Real-time Expression Analysis Research Articles

Isolation, Characterization, and Expression Analysis of NAC Transcription Factor from Andrographis paniculata (Burm. f.) Nees and Their Role in Andrographolide Production.

Andrographis paniculata (Burm. f.) Nees is an important medicinal plant known for its bioactive compound andrographolide. NAC transcription factors (NAM, ATAF1/2, and CUC2) play a crucial role in secondary metabolite production, stress responses, and plant development through hormonal signaling. In this study, a putative partial transcript of three NAC family genes (ApNAC83, ApNAC21 22 and ApNAC02) was used to isolate full length genes using RACE. Bioinformatics analyses such as protein structure prediction, cis-acting regulatory elements, and gene ontology analysis were performed. Based on in silico predictions, the diterpenoid profiling of the plant's leaves (five-week-old) and the real-time PCR-based expression analysis of isolated NAC genes under abscisic acid (ABA) treatment were performed. Additionally, the expression analysis of isolated NAC genes under MeJA treatment and transient expression in Nicotiana tabacum was performed. Full-length sequences of three members of the NAC transcription factor family, ApNAC83 (1102 bp), ApNAC21 22 (996 bp), and ApNAC02 (1011 bp), were isolated and subjected to the promoter and gene ontology analysis, which indicated their role in transcriptional regulation, DNA binding, ABA-activated signaling, and stress management. It was observed that ABA treatment leads to a higher accumulation of andrographolide and 14-deoxyandrographolide content, along with the upregulation of ApNAC02 (9.6-fold) and the downregulation of ApNAC83 and ApNAC21 22 in the leaves. With methyl jasmonate treatment, ApNAC21 22 expression decreased, while ApNAC02 increased (1.9-fold), with no significant change being observed in ApNAC83. The transient expression of the isolated NAC genes in a heterologous system (Nicotiana benthamiana) demonstrated their functional transcriptional activity, leading to the upregulation of the NtHMGR gene, which is related to the terpene pathway in tobacco. The expression analysis and heterologous expression of ApNAC21 22 and ApNAC02 indicated their role in andrographolide biosynthesis.

Genome-wide profiling of CBL interacting protein kinases (CIPKs) in banana unveils their role in abiotic stress signaling and stress tolerance enhancement

Calcineurin B-like (CBL)-interacting protein kinases (CIPKs) play a crucial role in the complex molecular systems of plants, acting as coordinators in different plant development processes, manage hormone signaling, and respond to environmental stress. Despite their well-established importance in various physiological process, the CIPK gene family in banana has remained an unexplored, creating a knowledge gap that this research aims to address. Through comprehensive analysis, we identified and characterized 34 CIPK genes in the banana genome. Structural diversity analysis revealed distinct clades characterized by homologs with varying intron-rich and intron-poor members. Particularly, evolutionary conservation was observed in intron-less members. Exploring conserved motifs and physicochemical properties provided insights into the detailed structure of specific homologs. Subcellular localization predictions indicated that most CIPK proteins primarily reside in the cytoplasm and nucleus, indicating their involvement in essential cellular activities. The predicted chromosomal distribution pattern suggests that the CIPK gene family largely expanded through segmental duplications in the banana genome. The phylogenetic analysis has uncovered two monophyletic clades for intron-less and intron-containing homologs, providing insights into two separate evolutionary history and expansion of the CIPK gene family. The clustering pattern in the phylogeny suggest that sequence of CIPKs members in the different genomes are largely conserved during course of evolution after evolution of higher plants. However, a few members having diverse sequences that these homologs are evolving with higher rate of evolution. Diverse physiological roles of banana CIPKs members are further supported by the presence of numerous cis-acting elements associated with growth, development, hormone responsiveness, and stress, with certain MaCIPK promoters exhibiting multiple hormone-sensitive elements. The protein-protein interaction network sheds light on the functional associations between MaCIPKs and MaCBLs, highlighting specific clusters of interactions. 3D protein structure predictions provide additional insights, revealing variations among the MaCIPK proteins. Real-time expression analysis across various tissues and developmental stages emphasizes the significant roles played by CIPKs in plant development. Under stress conditions, 16 in leaves and 22 in root MaCIPK gene transcript levels were up –regulated in response to drought and cold stress. These insights into the dynamic expression and functional roles of CIPK genes provide valuable information for understanding the mechanisms underlying banana's resilience to stress, with direct implications for the development of stress-resistant banana varieties. Overall this research offers promising prospects for enhancing global food security by ensuring the sustainability of banana production in the face of changing environmental conditions.

