Targeting OsNIP3;1 via CRISPR/Cas9: A strategy for minimizing arsenic accumulation and boosting rice resilience.

Arsenic (As) contamination in rice poses a significant threat to human health due to its toxicity and widespread consumption. Identifying and manipulating key genes governing As accumulation in rice is crucial for reducing this threat. The large NIP gene family of aquaporins in rice presents a promising target due to functional redundancy, potentially allowing for gene manipulation without compromising plant growth. This study aimed to utilize genome editing to generate knock-out (KO) lines of genes of NIP family (OsLsi1, OsNIP3;1) and an anion transporter family (OsLsi2), in order to assess their impact on As accumulation and stress tolerance in rice. KO lines were created using CRISPR/Cas9 technology, and the As accumulation patterns, physiological performance, and grain yield were compared against wild-type (WT) under As-treated conditions. KO lines exhibited significantly reduced As accumulation in grain compared to WT. Notably, Osnip3;1 KO line displayed reduced As in xylem sap (71-74%) and grain (32-46%) upon treatment. Additionally, these lines demonstrated improved silicon (23%) uptake, photosynthetic pigment concentrations (Chl a: 77%; Chl b: 79%, Total Chl: 79% & Carotenoid: 49%) overall physiological and agronomical performance under As stress compared to WT. This study successfully utilized genome editing for the first time to identify OsNIP3;1 as a potential target for manipulating As accumulation in rice without compromising grain yield or plant vigor.

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.].

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.

Comprehensive illustration of transcriptomic and proteomic dataset for mitigation of arsenic toxicity in rice (Oryza sativa L.) by microbial consortium.

The present article represents the data for analysis of microbial consortium (P.putida+C.vulgaris) mediated amelioration of arsenic toxicity in rice plant. In the current study the transcriptome profiling of treated rice root and shoot was performed by illumina sequencing (Platform 2000). To process the reads and to analyse differential gene expression, Fastxtoolkit, NGSQCtoolkit, Bowtie 2 (version 2.1.0), Tophat program (version 2.0.8), Cufflinks and Cuffdiff programs were used. For Proteome profiling, total soluble proteins in shoot of rice plant among different treatments were extracted and separated by 2D poly acrylamide gel electrophoresis (PAGE) and then proteins were identified with the help of MALDI-TOF/TOF. In gel based method of protein identification, the isoelectric focusing machine (IPGphor system,Bio-Rad USA), gel unit (SDS-PAGE) and MALDI-TOF/TOF (4800 proteomic analyzer Applied Biosystem, USA) were used for successful separation and positive identification of proteins. To check the differential abundance of proteins among different treatments, PDQuest software was used for data analysis. For protein identification, Mascot search engine (http://www.matrixscience.com) using NCBIprot/SwissProt databases of rice was used. The analyzed data inferred comprehensive picture of key genes and their respective proteins involved in microbial consortium mediated improved plant growth and amelioration of As induced phyto-toxicity in rice. For the more comprehensive information of data, the related full-length article entitled "Microbial consortium mediated growth promotion and Arsenic reduction in Rice: An integrated transcriptome and proteome profiling" may be accessed.

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.

Root system architecture, physiological analysis and dynamic transcriptomics unravel the drought-responsive traits in rice genotypes.

