Correction to: Identification and validation of reference genes in vetiver (Chrysopogon zizanioides) root transcriptome.

[This corrects the article DOI: 10.1007/s12298-023-01315-7.].

Functional characterization of rice metallothionein OsMT-I-Id: Insights into metal binding and heavy metal tolerance mechanisms.

Metallothioneins (MTs) are cysteine-rich proteins known for their strong metal-binding capabilities, making them effective in detoxifying heavy metals (HMs). This study focuses on characterizing the functional properties of OsMT-I-Id, a type-I Metallothionein found in rice. Using a HM-responsive yeast cup1Δ (DTY4), ycf1∆ (for cadmium), and acr3∆ mutants (for trivalent arsenic), we assessed the impact of OsMT-I-Id on metal accumulation and cellular resilience. Our results demonstrated that yeast cells expressing OsMT-I-Id showed increased tolerance and accumulated higher levels of copper (Cu), arsenic (As), and cadmium (Cd), compared to control cells. This can be attributed to the protein's ability to chelate and bind HMs. Site-directed mutagenesis was employed to investigate the specific contributions of cysteine residues. The study revealed that yeast cells with a mutated C-domain displayed heightened HM sensitivity, while cells with a mutated N-domain exhibited reduced sensitivity. This underscores the critical role of C-cysteine-rich domains in metal binding and tolerance of type-I rice MTs. Furthermore, the study identified the significance of the 12th cysteine position at the N-domain and the 68th and 72nd cysteine positions at the C-domain in influencing OsMT-I-Id metal-binding capacity. This research provides novel insights into the structure-function relationship and metal binding properties of type-I plant MTs.

Identification and validation of reference genes in vetiver (Chrysopogon zizanioides) root transcriptome.

Vetiver [Vetiveria zizanioides (L.) Roberty] is a perennial C-4 grass traditionally valued for its aromatic roots/root essential oil. Owing to its deep penetrating web-forming roots, the grass is now widely used across the globe for phytoremediation and the conservation of soil and water. This study has used the transcriptome data of vetiver roots in its two distinct geographic morphotypes (North Indian type A and South Indian type B) for reference gene(s) identification. Further, validation of reference genes using various abiotic stresses such as heat, cold, salt, and drought was carried out. The de novo assembly based on differential genes analysis gave 1,36,824 genes (PRJNA292937). Statistical tests like RefFinder, NormFinder, BestKeeper, geNorm, and Delta-Ct software were applied on 346 selected contigs. Eleven selected genes viz., GAPs, UBE2W, RP, OSCam2, MUB, RPS, Core histone 1, Core histone 2, SAMS, GRCWSP, PLDCP along with Actin were used for qRT-PCR analysis. Finally, the study identified the five best reference genes GAPs, OsCam2, MUB, Core histone 1, and SAMS along with Actin. The two optimal reference genes SAMS and Core histone 1 were identified with the help of qbase + software. The findings of the present analyses have value in the identification of suitable reference gene(s) in transcriptomic and molecular data analysis concerning various phenotypes related to abiotic stress and developmental aspects, as well as a quality control measure in gene expression experiments. Identifying reference genes in vetiver appears important as it allows for accurate normalization of gene expression data in qRT-PCR experiments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01315-7.

Agrobacterium-mediated gene transfer: recent advancements and layered immunity in plants.

MAIN CONCLUSION: Plant responds to Agrobacterium via three-layered immunity that determines its susceptibility or resistance to Agrobacterium infection. Agrobacterium tumefaciens is a soil-borne Gram-negative bacterium that causes crown gall disease in plants. The remarkable feat of interkingdom gene transfer has been extensively utilised in plant biotechnology to transform plant as well as non-host systems. In the past two decades, the molecular mode of the pathogenesis of A. tumefaciens has been extensively studied. Agrobacterium has also been utilised as a premier model to understand the defence response of plants during plant-Agrobacterium interaction. Nonetheless, the threat of Agrobacterium-mediated crown gall disease persists and is associated with a huge loss of plant vigour in agriculture. Understanding the molecular dialogues between these two interkingdom species might provide a cure for crown gall disease. Plants respond to A. tumefaciens by mounting a three-layered immune response, which is manipulated by Agrobacterium via its virulence effector proteins. Comparative studies on plant defence proteins versus the counter-defence of Agrobacterium have shed light on plant susceptibility and tolerance. It is possible to manipulate a plant's immune system to overcome the crown gall disease and increase its competence via A. tumefaciens-mediated transformation. This review summarises the recent advances in the molecular mode of Agrobacterium pathogenesis as well as the three-layered immune response of plants against Agrobacterium infection.

