Molecular Characterization of Lineage-IV Peste Des Petits Ruminants Virus and the Development of In-House Indirect Enzyme-Linked Immunosorbent Assay (IELISA) for its Rapid Detection".

Peste des petits ruminants (PPRV), a highly contagious viral disease, causes significant economic losses concerning sheep and goats. Recently, PPR viruses (PPRVs), have adopted new hosts and lineage IV of PPRVs represents genetic diversity within the same lineage. 350 samples, including blood, swabs, and tissues from sheep/goats, were collected during the 2020-2021 disease outbreaks in Pakistan. These samples were analysed through RT-PCR and three isolates of PPRV with accession numbers, MW600920, MW600921, and MW600922, were submitted to GenBank, based on the partial N-gene sequencing. This analysis provides a better understanding of genetic characterizations and a targeted RT-PCR approach for rapid PPRV diagnosis. An IELISA test was developed using the semi-purified antigen MW600922 isolate grown in Vero cells. The PPRV isolates currently present high divergence with the Turkish strain; conversely, similarities equivalent to 99.73% were observed for isolates collected from Pakistan. The developed indirect ELISA (IELISA) test demonstrated antibody detection rates at dilutions of 1:200 for antibodies (serum) and 1:32 for antigens. In comparison to cELISA, high specificity (85.23%) and sensitivity (90.60%) rates were observed. In contrast to the virus neutralization test (VNT), IELISA was observed to be 100% specific and 82.14% sensitive in its results. Based on these results, serological surveys conducted for PPR antibodies using IELISA can be a more effective strategy on a larger scale. Furthermore, our results demonstrate a significant breakthrough in the research in terms of cost-effectiveness and storage efficiency, and the developed IELISA test is highly recommended for use in developing countries.

Peste des petits ruminants (PPRV) is a transboundary, highly contagious, and economically significant viral disease affecting small ruminants and wildlife. PPRV, a disease that only targets animals, is the focus of the Global Eradication Programme (PPRV GEP), which aims to eradicate the disease by 2030. Following the completion of the first phase of the GEP (2017­2021), Pakistan has initiated the second phase: PPRV presence and the implementation of a control strategy. Rapid and accurate laboratory diagnosis is vital to the disease's effective control and eradication. In the present study, we have improved diagnosis by reverse transcriptase polymerase chain reaction (RT-PCR), which not only can detect low viral concentrations but also contributes to the genetic analysis of lineage-IV viruses. However, the development of cost-effective indirect ELISA (iELISA) may allow for the analysis of serum samples obtained from larger populations of small ruminants.

G-ACP: a machine learning approach to the prediction of therapeutic peptides for gastric cancer.

Conventional Gastrointestinal (GI) cancer treatments are quite expensive and have major hazards. Nowadays, a different strategy places more emphasis on creating tiny biologically active peptides that do not cause severe poisoning. Anticancer peptides (ACPs) are found through experimental screening, which is time-dependent and frequently fraught with difficulties. Gastric ACPs are emerging as a promising GI cancer treatment in the current day. It is crucial to identify novel gastric ACPs to have an improved knowledge of their functioning processes and treatment of gastric cancer. As a result of the post-genomic era's massive production of peptide sequences, rapid and effective ACPs using a computational method are essential. Several adaptive statistical techniques for distinguishing ACPs and non-ACPs have recently been developed. A variety of adapted statistically significant methods have been developed to differentiate between ACPs and non-ACPs. Despite significant progress, there is no specific model for the prediction of gastric ACPs because the specific model will predict a particular type of peptide more accurately and quickly. To overcome this, an initiative is taken for the creation of a reliable framework for the accurate identification of gastric ACPs. The current technique in particular contains four possible features along with one hybrid feature encoding mechanisms which are the target-class motif previously indicated by Amino Acid Composition, Dipeptide Composition, Tripeptide Composition (TPC), Pseudo Amino Acid Composition (PAAC), and their Hybrid. Machine Learning algorithms make high-performance and accurate prediction tools. Moreover, highly variable and ideal deep feature selection is done using an ANOVA-based F score for feature pruning. Experiments on a range of algorithms are carried out to identify the optimal operating strategy due to the diverse nature of learning. Following analysis of the empirical results, Naïve Bayes with TPC and Hybrid feature space outperforms other methods with 0.99 accuracy score on the testing dataset. To find the model generalization an external validation is carried out. In external datasets, the Extra Trees with PAAC features outperforms with the accuracy of 0.94. The comparison study shows that our suggested model will predict gastric ACPs more accurately and will be useful in drug development and gastric cancer. The predictive model can be freely accessed at https://github.com/humeraazad10/G-ACP.git.Communicated by Ramaswamy H. Sarma.