Drought is the major abiotic factors that limit crop productivity worldwide. To withstand stress conditions, plants alter numerous mechanisms for adaption and tolerance. Therefore, in the present study, 106 rice varieties were screened for drought tolerance phenotype via exposing different concentrations of polyethylene glycol 6000 (PEG) in the hydroponic nutrient medium at the time interval of 1, 3, and 7 days to evaluate the changes in their root system architecture. Further, based on root phenotype obtained after PEG-induced drought, two contrasting varieties drought-tolerant Heena and -sensitive Kiran were selected to study transcriptional and physiological alterations at the same stress durations. Physiological parameters (photosynthesis rate, stomatal conductance, transpiration), and non-enzymatic antioxidants (carotenoids, anthocyanins, total phenol content) production indicated better performance of Heena than Kiran. Comparatively higher accumulation of carotenoid and anthocyanin content and the increased photosynthetic rate was also observed in Heena. Root morphology (length, numbers of root hairs, seminal roots and adventitious roots) and anatomical data (lignin deposition, xylem area) enable tolerant variety Heena to better maintain membrane integrity and relative water content, which also contribute to comparatively higher biomass accumulation in Heena under drought. In transcriptome profiling, significant drought stress-associated differentially expressed genes (DEGs) were identified in both the varieties. A total of 1033 and 936 uniquely upregulated DEGs were found in Heena and Kiran respectively. The significant modulation of DEGs that were mainly associated with phytohormone signaling, stress-responsive genes (LEA, DREB), transcription factors (TFs) (AP2/ERF, MYB, WRKY, bHLH), and genes involved in photosynthesis and antioxidative mechanisms indicate better adaptive nature of Heena in stress tolerance. Additionally, the QTL-mapping analysis showed a very high number of DEGs associated with drought stress at AQHP069 QTL in Heena in comparison to Kiran which further distinguishes the drought-responsive traits at the chromosomal level in both the contrasting varieties. Overall, results support the higher capability of Heena over Kiran variety to induce numerous genes along with the development of better root architecture to endure drought stress.

Over-expression of rice R1-type MYB transcription factor confers different abiotic stress tolerance in transgenic Arabidopsis.

Among various abiotic stresses, water deficit hit the first in the list followed by heavy metal stresses as a serious environmental growth-limiting factor that restricts the global crop yield. Molecular approaches will help us to trace key regulators which are involved in stress-related phenomenon to enhance crop productivity. The present study functionally characterized one of the key regulators, OsMYB-R1 in Arabidopsis. Phylogenetic analyses indicated that OsMYB-R1 had a close relationship with Sorghum bicolour and Zea mays. Ectopic expression of OsMYB-R1 in Arabidopsis resulted in improved tolerance to PEG/drought and chromium stress in addition to conferring no tolerance to salinity stress. Further RNA seq. data revealed that OsMYB-R1 regulates the expression of key genes that improve the root architecture and maintain the cellular homeostasis of transgenic lines through an efficient anti-oxidant system. It also reveals the differential gene expression of stress-responsive and hormone-responsive genes, which indicate the intricate network of defense regulatory machinery activated in transgenic lines. Additionally, salicylic acid (SA) plays a significant role in promoting the growth of the OsMYB-R1 over-expressing plants and increased GUS intensity in SA treated OsMYB-R1 promoter plants demonstrate the explicit role of SA signaling in overcoming stress tolerance. Whereas no significant change was observed in OsMYB-R1 over-expressing plants after ABA and MeJA treatment. Overall, OsMYB-R1 is a promising gene resource for improving abiotic stress tolerance in other crops, especially in dicotyledon plants.

Auxin-salicylic acid cross-talk ameliorates OsMYB-R1 mediated defense towards heavy metal, drought and fungal stress.

The MYB TF family is an immensely large and functionally diverse class of proteins involved in the regulation of cell cycle, cell morphogenesis to stress signaling mechanism. The present study deciphered the hormonal cross-talk of wound inducible and stress-responsive OsMYB-R1 transcription factor in combating abiotic [Cr(VI) and drought/PEG] as well as biotic (Rhizoctonia solani) stress. OsMYB-R1 over-expressing rice transgenics exhibit a significant increase in lateral roots, which may be associated with increased tolerance under Cr(VI) and drought exposure. In contrast, its loss-of-function reduces stress tolerance. Higher auxin accumulation in the OsMYB-R1 over-expressed lines further strengthens the protective role of lateral roots under stress conditions. RNA-seq. data reveals over-representation of salicylic acid signaling molecule calcium-dependent protein kinases, which probably activate the stress-responsive downstream genes (Peroxidases, Glutathione S-transferases, Osmotins, Heat Shock Proteins, Pathogenesis Related-Proteins). Enzymatic studies further confirm OsMYB-R1 mediated robust antioxidant system as catalase, guaiacol peroxidase and superoxide dismutase activities were found to be increased in the over-expressed lines. Our results suggest that OsMYB-R1 is part of a complex network of transcription factors controlling the cross-talk of auxin and salicylic acid signaling and other genes in response to multiple stresses by modifying molecular signaling, internal cellular homeostasis and root morphology.