"Genome-wide identification of bZIP gene family in Pearl millet and transcriptional profiling under abiotic stress, phytohormonal treatments; and functional characterization of PgbZIP9".

Abiotic stresses are major constraints in crop production, and are accountable for more than half of the total crop loss. Plants overcome these environmental stresses using coordinated activities of transcription factors and phytohormones. Pearl millet an important C4 cereal plant having high nutritional value and climate resilient features is grown in marginal lands of Africa and South-East Asia including India. Among several transcription factors, the basic leucine zipper (bZIP) is an important TF family associated with diverse biological functions in plants. In this study, we have identified 98 bZIP family members (PgbZIP) in pearl millet. Phylogenetic analysis divided these PgbZIP genes into twelve groups (A-I, S, U and X). Motif analysis has shown that all the PgbZIP proteins possess conserved bZIP domains and the exon-intron organization revealed conserved structural features among the identified genes. Cis-element analysis, RNA-seq data analysis, and real-time expression analysis of PgbZIP genes suggested the potential role of selected PgbZIP genes in growth/development and abiotic stress responses in pearl millet. Expression profiling of selected PgbZIPs under various phytohormones (ABA, SA and MeJA) treatment showed differential expression patterns of PgbZIP genes. Further, PgbZIP9, a homolog of AtABI5 was found to localize in the nucleus and modulate gene expression in pearl millet under stresses. Our present findings provide a better understanding of bZIP genes in pearl millet and lay a good foundation for the further functional characterization of multi-stress tolerant PgbZIP genes, which could become efficient tools for crop improvement.

Induction of defense-related enzymes and enhanced disease resistance in rice against Sarocladium oryzae by Bacillus cereus RBS-57

Bacillus species have been identified as effective bioagents and plant growth boosters in rice. Currently, there is limited research on the effectiveness of Bacillus strains in managing the sheath rot disease of rice caused by Sarocladium oryzae, a significant threat to rice-cultivating areas in India. The aim of this study was to evaluate the efficacy of Bacillus spp. to control the sheath rot disease and promote growth in rice plants. We investigated the in vitro antagonistic activity and growth-promoting potential of eighty-four Bacillus spp. against S. oryzae. Ten efficient strains were carefully selected and analyzed at the molecular level based on their capacity for antagonism and growth promotion. A PCR study of the potential strains has revealed the presence of lipopeptide genes that produce bacilysin, bacillomycin, iturin, surfactin, subtilin, subtilosin and fengycin. Among the strains, RBS-57 had the highest mycelial growth inhibition (74.44 %) of the pathogen as well as the highest number of antibiotic genes (6). The efficacy of talc and liquid formulations of Bacillus strains BS-5, RBS-57, and RBS-3 was further tested against sheath rot disease. Results indicated that a combined application of seed treatment + seedling dip + foliar spray of RBS-57 liquid formulation under pot culture conditions, showed a considerable reduction in the incidence of sheath rot disease, followed by BS-5 and RBS-3. Similarly, a significant increase in the plant growth attributes and yield was observed with RBS-57, when compared to the other treatments. Enzymatic activities such as PO, PPO, PAL, CAT and SOD were induced in the rice sheath by B. cereus (RBS-57) treatment. Real-time expression analysis was performed to validate the fold changes in defense enzymes and reactive oxygen species. The effectiveness of RBS-57 liquid formulation in reducing disease incidence was demonstrated when applied as a combination of seed treatment @ 10 ml/l + seedling dip @ 10 ml/l + foliar spray @ 10 ml/l. Moreover, higher defense enzyme activities, maximum plant growth promotion and higher yield were reported with RBS-57 treatment. This study has the potential to be exploited to manage sheath rot disease in an eco-friendly and sustainable manner.