The comparative transcriptome analysis of two green super rice genotypes with varying tolerance to salt stress.

BACKGROUND: Salinity is one of the main abiotic factors that restrict plant growth, physiology, and crop productivity is salt stress. About 33% of the total irrigated land suffers from severe salinity because of intensive underground water extraction and irrigation with brackish water. Thus, it is important to understand the genetic mechanism and identify the novel genes involved in salt tolerance for the development of climate-resilient rice cultivars. METHODS AND RESULTS: In this study, two rice genotypes with varying tolerance to salt stress were used to investigate the differential expressed genes and molecular pathways to adapt under saline soil by comparative RNA sequencing at 42 days of the seedling stage. Salt-susceptible (S3) and -tolerant (S13) genotypes revealed 3982 and 3463 differentially expressed genes in S3 and S13 genotypes. The up-regulated genes in both genotypes were substantially enriched in different metabolic processes and binding activities. Biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and plant signal transduction mechanisms were highly enriched. Salt-susceptible and -tolerant genotypes shared the same salt adaptability mechanism with no significant quantitative differences at the transcriptome level. Moreover, bHLH, ERF, NAC, WRKY, and MYB transcription factors were substantially up-regulated under salt stress. 391 out of 1806 identified novel genes involved in signal transduction mechanisms. Expression profiling of six novel genes further validated the findings from RNA-seq data. CONCLUSION: These findings suggest that the differentially expressed genes and molecular mechanisms involved in salt stress adaptation are conserved in both salt-susceptible and salt-tolerant rice genotypes. Further molecular characterization of novel genes will help to understand the genetic mechanism underlying salt tolerance in rice.

Bacopa monnieri: A promising herbal approach for neurodegenerative disease treatment supported by in silico and in vitro research.

Neurodegenerative disorders, caused by progressive neuron loss, are a global health issue. Among the various factors implicated in their pathogenesis, dysregulation of acetylcholinesterase activity has been recognized as a key contributor. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, important for neural transmission. Evaluating phyto-compounds from Bacopa monnieri Linn. through in vitro and in silico analysis may expand their role as alternative therapeutic agents by modulating the function of acetylcholinesterase and complementing existing treatments. To accomplish this objective, chemical structures of phyto-compounds were retrieved from PubChem database and subjected to in silico and in vitro approaches. Virtual screening was performed through molecular docking and molecular dynamic simulation resulting in four top hit compounds including quercetin, apigenin, wogonin, and bacopaside X (novel lead compound for acetylcholinesterase inhibitor) with least binding score. Further, dose dependent acetylcholinesterase inhibition biochemical assay depicted that bacopaside X, apigenin, quercetin, and wogonin exhibited strong potential against acetylcholinesterase with IC50 values of 12.78 µM, 13.83 µM, 12.73 µM and 15.48 µM respectively, in comparison with the donepezil (IC50: 0.0204 µM). The in silico and in vitro research suggests that B. monnieri phyto-compounds have the potential to modulate molecular targets associated with neurodegenerative diseases and have a role in neuroprotection.

Synergistic neuroprotection by phytocompounds of Bacopa monnieri in scopolamine-induced Alzheimer's disease mice model.

BACKGROUND: Millions of people around the globe are affected by Alzheimer's disease (AD). This crippling condition has no treatment despite intensive studies. Some phytocompounds have been shown to protect against Alzheimer's in recent studies. METHODS: Thus, this work aimed to examine Bacopa monnieri phytocompounds' synergistic effects on neurodegeneration, antioxidant activity, and cognition in the scopolamine-induced AD mice model. The toxicity study of two phytocompounds: quercetin and bacopaside X revealed an LD50 of more than 2000 mg/kg since no deaths occurred. RESULTS: The neuroprotection experiment consists of 6 groups i.e., control (saline), scopolamine (1 mg/kg), donepezil (5 mg/kg), Q (25 mg/kg), BX (20 mg/kg), and Q + BX (25 mg/kg + 20 mg/kg). Visual behavioral assessment using the Morris water maze showed that animals in the diseased model group (scopolamine) moved more slowly toward the platform and exhibited greater thigmotaxis behavior than the treatment and control groups. Likewise, the concentration of biochemical NO, GSH, and MDA improved in treatment groups concerning the diseased group. mRNA levels of different marker genes including ChAT, IL-1α, IL-1 ß, TNF α, tau, and ß secretase (BACE1) improved in treatment groups with respect to the disease group. CONCLUSION: Both bacopaside X and quercetin synergistically have shown promising results in neuroprotection. Therefore, it is suggested that Q and BX may work synergistically due to their antioxidant and neuroprotective property.

