmium (Cd) is toxic to plants and animals, making it necessary to develop strategies that seek to reduce its introduction into food chains. Thus, the aim of this study was to investigate whether silicon (Si) and selenium (Se) reduce Cd concentrations in Pfaffia glomerata medicinal plant and attenuate the oxidative stress promoted by this metal. These plants were cultivated in hydroponics under the following treatments: control (nutrient solution), 2.5 µM Se, 2.5 mM Si, 50 µM Cd, 50 µM Cd + 2.5 µM Se, 50 µM Cd + 2.5 mM Si. After 14 days of exposure to treatments, leaves and roots were collected for the determination of dry weight of shoot and roots, Cd concentrations, chlorophyll and carotenoids content, and biochemical parameters (lipid peroxidation and guaiacol peroxidase and superoxide dismutase activities). The data were submitted to analysis of variance and means were compared with Scott-Knott test at 5% error probability. Roots of P. glomerata plants showed a significant reduction on dry weight accumulation when exposed to Cd. However, both Se and Si promoted a significant reduction of deleterious effects of Cd. The Cd concentrations in the tissues were reduced in the presence of Se or Si. Plants treated with Cd together with Se or Si presented higher pigment content than those with only Cd, thus showing a reduction in the negative effects caused by this element. In the treatments in which Se and Si were added in the growth medium together with Cd, an activation of superoxide dismutase and guaiacol peroxidase enzymes was observed in the roots and shoot, which may have contributed to lower lipid peroxidation. Thus, Se and Si reduce Cd concentrations and have potential to ameliorate Cd toxicity in P. glomerata plants, which can be used to increase productivity and quality of medicinal plants.

Changes in redox regulation during transition from C3 to single cell C4 photosynthesis in Bienertia sinuspersici.

Bienertia sinuspersici performs single cell C4 photosynthesis without Kranz anatomy. Peripheral and central cytoplasmic compartments in a single chlorenchyma cell act as mesophyll cells and bundle sheath cells. Development of this specialized mechanism is gradual during plant development. Young leaves perform C3 photosynthesis, while mature leaves have complete C4 cycle. The aim of this work was to investigate changes in redox regulation and antioxidant defence during transition from C3 to single cell C4 photosynthesis in B. sinuspersici leaves. First, we confirmed gradual development of C4 with protein blot and qRT-PCR analysis of C4 enzymes. After this activities and isoenzymes of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and H2O2 and TBARS and glutathione pool and redox status (GSH/GSSG) were determined in young, developing and mature leaves during transition from C3 to single cell C4 photosynthesis. Activities of SOD, APX and POX decrease, while GR and DHAR were increased. However, most striking results were the changes in isoenzyme patterns of SOD, CAT and GR which were gradual through transition to C4 photosynthesis.

Hexavalent chromium reduction, uptake and oxidative biomarkers in Halimione portulacoides.

The in situ reduction of Cr (VI) to its less toxic form Cr (III) may be a useful detoxification mechanism for phytoremediation. Using a hydroponics mesocosmos approach, we evaluated the ability of Halimione portulacoides to reduce and uptake Cr (VI) and its anti-oxidative feedback and biomarkers. It was found that this specie can, not only reduce large amounts of Cr (VI) in the external medium, but also withdrawn and accumulate this element in its roots and aboveground organs. Both these mechanisms were found to be dose dependent. Jointly with this phytoremediative potential the oxidative feedback was also assessed. Chromium uptake had its major implications on the chlorophyll content and flavonoid content, with potential consequences in the photosynthetic and photo-protective mechanisms. Although the high Cr root accumulation in H. portulacoides, there were no inactivation of the enzymatic defenses, allowing a continuous defense against reactive oxygen species. In fact, GPX and specially SOD revealed to be an excellent dose-related biomarker of Cr induced stress. All these aspects make this specie suitable for Cr (VI) phytoremediation processes, either by phytoextraction or by reduction of Cr (VI) to Cr (III) and also for monitoring programs using SOD and GPX as biomarkers of Cr environmental contamination.

Overexpression of the halophyte Kalidium foliatum H⁺-pyrophosphatase gene confers salt and drought tolerance in Arabidopsis thaliana.

According to sequences of H(+)-pyrophosphatase genes from GenBank, a new H(+)-pyrophosphatase gene (KfVP1) from the halophyte Kalidium foliatum, a very salt-tolerant shrub that is highly succulent, was obtained by using reverse transcription PCR and rapid amplification of cDNA ends methods. The obtained KfVP1 cDNA contained a 2295 bp ORF and a 242 bp 3'-untranslated region. It encoded 764 amino acids with a calculated molecular mass of 79.78 kDa. The deduced amino acid sequence showed high identity to those of H(+)-PPase of some Chenopodiaceae plant species. Semi-quantitative PCR results revealed that transcription of KfVP1 in K. foliatum was induced by NaCl, ABA and PEG stress. Transgenic lines of A. thaliana with 35S::KfVP1 were generated. Three transgenic lines grew more vigorous than the wild type (ecotype Col-0) under salt and drought stress. Moreover, the transgenic plants accumulated more Na(+) in the leaves compared to wild type plants. These results demonstrated that KfVP1 from K. foliatum may be a functional tonoplast H(+)-pyrophosphatase in contributing to salt and drought tolerance.

