Beneath the rind: A review on the remarkable health benefits and applications of the wood apple fruit.

Limonia acidissima Groff, commonly referred to as the Wood apple, is a tropical fruit belonging to Rutaceae family. Indigenous to Sri Lanka, India, and Myanmar, it is extensively cultivated throughout Southeast Asia. This fruit holds a profound historical significance in traditional medicine due to its exceptional nutritional and therapeutic attributes. Wood apple pulp is significantly abundant in ß-carotene, a precursor to vitamin A, and contains a substantial amount of vitamin B, including riboflavin and thiamine, as well as trace amounts of ascorbic acid (vitamin C). Moreover health-benefitting properties associated with L. acidissima, such as, antioxidant, hepatoprotective, antimicrobial, neuroprotective, antidiabetic, anti-inflammatory, anti-spermatogenic, analgesic, antiulcer, and antihyperlipidemic properties, are attributed to a diverse range of phytochemicals. These encompass polyphenolic compounds, saponins, phytosterols, tannins, triterpenoids, coumarins, amino acids, tyramine derivatives, and vitamins. From the findings of the various studies, it was observed that wood apple fruit shows significant anticancer activity by inhibiting the proliferation of cancer. Furthermore, wood apple finds wide-ranging commercial applications in the formulation of ready-to-serve beverages, syrups, jellies, chutneys, and various other food products. In summary, this review highlights the nutritional and phytochemical constituents of wood apple, depicts its antioxidant, anti-inflammatory, and anti-diabetic capabilities, and explores its potential in value-added product development. Nevertheless, it is crucial to acknowledge that the molecular mechanisms supporting these properties remain an underexplored domain. To ensure the safe integration of wood apple fruit into the realms of the food, cosmetics, and pharmaceutical sectors, rigorous clinical trials, including toxicity assessments, are required. These endeavors hold the potential to promote innovation and contribute significantly to both research and industrial sectors.

Innovations in textile wastewater management: a review of zero liquid discharge technology.

Zero liquid discharge (ZLD) technology emerges as a transformative solution for sustainable wastewater management in the textile industry, emphasizing water recycling and discharge minimization. This review comprehensively explores ZLD's pivotal role in reshaping wastewater management practices within the textile sector. With a primary focus on water recycling and minimized discharge, the review thoroughly examines the economic and environmental dimensions of ZLD. Additionally, it includes a comparative cost analysis against conventional wastewater treatment methods and offers a comprehensive outlook on the global ZLD market. Presently valued at US $0.71 billion, the market is anticipated to reach US $1.76 billion by 2026, reflecting a robust annual growth rate of 12.6%. Despite ZLD's efficiency in wastewater recovery, environmental challenges, such as heightened greenhouse gas emissions, increased carbon footprint, elevated energy consumption, and chemical usage, are discussed. Methodologies employed in this review involve an extensive analysis of existing literature, empirical data, and case studies on ZLD implementation in the textile industry worldwide. While acknowledging existing adoption barriers, the review underscores ZLD's potential to guide the textile industry toward a more sustainable and environmentally responsible future.

Immunological depiction of synthetic B-cell epitopes of Mycobacterium tuberculosis.

Background: To combat the tuberculosis (TB) epidemic, the development of a better and faster diagnosis or more effective vaccine is essential. Pulmonary TB (PTB) is one of the major causes of morbidity and mortality. Early diagnosis of TB is difficult. Serological assays have been performed with several antigens of laboratory strains such as Mycobacterium tuberculosis H37Rv which have not been found to be highly sensitive. In the present study, various peptides were synthesized which were predicted on the basis of immunoreactivity and differential expression in clinical isolates of M. tuberculosis compared to their expression in a laboratory strain of M. tuberculosis. Therefore, the aim of this study was to compare the antibody levels in PTB and healthy controls against these peptides. Methods: An effort was made to evaluate antibody response to peptides derived from proteins Rv2588c, Rv0512, Rv0148, Rv0896, and Rv0635 of M. tuberculosis in PTB patients and healthy individuals through enzyme-linked immunosorbent assay. Five milliliters of venous blood samples was collected from each participant, and serum was separated and stored until use. Results: Antibody levels against these peptides, Rv2588c, Rv0512, Rv0148, Rv0896. and Rv0635 in 139 PTB patients and 52 healthy controls were evaluated. Higher immune response was observed in PTB patients when compared with healthy individuals. Strong immunoglobulin G responses with high percentage, considerable difference among patients and healthy controls was observed with P < 0.0001. Conclusion: In our study, we found significant statistical differences in antibody levels in PTB patients and healthy individuals against these peptides. These peptides are suggestive of being a potential new candidate (s) for early diagnosis of TB.

