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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.

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, β-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, β-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, β-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.

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.

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.

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.

Genetically engineered plants have varied applications in agriculture for enhancing the values of food and feed. Genetic engineering aims to introduce selected genetic regions with desirable traits into target plants for both spatial and temporal expressions. Promoters are the key elements responsible for regulating gene expressions by modulating the transcription factors (TFs) through recognition of RNA polymerases. Based on their recognition and expression, RNA polymerases were categorized into RNA pol II and pol III promoters. Promoter activity and specificity are the two prime parameters in regulating the transgene expression. Since the use of constitutive promoters like Cauliflower mosaic virus (CaMV) 35S may lead to adverse effects on non-target organisms or ecosystem, inducible/tissue specific promoters and/or the RNA pol III promoters provide myriad opportunities for gene expressions with controlled regulation and with minimum adverse effects. Besides their role in transgene expression, their influence in synthetic biology and genome editing are also discussed. This review provides an update on the importance, current prospects, and insight into the advantages and disadvantages of promoters reported thus far would help to utilize them in the endeavour to develop nutritionally and agronomically improved transgenic crops for commercialization.

Water-saving traits are critical for the adaptation of pearl millet ( Pennisetum glaucum ) to arid environments. This study investigates how breeding history and specifically selection in High Rainfall (HR) versus Low Rainfall (LR) zones influences root hydraulics and the molecular regulation of aquaporins (AQP). The study utilized four contrasting hybrids to integrate physiological assays with anatomical and molecular phenotyping. Key methodologies included measuring transpiration responses to vapor pressure deficit (VPD), root pressurization, and pharmacological inhibition. These were coupled with anatomical characterization of root systems, aerial growth and transcriptional profiling of PIP and TIP (Plasma membrane/Tonoplast Intrinsic Protein) genes. LR hybrids exhibited an \"opportunistic\" strategy characterized by superior root and canopy growth supported by extensive metaxylem proliferation. However, these genotypes showed lower constitutive root hydraulic conductivity and a steeper transpiration increase during pressurization, suggesting a radial transport bottleneck. At the molecular level, HR hybrids significantly downregulated PIP2;3 in roots under high VPD. In contrast, LR hybrids maintained PIP2;3 expression, facilitating radial flow to match their high transpiration demand. The findings demonstrate that breeding for specific rainfall zones has shaped divergent hydraulic strategies. HR genotypes utilize a conservative \"hydraulic brake\" mechanism driven by AQP downregulation to conserve water. Conversely, LR genotypes employ an acquisitive strategy, relying on coordinated anatomical capacity and molecular gating to meet high transpiration demands. These distinct mechanisms provide a blueprint for selecting resilient crops based on specific environmental pressures.

Late embryogenesis abundant (LEA) proteins, the space fillers or molecular shields, are the hydrophilic protective proteins which play an important role during plant development and abiotic stress. The systematic survey and characterization revealed a total of 68 LEA genes, belonging to 8 families in Sorghum bicolor. The LEA-2, a typical hydrophobic family is the most abundant family. All of them are evenly distributed on all 10 chromosomes and chromosomes 1, 2, and 3 appear to be the hot spots. Majority of the S. bicolor LEA (SbLEA) genes are intron less or have fewer introns. A total of 22 paralogous events were observed and majority of them appear to be segmental duplications. Segmental duplication played an important role in SbLEA-2 family expansion. A total of 12 orthologs were observed with Arabidopsis and 13 with Oryza sativa. Majority of them are basic in nature, and targeted by chloroplast subcellular localization. Fifteen miRNAs targeted to 25 SbLEAs appear to participate in development, as well as in abiotic stress tolerance. Promoter analysis revealed the presence of abiotic stress-responsive DRE, MYB, MYC, and GT1, biotic stress-responsive W-Box, hormone-responsive ABA, ERE, and TGA, and development-responsive SKn cis-elements. This reveals that LEA proteins play a vital role during stress tolerance and developmental processes. Using microarray data, 65 SbLEA genes were analyzed in different tissues (roots, pith, rind, internode, shoot, and leaf) which show clear tissue specific expression. qRT-PCR analysis of 23 SbLEA genes revealed their abundant expression in various tissues like roots, stems and leaves. Higher expression was noticed in stems compared to roots and leaves. Majority of the SbLEA family members were up-regulated at least in one tissue under different stress conditions. The SbLEA3-2 is the regulator, which showed abundant expression under diverse stress conditions. Present study provides new insights into the formation of LEAs in S. bicolor and to understand their role in developmental processes under stress conditions, which may be a valuable source for future research.

