Synergistic Al-Al Dual-Atomic Site for Efficient Artificial Nitrogen Fixation.

Synthesis of ammonia by electrochemical nitrogen reduction reaction (NRR) is a promising alternative to the Haber-Bosch process. However, it is commonly obstructed by the high activation energy. Here, we report the design and synthesis of an Al-Al bonded dual atomic catalyst stabilized within an amorphous nitrogen-doped porous carbon matrix (Al2NC) with high NRR performance. The dual atomic Al2-sites act synergistically to catalyze the complex multiple steps of NRR through adsorption and activation, enhancing the proton-coupled electron transfer. This Al2NC catalyst exhibits a high Faradaic efficiency of 16.56±0.3 % with a yield rate of 29.22±1.2 µg h-1 mgcat -1. The dual atomic Al2NC catalyst shows long-term repeatable, and stable NRR performance. This work presents an insight into the identification of synergistic dual atomic catalytic site and mechanistic pathway for the electrochemical conversion of N2 to NH3.

Diagnosing arsenic-mediated biochemical responses in rice cultivars using Raman spectroscopy.

Rice (Oryza sativa) is the primary crop for nearly half of the world's population. Groundwater in many rice-growing parts of the world often has elevated levels of arsenite and arsenate. At the same time, rice can accumulate up to 20 times more arsenic compared to other staple crops. This places an enormous amount of people at risk of chronic arsenic poisoning. In this study, we investigated whether Raman spectroscopy (RS) could be used to diagnose arsenic toxicity in rice based on biochemical changes that were induced by arsenic accumulation. We modeled arsenite and arsenate stresses in four different rice cultivars grown in hydroponics over a nine-day window. Our results demonstrate that Raman spectra acquired from rice leaves, coupled with partial least squares-discriminant analysis, enabled accurate detection and identification of arsenic stress with approximately 89% accuracy. We also performed high-performance liquid chromatography (HPLC)-analysis of rice leaves to identify the key molecular analytes sensed by RS in confirming arsenic poisoning. We found that RS primarily detected a decrease in the concentration of lutein and an increase in the concentration of vanillic and ferulic acids due to the accumulation of arsenite and arsenate in rice. This showed that these molecules are detectable indicators of biochemical response to arsenic accumulation. Finally, a cross-correlation of RS with HPLC and ICP-MS demonstrated RS's potential for a label-free, non-invasive, and non-destructive quantification of arsenic accumulation in rice.

Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.

Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.

Increasing the level of resistant starch in 'Presidio' rice through multiplex CRISPR-Cas9 gene editing of starch branching enzyme genes.

Rice (Oryza sativa L.) is an excellent source of starch, which is composed of amylopectin and amylose. Resistant starch (RS) is a starch product that is not easily digestible and absorbed in the stomach or small intestine and instead is passed on directly to the large intestine. Cereals high in RS may be beneficial to improve human health and reduce the risk of diet-related chronic diseases. It has been reported through chemical mutagenesis and RNA interference studies that starch branching enzymes (SBEs) play a major role in contributing to higher levels of RS in cereal crops. In this study, we used multiplex clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) genome editing to simultaneously target all four SBE genes in rice using the endogenous transfer RNA (tRNA)-processing system for expressing the single-guide RNAs (sgRNAs) targeting these genes. The CRISPR-Cas9 vector construct with four SBE gene sgRNAs was transformed into the U.S. rice cultivar Presidio using Agrobacterium-mediated transformation. Knockout mutations were identified at all four SBE genes across eight transgene-positive T0 plants. Transgene-free T1 lines with different combinations of disrupted SBE genes were identified, with several SBE-edited lines showing significantly increased RS content up to 15% higher than the wild-type (WT) cultivar Presidio. Although further efforts are needed to fix all of the mutant alleles as homozygous, our study demonstrated the potential of multiplex genome editing to develop high-RS lines.

Salt tolerance QTLs of an endemic rice landrace, Horkuch at seedling and reproductive stages.

