Anindita Seal

Isolation and expression analysis of partial sequences of heavy metal transporters from Brassica juncea by coupling high throughput cloning with a molecular fingerprinting technique

Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. These are important candidate genes to study in metal tolerant and accumulator plants for their potential use in environmental clean up. We coupled a degenerate primer-based RT-PCR approach with a molecular fingerprinting technique based on amplified rDNA restriction analysis (ARDRA) to identify novel ESTs corresponding to heavy metal transporters from metal accumulator Brassica juncea. We utilized this technique to clone several family members of natural resistance-associated macrophage proteins (NRAMP) and yellow stripe-like proteins (YSL) in a high throughput manner to distinguish between closely related isoforms and/or allelic variants from the allopolyploid B. juncea. Partial clones of 23 Brassica juncea NRAMPs and 27 YSLs were obtained with similarity to known Arabidopsis thaliana and Noccaea (Thlaspi) caerulescens NRAMP and YSL genes. The cloned transporters showed Brassica-specific changes in domains, which can have important functional consequences. Semi-quantitative RT-PCR-based expression analysis of chosen members indicated that even closely related isoforms/allelic variants of BjNRAMP and BjYSL have distinct tissue-specific and metal-dependent expressions which might be essential for adaptive fitness and heavy metal tolerance. Consistent to this, BjYSL6.1 and BjYSL5.8 were found to show elevated expressions specifically in cadmium-treated shoots and lead-treated roots of B. juncea, respectively.

Manganese and iron both influence the shoot transcriptome of Typha angustifolia despite distinct preference towards manganese accumulation

Typha angustifolia is a metal hypertolerant grass that predominates the wetlands of uranium tailings in Jaduguda, India, contaminated with extreme levels of iron (Fe) and manganese (Mn). In the paper investigations were carried out to understand the molecular mechanism of metal tolerance in this tolerant macrophyte. Metal analysis was coupled with fluorescent differential display (FDD) and reverse northern to compare the metal profile and transcriptome between Typha plants growing in the tailings versus a control wetland. The expressions of the validated transcripts in Mn and Fe stress were confirmed using reverse northern and semiquantitaive RT-PCR. Typha selectively accumulated and stored Mn in shoots but excluded/restricted Fe both in the field and in vitro. Differential expressions of 23 ESTs were validated among 118 FDD genes. Although Typha behaved as a Mn accumulator, these transcripts were found to be regulated in a complex fashion not only by Mn accumulated in the shoots but also by Fe excluded from the shoots. Our results provide the first report in the molecular understanding of metal tolerance in Typha angustifolia and indicate a complex regulation of genes in response to metals in a contaminated habitat.

A consortium of non-rhizobial endophytic microbes from Typha angustifolia functions as probiotic in rice and improves nitrogen metabolism.

Endophytic microbes isolated from plants growing in nutrient-deficient environments often possess properties that improve nutrition of agriculturally important plants. A consortium of non-rhizobial endophytic microbes isolated from a macrophyte Typha angustifolia growing in the marginal wetlands associated with a Uranium mine was characterized for their beneficial effect on rice and the mechanisms of growth promotion were investigated. The microbes were identified and characterized for their potential plant growth promoting (PGP) properties. Effect of these microbes on nitrogen (N)-metabolism of rice was tested as Typha endophytes were predominantly (N)-fixing. Relative N-use efficiency and expression of genes involved in N-uptake and assimilation were investigated in treated plants. Evidence of horizontal gene transfer (HGT) of dinitrogen reductase gene was observed within the consortium from a Pseudomonas stutzeri strain. The consortium behaved as plant probiotic and showed substantial growth benefits to Typha, their natural host as well as to rice. Typha endophytes colonized rice endosphere significantly increasing biomass, shoot length and chlorophyll content in rice plants both under N-sufficient and N-deficient conditions. N-uptake and assimilation genes were upregulated in plants treated with the endophytes even after three weeks post infection. Our results suggested, HGT of nitrogen-fixation trait to be highly prevalent among endophytes isolated from nutrient-poor habitats of the uranium mine. A long-term nitrogen deficiency response in the treated plants was elicited by the consortium improving N-uptake, assimilation and relative N-use efficiency of rice plants. This appeared to be at least one of the main strategies of plant growth promotion.