Phytochemicals, therapeutic benefits and applications of chrysanthemum flower: A review.

Chrysanthemum is a flowering plant belonging to a genus of the dicotyledonous herbaceous annual flowering plant of the Asteraceae (Compositae) family. It is a perpetual flowering plant, mostly cultivated for medicinal purposes; generally, used in popular drinks due to its aroma and flavor. It is primarily cultivated in China, Japan, Europe, and United States. These flowers were extensively used in various healthcare systems and for treating various diseases. Chrysanthemum flowers are rich in phenolic compounds and exhibit strong properties including antioxidant, antimicrobial, anti-inflammatory, anticancer, anti-allergic, anti-obesity, immune regulation, hepatoprotective, and nephroprotective activities. The main aim of the present review was to investigate the nutritional profile, phytochemistry, and biological activities of flowers of different Chrysanthemum species. Also, a critical discussion of the diverse metabolites or bioactive constituents of the Chrysanthemum flowers is highlighted in the present review. Moreover, the flower extracts of Chrysanthemum have been assessed to possess a rich phytochemical profile, including compounds such as cyanidin-3-O-(6″-O-malonyl) glucoside, delphinidin 3-O-(6" -O-malonyl) glucoside-3', rutin, quercetin, isorhamnetin, rutinoside, and others. These profiles exhibit potential health benefits, leading to their utilization in the production of supplementary food products and pharmaceutical drugs within the industry. However, more comprehensive research studies/investigations are still needed to further discover the potential benefits for human and animal utilization.

Data-driven approaches to improve water-use efficiency and drought resistance in crop plants.

With the increasing population, there lies a pressing demand for food, feed and fibre, while the changing climatic conditions pose severe challenges for agricultural production worldwide. Water is the lifeline for crop production; thus, enhancing crop water-use efficiency (WUE) and improving drought resistance in crop varieties are crucial for overcoming these challenges. Genetically-driven improvements in yield, WUE and drought tolerance traits can buffer the worst effects of climate change on crop production in dry areas. While traditional crop breeding approaches have delivered impressive results in increasing yield, the methods remain time-consuming and are often limited by the existing allelic variation present in the germplasm. Significant advances in breeding and high-throughput omics technologies in parallel with smart agriculture practices have created avenues to dramatically speed up the process of trait improvement by leveraging the vast volumes of genomic and phenotypic data. For example, individual genome and pan-genome assemblies, along with transcriptomic, metabolomic and proteomic data from germplasm collections, characterised at phenotypic levels, could be utilised to identify marker-trait associations and superior haplotypes for crop genetic improvement. In addition, these omics approaches enable the identification of genes involved in pathways leading to the expression of a trait, thereby providing an understanding of the genetic, physiological and biochemical basis of trait variation. These data-driven gene discoveries and validation approaches are essential for crop improvement pipelines, including genomic breeding, speed breeding and gene editing. Herein, we provide an overview of prospects presented using big data-driven approaches (including artificial intelligence and machine learning) to harness new genetic gains for breeding programs and develop drought-tolerant crop varieties with favourable WUE and high-yield potential traits.

Non-coding RNAs-mediated environmental surveillance determines male fertility in plants.

Plants are continuously exposed to environmental stresses leading to significant yield losses. With the changing climatic conditions, the intensity and duration of these stresses are expected to increase, posing a severe threat to crop productivity worldwide. Male gametogenesis is one of the most sensitive developmental stages. Exposure to environmental stresses during this stage leads to male sterility and yield loss. Elucidating the underlying molecular mechanism of environment-affected male sterility is essential to address this challenge. High-throughput RNA sequencing studies, loss-of-function phenotypes of sRNA biogenesis genes and functional genomics studies with non-coding RNAs have started to unveil the roles of small RNAs, long non-coding RNAs and the complex regulatory interactions between them in regulating male fertility under different growth regimes. Here, we discuss the current understanding of the non-coding RNA-mediated environmental stress surveillance and regulation of male fertility in plants. The candidate ncRNAs emerging from these studies can be leveraged to generate environment-sensitive male sterile lines for hybrid breeding or mitigate the impact of climate change on male fertility, as the situation demands.