Cross talk of tumor protein D52 (TPD52) with KLF9, PKCε, and MicroRNA 223 in ovarian cancer

BackgroundGynecologic cancers comprise malignancies in the female reproductive organs. Ovarian cancer ranks sixth in terms of incidence rates while seventh in terms of mortality rates. The stage at which ovarian cancer is diagnosed mainly determines the survival outcomes of patients. Various screening approaches are presently employed for diagnosing ovarian cancer; however, these techniques have low accuracy and are non-specific, resulting in high mortality rates of patients due to this disease. Hence, it is crucial to identify improved screening and diagnostic markers to overcome this cancer. This study aimed to find new biomarkers to facilitate the prognosis and diagnosis of ovarian cancer.MethodsBioinformatics approaches were used to predict the tertiary structure and cellular localization along with phylogenetic analysis of TPD52. Its molecular interactions were determined through KEGG analysis, and real-time PCR-based expression analysis was performed to assess its co-expression with another oncogenic cellular pathway (miR-223, KLF9, and PKCε) proteins in ovarian cancer.ResultsBioinformatics analysis depicted the cytoplasmic localization of TPD52 and the high conservation of its coiled-coil domains. Further study revealed that TPD52 mRNA and miRNA-223 expression was elevated, while the expression of KLF 9 and PKCε was reduced in the blood of ovarian cancer patients. Furthermore, TPD52 and miR-223 expression were upregulated in the early stages of cancer and non-metastatic cancers.ConclusionTPD52, miR-223, PKCε, and KLF9, can be used as a blood based markers for disease prognosis, metastasis, and treatment response. The study outcomes hold great potential to be translated at the clinical level after further validation on larger cohorts.

Rhubarb: A novel model plant to study the conundrum of calcium oxalate synthesis

The study investigated calcium oxalate (CaOx) crystal composition, accumulation, synthesis, and degradation in five rhubarb species from the North-Western Indian Himalayas. Techniques like optical and scanning electron microscopy (SEM), SEM-energy-dispersive X-ray spectroscopy (SEM-EDS), inductively coupled plasma-optical emission spectrometry (ICP-OES), X-ray diffraction spectroscopy (XRD), and real-time (qRT-PCR) expression analysis of strategic genes were used to understand the processes of oxalate synthesis and precipitation. Results showed crystals tend to accumulate around vascular bundles in all species, irrespective of size, indicating a consistent pattern. Crystal synthesis and accumulation were stress-driven, linked to substrate composition, and in planta soluble oxalate and calcium levels, paralleling oxalate precursors. Based on their availability, CaOx crystals precipitated heavy metals mostly associated with its weddellite form. Crystal content correlated positively with mRNA levels of calcium/oxalate/ascorbate-related and stress-responsive genes, and negatively with oxalate oxidation/decarboxylation genes. CaOx crystals were suggested as potential biominerals for addressing heavy metal stress in agriculture.

Sequence characterization and comparative expression profile of buffalo WNT10B gene in adult and fetal tissues

In this study, Wingless-type MMTV (mouse mammary tumor virus) integration site family member (WNT10B) gene was sequence characterized in the Indian water buffalo. Sequence analysis revealed an open reading frame of 1176 nucleotides in buffalo, encoding 391 amino acids long protein. Nineteen nucleotide variations were observed between cattle and buffalo resulting in six amino acid changes. Phylogenetic analysis showed the clustering of ruminant species together. Real-time expression analysis of WNT10B in tissues collected from different organs of fetal and adult buffalo, revealed, the gene being abundantly expressed in the rumen and liver of the fetus. The fetal ovary, heart, kidney, lung, testis and mammary gland showed moderate expression, while in adult tissues, expression was high in the ovary, testis, brain, kidney, small intestine and liver, whereas lower expression was observed in the adult rumen. Significant differences in WNT10B expression levels were found for the brain, small intestine, testes, kidney, heart, rumen, and ovary when adult and fetal tissues were compared. A moderate level of genetic variation was found between cattle and buffalo WNT10B and expression patterns in a variety of tissues in adult buffalo implies that in addition to possible roles in adipogenesis and hematopoiesis, the WNT10B gene might be playing a significant role in other regulatory pathways as well.