Salinity has a significant negative impact on production of rice. To cope with the increased soil salinity due to climate change, we need to develop salt tolerant rice varieties that can maintain their high yield. Rice landraces indigenous to coastal Bangladesh can be a great resource to study the genetic basis of salt adaptation. In this study, we implemented a QTL analysis framework with a reciprocal mapping population developed from a salt tolerant landrace Horkuch and a high yielding rice variety IR29. Our aim was to detect genetic loci that contributes to the salt adaptive responses of the two different developmental stages of rice which are very sensitive to salinity stress. We identified 14 QTLs for 9 traits and found that most are unique to specific developmental stages. In addition, we detected a significant effect of the cytoplasmic genome on the QTL model for some traits such as leaf total potassium and filled grain weight. This underscores the importance of considering cytoplasm-nuclear interaction for breeding programs. Finally, we identified QTLs co-localization for multiple traits that highlights the possible constraint of multiple QTL selection for breeding programs due to different contributions of a donor allele for different traits.

Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity.

Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a prime editing guide RNA (pegRNA). In addition, prime editing has a wider range of editing types than base editing and can produce nearly all types of edits. Although prime editing was first established in human cells, it has recently been applied to plants. As a relatively new technique, optimization will be needed to increase the editing efficiency in different crops. In this study, we successfully edited a mutant GFP in rice, peanut, chickpea, and cowpea protoplasts. In rice, up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors. Edited-mutant GFP protoplasts have also been obtained in peanut, chickpea, and cowpea after transformation with the dual pegRNA vectors, albeit with much lower editing efficiency than in rice, ranging from 0.2% to 0.5%. These initial results promise to expedite the application of prime editing in legume breeding programs to accelerate crop improvement.

Raman Spectroscopy Enables Non-invasive and Confirmatory Diagnostics of Aluminum and Iron Toxicities in Rice.

Metal toxicities can be detrimental to a plant health, as well as to the health of animals and humans that consume such plants. Metal content of plants can be analyzed using colorimetric, atomic absorption- or mass spectroscopy-based methods. However, these techniques are destructive, costly and laborious. In the current study, we investigate the potential of Raman spectroscopy (RS), a modern spectroscopic technique, for detection and identification of metal toxicities in rice. We modeled medium and high levels of iron and aluminum toxicities in hydroponically grown plants. Spectroscopic analyses of their leaves showed that both iron and aluminum toxicities can be detected and identified with ∼100% accuracy as early as day 2 after the stress initiation. We also showed that diagnostics accuracy was very high not only on early, but also on middle (day 4-day 8) and late (day 10-day 14) stages of the stress development. Importantly this approach only requires an acquisition time of 1 s; it is non-invasive and non-destructive to plants. Our findings suggest that if implemented in farming, RS can enable pre-symptomatic detection and identification of metallic toxins that would lead to faster recovery of crops and prevent further damage.

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement.

Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.

Morphologically Flex Sm-MOF Based Electrochemical Immunosensor for Ultrasensitive Detection of a Colon Cancer Biomarker.

Despite having the potential to synthesize stable metal-organic frameworks (MOFs), rare earth metal-based MOFs have not been exploited extensively. Owing to the high coordination numbers, the MOFs can generate a suitable coordination environment for various applications. Herein, samarium (Sm)-based MOFs were synthesized with three different organic linkers, namely, trimesic acid (TMA), meso-tetra(4-carboxyphenyl)porphine (TCPP), and 1,3,6,8-tetra(4-carboxylphenyl) pyrene(TBPy) by the solvothermal approach. The morphologies of Sm-TMA MOF, Sm-TCPP MOF, Sm-TBPy MOF were rod-shaped, cubic consisting of stacked 2D layers, and spherical made of small cubic structures, respectively. After the electrochemical properties of the synthesized MOFs were investigated, the MOFs were used to fabricate immunosensors for detection of carcinoembryonic antigen using a label-free signaling strategy. The immunosensors exhibited a wide linear detection range and a lower detection limit. The exhibited reproducibility and selectivity of the immunosensors were within the tolerable limits. The established label-free immunosensor has been successfully applied for detection of carcinoembryonic antigen in human serum samples, demonstrating that the rare earth metal-based MOFs are promising for construction of biosensors for medical diagnosis.

Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2.

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9.

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

Ultrasensitive Detection of Bacteria Using a 2D MOF Nanozyme-Amplified Electrochemical Detector.

