Ectopic expression of pigeonpea Orf147 gene imparts partial sterility in Cicer arietinum.

Orf147, a cytotoxic peptide, has been found to cause cytoplasmic male sterility (CMS) in Cajanus cajanifolius (pigeonpea). In our study, Orf147 was introduced into self-pollinating Cicer arietinum (chickpea) using Agrobacterium-mediated transformation for induction of CMS. The stable integration and expression of the transgene has been assessed through PCR and qRT-PCR analysis. In addition, phenotypic sterility analysis has been performed, considering developmental parameters like flower development, pod formation and flower drop. Transgene inheritance analysis demonstrates that out of the five PCR positive events in the T0 generation, two events have segregated according to the Mendelian segregation ratio (3:1) in the T2 generation. Further, pollen viability test using microscopic analysis confirms the induction of partial CMS in transgenic chickpea. The study holds significant value regarding the heterosis of self-pollinating legumes like chickpea. As a part of the prospect, exploring inducible promoters of species-specific or related legumes would be the next step to developing a two-line hybrid system.

Genome-wide identification and multiple abiotic stress transcript profiling of potassium transport gene homologs in Sorghum bicolor.

Potassium (K+) is the most abundant cation that plays a crucial role in various cellular processes in plants. Plants have developed an efficient mechanism for the acquisition of K+ when grown in K+ deficient or saline soils. A total of 47 K+ transport gene homologs (27 HAKs, 4 HKTs, 2 KEAs, 9 AKTs, 2 KATs, 2 TPCs, and 1 VDPC) have been identified in Sorghum bicolor. Of 47 homologs, 33 were identified as K+ transporters and the remaining 14 as K+ channels. Chromosome 2 has been found as the hotspot of K+ transporters with 9 genes. Phylogenetic analysis revealed the conservation of sorghum K+ transport genes akin to Oryza sativa. Analysis of regulatory elements indicates the key roles that K+ transport genes play under different biotic and abiotic stress conditions. Digital expression data of different developmental stages disclosed that expressions were higher in milk, flowering, and tillering stages. Expression levels of the genes SbHAK27 and SbKEA2 were higher during milk, SbHAK17, SbHAK11, SbHAK18, and SbHAK7 during flowering, SbHAK18, SbHAK10, and 23 other gene expressions were elevated during tillering inferring the important role that K+ transport genes play during plant growth and development. Differential transcript expression was observed in different tissues like root, stem, and leaf under abiotic stresses such as salt, drought, heat, and cold stresses. Collectively, the in-depth genome-wide analysis and differential transcript profiling of K+ transport genes elucidate their role in ion homeostasis and stress tolerance mechanisms.

RNA Pol III promoters-key players in precisely targeted plant genome editing.

The clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein Cas) system is a powerful and highly precise gene-editing tool in basic and applied research for crop improvement programs. CRISPR/Cas tool is being extensively used in plants to improve crop yield, quality, and nutritional value and make them tolerant to environmental stresses. CRISPR/Cas system consists of a Cas protein with DNA endonuclease activity and one CRISPR RNA transcript that is processed to form one or several short guide RNAs that direct Cas9 to the target DNA sequence. The expression levels of Cas proteins and gRNAs significantly influence the editing efficiency of CRISPR/Cas-mediated genome editing. This review focuses on insights into RNA Pol III promoters and their types that govern the expression levels of sgRNA in the CRISPR/Cas system. We discussed Pol III promoters structural and functional characteristics and their comparison with Pol II promoters. Further, the use of synthetic promoters to increase the targeting efficiency and overcome the structural, functional, and expressional limitations of RNA Pol III promoters has been discussed. Our review reports various studies that illustrate the use of endogenous U6/U3 promoters for improving editing efficiency in plants and the applicative approach of species-specific RNA pol III promoters for genome editing in model crops like Arabidopsis and tobacco, cereals, legumes, oilseed, and horticultural crops. We further highlight the significance of optimizing these species-specific promoters' systematic identification and validation for crop improvement and biotic and abiotic stress tolerance through CRISPR/Cas mediated genome editing.

Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)].