A tau class GST, OsGSTU5, interacts with VirE2 and modulates the Agrobacterium-mediated transformation in rice.

KEY MESSAGE: OsGSTU5 interacts and glutathionylates the VirE2 protein of Agrobacterium and its (OsGSTU5) overexpression and downregulation showed a low and high AMT efficiency in rice, respectively. During Agrobacterium-mediated transformation (AMT), T-DNA along with several virulence proteins such as VirD2, VirE2, VirE3, VirD5, and VirF enter the plant cytoplasm. VirE2 serves as a single-stranded DNA binding (SSB) protein that assists the cytoplasmic trafficking of T-DNA inside the host cell. Though the regulatory roles of VirE2 have been established, the cellular reaction of their host, especially in monocots, has not been characterized in detail. This study identified a cellular interactor of VirE2 from the cDNA library of rice. The identified plant protein encoded by the gene cloned from rice was designated OsGSTU5, it interacted specifically with VirE2 in the host cytoplasm. OsGSTU5 was upregulated during Agrobacterium infection and involved in the post-translational glutathionylation of VirE2 (gVirE2). Interestingly, the in silico analysis showed that the 'gVirE2 + ssDNA' complex was structurally less stable than the 'VirE2 + ssDNA' complex. The gel shift assay also confirmed the attenuated SSB property of gVirE2 over VirE2. Moreover, knock-down and overexpression of OsGSTU5 in rice showed increased and decreased T-DNA expression, respectively after Agrobacterium infection. The present finding establishes the role of OsGSTU5 as an important target for modulation of AMT efficiency in rice.

A tau class glutathione-S-transferase (OsGSTU5) confers tolerance against arsenic toxicity in rice by accumulating more arsenic in root.

Arsenic (As) considered as one of the hazardous metalloid that hampers various physiological activities in rice. To study the mechanism of As tolerance in rice, one differentially expressed tau class glutathione-S-transferase (OsGSTU5) has been selected and transgenic rice plants with knockdown (KD) and overexpressing (OE) OsGSTU5 were generated. Our results suggested that KD lines became less tolerant to As stress than WT plants, while OE lines showed enhanced tolerance to As. Under As toxicity, OE and KD lines showed enhanced and reduced antioxidant activities such as, SOD, PRX and catalase, respectively indicating its role in ROS homeostasis. In addition, higher malondialdehyde content, poor photosynthetic parameters and higher reactive oxygen species (ROS) in KD plant, suggests that knockdown of OsGSTU5 renders KD plants more susceptible to oxidative damage. Also, the relative expression profile of various transporters such as OsABCC1 (As sequestration), Lsi2 and Lsi6 (As translocaters) and GSH dependent activity of GSTU5 suggests that GSTU5 might help in chelation of As with GSH and sequester it into the root vacuole using OsABCC1 transporter and thus limits the upward translocation of As towards shoot. This study suggests the importance of GSTU5 as a good target to improve the As tolerance in rice.

Functional characterization of tau class glutathione-S-transferase in rice to provide tolerance against sheath blight disease.

Glutathione-S-transferase (GST) is an important defense gene that confers resistance against several abiotic and biotic stresses. The present study identifies a tau class GST in rice (Oryza sativa L.), OsGSTU5 (Os09g20220), which provided tolerance against sheath blight (SB) disease, caused by a necrotrophic fungus, Rhizoctonia solani (RS). Overexpression and knockdown rice transgenic lines of OsGSTU5 were generated and tested for the severity of infection during sheath blight disease. The results obtained after RS infection showed that the lesion cover area and hyphal penetration were more in knockdown line and lesser in the overexpression line. Analysis of reactive oxygen species (ROS) accumulation showed more spots of H2O2 and O2- in knockdown lines compared to overexpressed lines. Later, RS transcript level was analyzed in RS-infected transgenic lines, which manifested that the knockdown line had higher RS transcripts in comparison to the control line and least RS transcripts were observed in the overexpressed line. In conclusion, rice transgenic lines overexpressing OsGSTU5 were found to be more tolerant, while the knockdown lines were more prone to Rhizoctonia infection compared to control lines.

Nanonetwork of Coordination Polymer AHMT-Ag for the Effective and Broad Spectrum Detection of 6-Mercaptopurine in Urine and Blood Serum.