Bacterial infection is one of the major causes of human death worldwide. To prevent bacterial infectious diseases from spreading, it is of critical importance to develop convenient, ultrasensitive, and cost-efficient methods for bacteria detection. Here, an electrochemical detector of a functional two-dimensional (2D) metal-organic framework (MOF) nanozyme was developed for the sensitive detection of pathogenic Staphylococcus aureus. A dual recognition strategy consisting of vancomycin and anti-S. aureus antibody was proposed to specifically anchor S. aureus. The 2D MOFs with excellent peroxidase-like activity can efficiently catalyze o-phenylenediamine to 2,2-diaminoazobenzene, which is an ideal electrochemical signal readout for monitoring the bacteria concentration. Under optimal conditions, the present bioassay provides a wide detection range of 10-7.5 × 107 colony-forming units CFU/mL with a detection limit of 6 CFU/mL, which is better than most of the previous reports. In addition, the established electrochemical sensor can selectively and accurately identify S. aureus in the presence of other bacteria. The present work provides a new pathway for sensitive and selective detection of S. aureus and presents a promising potential in the realm of clinical diagnosis.

Label-Free Electrochemical Immunosensor for Ultrasensitive Detection of Carbohydrate Antigen 125 Based on Antibody-Immobilized Biocompatible MOF-808/CNT.

In this work, a nanocomposite of Zr-trimesic acid MOF (MOF-808) with carbon nanotube (CNT) was synthesized through an in situ formation of MOF-808 on the activated CNT. The synthesized materials were characterized by powder X-ray diffraction, transmission electron microscopy, X-ray photoluminescence spectroscopy, Brunauer-Emmett-Teller, Fourier transform infrared spectroscopy, and Raman spectroscopy. The protein compatible nature with high surface area and electrocatalytic ability of MOF-808 was utilized to construct an immunosensor for ultra low-level detection of the ovarian cancer biomarker, carbohydrate antigen 125 (CA 125). The mutual benefit of each constituent of the MOF-808/CNT composite was capable of producing highly enhanced electrochemical properties. A glassy carbon electrode modified with MOF-808/CNT was used as a platform to fabricate a label-free electrochemical immunosensor. The antibody binding sites of MOF-808/CNT were enriched by functionalization with streptavidin. The immunosensor exhibited two linear determination ranges of 0.001-0.1 and 0.1-30 ng·mL-1, and the calculated limit of detection was 0.5 pg·mL-1 (S/N 3). The immunosensor showed excellent reproducibility and selectivity. The patient serum sample analysis was cross-verified with the electrochemiluminescence method with a relative error of 105-110%.

Raman Spectroscopy Enables Non-invasive and Confirmatory Diagnostics of Salinity Stresses, Nitrogen, Phosphorus, and Potassium Deficiencies in Rice.

Proper management of nutrients in agricultural systems is critically important for maximizing crop yields while simultaneously minimizing the health and environmental impacts of pollution from fertilizers. These goals can be achieved by timely confirmatory diagnostics of nutrient deficiencies in plants, which enable precise administration of fertilizers and other supplementation in fields. Traditionally, nutrient diagnostics are performed by wet-laboratory analyses, which are both time- and labor-consuming. Unmanned aerial vehicle (UAV) and satellite imaging have offered a non-invasive alternative. However, these imaging approaches do not have sufficient specificity, and they are only capable of detecting symptomatic stages of nutrient deficiencies. Raman spectroscopy (RS) is a non-invasive and non-destructive technique that can be used for confirmatory detection and identification of both biotic and abiotic stresses on plants. Herein, we show the use of a hand-held Raman spectrometer for highly accurate pre-symptomatic diagnostics of nitrogen, phosphorus, and potassium deficiencies in rice (Oryza sativa). Moreover, we demonstrate that RS can also be used for pre symptomatic diagnostics of medium and high salinity stresses. A Raman-based analysis is fast (1 s required for spectral acquisition), portable (measurements can be taken directly in the field), and label-free (no chemicals are needed). These advantages will allow RS to transform agricultural practices, enabling precision agriculture in the near future.

Polypyrrole merged zirconium-based metal-organic framework NU-1000 for detection of levodopa.

