Transgenic chickpea (Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit.

BACKGROUND: Chickpea (Cicer arietinum L.) is the second most widely grown pulse and drought (limiting water) is one of the major constraints leading to about 40-50% yield losses annually. Dehydration responsive element binding proteins (DREBs) are important plant transcription factors that regulate the expression of many stress-inducible genes and play a critical role in improving the abiotic stress tolerance. Transgenic chickpea lines harbouring transcription factor, Dehydration Responsive Element-Binding protein 1A from Arabidopsis thaliana (AtDREB1a gene) driven by stress inducible promoter rd29a were developed, with the intent of enhancing drought tolerance in chickpea. Performance of the progenies of one transgenic event and control were assessed based on key physiological traits imparting drought tolerance such as plant water relation characteristics, chlorophyll retention, photosynthesis, membrane stability and water use efficiency under water stressed conditions. RESULTS: Four transgenic chickpea lines harbouring stress inducible AtDREB1a were generated with transformation efficiency of 0.1%. The integration, transmission and regulated expression were confirmed by Polymerase Chain Reaction (PCR), Southern Blot hybridization and Reverse Transcriptase polymerase chain reaction (RT-PCR), respectively. Transgenic chickpea lines exhibited higher relative water content, longer chlorophyll retention capacity and higher osmotic adjustment under severe drought stress (stress level 4), as compared to control. The enhanced drought tolerance in transgenic chickpea lines were also manifested by undeterred photosynthesis involving enhanced quantum yield of PSII, electron transport rate at saturated irradiance levels and maintaining higher relative water content in leaves under relatively severe soil water deficit. Further, lower values of carbon isotope discrimination in some transgenic chickpea lines indicated higher water use efficiency. Transgenic chickpea lines exhibiting better OA resulted in higher seed yield, with progressive increase in water stress, as compared to control. CONCLUSIONS: Based on precise phenotyping, involving non-invasive chlorophyll fluorescence imaging, carbon isotope discrimination, osmotic adjustment, higher chlorophyll retention and membrane stability index, it can be concluded that AtDREB1a transgenic chickpea lines were better adapted to water deficit by modifying important physiological traits. The selected transgenic chickpea event would be a valuable resource that can be used in pre-breeding or directly in varietal development programs for enhanced drought tolerance under parched conditions.

Amino Acid Digestibility of Extruded Chickpea and Yellow Pea Protein is High and Comparable in Moderately Stunted South Indian Children with Use of a Dual Stable Isotope Tracer Method.

BACKGROUND: Legumes are an excellent plant source of the limiting indispensable amino acid (IAA) lysine in vegetarian, cereal-based diets. However, their digestibility is poor largely because of their antiprotease content. Extrusion can enhance digestibility by inactivating trypsin inhibitors and thus potentially improve the protein quality of legumes. OBJECTIVE: We measured the digestibility of extruded chickpea and yellow pea protein with use of a dual stable isotope method in moderately stunted South Indian primary school children. METHODS: Twenty-eight moderately stunted children (height-for-age z scores <-2.0 SD and >-3.0 SD) aged 6-11 y from low to middle socioeconomic status were randomly assigned to receive a test protein (extruded intrinsically [2H]-labeled chickpea or yellow pea) along with a standard of U-[13C]-spirulina protein to measure amino acid (AA) digestibility with use of a dual stable isotope method. Individual AA digestibility in the test protein was calculated by the ratios of AA enrichments in the test protein to the standard protein in the food and their appearance in blood plasma collected at 6 and 6.5 h during the experiment, representing a plateau state. RESULTS: The mean AA digestibility of extruded chickpea and yellow pea protein in moderately stunted children (HAZ; -2.86 to -1.2) was high and similar in both extruded test proteins (89.0% and 88.0%, respectively, P = 0.83). However, lysine and proline digestibilities were higher in extruded chickpea than yellow pea (79.2% compared with 76.5% and 75.0% compared with 72.0%, respectively, P < 0.02). CONCLUSION: Extruded chickpea and yellow pea protein had good IAA digestibility in moderately stunted children, which was 20% higher than an earlier report of their digestibility when pressure-cooked, measured by the same method in adults. Higher digestibility of lysine and proline highlights better retention of these AA in chickpea during extrusion-based processing. Extrusion might be useful for developing high-quality protein foods from legumes. This trial was registered at www.ctri.nic.in as CTRI/2018/03/012439.