Transformation and expressional studies of GaZnF gene to improve drought tolerance in Gossypium hirsutum

Drought stress is the major limiting factor in plant growth and production. Cotton is a significant crop as textile fiber and oilseed, but its production is generally affected by drought stress, mainly in dry regions. This study aimed to investigate the expression of Zinc finger transcription factor’s gene (GaZnF) to enhance the drought tolerance in Gossypium hirsutum. Sequence features of the GaZnF protein were recognized through different bioinformatics tools like multiple sequence alignment analysis, phylogenetic tree for evolutionary relationships, Protein motifs, a transmembrane domain, secondary structure and physio-chemical properties indicating that GaZnF is a stable protein. CIM-482, a local Gossypium hirsutum variety was transformed with GaZnF through Agrobacterium-mediated transformation method with 2.57% transformation efficiency. The integration of GaZnF was confirmed through Southern blot showing 531 bp, and Western blot indicated a 95 kDa transgene-GUS fusion band in transgenic plants. The normalized real-time expression analysis revealed the highest relative fold spatial expression of cDNA of GaZnF within leaf tissues at vegetative and flowering stages under drought stress. Morphological, physiological and biochemical parameters of transgenic cotton plants at 05- and 10-day drought stress was higher than those of non-transgenic control plants. The values of fresh and dry biomass, chlorophyll content, photosynthesis, transpiration rate, and stomatal conductance reduced in GaZnF transgenic cotton plants at 05- and 10-day drought stress, but their values were less low in transgenic plants than those of non-transgenic control plants. These findings showed that GaZnF gene expression in transgenic plants could be a valuable source for the development of drought-tolerant homozygous lines through breeding.

Pseudomonas fluorescens imparts cadmium stress tolerance in Arabidopsis thaliana via induction of AtPCR2 gene expression

BackgroundCadmium is a non-essential, third largest heavy metal contaminant with long retention time that poses environmental hazards. It emanating majorly from industrial processes and phosphate fertilizers. Cadmium is effortlessly assimilated by plants and leads to yield loss. Henceforth, identification of mechanisms to attenuate the heavy metal toxicity in crops is beneficial for enhanced yields. ResultsBeneficial soil bacteria have been known to combat both biotic and abiotic stress, thereby promoting plant growth. Amongst them, Pseudomonas fluorescens has been shown to enhance abiotic stress resistance in umpteen crops for instance maize and groundnut. Here, we investigated the role of P. fluorescens in conferring cadmium stress resistance in Arabidopsis thaliana. In silico analysis of PCR2 gene and promoter revealed the role, in cadmium stress resistance of A. thaliana. Real-time expression analysis employing qRT-PCR ratified the upregulation of AtPCR2 transcript under cadmium stress up to 6 folds. Total leaf (50%), biomass (23%), chlorophyll content (chlorophyll-a and b 40%, and 36 %) silique number (50%), and other growth parameters significantly improved on bacterial treatment of the 2mM Cd-stressed plants. ConclusionMoreover, generated 35s-promoter driven AtPCR2 over-expressing transgenic lines that exhibited resistance to cadmium and other heavy metal stress. Taken together, a crucial interplay of P. fluorscens mediated enhanced expression of AtPCR2 significantly induced cadmium stress resistance in Arabidopsis plants.

Comparative transcriptomics of Pinus massoniana organs provides insights on terpene biosynthesis regulation.

Terpenoids are the most important natural products collected from conifer species. However, the molecular mechanisms and core factors underlying terpenoid biosynthesis in Pinus massoniana remain unclear. To clarify these mechanisms, this study aimed to identify potential genes that might participate in the terpenoid biosynthesis of P. massoniana. In this study, single molecule real-time (SMRT) sequencing and expression analysis were used to confirm the expression patterns of genes involved in the cones, immature needles, mature needles, immature branches, and mature branches of P. massoniana. A total of 31,331 lncRNAs and 71,240 mRNAs were identified from these organs, and the greatest number of differentially expressed genes (DEGs) was discovered between needles and branches. Weighted gene coexpression network analysis (WGCNA) classified all expressed genes into nine typical modules with 11 kinds of transcription factors (TFs), namely, AP2-ERF, ARF, AUX-IAA, C2H2, Dof, F-box, SBP, WRKY, bHLH, bZIP, and GRAS, and seven kinds of functional genes, namely, ABC transporter, cellulose synthase (CesA), leucine-rich repeats (LRR), cytochrome P450 (CYT P450), pathogenesis-related protein (PR), terpene synthase (TPS), and chlorophyllase enzyme. A molecular network was constructed for hub genes, TFs, and functional genes in three modules. The potential function of eight candidate genes, including PmbHLH2, PmERF1, PmRGA, PmGAI, PmbZIP1, PmLOB1, PmMADS1, and PmMYB1, was validated through correlation analysis between terpenoid contents and expression levels, subcellular localization, and transcriptional activation activity, which provides us with probable regulators of terpenoid biosynthesis in conifers.