A post-synthetic integration of polypyrrole onto NU-1000 MOF (PPy@NU-1000) was done by pyrrole adsorption, followed by oxidative polymerization. The synthesized materials were characterized by XRD, SEM, BET, and FTIR. The ultra-high specific surface area with high-density catalytic sites of NU-1000 (2223 m2 g-1) was combined with the electrical conductivity of PPy (2-100 S cm-1). PPy@NU-1000 provides superior electrocatalytic activity and charge transfer properties compared to an individual component. The PPy@NU-1000-modified GCE was applied to detect the biomolecule Levodopa (LD). The DPV oxidation peak of LD was strongest at 272 ± 10 mV vs. Ag/AgCl reference electrode. Under the optimized experimental condition, the fabricated electrochemical sensor exhibited a wide quantification range of 0.005-70 µM with a sub-nanomolar detection limit of 0.0001 µM (S/N 3). The described sensor exhibits high sensitivity (2.08 µA µM-1 cm-2) with reasonable stability, reproducibility, and selectivity for the detection LD in the presence of potentially interfering compounds. Furthermore, human serum analysis showed excellent recovery values within the range 99.3-101.6%. Validation of the method was performed against HPLC.Graphical abstract.

Ultrasmall Au(0) Inserted Hollow PCN-222 MOF for The High-Sensitive Detection of Estradiol.

Here, the effect of morphology on the electrocatalytic activity of metal-organic framework (MOF) was investigated. Post synthetic gold (Au) insertion was done into hollow PCN-222 (HPCN-222) and solid PCN-222 (SPCN-222) frameworks by a simple hydrothermal method. The crystalline nature, chemical composition, and morphologies of the synthesized MOFs were characterized by PXRD, XPS, and TEM. Electrochemical characterizations were done by cyclic voltammetry and electrochemical impedance spectroscopy. The excellent electrocatalytic activity of highly small-sized Au(0) with the enhancement of electrical conductivity through a hopping mechanism combined with a hollow structure and high surface area in the HPCN-222 MOF hugely alters the electrochemical properties. Overall, a better electrocatalytic surface area, charge transfer coefficient, and catalytic activity were generated at the modified electrode. The hollow structure MOF showed better electrocatalytic activity than solid structured MOF. The AuHPCN-222 modified glassy carbon electrode (AuHPCN-222/GCE) electrochemical sensor was employed for the analytical analysis of estradiol (ED) in an optimum experimental condition. AuHPCN-222 showed selective and better electrocatalytic activity toward ED than GCE. The differential pulse voltammetric measurements by the fabricated sensor showed two linear determination ranges for ED; those are 0.01-1 and 1-220 µM. The measured limit of detection (m-LOD) was found to be 0.5 nM with the highest sensitivity of 2.2 µA µM cm-2. The analytical performances for the detection of ED in human urine and serum samples were done with reasonable recoveries (98%-103%) using the standard addition method. Also, the sensor showed better reproducibility and repeatability with stability.

Simultaneous voltammetric determination of Adrenaline and Tyrosine in real samples by neodymium oxide nanoparticles grafted graphene.

Nd2O3 nanoparticle grafted graphene nanocomposite (NOGG) was synthesized by sonochemical treatment of an ethanolic dispersion of Nd2O3 nanoparticle and graphene. All the synthesized materials were characterized by XRD, FESEM, TEM, and BET. The NOGG has a high specific surface area (272 m2g-1) with narrowly distributed pores with diameter centered at 2.8, 6.0 and 7.50 nm. A composite paste electrode of 1:1 (w/w) NOGG and graphite was showed better electrochemical properties. The NOGG/GP electrode showed all around better electrocatalytic activity towards Adrenaline (AD) and Tyrosine (TY) than TRG/GP and bare GP electrode. According to cyclic voltammograms, AD and TY oxidized irreversibly through adsorption control process. DPV peak currents were measured at 378 ±â€¯15 mV and 787 ±â€¯15 mV for determination AD and TY respectively using the NOGG/GP electrode as the peak intensities were highest at those potentials. Under the optimized experimental condition, the determination ranges for AD and TY showed two linear ranges, those were 0.1-5-130 µM and 0.1-3-120 µM, respectively. A detection limit of AD and TY was measured (m-LOD) to be 50 nM and 40 nM, respectively. The modified electrode was reproducible, selective, highly sensitive and also employed for analysis of AD and TY in biological and pharmaceutical samples with excellent recovery.

Overexpression of heterotrimeric G protein beta subunit gene (OsRGB1) confers both heat and salinity stress tolerance in rice.