Genetic dissection of drought tolerance in chickpea (Cicer arietinum L.).

KEY MESSAGE: Analysis of phenotypic data for 20 drought tolerance traits in 1-7 seasons at 1-5 locations together with genetic mapping data for two mapping populations provided 9 QTL clusters of which one present on CaLG04 has a high potential to enhance drought tolerance in chickpea improvement. Chickpea (Cicer arietinum L.) is the second most important grain legume cultivated by resource poor farmers in the arid and semi-arid regions of the world. Drought is one of the major constraints leading up to 50% production losses in chickpea. In order to dissect the complex nature of drought tolerance and to use genomics tools for enhancing yield of chickpea under drought conditions, two mapping populations-ICCRIL03 (ICC 4958 × ICC 1882) and ICCRIL04 (ICC 283 × ICC 8261) segregating for drought tolerance-related root traits were phenotyped for a total of 20 drought component traits in 1-7 seasons at 1-5 locations in India. Individual genetic maps comprising 241 loci and 168 loci for ICCRIL03 and ICCRIL04, respectively, and a consensus genetic map comprising 352 loci were constructed ( http://cmap.icrisat.ac.in/cmap/sm/cp/varshney/). Analysis of extensive genotypic and precise phenotypic data revealed 45 robust main-effect QTLs (M-QTLs) explaining up to 58.20% phenotypic variation and 973 epistatic QTLs (E-QTLs) explaining up to 92.19% phenotypic variation for several target traits. Nine QTL clusters containing QTLs for several drought tolerance traits have been identified that can be targeted for molecular breeding. Among these clusters, one cluster harboring 48% robust M-QTLs for 12 traits and explaining about 58.20% phenotypic variation present on CaLG04 has been referred as "QTL-hotspot". This genomic region contains seven SSR markers (ICCM0249, NCPGR127, TAA170, NCPGR21, TR11, GA24 and STMS11). Introgression of this region into elite cultivars is expected to enhance drought tolerance in chickpea.

Carbonyl Cytotoxicity Affects Plant Cellular Processes and Detoxifying Enzymes Scavenge These Compounds to Improve Stress Tolerance.

Oxidative stress is ubiquitous in environmental stresses and prevails over the cellular metabolic and phenotypic responses in plants. Reactive oxygen species (ROS) generated under stress affect macromolecules to form another group of toxic compounds called reactive carbonyl compounds (RCCs). These molecules have a longer half-life than ROS and cause carbonyl stress that affects cellular metabolism, cellular homeostasis, and crop productivity. The later effect of oxidative stress in terms of the generation of RCCs and glycation products and their effects on plant processes have not been explored well in plant biology. Therefore, how these molecules are produced and a few important effects of RCCs on plants have been discussed in this review article. Further, the plant adaptive detoxification mechanisms of RCCs have been discussed. The enzymes that were identified in plants to detoxify these cytotoxic compounds have broad substrate specificity and the potential for use in breeding programs. The review should provide a comprehensive understanding of the cytotoxic compounds beyond ROS and subsequently their mitigation strategies for crop improvement programs.

Protein-quality evaluation of complementary foods in Indian children.