The extent of the abundance of exosomal and non‐exosomal extracellular miRNAs in the bovine follicular fluid

Follicular fluid (FF) plays an important role during follicular development and it contains several bioactive molecules including extracellular microRNAs (ECmiRNAs) that may mediate cell-cell communication during follicular development. Yet, the distribution patterns of ECmiRNAs in FF is not well characterized. This study aims to investigate the distribution of ECmiRNAs in two major fractions, namely exosomal and non-exosomal, of bovine follicular fluid (bFF). Exosomal and non-exosomal fractions from bFF were separated using Exoquick™ exosomes precipitation kit. miRNA expression was evaluated using the human miRCURY LNA™ Universal RT miRNA PCR array system. Transmission electron microscopy and immunoblotting revealed that the isolated vesicles were exosomes. The real-time PCR-based expression analysis revealed that 516 miRNAs were detected in the exosomal fraction of bFF, while 393 miRNAs were detected in the non-exosomal fraction. Among the detected miRNAs, a total of 370 miRNAs were detected in both fractions, while 145 miRNAs and 23 miRNAs were solely detected in exosomal and non-exosomal fractions, respectively. Exploratory pathway analysis showed that the genes targeted by exosomal and non-exosomal miRNAs to be involved in MAPK, Wnt, FoxO, TGF-beta, Oxytocin, ErbB, PI3K-Akt, Neurotrophin signalling pathways which are believed to be involved in follicular development, cell proliferation, and meiotic resumption. The results of our study demonstrated that besides the exosomal fraction, non-exosomal fractions can carry a significant amount of miRNAs in bFF where the exosomal fraction carries a significantly higher number of detectable miRNAs.

Genome-wide exploration of auxin response factors (ARFs) and their expression dynamics in response to abiotic stresses and growth regulators in coconut (Cocos nucifera L.)

Auxin response factors (ARFs) are a class of transcription factors with the ability to bind specifically to the promoter region of auxin-responsive genes in plants and thereby modulate various aspects of biological processes and responses to diverse environmental conditions. Identification and characterization of ARF gene families have been undertaken in various plant species; however, no such data, delineating ARFs in coconut, is available. Through genome-wide analysis, we have identified 20 CnARF (Cocos nucifera ARF) genes in the genome of a dwarf cultivar, Chowghat Green Dwarf. Evolutionary analysis of CnARFs, along with ARFs from oil palm and date palm, showed that palm ARFs could be classified into three major clades. Multiple sequence alignment and analysis of conserved motifs revealed that majority of the CnARFs harbored all the three principal domains (i.e. N-terminus DNA binding domain, an ARF sub-domain and an AUX/IAA sub-domain), which are characteristic of all plant ARFs. Quantitative real-time PCR-based expression analysis of ARFs of mature zygotic embryos, exposed to osmotic and temperature stresses and growth regulators, suggest a complex and stress-specific transcriptional response of coconut embryo-derived ARFs. Collectively, these findings offer valuable information and the first molecular insights into the CnARFs, laying the foundation for further functional characterization in developmental and reproductive pathways and their inclusion in breeding for stress tolerance in coconut.