Transcriptome and proteome analyses reveal selenium mediated amelioration of arsenic toxicity in rice (Oryza sativa L.).

Arsenic (As), a chronic poison and non-threshold carcinogen, is a food chain contaminant in rice, posing yield losses as well as serious health risks. Selenium (Se), a trace element, is a known antagonist of As toxicity. In present study, RNA seq. and proteome profiling, along with morphological analyses were performed to explore molecular cross-talk involved in Se mediated As stress amelioration. The repair of As induced structural deformities involving disintegration of cell wall and membranes were observed upon Se supplementation. The expression of As transporter genes viz., NIP1;1, NIP2;1, ABCG5, NRAMP1, NRAMP5, TIP2;2 as well as sulfate transporters, SULTR3;1 and SULTR3;6, were higher in As + Se compared to As alone exposure, which resulted in reduced As accumulation and toxicity. The higher expression of regulatory elements like AUX/IAA, WRKY and MYB TFs during As + Se exposure was also observed. The up-regulation of GST, PRX and GRX during As + Se exposure confirmed the amelioration of As induced oxidative stress. The abundance of proteins involved in photosynthesis, energy metabolism, transport, signaling and ROS homeostasis were found higher in As + Se than in As alone exposure. Overall, present study identified Se responsive pathways, genes and proteins involved to cope-up with As toxicity in rice.

Nitric oxide mediated transcriptional modulation enhances plant adaptive responses to arsenic stress.

Arsenic (As) contamination in rice leads to yield decline and causes carcinogenic risk to human health. Although the role of nitric oxide (NO) in reducing As toxicity is known, NO-mediated genetic modulation in the plant during arsenic toxicity has not yet been established. We analyzed the key components of NO metabolism and the correlations between NO interaction and arsenic stress using rice as a relevant model plant. Illumina sequencing was used to investigate the NO-mediated genome-wide temporal transcriptomic modulation in rice root upon AsIII exposure during 12 days (d) of the growth period. Sodium nitroprusside (SNP) was used as NO donor. SNP supplementation resulted in marked decrease in ROS, cell death and As accumulation during AsIII stress. NO was found to modulate metal transporters particularly NIP, NRAMP, ABC and iron transporters, stress related genes such as CytP450, GSTs, GRXs, TFs, amino acid, hormone(s), signaling and secondary metabolism genes involved in As detoxification. We detected NO-mediated change in jasmonic acid (JA) content during AsIII stress. The study infers that NO reduces AsIII toxicity through modulating regulatory networks involved in As detoxification and JA biosynthesis.

Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies.

Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells.

De novo assembly and characterization of root transcriptome in two distinct morphotypes of vetiver, Chrysopogon zizaniodes (L.) Roberty.

Vetiver, a perennial C4 grass, has long been known for its multifarious uses in perfumery, medicine and environmental protection. Two distinct vetiver morphotypes have been identified in India, i.e., A. North Indian type characterized by thick and smooth fast growing roots that produce superior quality of laevorotatory oil; and B. South Indian type with more number of thin and hairy roots that produce inferior quality of dextrorotatory oil. The two morphotypes were targeted for transcriptome analysis to understand the contribution of genetic background on oil quality and root morphology. Sample A showed enhanced activity of flavonoid and terpenoid biosynthesis related genes, i.e. ERF, MYB, bHLH, bZIP and WRKY. Interestingly, expression analysis revealed that the genes involved in sesquiterpene biosynthesis pathway were up regulated in Sample A. Moreover, some of the genes involved in mevalonate pathway of sesquiterpene biosynthesis were unique to Sample A. Our results also demonstrated several transcripts involved in root development and hormonal regulation being up regulated in Sample A. To validate gene expression results of RNA-seq data, 20 transcripts were validated by qRT-PCR experiment. The present study provided an important start point for further discovery of genes related to root oil quality in different ecotypes of vetiver.

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.