Rubisco evolution in C4 eudicots: an analysis of Amaranthaceae sensu lato.

BACKGROUND: Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the key reaction in the photosynthetic assimilation of CO2. In C4 plants CO2 is supplied to Rubisco by an auxiliary CO2-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C4 Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C3 enzyme. Specific amino-acids in Rubisco are associated with C4 photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots. METHODOLOGY/PRINCIPAL FINDINGS: We investigated Rubisco evolution in Amaranthaceae sensu lato (including Chenopodiaceae), the third-largest family of C4 plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C4 Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C4 plant groups, such as C4 monocots, illustrating a striking parallelism in molecular evolution. CONCLUSIONS/SIGNIFICANCE: Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco.

Resolving the compartmentation and function of C4 photosynthesis in the single-cell C4 species Bienertia sinuspersici.

Bienertia sinuspersici is a land plant known to perform C(4) photosynthesis through the location of dimorphic chloroplasts in separate cytoplasmic domains within a single photosynthetic cell. A protocol was developed with isolated protoplasts to obtain peripheral chloroplasts (P-CP), a central compartment (CC), and chloroplasts from the CC (C-CP) to study the subcellular localization of photosynthetic functions. Analyses of these preparations established intracellular compartmentation of processes to support a NAD-malic enzyme (ME)-type C(4) cycle. Western-blot analyses indicated that the CC has Rubisco from the C(3) cycle, the C(4) decarboxylase NAD-ME, a mitochondrial isoform of aspartate aminotransferase, and photorespiratory markers, while the C-CP and P-CP have high levels of Rubisco and pyruvate, Pidikinase, respectively. Other enzymes for supporting a NAD-ME cycle via an aspartate-alanine shuttle, carbonic anhydrase, phosophoenolpyruvate carboxylase, alanine, and an isoform of aspartate aminotransferase are localized in the cytosol. Functional characterization by photosynthetic oxygen evolution revealed that only the C-CP have a fully operational C(3) cycle, while both chloroplast types have the capacity to photoreduce 3-phosphoglycerate. The P-CP were enriched in a putative pyruvate transporter and showed light-dependent conversion of pyruvate to phosphoenolpyruvate. There is a larger investment in chloroplasts in the central domain than in the peripheral domain (6-fold more chloroplasts and 4-fold more chlorophyll). The implications of this uneven distribution for the energetics of the C(4) and C(3) cycles are discussed. The results indicate that peripheral and central compartment chloroplasts in the single-cell C(4) species B. sinuspersici function analogous to mesophyll and bundle sheath chloroplasts of Kranz-type C(4) species.

Purification and gene cloning of alpha-methylserine aldolase from Ralstonia sp. strain AJ110405 and application of the enzyme in the synthesis of alpha-methyl-L-serine.

By screening microorganisms that are capable of assimilating alpha-methyl-DL-serine, we detected alpha-methylserine aldolase in Ralstonia sp. strain AJ110405, Variovorax paradoxus AJ110406, and Bosea sp. strain AJ110407. A homogeneous form of this enzyme was purified from Ralstonia sp. strain AJ110405, and the gene encoding the enzyme was cloned and expressed in Escherichia coli. The enzyme appeared to be a homodimer consisting of identical subunits, and its molecular mass was found to be 47 kDa. It contained 0.7 to 0.8 mol of pyridoxal 5'-phosphate per mol of subunit and could catalyze the interconversion of alpha-methyl-L-serine to L-alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from alpha-methyl-L-serine but not from alpha-methyl-D-serine, L-serine, or D-serine. Alpha-methyl-L-serine synthesis activity was detected when L-alanine was used as the substrate. In contrast, no activity was detected when D-alanine was used as the substrate. In the alpha-methyl-L-serine synthesis reaction, the enzymatic activity was inhibited by an excess amount of formaldehyde, which was one of the substrates. We used cells of E. coli as a whole-cell catalyst to express the gene encoding alpha-methylserine aldolase and effectively obtained a high yield of optically pure alpha-methyl-L-serine using L-alanine and formaldehyde.

Seasonal variation of extracellular enzymatic activity (EEA) and its influence on metal speciation in a polluted salt marsh.