VRILLE shows high divergence among Higher Diptera flies but may retain role as transcriptional repressor of Clock

In the circadian system, the clock gene vrille (vri) is an essential component of the second feedback loop, being responsible in Drosophila for the rhythmicity of the Clock (Clk) gene transcription by its repression. Here we studied vri in a fruit fly pest, the Tephritidae Anastrepha fraterculus, aimingtoinvestigate its molecular evolution and expression patterns from whole-head extracts. We used a combination of transcriptomic, genomic and gene walking strategies to sequence and characterize Afravri in male and female head transcriptomes of A. fraterculus and detected two putative isoforms that may correspond to A and D vri isoforms of Drosophila. Both isoforms produced a full-length sequence that translates to 842 amino acids. While the protein sequence showed significant divergence to orthologous sequences from other organisms, the bZIP domain was highly conserved. Molecular evolutionary analyses showed that vri in higher Diptera flies has been evolving under positive selection. A more detailed analysis showed positive selection also in Tephritidae with 29 sites evolving under positive selection in comparison with Drosophilidae. Real time expression analysis in LD and DD conditions showed cyclic expression of Afravri mRNA with oscillation opposite to AfraClk, suggesting that VRI may also behave in Anastrepha as a transcriptional repressor of Clk, providing another indication that higher Diptera might share common interlocked transcript-translation feedback loops (TTFLs) mechanisms that differ from other insects in target genes.

Overexpression of EcDREB2A transcription factor from finger millet in tobacco enhances tolerance to heat stress through ROS scavenging

An extreme temperature regime beyond desired level imposes significant stress in crop plants. The low and high temperature stresses are one of the primary constraints for plant development and yield. Finger millet, being a climate resilient crop, is a potential source of novel stress tolerant genes. In this study, functional characterization of finger millet DREB2A gene in different abiotic stress conditions was done. This novel EcDREB2A transcription factor isolated from finger millet is a truncated version of DREB2A gene compared to previously reported DREB genes from other plant species. The overexpression of EcDREB2A in transgenic tobacco exhibits improved tolerance against heat stress 42 °C for up to 7 days, by altering physiology and biochemical means. However, same transgenic lines were unable to provide tolerance to 200 mM NaCl and 200 mM Mannitol stress. Under heat stress conditions, increased seed germination with improved lateral roots, fresh and dry weight relative to wild type (WT) was observed. The EcDREB2A transgenics exposed to heat stress showed improved rate of stomatal conductance, chlorophyll and carotenoids contents, and other photosynthesis parameters compared to WT plants. EcDREB2A overexpression also resulted in increased antioxidant enzyme activity (SOD, CAT, GR, POD and, APX) with decreased electrolyte leakage (EL), H2O2, and malondialdehyde (MDA) content than WT plants under heat stress. Quantitative real time expression analysis demonstrated that all eight downstream genes were significantly upregulated in transgenic plants only after heat stress. Our data provide a clear demonstration of the positive impact of overexpression of EcDREB2A providing heat stress tolerance to plants.

High throughput identification of miRNAs reveal novel interacting targets regulating chickpea-rhizobia symbiosis

Legumes developed symbiotic associations to meet its nitrogen requirement. The nitrogen fixation takes place in root nodules which involves bacterial colonization, organogenesis and nitrogen fixation. In order to unravel the miRNA mediated regulation of chickpea symbiosis, one microRNA and four parallel analysis of RNA ends (PARE) libraries were sequenced. Analysis of microRNA library identified a set of 91 miRNAs comprising of 84 conserved and 7 novel miRNAs. Additionally, PARE library analysis revealed 564 genes being targeted by 85 miRNAs. Phylogenetic analysis of the precursor sequences of the 91 miRNAs was carried out which revealed their ancestral relationships. Further, the mechanism of miRNAs biogenesis was predicted using the miRNAs information from other legumes. Reads from the nodule library were mapped to bacterial genomes to predict bacterial-encoded small RNAs. Real time expression analysis was used to validate the antagonistic expression pattern of important miRNA-mRNA target pairs. Four candidate miRNAs were selected for in planta study based on the antagonistic expression profiling as well as the novelty of their respective targets. miR171f, miR172c, miR394 and miR1509 targeted nodulation receptor kinase, Apetala2, histidine phosphotransferase, adenylate kinase respectively and were ectopically expressed in chickpea roots. The overexpression lines showed significant change in nodule numbers, the miR172c, miR394 and miR1509 resulted in an increase in nodule number whereas, miR171f overexpression led to a decrease in nodule number. Our analysis lays the foundation for functional characterization of novel miRNAs and their respective target pairs which control nodulation in chickpea and other leguminous crops.

