Are TaNAC Transcription Factors Involved in Promoting Wheat Yield by cis-Regulation of TaCKX Gene Family?

NAC transcription factors (TFs) are one of the largest TF families in plants, and TaNACs have been known to participate in the regulation of the transcription of many yield-regulating genes in bread wheat. The TaCKX gene family members (GFMs) have already been shown to regulate yield-related traits, including grain mass and number, leaf senescence, and root growth. The genes encode cytokinin (CK) degrading enzymes (CKXs) and are specifically expressed in different parts of developing wheat plants. The aim of the study was to identify and characterize TaNACs involved in the cis-regulation of TaCKX GFMs. After analysis of the initial transcription factor data in 1.5 Kb cis-regulatory sequences of a total of 35 homologues of TaCKX GFMs, we selected five of them, namely TaCKX1-3A, TaCKX22.1-3B, TaCKX5-3D, TaCKX9-1B, and TaCKX10, and identified five TaNAC genes: TaNACJ-1, TaNAC13a, TaNAC94, TaNACBr-1, and TaNAC6D, which are potentially involved in the cis-regulation of selected TaCKX genes, respectively. Protein feature analysis revealed that all of the selected TaNACs have a conserved NAC domain and showed a stable tertiary structure model. The expression profile of the selected TaNACs was studied in 5 day-old seedling roots, 5-6 cm inflorescences, 0, 4, 7, and 14 days-after-pollination (DAP) spikes, and the accompanying flag leaves. The expression pattern showed that all of the selected TaNACs were preferentially expressed in seedling roots, 7 and 14 DAP spikes, and flag leaves compared to 5-6 cm inflorescence and 0 and 4 DAP spikes and flag leaves in Kontesa and Ostka spring wheat cultivars (cvs.). In conclusion, the results of this study highlight the potential role of the selected TaNACs in the regulation of grain productivity, leaf senescence, root growth, and response to various stresses.

Over-expression of GhACTIN1 under the control of GhSCFP promoter improves cotton fiber and yield.

Actin dynamics is pivotal in controlling cotton fiber elongation and the onset of secondary wall biosynthesis. We report that overexpression of GhACTIN1 under fiber fiber-specific promoter, GhSCFP, improves cotton fiber length, strength, and micronaire value. However, the effect of transgene has a more positive effect on fiber strength and micronaire value than fiber length. F-actin quantification and cellulose contents measurement in transgenic developing cotton fiber during the elongation phase showed an increase of up to 8.7% and 4.7% respectively. Additionally, physiological factors such as water use efficiency showed no significant change in transgenic cotton lines, while stomatal conductance and photosynthetic rate were significantly increased. Moreover, agronomical data determined that lint percentage (GOT) and seed cotton yield also increased up to 4.6% and 29.5% respectively, in transgenic cotton lines compared to the control lines. Our data demonstrate that the GhACTIN1 gene is a strong candidate gene for cotton fiber and yield improvement.

Role of Actin Dynamics and GhACTIN1 Gene in Cotton Fiber Development: A Prototypical Cell for Study.

Cotton crop is considered valuable for its fiber and seed oil. Cotton fiber is a single-celled outgrowth from the ovule epidermis, and it is a very dynamic cell for study. It has four distinct but overlapping developmental stages: initiation, elongation, secondary cell wall synthesis, and maturation. Among the various qualitative characteristics of cotton fiber, the important ones are the cotton fiber staple length, tensile strength, micronaire values, and fiber maturity. Actin dynamics are known to play an important role in fiber elongation and maturation. The current review gives an insight into the cotton fiber developmental stages, the qualitative traits associated with cotton fiber, and the set of genes involved in regulating these developmental stages and fiber traits. This review also highlights some prospects for how biotechnological approaches can improve cotton fiber quality.

Tissue specific expression of bacterial cellulose synthase (Bcs) genes improves cotton fiber length and strength.