Novel quantitative trait loci from an interspecific Brassica rapa derivative improve pod shatter resistance in Brassica napus.

Pod shatter is a trait of agricultural relevance that ensures plants dehisce seeds in their native environment and has been subjected to domestication and selection for non-shattering types in several broadacre crops. However, pod shattering causes a significant yield reduction in canola (Brassica napus L.) crops. An interspecific breeding line BC95042 derived from a B. rapa/B. napus cross showed improved pod shatter resistance (up to 12-fold than a shatter-prone B. napus variety). To uncover the genetic basis and improve pod shatter resistance in new varieties, we analysed F2 and F2:3 derived populations from the cross between BC95042 and an advanced breeding line, BC95041, and genotyped with 15,498 DArTseq markers. Through genome scan, interval and inclusive composite interval mapping analyses, we identified seven quantitative trait loci (QTLs) associated with pod rupture energy, a measure for pod shatter resistance or pod strength, and they locate on A02, A03, A05, A09 and C01 chromosomes. Both parental lines contributed alleles for pod shatter resistance. We identified five pairs of significant epistatic QTLs for additive x additive, additive dominance and dominance x dominance interactions between A01/C01, A03/A07, A07/C03, A03/C03, and C01/C02 chromosomes for rupture energy. QTL effects on A03/A07 and A01/C01 were in the repulsion phase. Comparative mapping identified several candidate genes (AG, ABI3, ARF3, BP1, CEL6, FIL, FUL, GA2OX2, IND, LATE, LEUNIG, MAGL15, RPL, QRT2, RGA, SPT and TCP10) underlying main QTL and epistatic QTL interactions for pod shatter resistance. Three QTLs detected on A02, A03, and A09 were near the FUL (FRUITFULL) homologues BnaA03g39820D and BnaA09g05500D. Focusing on the FUL, we investigated putative motifs, sequence variants and the evolutionary rate of its homologues in 373 resequenced B. napus accessions of interest. BnaA09g05500D is subjected to purifying selection as it had a low Ka/Ks ratio compared to other FUL homologues in B. napus. This study provides a valuable resource for genetic improvement for yield through an understanding of the genetic mechanism controlling pod shatter resistance in Brassica species.

Comparative transcriptome analysis reveals molecular regulation of salt tolerance in two contrasting chickpea genotypes.

Salinity is a major abiotic stress that causes substantial agricultural losses worldwide. Chickpea (Cicer arietinum L.) is an important legume crop but is salt-sensitive. Previous physiological and genetic studies revealed the contrasting response of two desi chickpea varieties, salt-sensitive Rupali and salt-tolerant Genesis836, to salt stress. To understand the complex molecular regulation of salt tolerance mechanisms in these two chickpea genotypes, we examined the leaf transcriptome repertoire of Rupali and Genesis836 in control and salt-stressed conditions. Using linear models, we identified categories of differentially expressed genes (DEGs) describing the genotypic differences: salt-responsive DEGs in Rupali (1,604) and Genesis836 (1,751) with 907 and 1,054 DEGs unique to Rupali and Genesis836, respectively, salt responsive DEGs (3,376), genotype-dependent DEGs (4,170), and genotype-dependent salt-responsive DEGs (122). Functional DEG annotation revealed that the salt treatment affected genes involved in ion transport, osmotic adjustment, photosynthesis, energy generation, stress and hormone signalling, and regulatory pathways. Our results showed that while Genesis836 and Rupali have similar primary salt response mechanisms (common salt-responsive DEGs), their contrasting salt response is attributed to the differential expression of genes primarily involved in ion transport and photosynthesis. Interestingly, variant calling between the two genotypes identified SNPs/InDels in 768 Genesis836 and 701 Rupali salt-responsive DEGs with 1,741 variants identified in Genesis836 and 1,449 variants identified in Rupali. In addition, the presence of premature stop codons was detected in 35 genes in Rupali. This study provides valuable insights into the molecular regulation underpinning the physiological basis of salt tolerance in two chickpea genotypes and offers potential candidate genes for the improvement of salt tolerance in chickpeas.

Acacia catechu (L.f.) Willd.: A Review on Bioactive Compounds and Their Health Promoting Functionalities.