Transcriptomic Profiling of Buffalo Spermatozoa Reveals Dysregulation of Functionally Relevant mRNAs in Low-Fertile Bulls.

Although, it is known that spermatozoa harbor a variety of RNAs that may influence embryonic development, little is understood about sperm transcriptomic differences in relation to fertility, especially in buffaloes. In the present study, we compared the differences in sperm functional attributes and transcriptomic profile between high- and low-fertile buffalo bulls. Sperm membrane and acrosomal integrity were lower (P < 0.05), while protamine deficiency and lipid peroxidation were higher (P < 0.05) in low- compared to high-fertile bulls. Transcriptomic analysis using mRNA microarray technology detected a total of 51,282 transcripts in buffalo spermatozoa, of which 4,050 transcripts were differentially expressed, and 709 transcripts were found to be significantly dysregulated (P < 0.05 and fold change >1) between high- and low-fertile bulls. Majority of the dysregulated transcripts were related to binding activity, transcription, translation, and metabolic processes with primary localization in the cell nucleus, nucleoplasm, and in cytosol. Pathways related to MAPK signaling, ribosome pathway, and oxidative phosphorylation were dysregulated in low-fertile bull spermatozoa. Using bioinformatics analysis, we observed that several genes related to sperm functional attributes were significantly downregulated in low-fertile bull spermatozoa. Validation of the results of microarray analysis was carried out using real-time qPCR expression analysis of selected genes (YBX1, ORAI3, and TFAP2C). The relative expression of these genes followed the same trend in both the techniques. Collectively, this is the first study to report the transcriptomic profile of buffalo spermatozoa and to demonstrate the dysregulation of functionally relevant transcripts in low-fertile bull spermatozoa. The results of the present study open up new avenues for understanding the etiology for poor fertility in buffalo bulls and to identify fertility biomarkers.

Genome-Wide Analysis of Heat Shock Transcription Factors in Ziziphus jujuba Identifies Potential Candidates for Crop Improvement Under Abiotic Stress.

Plant heat shock transcription factors (Hsfs) play a significant role in adoption under abiotic stress conditions by modulating the expression of several stress-responsive genes. Analysis of the Hsf gene family will serve to understand the molecular mechanism which is involved in response to abiotic stress. The Ziziphus species grows in warm and dry regions and is inherently tolerant to abiotic stress conditions; thus, Ziziphus is a highly enriched source of genes conferring abiotic stress tolerance. Therefore, the present study provides a comprehensive genome-wide analysis of the Hsf gene family in Z. jujuba. Identified 21 non-redundant Hsf genes were grouped into three major classes (classes A, B, and C) based on the phylogenetic analysis. Promoter and gene ontology analysis suggested that ZjHsfs perform diverse functions in response to abiotic stress conditions. Two paralogous pairs resulting from tandem gene duplication events were identified. Also, physio-chemical properties of chromosomal locations, gene structure, motifs, and protein domain organization of Hsfs were analyzed. Real-time PCR expression analyses revealed that most of the Z. jujuba Hsf genes are differentially expressed in response to heat stress. The analysis suggested ZjHsf-2, ZjHsf-3, ZjHsf-5, ZjHsf-7, ZjHsf-8, ZjHsf-10, ZjHsf-12, ZjHsf-17, and ZjHsf-18 were the outstanding candidate genes for imparting heat stress tolerance and for future functional analysis. The present analysis laid the foundation for understanding the molecular mechanism of the Hsf gene family regulating Z. jujuba development and tolerance to abiotic stress conditions.