BACKGROUND: The types of food in complementary feeding of infants and young children are important for growth and development. Food protein quality, as measured by the Digestible Indispensable Amino Acid Score (DIAAS), requires the determination of true ileal digestibility of indispensable amino acids (IAAs) in children. OBJECTIVES: First, the aim of this study was to measure the true ileal IAA digestibility of 4 (rice, finger millet, mung bean, and hen egg) commonly consumed complementary foods in children aged <2 y using the dual-isotope tracer method. Second, we calculated the DIAAS of complementary feeding diets and their relation to stunting in a representative Indian rural population. DESIGN: Rice, finger millet, and mung bean were intrinsically labeled with deuterium oxide (2H2O), whereas egg was labeled through oral dosing of hens with a uniformly 2H-labeled amino acid mixture. True ileal IAA digestibility was determined by the dual-isotope tracer technique. The DIAAS of complementary food protein was calculated in children aged 1-3 y from a nationally representative survey to evaluate its relation with stunting. RESULTS: True ileal IAA digestibility was lowest in mung bean (65.2% ± 7.1%), followed by finger millet (68.4 %± 5.3%) and rice (78.5% ± 3.5%), and was highest for egg (87.4% ± 4.0%). There was a significant inverse correlation of complementary food DIAAS with stunting in survey data (r = -0.66, P = 0.044). The addition of egg or milk to nationally representative complementary diets theoretically improved the DIAAS from 80 to 100. CONCLUSIONS: The true ileal IAA digestibility of 4 foods commonly consumed in complementary diets showed that the DIAAS was associated with stunting and reinforces the importance of including animal source food (ASF) in diets to improve growth. This trial was registered at http://ctri.nic.in/clinicaltrials/login.php as CTRI/2017/02/007921.

Measurement of protein digestibility in humans by a dual-tracer method.

Background: Recent evaluations of the risk of dietary protein deficiency have indicated that protein digestibility may be a key limiting factor in the provision of indispensable amino acids (IAAs), particularly for vulnerable populations living in challenging environments where intestinal dysfunction may exist. Since the digestion of protein occurs only in the small intestine, and the metabolic activity of colonic bacteria confounds measurements at the fecal level, there is a need to develop noninvasive protein digestibility measurements at the ileal level. Objective: We used a dual-tracer method with stable isotopes to characterize the digestibility of uniformly labeled [13C]-spirulina protein as a standard protein, in comparison to a mixture of 2H-labeled crystalline amino acids, and then demonstrated the use of this standard protein to measure the digestibility of selected legumes (chick pea and mung bean) through the use of proteins that were intrinsically labeled with 2H. Design: The digestibility of uniformly labeled [13C]-spirulina was first measured in 6 healthy volunteers (3 males and 3 females) by feeding it along with a standard mixture of 2H-labeled amino acids, in a dual-tracer, plateau-fed test meal approach. Next, intrinsically labeled legume protein digestibility was studied with a similar dual-tracer approach, with uniformly labeled [13C]-spirulina as the standard, when processed differently before consumption. Results: The average digestibility of IAA in spirulina protein was 85.2%. The average IAA digestibility of intrinsically 2H-labeled chick pea and mung bean protein was 56.6% and 57.7%, respectively. Dehulling of mung bean before ingestion increased the average IAA digestibility by 9.9% in comparison to whole mung bean digestibility. Conclusions: An innovative, minimally invasive "dual-stable-isotope" method was developed to measure protein digestibility, in which the ingestion of an intrinsically 2H-labeled test protein along with a 13C-labeled standard protein of known digestibility allows for an accurate measure of digestion and absorption of the intrinsically labeled protein. This minimally invasive method is critical to redefining protein quality and will aid in revisiting human protein requirements in different settings and in vulnerable populations. This trial was registered at Clinical Trials Registry-India as CTRI/2017/11/010468.

Whole Genome Characterization of a Few EMS-Induced Mutants of Upland Rice Variety Nagina 22 Reveals a Staggeringly High Frequency of SNPs Which Show High Phenotypic Plasticity Towards the Wild-Type.