With the advent of pandemics and infectious diseases, numerous research activities on natural products have been carried out to combat them. Researchers are investigating natural products for the treatment and/or management of various infectious diseases and/or disorders. Acacia catechu (L.f.) Willd. belongs to the family Fabaceae (subfamily Mimosoideae) known as Khair or Cutch tree, possesses diverse pharmacological actions, and has been widely used in Asia and different parts of the world. The purpose of the present study is to highlight the phytochemical profile of different parts of A. catechu, the different biological activities of A. catechu extract, and the utilization of A. catechu as food and beverage. The present work constitutes a review of A. catechu; we performed searches (books, Google, Google Scholar, and Scopus publications) to compile the work/investigations made on A. catechu to the present. From our survey, it was concluded that the main phytochemicals compounds in A. catechu are protocatechuic acid, taxifolin, epicatechin, epigallocatechin, catechin, epicatechin gallate, procyanidin, phloroglucin, aldobiuronic acid, gallic acid, D-galactose, afzelchin gum, L-arabinose, D-rhamnose, and quercetin. The whole plant of A. catechu possesses a comprehensive variety of medicinal potentials such as antimicrobial, antidiarrheal, antinociceptive, antihyperlipidemic, antiulcer, antioxidant, antidiabetic, antiproliferative, haemolytic, and anti-inflammatory properties due to the presence of bioactive compounds like flavonoids, alkaloids, and tannins. However, even though the plant's metabolites were reported to have many different pharmacological uses, there is limited information about their toxicity or clinical trials. Further research on diverse metabolites of A. catechu should be carried out to ensure the safety or utilization of this plant in the pharma or food industries and in the development of potent plant-based drugs.

Aegle marmelos (L.) Correa: An Underutilized Fruit with High Nutraceutical Values: A Review.

Aegle marmelos (L.) Correa (Bael) fruit, a member of the Rutaceae family, is a major cultivated fruit plant in tropical and subtropical regions in countries of southeast Asia. Bael fruit has been a major topic for studies in recent years mainly due to its high nutritional (carbohydrates, proteins, minerals, and vitamins) value and presence of various phytochemicals, which attributed to its high medicinal value. These phytochemicals include various compounds, e.g., alkaloids, flavonoids, and phenolic acids (protocatechuic acid, gallic, and ellagic acid). The fruit extract of bael has been also an important study area for its pharmacological activities, including antidiarrheal, antioxidant, antidiabetic, hepatoprotective, radioprotective, anticancer, antiulcer properties. The current review mainly highlighted the nutritional and pharmacological activities of bael fruit. The nutritional profile and phytochemical profile were discussed in the review, along with their concentration in the fruit. Moreover, the experiments carried out in vivo and in vitro of bael fruit extracts with respect to their pharmacological activities were also discussed in the article. The recent literature based on nutritional and pharmacological values of bael fruit showed its high potential as a food and pharmaceutical product. Despite having high nutritional and pharmacological value, research related to molecular mechanisms of bael fruit is still limited, and clinical trials are needed to ensure its safety as a product in the food and pharma industries.

Electrochemical, Photophysical, Morphological and DFT Study of Polymorphic Sn(IV)-Porphyrins Containing Fluorinated Axial Ligand.

In this study, we report the polymorphism of six coordinated Sn(IV)- tetrabromophenyl porphyrins axially armed with fluorine-substituted phenolate ligands (structural formula [Sn(TBrPP)2+ (A- )2 ], where A is the axial ligand=3,5-difluoro phenol, compound 1). One form stabilizes in triclinic system (namely, 1α), and the other stabilizes in monoclinic system (namely, 1ß). The two 1α and 1ß polymorphs display distinct photophysical and morphological properties in the solid state. X-ray diffraction study reveals that these polymorphs 1α and 1ß significantly differ in their supramolecular architecture, different axial phenolate conformations, and noncovalent interactions, which are responsible for their distinct solid-state properties. The crystal packing of these polymorphs dominates by intermolecular C-H⋅⋅⋅F, C-H⋅⋅⋅π and C-Br⋅⋅⋅F interhalogen interactions. Furthermore, the solid-state emission spectra of 1α showed red-shifted emission bands with respect to 1ß, in addition the redox behavior of 1α is slightly different in comparison to 1ß. Complementary theoretical studies with Hirshfeld surface analysis show the definite role of Br⋅⋅⋅F interhalogen interactions in the overall stability. Mapping the electrostatic potential isosurfaces with the aid of density functional theory in compound 1 clearly shows the presence of σ-hole, a requisite feature to show halogen interactions in the crystalline state. In addition, lattice energy and single point energy calculation shows that 1α was found to be energetically more favorable and thermodynamically more stable compare to 1ß.