Fiber growing inside the cotton bolls is a highly demandable product and its quality is key to the success of the textile industry. Despite the various efforts to improve cotton fiber staple length Pakistan has to import millions of bales to sustain its industrial needs. To improve cotton fiber quality Bacterial cellulose synthase (Bcs) genes (acsA, acsB) were expressed in a local cotton variety CEMB-00. In silico studies revealed a number of conserved domains both in the cotton-derived and bacterial cellulose synthases which are essential for the cellulose synthesis. Transformation efficiency of 1.27% was achieved by using Agrobacterium shoot apex cut method of transformation. The quantitative mRNA expression analysis of the Bcs genes in transgenic cotton fiber was found to be many folds higher during secondary cell wall synthesis stage (35 DPA) than the expression during elongation phase (10 DPA). Average fiber length of the transgenic cotton plant lines S-00-07, S-00-11, S-00-16 and S-00-23 was calculated to be 13.02% higher than that of the non-transgenic control plants. Likewise, the average fiber strength was found to be 20.92% higher with an enhanced cellulose content of 22.45%. The mutated indigenous cellulose synthase genes of cotton generated through application of CRISPR/Cas9 resulted in 6.03% and 12.10% decrease in fiber length and strength respectively. Furthermore, mature cotton fibers of transgenic cotton plants were found to have increased number of twists with smooth surface as compared to non-transgenic control when analyzed under scanning electron microscope. XRD analysis of cotton fibers revealed less cellulose crystallinity index in transgenic cotton fibers as compared to control fibers due to deposition of more amorphous cellulose in transgenic fibers as a result of Bcs gene expression. This study paved the way towards unraveling the fact that Bcs genes influence cellulose synthase activity and this enzyme helps in determining the fate of cotton fiber length and strength.

Cis-Regulation by NACs: A Promising Frontier in Wheat Crop Improvement.

Crop traits are controlled by multiple genes; however, the complex spatio-temporal transcriptional behavior of genes cannot be fully understood without comprehending the role of transcription factors (TFs) and the underlying mechanisms of the binding interactions of their cis-regulatory elements. NAC belongs to one of the largest families of plant-specific TFs and has been associated with the regulation of many traits. This review provides insight into the cis-regulation of genes by wheat NACs (TaNACs) for the improvement in yield-related traits, including phytohormonal homeostasis, leaf senescence, seed traits improvement, root modulation, and biotic and abiotic stresses in wheat and other cereals. We also discussed the current potential, knowledge gaps, and prospects of TaNACs.

Barley Resistance to Fusarium graminearum Infections: From Transcriptomics to Field with Food Safety Concerns.

Global climate change and the urgency to transform food crops require substantial breeding efforts to meet the food security challenges. Barley, an important cereal, has remained a preferential host of phytotoxic diseases caused by the Fusarium graminearum that not only severely reduces the crop yield but also compromises its food quality due to the accumulation of mycotoxins. To develop resistance against Fusarium infections, a better understanding of the host-pathogen interaction is inevitable and could be tracked through molecular insights. Here, we focused precisely on the potential gene targets that are exclusive to this devastating pathosystem and could be harnessed for fast breeding of barley. We also discuss the eco-friendly applications of nanobio hybrid and the CRISPR technology for barley protection. This review covers the critical information gaps within the subject and may be useful for the sustainable improvement of barley from the perspective of food and environmental safety concerns.

Assessment of COVID-19 vaccine booster dose acceptance, reluctance and concerns among elderly diabetes patients in Pakistan.

People around the globe rumored so many things about the safety and efficacy of initial two doses and booster dose of Covid-19 vaccine, which eventually affected the acceptance of the only tool available against the fight between humans and virus. The aim of current study is to evaluate the acceptance and reluctance level among the population specifically elderly diabetes patients. The cross-sectional study was conducted during a time period of 3 months i.e. from July 2021 until September 2021. A 16-item questionnaire was used to assess the acceptance, reluctance and concerns of the Covid-19 vaccine booster dose among elderly diabetes patients. A 16-item questionnaire was used to assess the acceptance, reluctance and concerns of the Covid-19 vaccine booster dose among elderly diabetes patients. A total of 497 responses were collected and analyzed. Approximately 32% of respondents believed that they need additional information about the Covid-19 vaccine booster dose will be ineffective due to not enough information about the potential side effects of the vaccine dose, while around 80% of respondents showed concerns about safety, efficacy, newness and not enough information about vaccine contents. Around 47.1% of respondents expressed robust concerns about possible side effects of the Covid-19 vaccine booster dose.

Engineered resistance and risk assessment associated with insecticidal and weeds resistant transgenic cotton using wister rat model.

Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and weeds is a promising tool to save crop from losses. Transgenic cotton variety, VH-289, with double Bt and cp4EPSPS genes under the control of 35S promoter was used for the expression analyses and biosafety studies. The transgenic cotton plants were screened through PCR amplification of fragments, 1.7 kb for Cry1Ac, 582 bp for Cry2A and 250 bp for cp4EPSPS; which confirmed the presence of all genes transformed in transgenic cotton. The Cry1Ac + Cry2A and cp4EPSPS proteins were quantified through ELISA in transgenic cotton plants. The Glyphosate assay performed by spraying 1900 mL per acre of glyphosate Roundup further confirmed complete survival of transgenic cotton plants as compared to the non-transgenic cotton plants and all weeds. Similarly, insect infestation data determined that almost 99% insect mortality was observed in controlled field grown transgenic cotton plants as compared to the non-transgenic control plants. Evaluation of effect of temperature and soil nutrients availability on transgene expression in cotton plants was done at two different cotton growing regions, Multan and Lahore, Pakistan and results suggested that despite of higher temperature in Multan field, an increased level of Cry and cp4EPSPS proteins was recorded due to higher soil organic matter availability compared to Lahore field. Before commercialization of any transgenic variety its biosafety study is mandatory so, a 90 days biosafety study of the transgenic cotton plants with 40% transgenic cottonseeds in standard diet showed no harmful effect on wister rat model when studied for liver function, renal function and serum electrolyte.

Cepharanthine action in preventing obesity and hyperlipidemia in rats on a high-fat high sucrose diet.

Background: It was demonstrated that cepharanthine (CEP), derived from Stephania cepharantha hayata, is a potent inhibitor of the ABCC10 transmembrane protein. It is approved to be a natural product or remedy. The present study focuses on investigating whether cepharanthine effectively reduces hyperlipidemia and obesity in an experimental hyperlipidemic rat model. Method: Four groups of Wistar rats were assigned randomly to the following groups: a high-fat high sucrose diet (HFHS), normal-fat diet (NFD), HFHS plus cepraranthine (10 mg/kg) (HFHS-C), and a HFHS diet with atorvastatin (HFHS-A). The responses of rats were observed on the basis of serum and hepatic biochemical parameters, food intake, and body weight after CEP treatment, and assessing the histopathological modifications by the optical microscope in the liver and its cells. Results: Significant improvement in the serum total cholesterol (TC), serum triglycerides (TG), and serum low-density lipoprotein (LDL) levels were observed following CEP treatment. We have also observed significant improvement in the structure of liver tissue and reduced-fat droplets in the cytoplasm. Moreover, CEP had a significant effect in preventing the gain in body weight of animals, and food intake was not significantly affected. Conclusion: Our research results revealed that CEP significantly improved dyslipidemia and prevented the accumulation of fatty deposits in the rats' liver tissue fed an HFHS diet. In addition, CEP exerted an anti-obesity effect.

Assessment of empagliflozin add-on therapy to metformin and glimepiride in patients with inadequately controlled type-2 diabetes mellitus.

Diabetes mellitus is a multifaceted metabolic disorder, which often required frequent blood glucose monitoring, poly-pharmacy and timely adjustments for its management. The present study focuses to check the effectiveness of empagliflozin add-on therapy in diabetic patients already taking metformin and glimepiride. This was observational, comparative and follow-up cohort study, conducted in a tertiary care hospital of Pakistan. Ninety subjects were enrolled and evenly distributed in Group A (patients on oral therapy of Metformin & Glimepiride) and Group B (patients on oral therapy of metformin, glimepiride and empagliflozin) randomly. The results showed that the addition of empagliflozin to metformin and glimepiride standard therapy provided better control over blood sugar with a significant decrease in HbA1c (16.1% decrease in HbA1c for Group B patients against 8.2% in Group A patients), FBS (23.8% decrease as compared to 14.6% decrease) and BMI (1.5% decrease in Group B patients against 0.06% increase in Group A). The addition of empagliflozin did not exacerbate the toxicity of the existing regimen and is safe to be included in multiple drug regimens. Empagliflozin addition to standard antidiabetic therapy might possess beneficial impacts in managing poorly controlled Type-2 Diabetes Mellitus in the Pakistani population.

Biochemical evidence of epicuticular wax compounds involved in cotton-whitefly interaction.