The influence of salt marsh sediment extracellular enzymatic activity (EEA) on metal fractions and organic matter cycling was evaluated on a seasonal basis, in order to study the relation between organic matter cycles and the associated metal species. Metals in the rhizosediment of Halimione portulacoides were fractioned according to the Tessier's scheme and showed a similar pattern regarding the organic-bound fraction, being always high in Autumn, matching the season when organic matter presented higher values. Both organic-bound and residual fractions were always dominant, being the seasonal variations due to interchanges between these two fractions. Phenol oxidase and beta-N-acetylglucosaminidase had higher activities during the Spring and Summer, contrarily to peroxidase which had higher activity during Winter. Protease showed high activities in both Spring and Winter. These different periods of high organic matter hydrolysis caused two periods of organic metal bound decrease. Sulphatase peaks (Spring and Winter) matched the depletion of exchangeable metal forms, probably due to sulphides formation and consequent mobilization. This showed an interaction between several microbial activities affecting metal speciation.

Physiological responses of Alternanthera philoxeroides (Mart.) Griseb leaves to cadmium stress.

Aquatic macrophytes were found to be the potential scavengers of heavy metals from aquatic environment. In this study, several physiological responses of Alternanthera philoxeroides (Mart.) Griseb leaves to elevated concentrations of cadmium (up to 10mM) were investigated. It was found that A. philoxeroides was able to accumulate cadmium in its leaves. The pigment contents decreased with the increase of the Cd concentrations. The Cd could induce rise of the activity of peroxidase (POD) at lower concentration (<5mM), however, when the concentration of Cd rose up to 10mM, the POD activity declined. The changes of superoxide dismutase (SOD) and Catalase (CAT) activities were exactly opposite to that of POD. In the leaves of Cd-treated fronds, the amounts of three polypeptides with apparent molecular weights 80, 39 and 28kDa, respectively, were became visible in SDS-PAGE. The nature of these polypeptides remains to be determined.

Evolution of C(4) phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C(4) isozyme and its orthologues in C(3) and C(3)/C(4) Alternantheras.

Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C(4) photosynthesis. It has evolved from ancestral non-photosynthetic (C(3)) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C(4) PEPCases were adapted to their new function in C(4) photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C(4) plant Alternanthera pungens H.B.K., the C(3)/C(4) intermediate plant A. tenella Colla, and the C(3) plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K (0.5) PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C(4)) as a C(4) PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C(3)) and A. tenella (C(3)/C(4)) were found to be typical C(3) PEPCase isozymes. The C(4) characteristics of the PEPCase of A. pungens were accompanied by the presence of the C(4)-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C(4) PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3' untranslated regions of these genes indicated the existence of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC.

Immunological characteristics of PEP carboxylase from leaves of C3-, C4- and C3-C4 intermediate species of Alternanthera--comparison with selected C3- and C4- plants.

Immunological cross-reactivity of phosphoenolpyruvate carboxylase (PEPC) in leaf extracts of C3-, C4- and C3-C4 intermediate species of Alternanthera (along with a few other C3- and C4- plants) was studied using anti-PEPC antibodies raised against PEPC of Amaranthus hypochondriacus (belonging to the same family as that of Alternanthera, namely Amaranthaceae). Antibodies were also raised in rabbits against the purified PEPC from Zea mays (C4- monocot-Poaceae) as well as Alternanthera pungens (C4- dicot-Amaranthaceae). Monospecificity of PEPC-antiserum was confirmed by immunoprecipitation. Amount of PEPC protein in leaf extracts of A. hypochondriacus could be quantified by single radial immunodiffusion. Cros- reactivity of PEPC in leaf extracts from selected C3-, C4-, and C3-C4 intermediate species (including those of Alternanthera) was examined using Ouchterlony double diffusion and Western blots. Anti-PEPC antiserum raised against A. hypochondriacus enzyme showed high cross-reactivity with PEPC in leaf extracts of A. hypochondriacus or Amaranthus viridis or Alternanthera pungens (all C4 dicots), but limited cross-reactivity with that of Zea mays, Sorghum or Pennisetum (all C4 monocots). Interestingly, PEPC in leaf extracts of Alternanthera tenella, A. ficoides, Parthenium hysterophorus (C3-C4 intermediates) exhibited stronger cross-reactivity (with anti-serum raised against PEPC from Amaranthus hypochondriacus) than that of Pisum sativum, Commelina benghalensis, Altenanthera sessilis (C3 plants). Further studies on cross-reactivities of PEPC in leaf extracts of these plants with anti-PEPC antisera raised against PEPC from leaves of Zea mays or Alternanthera pungens confirmed two points--(i) PEPC of C3-C4 intermediate is distinct from C3 species and intermediate between those of C3- and C4-species; and (ii) PEPC of C4-dicots was closer to that of C3-species or C3-C4 intermediates (dicots) than to that of C4-monocots.