Genome-Wide Association Analyses Track Genomic Regions for Resistance to Ascochyta rabiei in Australian Chickpea Breeding Germplasm.

Ascochyta blight (AB), caused by a necrotrophic fungus, Ascochyta rabiei (syn. Phoma rabiei) has the potential to destroy the chickpea industry worldwide, due to limited sources of genetic resistance in the cultivated gene pool, high evolutionary potential of the pathogen and challenges with integrated disease management. Therefore, the deployment of stable genetic resistance in new cultivars could provide an effective disease control strategy. To investigate the genetic basis of AB resistance, genotyping-by-sequencing based DArTseq-single nucleotide polymorphism (SNP) marker data along with phenotypic data of 251 advanced breeding lines and chickpea cultivars were used to perform genome-wide association (GWAS) analysis. Host resistance was evaluated seven weeks after sowing using two highly aggressive single spore isolates (F17191-1 and TR9571) of A. rabiei. GWAS analyses based on single-locus and multi-locus mixed models and haplotyping trend regression identified twenty-six genomic regions on Ca1, Ca4, and Ca6 that showed significant association with resistance to AB. Two haplotype blocks (HB) on chromosome Ca1; HB5 (992178-1108145 bp), and HB8 (1886221-1976301 bp) were associated with resistance against both isolates. Nine HB on the chromosome, Ca4, spanning a large genomic region (14.9-56.6 Mbp) were also associated with resistance, confirming the role of this chromosome in providing resistance to AB. Furthermore, trait-marker associations in two F3 derived populations for resistance to TR9571 isolate at the seedling stage under glasshouse conditions were also validated. Eighty-nine significantly associated SNPs were located within candidate genes, including genes encoding for serine/threonine-protein kinase, Myb protein, quinone oxidoreductase, and calmodulin-binding protein all of which are implicated in disease resistance. Taken together, this study identifies valuable sources of genetic resistance, SNP markers and candidate genes underlying genomic regions associated with AB resistance which may enable chickpea breeding programs to make genetic gains via marker-assisted/genomic selection strategies.

A survey on ethnoveterinary medicines used by the tribal migratory shepherds of Northwestern Himalaya.

ETHNOPHARMACOLOGICAL RELEVANCE: Tribal migratory shepherds (Gaddi) living in the Northwestern Himalayan region are well known for their nomadic lifestyle in which tribal migratory shepherds along with their livestock which mainly include sheep and goat migrate seasonally in the Northwestern Himalayan region from high hills to low hills for the survival of their livestock from cold winters and hot summer. Due to harsh environmental conditions, heavy snowfall, heavy rainfall, wild animal attacks, no medical facilities, no transportation, and no electricity facilities tribal migratory shepherds mostly rely on plant species mentioned for ethnoveterinary use to treat livestock disease. AIM OF STUDY: The aim of our study is to conduct ethnoveterinary survey for the first time to document the contemporary oral ethnoveterinary knowledge of Gaddi shepherds living in Northwestern Himalayan region. METHODOLOGY: The documentation of plant species mentioned for ethnoveterinary use was executed through extensive field surveys from 2017 to 2019. Data were collected through direct interviews by administrating questionnaire among tribal migratory shepherds. RESULTS: A total of 181 plant species mentioned for ethnoveterinary use belonging to the same or different families were documented during the seasonal migration of shepherds from Northwestern Himalayan region. Most frequently occurring family of plant species mentioned for ethnoveterinary was Poaceae, leaves and roots were reported to be the most frequently used plant parts. Most frequently documented plant species for ethnoveterinary use were Cynodon dactylon, Chenopodium album, Zanthoxylum armatum, Aloe vera, Azadirachta indica and Cannabis sativa. Chrysopogon serrulatus, Cynodon dactylon, and Vitex negundo were reported with high use reports. Some of the endemic species of Northwestern Himalayan region such as Elymus himalayanus and Euphorbia pilosa and one endangered species Picrorhiza kurrooa were reported in current study. CONCLUSION: It is observed that there is a need to raise awareness among the tribal migratory shepherds about the sustainable use and conservation of some of the plant species mentioned for ethnoveterinary use. This study provided an inventory of plant species mentioned for ethnoveterinary use having significant ethnoveterinary potential, however there is need of scientific evaluations by in vitro, in vivo and clinical studies.