Screening and validation of salt-stress responsive cg-SSR markers in wheat (Triticum aestivum L.) germplasm of Pakistan.

BACKGROUND: Soil salinity has been affecting wheat production worldwide over past few decades. Evaluation of wheat genotypes for salinity tolerance at germination and vegetative growth level is crucial. Marker assisted selection is a technique used extensively for choosing salt-tolerant genotypes from breeding populations to introduce novel genes. METHODS AND MATERIALS: The current study's main goal was to discover salt-stress resistant genes; genetic divergence and genome-wide connection by using recently designed candidate gene-based simple-sequence-repeat markers (cg-SSRs). The phenotypic connection of morphological features during the germination growth stage i.e., germination period, root length/weight and shoot length/weight, and vegetative growth stages i.e., root length/weight and shoot length/weight were tested in a group of 50 wheat genotypes. Significant difference was observed in germination rate, root length and weight among control and saline treatments. CONCLUSION: Total 30 SSR markers were utilized to test salinity resistance genes in wheat genotypes. Three (10%) of which were monomorphic, one (3.34%) showed no result, and the other 26 (86%) were polymorphic. Using 30 polymorphic markers discovered total 37 alleles. The polymorphic information content (PIC), quantifies each SSR locus capacity to discriminate between wheat, varied from 0.00 to 0.38 with an average of 0.19. Association analysis revealed that 26 primers were associated with morphological features, 03 with root length and the remaining 23 with germination. Utilizing morphological data, stress tolerance index (STI) was designed concluding that Auqab-2000, Margala-99 and Ufaq showed better resistance against salinity among other wheat genotypes. Cluster analysis demonstrated that wheat genotypes have vast genetic variability.

CRISPR/Cas9 editing of wheat Ppd-1 gene homoeologs alters spike architecture and grain morphometric traits.

Mutations in Photoperiod-1 (Ppd-1) gene are known to modify flowering time and yield in wheat. We cloned TaPpd-1 from wheat and found high similarity among the three homoeologs of TaPpd-1. To clarify the characteristics of TaPpd-1 homoeologs in different photoperiod conditions for inflorescence architecture and yield, we used CRISPR/Cas9 system to generate Tappd-1 mutant plants by simultaneous modification of the three homoeologs of wheat Ppd-1. Tappd-1 mutant plants showed no off-target mutations. Four T0-edited lines under short-day length and three lines under long-day length conditions with the mutation frequency of 25% and 21%, respectively. These putative transgenic plants of all the lines were self-fertilized and generated T1 and T2 progenies and were evaluated by phenotypic and expression analysis. Results demonstrated that simultaneously edited TaPpd-1- A1, B1, and D1 homoeologs gene copies in T2_SDL-8-4, T2_SDL-4-5, T2_SDL-3-9, and T2_LDL-10-9 showed similar spike inflorescence, flowering time, and significantly increase in 1000-grain weight, grain area, grain width, grain length, plant height, and spikelets per spike due to mutation in both alleles of Ppd-B1 and Ppd-D1 homoeologs but only spike length was decreased in T2_SDL-8-4, T2_SDL-4-5, and T2_LDL-13-3 mutant lines due to mutation in both alleles of Ppd-A1 homoeolog under both conditions. Our results indicate that all TaPpd1 gene homoeologs influence wheat spike development by affecting both late flowering and earlier flowering but single mutant TaPpd-A1 homoeolog affect lowest as compared to the combination with double mutants of TaPpd-B1 and TaPpd-D1, TaPpd-A1 and TaPpd-B1, and TaPpd-A1 and TaPpd-D1 homoeologs for yield enhancement. Our findings further raised the idea that the relative expression of the various genomic copies of TaPpd-1 homoeologs may have an impact on the spike inflorescence architecture and grain morphometric features in wheat cultivars.

Temporal Comparative Transcriptome Analysis on Wheat Response to Acute Cd Toxicity at the Seedling Stage.