Root associated iron oxidizing bacteria increase phosphate nutrition and influence root to shoot partitioning of iron in tolerant plant Typha angustifolia

AimsTypha angustifolia is a heavy metal tolerant plant that grows in a uranium mine tailings highly contaminated with iron. In this study three iron oxidizing microbes (FeOBs) isolated from Typha rhizoplane were investigated for their role in plant growth promotion (PGP). Their effect on iron nutrition in Typha under iron replete and excess condition was also evaluated.MethodsThe PGP activities of the FeOBs were studied by measuring their influence on plant growth. To investigate the mechanism of growth promotion their ability to solubilize phosphate, and to produce Indole acetic acid and siderophores were studied. The influence of the FeOBs on root to shoot partitioning of iron was tested by measuring total iron content in roots and shoots treated with microbes.ResultsThe FeOBs were named as Paenibacillus cookii JGR8, (MTCC12002), Pseudomonas jaduguda JGR2 (LMG25820) and Bacillus megaterium JGR9 (MTCC12001). The siderophore producers, influenced iron accumulation in the plant root. Additionally P. pseudoalcaligenes JGR2 increased shoot iron content overcoming the root- shoot barrier that allows Typha to exclude metals from its shoot. Among the PGP mechanisms tested, ability to solubilize phosphate appeared to be most significant for increasing the plant biomass.ConclusionFeOBs that produce siderophore increased iron content in plant and therefore can be of immense biotechnological importance. However Biomass increase was directly correlated with increased phosphate acquisition and not with enhanced iron accumulation in Typha.

Protein Turnover in Response to Transient Exposure to Exogenous Auxin is Necessary for Restoring Auxin Autotrophy in a Stressed Arachis hypogea Cell Culture

An auxin autotrophic Arachis hypogea cell culture was sensitive to stress treatments leading to water loss whereas the growth of its auxin-supplemented counterpart was unaffected under similar conditions. Here we show that an hour of transient auxin treatment in the post stress period was sufficient for restoring the auxin autotrophic growth potential of the stress driven quiescent Arachis cells. Qualitative proteome analysis revealed protein turnover to have a role in mediating auxin-originated signals in these cells. In consonance, MG132 a cell permeable inhibitor of the ubiquitin mediated protein turnover completely inhibited the auxin dependent growth restoration of the stressed Arachis cells. Thus protein turnover is a necessary downstream event in exogenous auxin mediated stress tolerance in Arachis cells.

Exogenous auxin depletion renders an Arachis hypogea suspension culture sensitive to water loss without affecting cell growth

Abstract. A suspension cell culture was initiated from callus cells originating from Arachis hypogea cotyledon explants in the presence of 1-napthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Exogenous NAA depletion did not affect the growth of these cells, indicating them to be auxin-autotrophic in nature. Unlike the NAA-supplemented counterparts, the NAA-depleted cells were unable to grow after being exposed to stress conditions that led to the loss of water. The rate and amount of water loss from both the NAA-supplemented and the NAA-deprived cells were found to be the same, indicating that the presence of NAA did not enable stress avoidance. Our results suggest that NAA contributed tolerance to these cells by allowing them to recover after water loss.

Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea.

Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein–protein interaction.

Early changes in shoot transcriptome of rice in response to Rhodotorula mucilaginosa JGTA-S1.

Yeasts of Rhodotorula genus have been reported to show endophytic colonization in different plants. Some of the Rhodotorula species are found to exhibit plant growth promoting activities and also have been reported to protect plants against invading pathogens. A yeast strain closely related to Rhodotorula mucilaginosa was isolated from the endosphere of Typha angustifolia collected from a Uranium mine. A microarray analysis was performed to investigate the early changes in rice shoot transcripts in response to this yeast (R. mucilaginosa JGTA-S1). Transcriptional changes were monitored in 6 h and 24 h treated rice plant shoots as compared to 0 h control. The microarray data has been submitted to the NCBI GEO repository under the accession number of GSE64321.

An Endophytic Bacterial Consortium modulates multiple strategies to improve Arsenic Phytoremediation Efficacy in Solanum nigrum

Endophytic microbes isolated from plants growing in contaminated habitats possess specialized properties that help their host detoxify the contaminant/s. The possibility of using microbe-assisted phytoremediation for the clean-up of Arsenic (As) contaminated soils of the Ganga-Brahmaputra delta of India, was explored using As-tolerant endophytic microbes from an As-tolerant plant Lantana camara collected from the contaminated site and an intermediate As-accumulator plant Solanum nigrum. Endophytes from L. camara established within S. nigrum as a surrogate host. The microbes most effectively improved plant growth besides increasing bioaccumulation and root-to-shoot transport of As when applied as a consortium. Better phosphate nutrition, photosynthetic performance, and elevated glutathione levels were observed in consortium-treated plants particularly under As-stress. The consortium maintained heightened ROS levels in the plant without any deleterious effect and concomitantly boosted distinct antioxidant defense mechanisms in the shoot and root of As-treated plants. Increased consortium-mediated As(V) to As(III) conversion appeared to be a crucial step in As-detoxification/translocation. Four aquaporins were differentially regulated by the endophytes and/or As. The most interesting finding was the strong upregulation of an MRP transporter in the root by the As + endophytes, which suggested a major alteration of As-detoxification/accumulation pattern upon endophyte treatment that improved As-phytoremediation.