Constitutive overexpression of the rice heterotrimeric G protein beta subunit gene (RGB1) in the commercial rice cultivar BRRI Dhan 55 resulted in improved tolerance to heat or salinity or their combination. Two independently in planta transformed plants with the gene confirmed to be integrated at T2 by Southern hybridization and showing high expression at the T3 seedling stage showed better physiological performance after 8 days in 120 mM salt stress than the wild type. The plants had significantly lower electrolyte leakage and malondialdehyde production, while showing higher levels of chlorophyll. Significantly higher germination at 48 °C or with combined stresses of 42/40 °C day/night stress in the presence of 120 mM salt for 2 days was also observed. Stress responsive genes such as OsAPX1, OsSOD, OsHKT1, OsHSP1, OsHSP2 and OsCOR47 showed higher expression in the RGB1 positive plants. These RGB1 transgenic plants can likely provide a strong defense against climate change.

Integrating polythiophene derivates to PCN-222(Fe) for electrocatalytic sensing of L-dopa.

Porphyrinic Metal-Organic Frameworks (porph-MOFs) are attracting attention due to the redox activity in the porphyrin subunit. Herein, we report the design of a novel core-shell structure hybrid material with a sea-cucumber morphology, namely PMeTh, containing the poly(3-methythiophene) conducting polymer coated on the surface of iron-based porph-MOFs PCN-222(Fe) via in-situ oxidative chemical polymerization. The porous PCN-222(Fe) serves as the electrocatalytic sites, while the poly(3-methythiophene) conducting polymer functions as the charge collector to facilitate the charge transport to the redox active sites. The resulting PMeTh composite demonstrates an excellent electrochemical response towards the levodopa detection. The sensitivity towards the L-dopa detection is estimated to be 1.868 µA ⋅ µM-1 ⋅ cm-2 in the linear concentration of 0.05-7.0 µmol ⋅ L-1 and 0.778 µA ⋅ µM-1 ⋅ cm-2 in the linear concentration of 7.00-100 µmol ⋅ L-1, respectively. Additionally, the levodopa sensor exhibits a low detection limit of 2 nmol L-1 as well as excellent stability after 120 cycles in 10 µmol L-1 levodopa. The feasibility of this novel L-dopa sensor was evaluated in human urine samples by standard addition. The satisfactory recoveries were in the range of 97.0-104.5% with the R.S.D. value lower than 4.4%. The method of intergrating porph-MOFs and conducting polymers can efficiently expand the porph-MOFs based composites in bioanalysis.

Gene Expression analysis associated with salt stress in a reciprocally crossed rice population.

The rice landrace Horkuch, endemic to the southern saline coast of Bangladesh, is known to have salt tolerance traits and can therefore contribute to a high yielding recipient for breeding purposes. In this study, we reciprocally crossed Horkuch with high yielding but salt sensitive IR29 to detect the complement of genes that were responsible for conferring salt tolerance versus sensitivity at the seedling developmental stage. We looked at tolerant and sensitive F3 families from individual F2 segregating plants and analyzed them for differential gene expressions using RNAseq. In general, we observed higher numbers of genes differentially expressed in leaves compared to root tissues. This included both upregulation and downregulation of gene expression across our experimental factors. Gene expression decreased in sensitive leaf after stress exposure where tolerant plants showed the opposite trend. In root, tolerant plants expression decreased at higher time points of stress exposure. We also observed a strong maternal cytoplasmic effect on gene expression and this was most evident in roots where there was upregulation in functional enrichments related to phosphorylation, electron carriers, transporter and cation transmembrane activities. Stress groups (tolerant and sensitive) response in F3 families were distinctive in both cytoplasmic backgrounds and involved uniquely upregulated genes in tolerant progenies including membrane sensor proteins, enzymes involved with signaling pathways, such as those producing trehalose and G-protein coupled receptor proteins, photosynthesis-related enzymes and golgi body recycling as well as prolamin precursor proteins involved in refolding of proteins. On the other hand, sensitivity was found to be associated with differential upregulation of only a few redox proteins and higher number of apoptosis related genes compared to the tolerant response. Overall, our highly replicated experimental design was powerful and allowed the detection of relatively subtle differential expression. Our future goal is to correlate these expression differences with QTLs in this population, which would help identify the relative importance of specific genetic loci and provide a direct avenue for combining higher levels of salt tolerance with better agronomic traits in rice.