Cadmium (Cd) is a non-essential and toxic metal that accumulates in plant's tissues and diminishes plant growth and productivity. In the present study, differential root transcriptomic analysis was carried out to identify Cd stress-responsive gene networks and functional annotation under Cd stress in wheat seedlings. For this purpose, the Yannong 0428 wheat cultivar was incubated with 40 µm/L of CdCl2·2.5H2O for 6 h at three different seedling growth days. After the quality screening, using the Illumina Hiseq 2000 platform, more than 2482 million clean reads were retrieved. Following this, 84.8% to 89.3% of the clean reads at three time points under normal conditions and 86.5% to 89.1% of the reads from the Cd stress condition were mapped onto the wheat reference genome. In contrast, at three separate seedling growth days, the data analysis revealed a total of 6221 differentially expressed genes (DEGs), including 1543 (24.8%) up-regulated genes and 4678 (75.8%) down-regulated genes. In total, 120 DEGs were co-expressed throughout all the growth days, whereas 1096, 1088, and 2265 DEGs were found to be selectively up-/down-regulated at 7d, 14d, and 30d, respectively. However, the clustering of DEGs, through utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG), revealed that the DEGs in the metabolic category were frequently annotated for phenylpropanoid biosynthesis. In comparison, a considerable number of DEGs were linked to protein processing in the endoplasmic reticulum under the process of genetic information processing. Similarly, in categories in organismal systems and cellular processes, DEGs were found in plant hormone signal transduction pathways, and DEGs were identified in the plant-pathogen interaction pathway, respectively. However, DEGs in "endocytosis pathways" were enriched in environmental information processing. In addition, in-depth annotations of roughly specific heavy metal stress-response genes and pathways were also mined, and the expression patterns of eight DEGs were studied using quantitative real-time PCR. The results were congruent with the findings of RNA sequencing regarding transcript abundance in the studied wheat cultivar.

High Genetic Diversity in the Himalayan Common Bean (Phaseolus vulgaris) Germplasm with Divergence from Its Center of Origin in the Mesoamerica and Andes.

The common bean is found in the Himalayan region of Pakistan with substantial morphological variability. Genetic diversity within any crop species is a precursor for genetic improvement; however, little is known about common bean genetic diversity in this region. We explored the genetic diversity in the common bean from the Himalayan region (Khyber Pakhtunkhwa, Gilgit-Baltistan, Kashmir) of Pakistan. Microsatellite genotyping was carried out for 147 samples with 40 simple sequence repeat (SSR) markers. The results revealed a clear divergence of the Pakistani population from the primary gene pool (with FST values of 0.2 with Andes and 0.27 with Mesoamerica). However, within the Himalayan germplasm, no clear evidence of spatial structure was observed (with the maximum FST values of only 0.025), probably due to the dispersal of seeds by human activity within the region. This was further elucidated by the discriminant analyses of principal components. Considering the diversity parameters, high genotypic diversity was observed for the indigenous lines (0.990), comparable to the primary gene pool (0.976 for Mesoamerica and 0.976 for Andes populations). A high genotypic diversity was observed within the Himalayan population (ranging from 0.500 for Upper Dir to 0.952 for Mansehra). Gene diversity across loci varied between 0.28 for Chitral to 0.38 for Kurram. Our results suggested a divergent and independent evolution of the Himalayan population, which might have led to the diversification of the common bean germplasm in the region postintroduction into the region. The diversity observed could also be exploited in future breeding programs for the development and introduction of climate-resilient varieties.

Genome-Wide Identification of Potential mRNAs in Drought Response in Wheat (Triticum aestivum L.).

Plant cell metabolism inevitably forms an important drought-responsive mechanism, which halts crop productivity. Globally, more than 30% of the total harvested area was affected by dehydration. RNA-seq technology has enabled biologists to identify stress-responsive genes in relatively quick times. However, one shortcoming of this technology is the inconsistent data generation compared to other parts of the world. So, we have tried, here, to generate a consensus by analyzing meta-transcriptomic data available in the public microarray database GEO NCBI. In this way, the aim was set, here, to identify stress genes commonly identified as differentially expressed (p < 0.05) then followed by downstream analyses. The search term "Drought in wheat" resulted in 233 microarray experiments from the GEO NCBI database. After discarding empty datasets containing no expression data, the large-scale meta-transcriptome analytics and one sample proportional test were carried out (Bonferroni adjusted p < 0.05) to reveal a set of 11 drought-responsive genes on a global scale. The annotation of these genes revealed that the transcription factor activity of RNA polymerase II and sequence-specific DNA-binding mechanism had a significant role during the drought response in wheat. Similarly, the primary root differentiation zone annotations, controlled by TraesCS5A02G456300 and TraesCS7B02G243600 genes, were found as top-enriched terms (p < 0.05 and Q < 0.05). The resultant standard drought genes, glycosyltransferase; Arabidopsis thaliana KNOTTED-like; bHLH family protein; Probable helicase MAGATAMA 3; SBP family protein; Cytochrome c oxidase subunit 2; Trihelix family protein; Mic1 domain-containing protein; ERF family protein; HD-ZIP I protein; and ERF family protein, are important in terms of their worldwide proved link with stress. From a future perspective, this study could be important in a breeding program contributing to increased crop yield. Moreover, the wheat varieties could be identified as drought-resistant/sensitive based on the nature of gene expression levels.