The Indian initiative, in creating mutant resources for the functional genomics in rice, has been instrumental in the development of 87,000 ethylmethanesulfonate (EMS)-induced mutants, of which 7,000 are in advanced generations. The mutants have been created in the background of Nagina 22, a popular drought- and heat-tolerant upland cultivar. As it is a pregreen revolution cultivar, as many as 573 dwarf mutants identified from this resource could be useful as an alternate source of dwarfing. A total of 541 mutants, including the macromutants and the trait-specific ones, obtained after appropriate screening, are being maintained in the mutant garden. Here, we report on the detailed characterizations of the 541 mutants based on the distinctness, uniformity, and stability (DUS) descriptors at two different locations. About 90% of the mutants were found to be similar to the wild type (WT) with high similarity index (>0.6) at both the locations. All 541 mutants were characterized for chlorophyll and epicuticular wax contents, while a subset of 84 mutants were characterized for their ionomes, namely, phosphorous, silicon, and chloride contents. Genotyping of these mutants with 54 genomewide simple sequence repeat (SSR) markers revealed 93% of the mutants to be either completely identical to WT or nearly identical with just one polymorphic locus. Whole genome resequencing (WGS) of four mutants, which have minimal differences in the SSR fingerprint pattern and DUS characters from the WT, revealed a staggeringly high number of single nucleotide polymorphisms (SNPs) on an average (16,453 per mutant) in the genic sequences. Of these, nearly 50% of the SNPs led to non-synonymous codons, while 30% resulted in synonymous codons. The number of insertions and deletions (InDels) varied from 898 to 2,595, with more than 80% of them being 1-2 bp long. Such a high number of SNPs could pose a serious challenge in identifying gene(s) governing the mutant phenotype by next generation sequencing-based mapping approaches such as Mutmap. From the WGS data of the WT and the mutants, we developed a genic resource of the WT with a novel analysis pipeline. The entire information about this resource along with the panicle architecture of the 493 mutants is made available in a mutant database EMSgardeN22 (http://14.139.229.201/EMSgardeN22).

Overexpression of Pea DNA Helicase 45 (PDH45) imparts tolerance to multiple abiotic stresses in chili (Capsicum annuum L.).

Imparting tolerance to abiotic stresses is of global importance as they inflict significant yield losses in field as well as in vegetable crops. Transcriptional activators, including helicases are identified to play a pivotal role in stress mitigation. Helicases, also known as molecular motors, are involved in myriad cellular processes that impart intrinsic tolerance to abiotic stresses in plants. Our study demonstrates the potential of a Pea DNA Helicase 45 (PDH45), in combating multiple abiotic stresses in chili. We harnessed Agrobacterium-mediated in planta transformation strategy for the generation of stable, single copy transgenic events. Precise molecular detection of the transgenes by sqRT-PCR coupled with genomic Southern analysis revealed variation in the integration of PDH45 at distinct loci in independent transgenic events. Characterization of five promising transgenic events showed both improved response to an array of simulated abiotic stresses and enhanced expression of several stress-responsive genes. While survival and recovery of transgenic events were significantly higher under gradual moisture stress conditions, under imposition of moderate stress, the transgenic events exhibited invigorated growth and productivity with concomitant improvement in water use efficiency (WUE). Thus, our study, unequivocally demonstrated the cardinal role of PDH45 in alleviating multiple abiotic stresses in chili.

Development and Genetic Characterization of A Novel Herbicide (Imazethapyr) Tolerant Mutant in Rice (Oryza sativa L.).