Multienvironment QTL analysis delineates a major locus associated with homoeologous exchanges for water-use efficiency and seed yield in canola.

Canola varieties exhibit variation in drought avoidance and drought escape traits, reflecting adaptation to water-deficit environments. Our understanding of underlying genes and their interaction across environments in improving crop productivity is limited. A doubled haploid population was analysed to identify quantitative trait loci (QTL) associated with water-use efficiency (WUE) related traits. High WUE in the vegetative phase was associated with low seed yield. Based on the resequenced parental genome data, we developed sequence-capture-based markers and validated their linkage with carbon isotope discrimination (Δ13 C) in an F2 population. RNA sequencing was performed to determine the expression of candidate genes underlying Δ13 C QTL. QTL contributing to main and QTL × environment interaction effects for Δ13 C and yield were identified. One multiple-trait QTL for Δ13 C, days to flower, plant height, and seed yield was identified on chromosome A09. Interestingly, this QTL region overlapped with a homoeologous exchange (HE) event, suggesting its association with the multiple traits. Transcriptome analysis revealed 121 significantly differentially expressed genes underlying Δ13 C QTL on A09 and C09, including in HE regions. Sorting out the negative relationship between vegetative WUE and seed yield is a priority. Genetic and genomic resources and knowledge so developed could improve canola WUE and yield.

A Major Locus on Wheat Chromosome 7B Associated With Late-Maturity α-Amylase Encodes a Putative ent-Copalyl Diphosphate Synthase.

Many wheat varieties have the potential to develop unacceptably high levels of α-amylase in the grains if exposed to a cool temperature shock or simply cool temperature during the early to middle stages of grain filling. This phenomenon is referred to as late maturity α-amylase (LMA). The enzyme persists in the grain until harvest and may result in wheat with a low Falling Number that does not meet receival and export specifications. Resistance to LMA is therefore a valuable target for wheat breeders and wheat industries in general. Genetic evidence implicating a locus on the long arm of chromosome 7B in variation in LMA phenotype was confirmed in this investigation. Through intensive fine-mapping an ent-copalyl diphosphate synthase (CPS), hitherto named LMA-1, was identified as the likely candidate gene associated with variation in LMA phenotype. Single Nucleotide Polymorphisms (SNPs) within the LMA-1 coding sequence of Chinese Spring, Maringa and Halberd result in either prematurely terminated or functionally altered proteins that are associated with useful levels of resistance to LMA. LMA-1 transcripts detected in de-embryonated grain tissue from around 15 days after anthesis, several days before the synthesis of α-amylase, were low in the resistant varieties Chinese Spring and Maringa compared with LMA susceptible genotype Spica. This was associated with a dramatic reduction in the concentrations of intermediates in the gibberellin biosynthesis pathway such as GA19, evidence that LMA-1 was functioning as CPS in the gibberellin biosynthesis pathway. A survey of a large collection of Australian and international wheat varieties distinguished 9 major haplotypes at the LMA-1 locus. Generally, within classes, there was notable variation for LMA phenotype and evidence for genotypes whose resistance is presumed to be due to genetic loci located elsewhere on the wheat genome. Further investigation is required to characterize the sequence of steps between LMA-1 and α-amylase synthesis as well as to gain a better understanding of the role and potential impact of other genetic loci. Diagnostic markers for sources of resistance and SNP variation reported in this study should assist breeders to deploy resistance associated with LMA-1 variants in breeding programs.

Improving Nitrogen Use Efficiency Through Overexpression of Alanine Aminotransferase in Rice, Wheat, and Barley.

Nitrogen is an essential nutrient for plants, but crop plants are inefficient in the acquisition and utilization of applied nitrogen. This often results in producers over applying nitrogen fertilizers, which can negatively impact the environment. The development of crop plants with more efficient nitrogen usage is, therefore, an important research goal in achieving greater agricultural sustainability. We utilized genetically modified rice lines over-expressing a barley alanine aminotransferase (HvAlaAT) to help characterize pathways which lead to more efficient use of nitrogen. Under the control of a stress-inducible promoter OsAnt1, OsAnt1:HvAlaAT lines have increased above-ground biomass with little change to both nitrate and ammonium uptake rates. Based on metabolic profiles, carbon metabolites, particularly those involved in glycolysis and the tricarboxylic acid (TCA) cycle, were significantly altered in roots of OsAnt1:HvAlaAT lines, suggesting higher metabolic turnover. Moreover, transcriptomic data revealed that genes involved in glycolysis and TCA cycle were upregulated. These observations suggest that higher activity of these two processes could result in higher energy production, driving higher nitrogen assimilation, consequently increasing biomass production. Other potential mechanisms contributing to a nitrogen-use efficient phenotype include involvements of phytohormonal responses and an alteration in secondary metabolism. We also conducted basic growth studies to evaluate the effect of the OsAnt1:HvAlaAT transgene in barley and wheat, which the transgenic crop plants increased seed production under controlled environmental conditions. This study provides comprehensive profiling of genetic and metabolic responses to the over-expression of AlaAT and unravels several components and pathways which contribute to its nitrogen-use efficient phenotype.