Biogenic Synthesis of Silver Nanoparticles Using Catharanthus roseus and Its Cytotoxicity Effect on Vero Cell Lines.

Background: Type 2 diabetes mellitus (DM2) is a chronic and sometimes fatal condition which affects people all over the world. Nanotherapeutics have shown tremendous potential to combat chronic diseases­including DM2­as they enhance the overall impact of drugs on biological systems. Greenly synthesized silver nanoparticles (AgNPs) from Catharanthus roseus methanolic extract (C. AgNPs) were examined primarily for their cytotoxic and antidiabetic effects. Methods: Characterization of C. AgNPs was performed by UV−vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and atomic force microscopy (AFM). The C. AgNPs were trialed on Vero cell line and afterwards on an animal model (rats). Results: The C. AgNPs showed standard structural and functional characterization as revealed by FTIR and XRD analyses. The zetapotential analysis indicated stability while EDX analysis confirmed the formation of composite capping with Ag metal. The cytotoxic effect (IC50) of C. AgNPs on Vero cell lines was found to be 568 g/mL. The animal model analyses further revealed a significant difference in water intake, food intake, body weight, urine volume, and urine sugar of tested rats after treatment with aqueous extract of C. AgNPs. Moreover, five groups of rats including control and diabetic groups (NC1, PC2, DG1, DG2, and DG3) were investigated for their blood glucose and glycemic control analysis. Conclusions: The C. AgNPs exhibited positive potential on the Vero cell line as well as on experimental rats. The lipid profile in all the diabetic groups (DG1-3) were significantly increased compared with both of the control groups (p < 0.05). The present study revealed the significance of C. AgNPs in nanotherapeutics.

Impact of Pre-Anthesis Drought Stress on Physiology, Yield-Related Traits, and Drought-Responsive Genes in Green Super Rice.

Optimum soil water availability is vital for maximum yield production in rice which is challenged by increasing spells of drought. The reproductive stage drought is among the main limiting factors leading to the drastic reduction in grain yield. The objective of this study was to investigate the molecular and morphophysiological responses of pre-anthesis stage drought stress in green super rice. The study assessed the performance of 26 rice lines under irrigated and drought conditions. Irrigated treatment was allowed to grow normally, while drought stress was imposed for 30 days at the pre-anthesis stage. Three important physiological traits including pollen fertility percentage (PFP), cell membrane stability (CMS), and normalized difference vegetative index (NDVI) were recorded at anthesis stage during the last week of drought stress. Agronomic traits of economic importance including grain yield were recorded at maturity stage. The analysis of variance demonstrated significant variation among the genotypes for most of the studied traits. Correlation and principal component analyses demonstrated highly significant associations of particular agronomic traits with grain yield, and genetic diversity among genotypes, respectively. Our study demonstrated a higher drought tolerance potential of GSR lines compared with local cultivars, mainly by higher pollen viability, plant biomass, CMS, and harvest index under drought. In addition, the molecular basis of drought tolerance in GSR lines was related to upregulation of certain drought-responsive genes including OsSADRI, OsDSM1, OsDT11, but not the DREB genes. Our study identified novel drought-responsive genes (LOC_Os11g36190, LOC_Os12g04500, LOC_Os12g26290, and LOC_Os02g11960) that could be further characterized using reverse genetics to be utilized in molecular breeding for drought tolerance.

Genome-wide characterization and expression analysis of pseudo-response regulator gene family in wheat.