Sucking insects require a surface of plants on which the legs and the eggs of insects will adhere and to which insect mouthparts will access. The primary plant protection against insects is their surface property, which hinders the attachment of the insect's legs and eggs. The epicuticular waxes chemistry influences the fine structure of the cuticular surface. In current study, an attempt was made to investigate the variation of chemical compounds in epicuticular waxes of four cotton species that classify them resistant or susceptible i.e., Gossypium abroreum, G. hirsutum, G. arboreum wax deficient mutant (GaWM3) and G. harknessi which were evaluated for their interaction with whitefly and CLCuV transmission. Gossypium hirsutum an insect and CLCuV susceptible cotton variety, was found to have four compounds namely Trichloroacetic acid, hexadecylester, P-xylenolpthalein, 2-cyclopentene-1-ol, 1-phenyl-and Phenol, 2,5-bis [1,1- dimethyl] which could interact with chitin of whitefly while only two compounds in Gossypium arboreum an insect and CLCuV resistant cotton variety could interact with chitin of whitefly. Similarly, GaWM3 and Gossypium harkasnessi were found to have only a single compound. Number of whiteflies found on leaves of G. hirsutum was much higher as compared to other cotton species. Keeping this fact in mind a wax biosynthetic gene CER3, from Arabidopsis thaliana was transformed into G. hirsutum and the plants were evaluated for their resistance against whitefly and CLCuV transmission. In microscopic analysis transgenic plants clearly showed higher amounts of leaf waxes as compared to non-transgenics. The least whitefly population and CLCuV titer of <10,000 units was found in transgenic plants compared to non-transgenic cotton where it was ≈4.5X106 units that confirmed the role of wax in insect interaction and ultimately to CLCuV transmission. This study provides novel insight on wax related compounds involved in cotton-whitefly interaction, which potentially can help in developing more efficient control strategies for this destructive pest.

Overexpression of a Sucrose Synthase Gene Indirectly Improves Cotton Fiber Quality Through Sucrose Cleavage.

The study aims to improve fiber traits of local cotton cultivar through genetic transformation of sucrose synthase (SuS) gene in cotton. Sucrose synthase (SuS) is an important factor that is involved in the conversion of sucrose to fructose and UDP-glucose, which are essential for the synthesis of cell wall cellulose. In the current study, we expressed a synthetic SuS gene in cotton plants under the control of a CaMV35S promoter. Amplification of an 813-bp fragment using gene-specific primers confirmed the successful introduction of SuS gene into the genome of cotton variety CEMB-00. High SuS mRNA expression was observed in two transgenic cotton plants, MA0023 and MA0034, when compared to the expression in two other transgenic cotton plants, MA0035 and MA0038. Experiments showed that SuS mRNA expression was positively correlated with SuS activity at the vegetative (54%) and reproductive stages (40%). Furthermore, location of transgene was found to be at chromosome no. 9 in the form of single insertion, while no signal was evident in non-transgenic control cotton plant when evaluated through fluorescent in situ hybridization and karyotyping analysis. Fiber analyses of the transgenic cotton plants showed increases of 11.7% fiber length, 18.65% fiber strength, and up to 5% cellulose contents. An improvement in the micronaire value of 4.21 was also observed in the MA0038 transgenic cotton line. Scanning electron microscopy (SEM) revealed that the fibers of the SuS transgenic cotton plants were highly spiral with a greater number of twists per unit length than the fibers of the non-transgenic control plants. These results determined that SuS gene expression influenced cotton fiber structure and quality, suggesting that SuS gene has great potential for cotton fiber quality improvement.

Constitutive expression of Asparaginase in Gossypium hirsutum triggers insecticidal activity against Bemisia tabaci.