Nanocrystalline coordination polymers (NCCPs) have been considered as an incredible electrochemical sensor for the effective detection of biologically dynamic drug 6-mercaptopurine (6-MP). In the present report, a significantly stable infinite arrayed coordination polymeric network was self-assembled via metal with efficient organic tecton 4-amino-3-hydrazino-5-mercapto-1,2,4,-triazole (AHMT) in which silver(I) ions are coordinated by AHMT via hydrazino and exocyclic thiol linkage to form AHMT-Ag NCCP. An efficient and highly sensitive detection of 6-MP is attained owing to eminent electron channeling via polymeric nanocrystallite pores. An effective charge transfer leads to an interface of the AHMT-Ag nano-pores and electrolyte anchored electrode via π-π electron coupling and hydrophobic interaction. The voltammogram exposes acute redox behavior of 6-MP and discloses an impeccable illustration for the AHMT-Ag facilitated oxidation of 6-MP. This unique signature was applied in voltammetric detection of 6-MP in blood serum, human urine, and pharmaceutical formulation (tablet) by a considerable high sensitivity of 0.074, 0.058, and 0.036 µA/µM and a detection limit of 87, 97, and 37 nM, respectively. Thus, the prepared AHMT-Ag NCCP can provide a valuable platform for fabrication of highly sensitive electrochemical devices to assay biologically essential drug molecules.

Oryza sativa class III peroxidase (OsPRX38) overexpression in Arabidopsis thaliana reduces arsenic accumulation due to apoplastic lignification.

ClassIII peroxidases are multigene family of plant-specific peroxidase enzyme. They are involved in various physiological and developmental processes like auxin catabolism, cell metabolism, various biotic, abiotic stresses and cell elongation. In the present study, we identified a class III peroxidase (OsPRX38) from rice which is upregulated several folds in both arsenate (AsV) and arsenite (AsIII) stresses. The overexpression of OsPRX38 in Arabidopsis thaliana significantly enhances Arsenic (As) tolerance by increasing SOD, PRX GST activity and exhibited low H2O2, electrolyte leakage and malondialdehyde content. OsPRX38 overexpression also affect the plant growth by increasing total biomass and seeds production in transgenics than WT under As stress condition. Confocal microscopy revealed that the OsPRX38-YFP fusion protein was localized to the apoplast of the onion epidermal cells. In addition, lignification was positively correlated with an increase in cell-wall-associated peroxidase activities in transgenic plants. This study indicates the role of OsPRX38 in lignin biosynthesis, where lignin act as an apoplastic barrier for As entry in root cells leading to reduction of As accumulation in transgenic. Overall the study suggests that overexpression of OsPRX38 in Arabidopsis thaliana activates the signaling network of different antioxidant systems under As stress condition, enhancing the plant tolerance by reducing As accumulation due to high lignification.

A nanoporous palladium(II) bridged coordination polymer acting as a peroxidase mimic in a method for visual detection of glucose in tear and saliva.

A nanoporous coordination polymer (NPCP) was prepared from palladium(II) chloride and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole and is shown to act as a peroxidase mimetic. It can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 which is formed on enzymatic oxidation of glucose by glucose oxidase. Based on these findings, a sensitive glucose test was worked out at 652 nm where the intensity if the greenish-blue product is related to the actual concentration of glucose. Figures of merit include (a) rather low Km value (30 µM) which evidences the strong binding affinity of the NPCP toward glucose, (b) a high v(max) (8.5 M·s-1), (c) a 47 nM detection limit, (d) a lifetime of a month, (e) a wide working pH range (2-10), and (f) a 25-80 °C temperature range. The assay was applied to non-invasive determination of glucose assay in tear, saliva where the detection limits are found to be 61 and 91 nM, respectively. Graphical abstract DSchematic of the mechanism of the peroxidase like catalytic activity of AHMT-Pd NPCP that was applied in a selective colorimetric method for glucose detection based on TMB oxidation in the presence of enzymatically generated H2O2.

Donor-π-Acceptor-Type Configured, Dimethylamino-Based Organic Push-Pull Chromophores for Effective Reduction of Mild Steel Corrosion Loss in 1 M HCl.

In this work, donor-π-acceptor-type four crystalline compounds have been tested for the first time to restrict the corrosion of mild steel in 1 M HCl. The details of the compounds are: C1, 4-N,N-dimethylamino-ß-nitrostyrene; C2, 2-(4-(dimethylamino) benzylidene)malononitrile; C3, ethyl 2-cyano-3-(4-(dimethylamino) phenyl)acrylate; and C4, methyl 2-cyano-3-(4-(dimethylamino)phenyl)acrylate. The corrosion inhibition potentials of the compounds have been primarily investigated by electrochemical techniques, such as linear polarization resistance, Tafel polarization curves, and electrochemical impedance spectroscopy. The secondary investigation is performed by scanning electron microscopy, fluorescence surface imaging, spectroscopic techniques (UV-visible and Fourier transform infrared spectroscopy), and X-ray diffraction patterns. The results disclosed that 50 mg L-1 of the compounds (1-4) in 1 M HCl provided the maximum inhibition efficiency as 93% (1), 88% (2), 82% (3), and 86% (4). The function of the compounds as corrosion inhibitors is explained with equilibrium corrosion potential, adsorption isotherms, and the frontier molecular orbital energies of the compounds (E HOMO and E LUMO) estimated by cyclic voltammetry curves and UV-visible spectra.