Impact of nitric oxide on proline and putrescine biosynthesisin Chlamydomonas via transcriptional regulation

Nitric oxide plays an important role in regulating adaption of the model alga Chlamydomonas reinhardtii to various environmental stresses. One response to abiotic stresses is the accumulation of protective molecules such as proline and putrescine. The NO treatment led to a significant accumulation of proline in cells. Quantitative real-time expression analysis of proline metabolic genes in NO-treated cells showed a prolonged upregulation of the gene encoding γ-glutamyl kinase 1 (GGK1) in the glutamate biosynthetic pathway. Furthermore, truncated hemoglobin 2 (THB2)-underexpressing strains with an enhanced endogenous NO demonstrated a higher proline content and GGK1 mRNA abundances than the wild type. In contrast, transcription of the gene encoding ornithine δ-aminotransferase in the ornithine pathway of proline biosynthesis decreased after treatment with NO. This suggests the predominance of the glutamate pathway over the ornithine pathway. We also found that the expression of the proline dehydrogenase gene encoding a key enzyme in proline catabolism was downregulated in NO-treated cells. Chlamydomonas reinhardtii exposed to exogenous NO also showed an increased ornithine decarboxylase 2 mRNA and putrescine content. Our findings indicate a clear link between changes in NO application and proline and putrescine content via transcriptional regulation of respective enzymes.

Transcriptome wide identification and characterization of regulatory genes involved in EAA metabolism and validation through expression analysis in different developmental stages of finger millet spikes.

Finger millet is a rich source of seed storage proteins (SSPs). Various regulatory genes play an important role to maintain the quality and accumulation of SSPs in crop seeds. In the present study, nine regulatory genes of EAAs metabolic pathway, i.e., aspartate kinase, homoserine dehydrogenase, threonine synthase, threonine dehydratase, dihydrodipicolinate synthase, cystathionine γ synthase, anthranilate synthase, acetolactate synthase and lysine 2-oxoglutarato reductase/saccharopine dehydrogenase (LOR/SD) were identified from the transcriptomic data of developing spikes of two finger millet genotypes, i.e., GP-45 and GP-1. Results of sequence alignment search and motif/domain analysis showed high similarity of nucleotide sequences of identified regulatory genes with their respective homologs in rice. Results of promoter analysis revealed the presence of various cis-regulatory elements, like nitrogen responsive cis-elements (O2-site and GCN4), light responsive cis-elements, and stress responsive cis-elements. The presence of nine regulatory genes identified from the transcriptomic data of GP-45 and GP-1 was further confirmed by real time expression analysis in high and low protein containing genotypes, i.e., GE-3885 and GE-1437. Results of real time expression analysis showed significantly higher expression (p ≤ 0.01) of regulatory genes in GE-3885 rather than GE-1437 under control and treatment condition. Crude protein content of GE-3885 was found to be significantly higher (p ≤ 0.01) in comparison to GE-1437 under control condition, while under treatment condition GE-1437 was found to be more responsive to KNO3 treatment rather than GE-3885.

Gene Regulatory Networks in Peripheral Mononuclear Cells Reveals Critical Regulatory Modules and Regulators of Multiple Sclerosis

Multiple sclerosis (MS) is a complex, demyelinating disease with the involvement of autoimmunity and neurodegeneration. Increasing efforts have been made towards identifying the diagnostic markers to differentiate the classes of MS from other similar neurological conditions. Using a systems biology approach, we constructed four types of gene regulatory networks (GRNs) involved in peripheral blood mononuclear cells (PBMCs). The regulatory strength of each GRN across primary progressive MS (PPMS), relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), and control were evaluated by an integrity algorithm. Among the constructed GRNs (referred as TF_gene_miRNA), POU3F2_CDK6_hsa-miR-590-3p, MEIS1_CASC3_hsa-miR-1261, STAT3_OGG1_hsa-miR-298, and TCF4_FMR1_hsa-miR-301b were top-ranked and differentially regulated in all classes of MS compared to control. These GRNs showed potential involvement in regulating various molecular pathways such as interleukin, integrin, glypican, sphingosine phosphate, androgen, and Wnt signaling pathways. For validation, the qPCR analysis of the GRN components (TFs, gene, and miRNAs) in PBMCs of healthy controls (n = 30), RRMS (n = 14), PPMS (n = 13) and SPMS (n = 12) were carried out. Real-time expression analysis of GRNs showed a similar regulatory pattern as derived from our systems biology approach. Also, our study provided several novel GRNs that regulate unique and common molecular mechanisms between MS conditions. Hence, these regulatory components of GRNs will help to understand the disease mechanism across MS classes and further insight may though light towards diagnosis.