Whitefly infestation of cotton crop imparts enormous damage to cotton yield by severely affecting plant health, vigour and transmitting Cotton Leaf Curl Virus (CLCuV). Genetic modification of cotton helps to overcome both the direct whitefly infestation as well as CLCuV based cotton yield losses. We have constitutively overexpressed asparaginase (ZmASN) gene in Gossypium hirsutum to overcome the cotton yield losses imparted by whitefly infestation. We achieved 2.54% transformation efficiency in CIM-482 by Agrobacterium-mediated shoot apex transformation method. The relative qRT-PCR revealed 40-fold higher transcripts of asparaginase in transgenic cotton line vs. non-transgenic cotton lines. Metabolic analysis showed higher contents of aspartic acid and glutamic acid in seeds and phloem sap of the transgenic cotton lines. Phenotypically, the transgenic cotton lines showed vigorous growth and height, greater number of bolls, and yield. Among six representative transgenic cotton lines, line 14 had higher photosynthetic rate, stomatal conductance, smooth fiber surface, increased fiber convolutions (SEM analysis) and 95% whitefly mortality as compared to non-transgenic cotton line. The gene integration analysis by fluorescence in situ hybridization showed single copy gene integration at chromosome number 1. Collectively, asparaginase gene demonstrated potential to control whitefly infestation, post-infestation damages and improve cotton plant health and yield: a pre-requisite for farmer's community.

Screening of water distilled Rosa damascena Mill. flowers as hematopoietic agent in an animal model.

Pancytopenia is classified as low blood cell count. Low levels of hemoglobin, red blood cells, white blood cells and platelets are indicative of pancytopenic state. This pancytopenic state can be treatment (drug) or disease induced. Conventional approaches available to treat pancytopenia are usually associated with many undesirable adverse effects, are costly and parenterally administered. Interest in natural products has significantly increased due to their ability to stimulate cellular components of immune system. This study is designed to investigate the hematopoietic i.e. erythropoeitic, leucopoietic and thrombopoeitic potential of water distilled flowers of Rosa damascena Mill.

Medicinal Plants: A Complementary and Alternative Antidepressant Therapy.

There is enough data available now to believe that nature has provided cure of almost every ailment through herbal medicine or management. Therefore, now there is lot of emphasis on identification, evaluation, development and characterization of numerous plants and their active constituents against several diseases including depression. Depression is not only one of the most common ailments but also a highly complex condition to study. Even though several antidepressant drugs are available now, yet their effectiveness and usefulness are highly questionable especially because of their side effects. As herbal remedies are generally associated with favourable safety profiles therefore they have the possible potential to deliver effective replacements to currently available synthetic antidepressants. More recently, efforts have been focused on characterization of pharmacologically active ingredients and to identify the mode of action of herbal antidepressant medicines. This review describes a brief introduction of different animal models for depression and discusses the advantages and disadvantages for each approach. Then we have summarized possible plant phytochemicals as antidepressant drug and their underlying mechanisms. In the main body of the review, we have discussed in detail the most frequently used plants (21) being investigated for the treatment of depression. Additionally, we have provided the list of medicinal plants (92) representing their origin, parts used, extraction method, evaluation method and possible active ingredient. In the final part of the review we have presented the summary of clinical trials on the use of medical plants for depression and their active constituents.

Creation of Nepal's First Skin Bank: Challenges and Outcomes.

BACKGROUND: In Nepal, burn trauma causes more than 55,000 injuries each year. Burn-related mortality is high in Nepal, in part due to lack of allograft, leading to high infection rates. To address this challenge, our collaboration between Kirtipur Hospital, America Nepal Medical Foundation, Stanford University, and ReSurge International established Nepal's first skin bank. METHODS: We identified 3 major tasks to create a sustainable skin banking program: 1) identify and acquire the equipment and personnel needed to collect, process, store, and graft cadaveric skin for burn injuries; 2) develop safe donation protocols and documentation tools that remain feasible for low-resource settings; and 3) develop a long-term awareness program to educate the Nepali people on skin donation, a previously foreign concept. RESULTS: Kirtipur Hospital acquired the necessary equipment and materials for the skin bank through a combination of local and international fundraising efforts. Existing U.S. skin banking protocols were adapted for the Nepali setting and piloted on potential patients, donors, and physicians. For the first time in the hospital's history, patients with > 40% total body surface area burns were successfully treated with extensive allografts. CONCLUSIONS: It is feasible to create a skin bank in a country with no tradition of allograft skin use. Long-term sustainability now depends on spreading awareness and education in the Kathmandu Valley to overcome religious and cultural barriers that have hindered donor recruitment. Our low-cost and high-impact skin bank provides a model to expand this system to other hospitals both within Nepal and beyond.

Chloroplast localization of Cry1Ac and Cry2A protein--an alternative way of insect control in cotton.

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.