The nanocrystalline coordination polymer of AMT-Ag for an effective detection of ciprofloxacin hydrochloride in pharmaceutical formulation and biological fluid.

The present report highlights a cost effective and portable AMT-Ag nanocrystalline coordination polymer (NCCP) based electrochemical sensor for an efficient sensing of biologically active drug molecule ciprofloxacin hydrochloride (CFX). The AMT-Ag NCCP, is synthesized using an easily accessible organic ligand 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) with silver nitrate. In the infinite polymer array of AMT-Ag, silver (I) centers are bridged by tecton AMT through the exocyclic thiol and amino linkage. A successful ultra‒trace detection of CFX has been achieved due to the prominent electron channeling through the pores of polymeric nano-crystallites. The efficient charge transfer arises at the interface of electrolyte and AMT-Ag nano-crystals anchored electrode through hydrophobic interaction and π-π electron coupling. The voltammogram reveals the critical redox features of CFX and provides a clear representation about the steps involved in the AMT-Ag assisted oxidation of CFX. This specific signature further applied in the voltammetric assay of CFX in pharmaceutical formulation (eye drops) and biological fluid (urine) by a significantly high sensitivity (0.002µA/µM and 0.007µA/µM) and detection limit (22nM and 60nM) respectively without any interference. Therefore, the developed AMT-Ag NCCP could serve as a highly valuable platform for the fabrication of high-performance electrochemical sensors for the detection of biologically important drug molecules.

Transformed yeast (Schizosaccharomyces pombe) overexpressing rice Tau class glutathione S-transferase (OsGSTU30 and OsGSTU41) shows enhanced resistance to hexavalent chromium.

Extensive use of hexavalent chromium [Cr(VI)] in leather tanning, stainless-steel production, wood preservatives and electroplating industries has resulted in widespread environmental pollution and poses a serious threat to human health. A plant's response to Cr(VI) stress results in growth inhibition and toxicity leading to changes in components of antioxidant systems. In a previous study, we observed that a large number of glutathione S-transferase (GST) genes were up-regulated under Cr(VI) stress in rice. In this study, two rice root-specific Tau class GST genes (OsGSTU30 and OsGSTU41) were introduced into yeast (Schizosaccharomyces pombe). Transformed yeast cells overexpressing OsGSTU30 and OsGSTU41 had normal growth, but had much higher levels of GST activities and showed enhanced resistance to Cr(VI) as compared to control cells (transformed with empty vector). Also, a higher accumulation of chromium was found in the transformed yeast cells as compared to the control cells. Manipulation of glutathione biosynthesis by exogenous application of buthionine sulfoximine abolishes the protective effect of OsGSTs against Cr(VI) stress. These results suggest that Tau class OsGSTs play a significant role in detoxification of Cr(VI), probably by chelating and sequestrating glutathione-Cr(VI) complexes into vacuoles.

Gold nanoparticles incorporated 3-(trimethoxysilyl)propyl methacrylate modified electrode for non-enzymatic electro-sensing of urea.

We describe non-enzymatic electrochemical sensor for urea based on the electrocatalytic activity of gold nanoparticle (AuNPs) incorporated in 3-(Trimethoxysilyl)propyl methacrylate (TMSPMA) network. AuNPs incorporated in TMSPMA network on poly crystalline gold electrode show excellent electrocatalytic activity towards urea sensing. Uniform distribution and interaction of Au nanoparticles in nanospace of TMSPMA network are probably the key factors for catalytic effect of the nanocomposite. Nanocomposite is characterized by UV-visible diffuse reflectance spectroscopy, cyclic voltammetry, field emission scanning electron microscopy and X-ray photoelectron spectroscopy before using as urea sensor. The sensing platform is found to be highly sensitive and shows linear response in wide range for urea concentration with a sensitivity of 1.13 microA/microM and limit of detection as 2.08 microM respectively at S/N (signal-to-noise ratio): 3. It is also observed that the sensor does not suffer interference from phosphate and sulphate ions. Further, a possible mechanism for electro-sensing of urea with modified electrode has also been proposed.