The growth-regulating factor (GRF) and GRF-interacting factor (GIF) families encode plant-specific transcription factors and play vital roles in plant development and stress response processes. Although GRF and GIF genes have been identified in various plant species, there have been no reports of the analysis and identification of the GRF and GIF transcription factor families in chickpea (Cicer arietinum) and pigeonpea (Cajanus cajan). The present study identified seven CaGRFs, eleven CcGRFs, four CaGIFs, and four CcGIFs. The identified proteins were grouped into eight and three clades for GRFs and GIFs, respectively based on their phylogenetic relationships. A comprehensive in-silico analysis was performed to determine chromosomal location, sub-cellular localization, and types of regulatory elements present in the putative promoter region. Synteny analysis revealed that GRF and GIF genes showed diploid-polyploid topology in pigeonpea, but not in chickpea. Tissue-specific expression data at the vegetative and reproductive stages of the plant showed that GRFs and GIFs were strongly expressed in tissues like embryos, pods, and seeds, indicating that GRFs and GIFs play vital roles in plant growth and development. This research characterized GRF and GIF families and hints at their primary roles in the chickpea and pigeonpea growth and developmental process. Our findings provide potential gene resources and vital information on GRF and GIF gene families in chickpea and pigeonpea, which will help further understand the regulatory role of these gene families in plant growth and development.

Members of the plant Heme Activator Protein (HAP) or NUCLEAR FACTOR Y (NF-Y) are trimeric transcription factor complexes composed of the NF-YA, NF-YB and NF-YC subfamilies. They bind to the CCAAT box in the promoter regions of the target genes and regulate gene expressions. Plant NF-Ys were reported to be involved in adaptation to several abiotic stresses as well as in development. In silico analysis of Sorghum bicolor genome resulted in the identification of a total of 42 NF-Y genes, among which 8 code for the SbNF-YA, 19 for SbNF-YB and 15 for the SbNF-YC subunits. Analysis was also performed to characterize gene structures, chromosomal distribution, duplication status, protein subcellular localizations, conserved motifs, ancestral protein sequences, miRNAs and phylogenetic tree construction. Phylogenetic relationships and ortholog predictions displayed that sorghum has additional NF-YB genes with unknown functions in comparison with Arabidopsis. Analysis of promoters revealed that they harbour many stress-related cis-elements like ABRE and HSE, but surprisingly, DRE and MYB elements were not detected in any of the subfamilies. SbNF-YA1, 2, and 6 were found upregulated under 200 mM salt and 200 mM mannitol stresses. While NF-YA7 appeared associated with high temperature (40˚C) stress, NF-YA8 was triggered by both cold (4˚C) and high temperature stresses. Among NF-YB genes, 7, 12, 15, and 16 were induced under multiple stress conditions such as salt, mannitol, ABA, cold and high temperatures. Likewise, NF-YC 6, 11, 12, 14, and 15 were enhanced significantly in a tissue specific manner under multiple abiotic stress conditions. Majority of the mannitol (drought)-inducible genes were also induced by salt, high temperature stresses and ABA. Few of the high temperature stress-induced genes are also induced by cold stress (NF-YA2, 4, 6, 8, NF-YB2, 7, 10, 11, 12, 14, 16, 17, NF-YC4, 6, 12, and 13) thus suggesting a cross talk among them. This work paves the way for investigating the roles of diverse sorghum NF-Y proteins during abiotic stress responses and provides an insight into the evolution of diverse NF-Y members.