Efficient lead preconcentration using two chemically functionalized carbon nanotubes in hyphenated flow injection-flame atomic absorption spectrometry system.

Lead contamination in drinking and natural water has reached alarming concentrations, thus necessitating the development of accurate and rapid determination systems for Pb(II) in aqueous systems. Two hyphenated flow injection-solid phase extraction- FAAS (FI-SPE-FAAS) systems using oxidized and m-phenylenediamine functionalized multiwalled carbon nanotubes for Pb(II) preconcentration from industrially contaminated real water samples have been proposed. The chemical and hydrodynamic parameters affecting Pb(II) sorption/desorption were optimized. The effect of common interfering ions in water was also studied. Different figures of merit such as preconcentration factor (> 70), detection limit (≤ 1.5 µg L-1), and relative standard deviation (≤ 1.3%) were achieved at the preconcentration time of 120 s for both the preconcentration systems. The method was applied to industrially contaminated real water samples and the spike recovery tests were carried out using standard Pb(II) solution traceable to NIST. The proposed method was validated using standard reference material 1640a supplied by NIST Gaithersburg, MD, USA.

gRNA validation for wheat genome editing with the CRISPR-Cas9 system.

BACKGROUND: The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate. RESULTS: The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat. CONCLUSIONS: We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

CRISPR/Cas9-mediated knockout of Ms1 enables the rapid generation of male-sterile hexaploid wheat lines for use in hybrid seed production.

The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male-sterile lines were potentially non-transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.

Allelic variants of OsSUB1A cause differential expression of transcription factor genes in response to submergence in rice.

BACKGROUND: Flooding during seasonal monsoons affects millions of hectares of rice-cultivated areas across Asia. Submerged rice plants die within a week due to lack of oxygen, light and excessive elongation growth to escape the water. Submergence tolerance was first reported in an aus-type rice landrace, FR13A, and the ethylene-responsive transcription factor (TF) gene SUB1A-1 was identified as the major tolerance gene. Intolerant rice varieties generally lack the SUB1A gene but some intermediate tolerant varieties, such as IR64, carry the allelic variant SUB1A-2. Differential effects of the two alleles have so far not been addressed. As a first step, we have therefore quantified and compared the expression of nearly 2500 rice TF genes between IR64 and its derived tolerant near isogenic line IR64-Sub1, which carries the SUB1A-1 allele. Gene expression was studied in internodes, where the main difference in expression between the two alleles was previously shown. RESULTS: Nineteen and twenty-six TF genes were identified that responded to submergence in IR64 and IR64-Sub1, respectively. Only one gene was found to be submergence-responsive in both, suggesting different regulatory pathways under submergence in the two genotypes. These differentially expressed genes (DEGs) mainly included MYB, NAC, TIFY and Zn-finger TFs, and most genes were downregulated upon submergence. In IR64, but not in IR64-Sub1, SUB1B and SUB1C, which are also present in the Sub1 locus, were identified as submergence responsive. Four TFs were not submergence responsive but exhibited constitutive, genotype-specific differential expression. Most of the identified submergence responsive DEGs are associated with regulatory hormonal pathways, i.e. gibberellins (GA), abscisic acid (ABA), and jasmonic acid (JA), apart from ethylene. An in-silico promoter analysis of the two genotypes revealed the presence of allele-specific single nucleotide polymorphisms, giving rise to ABRE, DRE/CRT, CARE and Site II cis-elements, which can partly explain the observed differential TF gene expression. CONCLUSION: This study identified new gene targets with the potential to further enhance submergence tolerance in rice and provides insights into novel aspects of SUB1A-mediated tolerance.