BACKGROUND: Increased water and labour scarcity in major rice growing areas warrants a shift towards direct seeded rice cultivation under which management of weeds is a major issue. Use of broad spectrum non-selective herbicides is an efficient means to manage weeds. Availability of rice genotypes with complete tolerance against broad-spectrum non-selective herbicides is a pre-requisite for advocating use of such herbicides. In the present study, we developed an EMS induced rice mutant, 'HTM-N22', exhibiting tolerance to a broad spectrum herbicide, 'Imazethapyr', and identified the mutations imparting tolerance to the herbicide. RESULTS: We identified a stable and true breeding rice mutant, HTM-N22 (HTM), tolerant to herbicide, Imazethapyr, from an EMS-mutagenized population of approximately 100,000 M2 plants of an upland rice variety, Nagina 22 (N22). Analysis of inheritance of herbicide tolerance in a cross between Pusa 1656-10-61/HTM showed that this trait is governed by a single dominant gene. To identify the causal gene for Imazethapyr tolerance, bulked segregant analysis (BSA) was followed using microsatellite markers flanking the three putative candidate genes viz., an Acetolactate Synthase (ALS) on chromosome 6 and two Acetohydroxy Acid Synthase (AHAS) genes, one on chromosomes 2 and another on chromosome 4. RM 6844 on chromosome 2 located 0.16 Mbp upstream of AHAS (LOC_Os02g30630) was found to co-segregate with herbicide tolerance. Cloning and sequencing of AHAS (LOC_Os02g30630) from the wild type, N22 and the mutant HTM and their comparison with reference Nipponbare sequence revealed several Single Nucleotide Polymorphisms (SNPs) in the mutant, of which eight resulted in non-synonymous mutations. Three of the eight amino acid substitutions were identical to Nipponbare and hence were not considered as causal changes. Of the five putative candidate SNPs, four were novel (at positions 30, 50, 81 and 152) while the remaining one, S627D was a previously reported mutant, known to result in Imidazolinone tolerance in rice. Of the novel ones, G152E was found to alter the hydrophobicty and abolish an N myristoylation site in the HTM compared to the WT, from reference based modeling and motif prediction studies. CONCLUSIONS: A novel mutant tolerant to the herbicide "Imazethapyr" was developed and characterized for genetic, sequence and protein level variations. This is a HTM in rice without any IPR (Intellectual Property Rights) infringements and hence can be used in rice breeding as a novel genetic stock by the public funded organizations in the country and elsewhere.

Genomewide association studies for 50 agronomic traits in peanut using the 'reference set' comprising 300 genotypes from 48 countries of the semi-arid tropics of the world.

Peanut is an important and nutritious agricultural commodity and a livelihood of many small-holder farmers in the semi-arid tropics (SAT) of world which are facing serious production threats. Integration of genomics tools with on-going genetic improvement approaches is expected to facilitate accelerated development of improved cultivars. Therefore, high-resolution genotyping and multiple season phenotyping data for 50 important agronomic, disease and quality traits were generated on the 'reference set' of peanut. This study reports comprehensive analyses of allelic diversity, population structure, linkage disequilibrium (LD) decay and marker-trait association (MTA) in peanut. Distinctness of all the genotypes can be established by using either an unique allele detected by a single SSR or a combination of unique alleles by two or more than two SSR markers. As expected, DArT features (2.0 alleles/locus, 0.125 PIC) showed lower allele frequency and polymorphic information content (PIC) than SSRs (22.21 alleles /locus, 0.715 PIC). Both marker types clearly differentiated the genotypes of diploids from tetraploids. Multi-allelic SSRs identified three sub-groups (K = 3) while the LD simulation trend line based on squared-allele frequency correlations (r2) predicted LD decay of 15-20 cM in peanut genome. Detailed analysis identified a total of 524 highly significant MTAs (p value > 2.1 × 10-6) with wide phenotypic variance (PV) range (5.81-90.09%) for 36 traits. These MTAs after validation may be deployed in improving biotic resistance, oil/ seed/ nutritional quality, drought tolerance related traits, and yield/ yield components.

Oxygen isotope enrichment (delta18O) as a measure of time-averaged transpiration rate.

Experimental evidence is presented to show that the 18O enrichment in the leaf biomass and the mean (time-averaged) transpiration rate are positively correlated in groundnut and rice genotypes. The relationship between oxygen isotope enrichment and stomatal conductance (g(s)) was determined by altering g(s) through ABA and subsequently using contrasting genotypes of cowpea and groundnut. The Peclet model for the 18O enrichment of leaf water relative to the source water is able to predict the mean observed values well, while it cannot reproduce the full range of measured isotopic values. Further, it fails to explain the observed positive correlation between transpiration rate and 18O enrichment in leaf biomass. Transpiration rate is influenced by the prevailing environmental conditions besides the intrinsic genetic variability. As all the genotypes of both species experienced similar environmental conditions, the differences in transpiration rate could mostly be dependent on intrinsic g(s). Therefore, it appears that the delta18O of leaf biomass can be used as an effective surrogate for mean transpiration rate. Further, at a given vapour pressure difference, delta18O can serve as a measure of stomatal conductance as well.