Pseudo-response regulator (PRR) gene family members play a significant role in plant circadian clocks, flowering time inflorescence architecture development during transition from vegetative growth phase to reproductive phase. In current study, we analyzed the expression profiling, phylogenetic relationship, and molecular characterization of PRR gene family members of common wheat by using IWGSC Ref seq v1.1 wheat genome database with a coverage rate of 90%. By using bioinformatic approach total 20 candidate gene sequences were identified and divided into six groups and four clades. It was found that mostly genes have same number of exons and introns showed similar features because they originated through duplication events during evolution processes. Although all the proteins have conserved PRR domains, but some are distinct in their sequences suggesting functional divergence. By comparative synteny analysis it was revealed that Group 1, 2, 3 and 11-D of group 4 have duplication events while group 5 and TaPRR9-B,10-D showed conservation with previously identified PRR members from rice. While expression variation of six groups from each analysis matches with each other. Five groups highly expressed in leaf, spike, and roots in pattern like leaf > spike > root at all three stages booting, heading and anthesis of spike development. This suggests that TaPRR genes play important roles in different photoperiod signaling pathways in different organs at different stages of spike development and flowering via unknown pathway. These findings will also provide comprehensive knowledge about future investigations on wheat PRR family members involved in complex network of circadian system for plant development.

Molecular Identification and Characterization of Bacillus sp. NIGAB-1 for Phenol Degradation Under Saline Conditions.

BACKGROUND: Phenol is an aromatic pollutant in industrial wastes that in combination with salts is highly toxic for all forms of life. Phenol elimination is the foremost challenge to meet the goal of pollutant-free environment. OBJECTIVE: The present study was carried out to isolate phenol degrading bacteria which can degrade phenol under saline conditions and to identify the isolated strains using 16S rRNA gene sequence analysis. MATERIAL AND METHODS: Sediment samples were collected from Rawal Lake, Islamabad, Pakistan and enriched in mineral salt medium (MSM) containing phenol (150 mg.L-1). Isolated strains were identified on the basis of 16S rRNA gene sequence analysis. Growth of strains were tested at different pH, NaCl concentrations and temperature using Tryptic Soy Agar (TSA). Tolerance to phenol (0-750 mg.L-1) was checked at 5% NaCl and phenol degrading experiment was performed at 4% NaCl, pH 7 and 30 oC. In both, phenol tolerance and degradation study, phenol was used a sole source of carbon and energy. RESULTS: Thirteen bacterial strains were isolated after enrichment among which, NIGAB-1 was found capable of degrading phenol in saline conditions. This strain was identified as Bacillus sp. NIGAB-1 on the basis of 16S rRNA gene sequence analysis and the closest match was Bacillus marisflavi with 99.71% sequence identity. The Bacillus sp. NIGAB-1 exhibited best growth at 30 oC at pH 7 with 10% NaCl. The optimum phenol concentration for growth was recorded as 300 mg.L-1. This strain degraded 300 mg.L-1 of phenol at 4% NaCl in 120 hours with the average degradation rate of 2.63 mg.L-1.h. CONCLUSION: These findings suggest that this strain could be efficient in phenol degradation at adverse environmental conditions and helpful in remediation of phenol where the salt concentration is high.

Evolution of Deeper Rooting 1-like homoeologs in wheat entails the C-terminus mutations as well as gain and loss of auxin response elements.

Root growth angle (RGA) in response to gravity controlled by auxin is a pertinent target trait for obtainment of higher yield in cereals. But molecular basis of this root architecture trait remain obscure in wheat and barley. We selected four cultivars two each for wheat and barley to unveil the molecular genetic mechanism of Deeper Rooting 1-like gene which controls RGA in rice leading to higher yield under drought imposition. Morphological analyses revealed a deeper and vertically oriented root growth in "NARC 2009" variety of wheat than "Galaxy" and two other barley cultivars "Scarlet" and "ISR42-8". Three new homoeologs designated as TaANDRO1-like, TaBNDRO1-like and TaDNDRO1-like corresponding to A, B and D genomes of wheat could be isolated from "NARC 2009". Due to frameshift and intronization/exonization events the gene structures of these paralogs exhibit variations in size. DRO1-like genes with five distinct domains prevail in diverse plant phyla from mosses to angiosperms but in lower plants their differentiation from LAZY, NGR and TAC1 (root and shoot angle genes) is enigmatic. Instead of IGT as denominator motif of this family, a new C-terminus motif WxxTD in the V-domain is proposed as family specific motif. The EAR-like motif IVLEM at the C-terminus of the TaADRO1-like and TaDDRO1-like that diverged to KLHTLIPNK in TaBDRO1-like and HvDRO1-like is the hallmark of these proteins. Split-YFP and yeast two hybrid assays complemented the interaction of TaDRO1-like with TOPLESS-a repressor of auxin regulated root promoting genes in plants-through IVLEM/KLHTLIPNK motif. Quantitative RT-PCR revealed abundance of DRO1-like RNA in root tips and spikelets while transcript signals were barely detectable in shoot and leaf tissues. Interestingly, wheat exhibited stronger expression of TaBDRO1-like than barley (HvDRO1-like), but TaBDRO1-like was the least expressing among three paralogs. The underlying cause of this expression divergence seems to be the presence of AuxRE motif TGTCTC and core TGTC with a coupling AuxRE-like motif ATTTTCTT proximal to the transcriptional start site in TaBDRO1-like and HvDRO1-like promoters. This is evident from binding of ARF1 to TGTCTC and TGTC motifs of TaBDRO1-like as revealed by yeast one-hybrid assay. Thus, evolution of DRO1-like wheat homoeologs might incorporate the C-terminus mutations as well as gain and loss of AuxREs and other cis-regulatory elements during expression divergence. Since root architecture is an important target trait for wheat crop improvement, therefore DRO1-like genes have potential applications in plant breeding for enhancement of plant productivity by the use of modern genome editing approaches.