Finger millet (Eleusine coracana L.) is an annual herbaceous self-pollinating C4 cereal crop of the arid and semi-arid regions of the world. Finger millet is a food security crop proven to have resilience to changing climate and scores very high in nutrition. In the current study, we have assessed sixteen candidate reference genes for their appropriateness for the normalization studies in finger millet subjected to experimental regimes and treatments. Ten candidate reference genes (GAPDH, ß-TUB, CYP, EIF4α, TIP41, UBC, G6PD, S24, MACP and MDH) were cloned and six (ACT, ELF1α, PP2A, PT, S21 and TFIID) were mined from the NCBI database as well as from the literature. Expression stability ranking of the finger millet reference genes was validated using four different statistical tools i.e., geNorm, NormFinder, BestKeeper, ΔCt and RefFinder. From the study, we endorse MACP, CYP, EIF4α to be most stable candidate reference genes in all 'tissues', whereas PT, TFIID, MACP ranked high across genotypes, ß-TUB, CYP, ELF1α were found to be best under abiotic stresses and 'all samples set'. The study recommends using minimum of two reference genes for RT-qPCR data normalizations in finger millet. All in all, CYP, ß-TUB, and EF1α, in combination were found to be best for robust normalizations under most experimental conditions. The best and the least stable genes were validated for confirmation by assessing their appropriateness for normalization studies using EcNAC1 gene. The report provides the first comprehensive list of suitable stable candidate reference genes for nutritional rich cereal finger millet that will be advantageous to gene expression studies in this crop.

Genome-Wide Identification and Analysis of Arabidopsis Sodium Proton Antiporter (NHX) and Human Sodium Proton Exchanger (NHE) Homologs in Sorghum bicolor.

Na⁺ transporters play an important role during salt stress and development. The present study is aimed at genome-wide identification, in silico analysis of sodium-proton antiporter (NHX) and sodium-proton exchanger (NHE)-type transporters in Sorghum bicolor and their expression patterns under varied abiotic stress conditions. In Sorghum, seven NHX and nine NHE homologs were identified. Amiloride (a known inhibitor of Na⁺/H⁺ exchanger activity) binding motif was noticed in both types of the transporters. Chromosome 2 was found to be a hotspot region with five sodium transporters. Phylogenetic analysis inferred six ortholog and three paralog groups. To gain an insight into functional divergence of SbNHX/NHE transporters, real-time gene expression was performed under salt, drought, heat, and cold stresses in embryo, root, stem, and leaf tissues. Expression patterns revealed that both SbNHXs and SbNHEs are responsive either to single or multiple abiotic stresses. The predicted protein⁻protein interaction networks revealed that only SbNHX7 is involved in the calcineurin B-like proteins (CBL)- CBL interacting protein kinases (CIPK) pathway. The study provides insights into the functional divergence of SbNHX/NHE transporter genes with tissue specific expressions in Sorghum under different abiotic stress conditions.

Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut.

Eukaryotic translational initiation factor 4A belong to family of helicases, involved in multifunctional activities during stress and non-stress conditions. The eIF4A gene was isolated and cloned from semi-arid cereal crop of Pennisetum glaucum. In present study, the PgeIF4A gene was expressed under the regulation of stress inducible Arabidopsis rd29A promoter in groundnut (cv JL-24) with bar as a selectable marker. The de-embryonated cotyledons were infected with Agrobacterium tumefaciens (LBA4404) carrying rd29A:PgeIF4A construct and generated high frequency of multiple shoots in phosphinothricin medium. Twenty- four T0 plants showed integration of both nos-bar and rd29A-PgeIF4A gene cassettes in genome with expected amplification products of 429 and 654 bps, respectively. Transgene copy number integration was observed in five T0 transgenic plants through Southern blot analysis. Predicted Mendelian ratio of segregation (3:1) was noted in transgenic plants at T1 generation. The T2 homozygous lines (L1-5, L8-2, and L16-2) expressing PgeIF4A gene were exhibited superior growth performance with respect to phenotypic parameters like shoot length, tap root length, and lateral root formation under simulated drought and salinity stresses compared to the wild type. In addition, the chlorophyll retention was found to be higher in these plants compared to the control plants. The quantitative real time-PCR results confirmed higher expression of PgeIF4A gene in L1-5, L8-3, and L16-2 plants imposed with drought/salt stress. Further, the salt stress tolerance was associated with increase in oxidative stress markers, such as superoxide dismutase accumulation, reactive oxygen species scavenging, and membrane stability in transgenic plants. Taken together our results confirmed that the PgeIF4A gene expressing transgenic groundnut plants exhibited better adaptation to stress conditions.

Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization.

Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.