Proteomic approach to address low seed germination in Cyclobalnopsis gilva.

Seeds play essential roles in plant life cycle and germination is a complex process which is associated with different phases of water imbibition. Upon imbibition, seeds begin utilization of storage substances coupled with metabolic activity and biosynthesis of new proteins. Regeneration of organelles and emergence of radicals lead to the establishment of seedlings. All these activities are regulated in coordinated manners. Translation is the requirement of germination of seeds via involvements of several proteins like beta-amylase, starch phosphorylase. Some important proteins involved in seed germination are discussed in this review. In the past decade, several proteomic studies regarding seed germination of various species such as rice, Arabidopsis have been conducted. We face A paucity of proteomic data with respect to woody plants e.g. Fagus, Pheonix etc. With particular reference to Cyclobalnopsis gilva, a woody plant having low seed germination rate, no proteomic studies have been conducted. The review aims to reveal the complex seed germination mechanisms from woody and herbaceous plants that will help in understanding different seed germination phases and the involved proteins in C. gilva.

Chilling tolerance in three tomato transgenic lines overexpressing CBF3 gene controlled by a stress inducible promoter.

Plants integrate and monitor low temperature signals to cope with the continual variations in their environment. Arabidopsis thaliana cold responsive-element binding factor 3 (AtCBF3) plays its role in various cellular activities by modulating multiple genes induced under chilling stress. In this work, AtCBF3 transcription was remarkably induced following chilling stress. AtCBF3-overexpressors namely AtCBF3-Rio Grande, AtCBF3-Moneymaker, and AtCBF3-Roma showed defensible response to various levels of chilling stress, while their isogenic wild type plants indicated hypersensitive response to chilling stress. Detailed photosynthetic studies revealed that AtCBF3 gene has harmonious influences on the expression of a large set of genes by virtue of improved stomatal conductance, transpiration rate, intercellular CO2 concentration, and photosynthetic rate compared to wild type plants. The AtCBF3 lines limited the water status-mediated hypersensitive response by lowering leaf osmotic potential due to overexpression of AtCBF3 under chilling stress. Biochemical analyses followed by phenotypic studies demonstrated that AtCBF3 plants exhibited membrane stability and lush green appearance by limiting membrane ions leakage and malondialdehyde contents and by accumulating more proline, soluble sugars, chlorophyll contents, carotenoid contents, and antioxidant enzymes relative to wild type plants. Hence, with a several lines of evidence, these findings support that tomato transgenic plants overexpressing Arabidopsis CBF3 show enhanced chilling tolerance.

What are farmers really planting? Measuring the presence and effectiveness of Bt cotton in Pakistan.

Genetically modified, insect-resistant Bacillus thuringiensis (Bt) cotton is cultivated extensively in Pakistan. Past studies, however, have raised concerns about the prevalence of Bt cotton varieties possessing weak or nonperforming insect-resistance traits conferred by the cry gene. We examine this issue using data drawn from a representative sample of cotton-growing households that were surveyed in six agroclimatic zones spanning 28 districts in Pakistan in 2013, as well as measurements of Cry protein levels in cotton tissue samples collected from the sampled households' main fields. The resultant dataset combines information from 593 sampled households with corresponding plant tissue diagnostics from 70 days after sowing, as well as information from 589 sampled households with corresponding diagnostics from 120 days after sowing. Our analysis indicates that 11 percent of farmers believed they were cultivating Bt cotton when, in fact, the Cry toxin was not present in the tested tissue at 70 days after sowing (i.e., a Type I error). The analysis further indicates that 5 percent of farmers believed they were cultivating non-Bt cotton when, in fact, the Cry toxin was present in the tested tissue (i.e., a Type II error). In addition, 17 percent of all sampled farmers were uncertain whether or not they were cultivating Bt cotton. Overall, 33 percent of farmers either did not know or were mistaken in their beliefs about the presence of the cry gene in the cotton they cultivated. Results also indicate that toxic protein levels in the plant tissue samples occurred below threshold levels for lethality in a significant percentage of cases, although these measurements may also be affected by factors related to tissue sample collection, handling, storage, and testing procedures. Nonetheless, results strongly suggest wide variability both in farmers' beliefs and in gene expression. Such variability has implications for policy and regulation in Pakistan's transgenic cotton seed market.

WSA206, a paralog of duplicated MPF2-like MADS-box family is recruited in fertility function in Withania.

Best known for their implication in calyx inflation, MPF2-like genes pertinent to the STMADS11 clade of the MADS-box family exert their functions in leaf development, flowering time, inflorescence architecture and floral reversion to just name but a few. However, our knowledge about their involvement in fertility function remained obscure. Therefore the major thrust of this study was to probe the recruitment of WSA206 (MPF2-like) protein in fertility function. The WSA206 functions were revealed by knocking down and overexpressing this protein in Withania somnifera. The WSA206 promoter functions were defined by stable integration in Arabidopsis using GUS tag. The interactions of WSA206 were investigated by screening Arabidopsis Oligo-dT yeast library and YFP-split analysis. WSA206 knockdown plants revealed fewer flowers, abortion in seed set, reduction in pollen number and deformed non-viable pollen in comparison with wild type counterparts. Overexpression of WSA206 in Withania generated more berries/seeds and healthier viable pollen grains. Remarkably, along with fertility control, the impairment in calyx inflation in knockdown Withania plants and extraordinary growth of sepals in overexpression lines is observed. Thus, fertility and calyx inflation are tightly coupled traits under the control of WSA206. Coding sequence revealed SNP mutations from arginine to lysine as well as a leucine-rich motif duplication at the C-terminus, a characteristic feature of pollen specific and fertility function proteins. The protein-protein interaction spectrum of WSA206 comprises 40% of those MADS and non-MADS-box proteins implicated in floral/anther expression and embryogenesis. Predominant WSA206 promoter:GUS expression accrued in the anthers/pollen may be attributed to of the presence of GAAATTGTTA pollen specific proximal motifs along with several other anther specific homotypic cis-clusters. MPF2-like protein WSA206 through interactions with MADS-box and non-MADS-domain proteins confers male fertility in Withania regulated by anther/pollen specific promoter motif GAAATTGTTA.

WsMAGO2, a duplicated MAGO NASHI protein with fertility attributes interacts with MPF2-like MADS-box proteins.

MAIN CONCLUSION: WsMAGO2 a duplicated protein in Withania through interactions with MPF2-like proteins affects male fertility by producing fewer flowers and aborted non-viable pollens/seeds regulated by anther-specific GAATTTGTGA motif. The MAGO NASHIs are highly conserved genes that encode proteins known to be involved in RNA physiology and many other developmental processes including germ cell differentiation in animals. However, their structural and functional implications in plants as fertility function proteins remained fragmented. MAGO (shorter name of MAGO NASHI) proteins form heterodimers with MPF2-like MADS-box proteins which are recruited in calyx identity and male fertility in Solanaceous plants. Four MAGO genes namely WsMAGO1 and WsMAGO2 and TaMAGO1 and TaMAGO2 were isolated from Withania somnifera and Tubocapsicum anomalum, respectively. These genes have duplicated probably due to whole genome duplication event. Dysfunction of WsMAGO2 through double-stranded RNAi in Withania revealed suppression of RNA transcripts, non-viable pollens, fewer flowers and aborted non-viable seeds in the developing berry suggesting a role of this protein in many traits particularly male fertility. WsMAGO2 flaunted stronger yeast 2-hybrid interactions with MPF2-like proteins WSA206, WSB206 and TAB201 than other MAGO counterparts. The native transcripts of WsMAGO2 culminated in stamens and seed-bearing berries though other MAGO orthologs also exhibited expression albeit at lower level. Coding sequences of the two orthologs are highly conserved, but they differ substantially in their upstream promoter regions. Remarkably, WsMAGO2 promoter is enriched with many anther-specific cis-motifs common in fertility function genes promoters. Among them, disruption of GAATTTGTGA abolished YFP/GUS gene expression in anthers alluding towards its involvement in regulating expression of MAGO in anther. Our findings support a possible recruitment of WsMAGO2 in fertility trait in Withania. These genes have practical application in hybrid production through cytoplasmic male sterility maintenance for enhancement in crops yield.