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Advances in biocontrol potentials and fungicide resistance are highly desirable for Trichoderma. Thus, it is profitable to use mutagenic agents to develop superior strains with enhanced biocontrol properties and fungicide tolerance in Trichoderma. This study investigates the N-methyl-n-nitro-N-nitrosoguanidine (NTG) (100 mg/L) induced mutants of Trichoderma asperellum. Six NTG (3 each from 1st & 2nd round) induced mutants were developed and evaluated their biocontrol activities and carbendazim tolerance. Among the mutant N2-3, N2-1, N1 and N2-2 gave the best antagonistic and volatile metabolite activities on inhibition of chickpea F. oxysporum f. sp. ciceri, B. cinerea and R. bataticola mycelium under in vitro condition. Mutant N2-2 (5626.40 μg/ml) showed the highest EC50 value against carbendazim followed by N2-3 (206.36 μg/ml) and N2-1 (16.41 μg/ml); and succeeded to sporulate even at 2000 μg/ml of carbendazim. The biocontrol activity of N2-2 and N2 with half-dose of carbendazim was evaluated on chickpea dry root rot under controlled environment. Disease reduction and progress of the dry root rot was extremely low in T7 (N2-2 + with half-dose of carbendazim) treatment. Further, carbendazim resistant mutants demonstrated mutation in tub2 gene of β-tubulin family which was suggested through the 37 and 183 residue changes in the superimposed protein structures encoded by tub2 gene in N2 and N2-2 with WT respectively. This study conclusively implies that the enhanced carbendazim tolerance in N2-2 mutant did not affect the mycoparasitism and plant growth activity of Trichoderma. These mutants were as good as the wild-type with respect to all inherent attributes.

Pearl millet is an important cereal crop of semi-arid regions since it is highly nutritious and climate resilient. However, pearl millet is underutilized commercially due to the rapid onset of hydrolytic rancidity of seed lipids post-milling. We investigated the underlying biochemical and molecular mechanisms of rancidity development in the flour from contrasting inbred lines under accelerated aging conditions. The breakdown of storage lipids (triacylglycerols; TAG) was accompanied by free fatty acid accumulation over the time course for all lines. The high rancidity lines had the highest amount of FFA by day 21, suggesting that TAG lipases may be the cause of rancidity. Additionally, the high rancidity lines manifested substantial amounts of volatile aldehyde compounds, which are characteristic products of lipid oxidation. Lipases with expression in seed post-milling were sequenced from low and high rancidity lines. Polymorphisms were identified in two TAG lipase genes (PgTAGLip1 and PgTAGLip2) from the low rancidity line. Expression in a yeast model system confirmed these mutants were non-functional. We provide a direct mechanism to alleviate rancidity in pearl millet flour by identifying mutations in key TAG lipase genes that are associated with low rancidity. These genetic variations can be exploited through molecular breeding or precision genome technologies to develop elite pearl millet cultivars with improved flour shelf life.

Technologies and innovations are critical for addressing the future food system needs where genetic resources are an essential component of the change process. Advanced breeding tools like “genome editing” are vital for modernizing crop breeding to provide game-changing solutions to some of the “must needed” traits in agriculture. CRISPR/Cas-based tools have been rapidly repurposed for editing applications based on their improved efficiency, specificity and reduced off-target effects. Additionally, precise gene-editing tools such as base editing, prime editing, and multiplexing provide precision in stacking of multiple traits in an elite variety, and facilitating specific and targeted crop improvement. This has helped in advancing research and delivery of products in a short time span, thereby enhancing the rate of genetic gains. A special focus has been on food security in the drylands through crops including millets, teff, fonio, quinoa, Bambara groundnut, pigeonpea and cassava. While these crops contribute significantly to the agricultural economy and resilience of the dryland, improvement of several traits including increased stress tolerance, nutritional value, and yields are urgently required. Although CRISPR has potential to deliver disruptive innovations, prioritization of traits should consider breeding product profiles and market segments for designing and accelerating delivery of locally adapted and preferred crop varieties for the drylands. In this context, the scope of regulatory environment has been stated, implying the dire impacts of unreasonable scrutiny of genome-edited plants on the evolution and progress of much-needed technological advances.

Aflatoxins are immunosuppressive and carcinogenic secondary metabolites, produced by the filamentous ascomycete Aspergillus flavus, that are hazardous to animal and human health. In this study, we show that multiplexed host-induced gene silencing (HIGS) of Aspergillus flavus genes essential for fungal sporulation and aflatoxin production (nsdC, veA, aflR, and aflM) confers enhanced resistance to Aspergillus infection and aflatoxin contamination in groundnut (Aspergillus infection and aflatoxin contamination. Fungal differentiation and pathogenicity proteins, including calmodulin, transcriptional activator-HacA, kynurenine 3-monooxygenase 2, VeA, VelC, and several aflatoxin pathway biosynthetic enzymes, were downregulated in Aspergillus infecting the HIGS lines. Additionally, in the resistant HIGS lines, a number of host resistance proteins associated with fatty acid metabolism were strongly induced, including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol Δ-7 desaturase, ceramide kinase-related protein, sphingolipid Δ-8 desaturase, and phospholipase-D. Combined, this knowledge can be used for groundnut pre-breeding and breeding programs to provide a safe and secure food supply.

Aspergillus flavus is an opportunistic fungal pathogen that produces carcinogenic aflatoxin, a serious constraint for food safety and human health. In this study, to better understand the molecular mechanism/s of peanut resistance to A. flavus growth and aflatoxin accumulation, comparative proteomic analysis was performed in two contrasting peanut genotypes, variety JL 24 (susceptible) and its near-isogenic resistant transgenic derivative expressing an alfalfa defensin gene. Several resistance proteins associated with secondary metabolic pathways were strongly induced in the resistant genotypes including phenylalanine ammonia lyase, cinnamic acid-4-hydroxylase, chalcone synthase, resveratrol synthase, flavanone-3-hydroxylase, lipoxygenase, diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase, β-ketoacyl-ACP-reductase, monoacylglycerol acyltransferase, and diacylglycerol acyltransferase, indicating their roles in resistance. Besides, several putative susceptibility-associated proteins were revealed providing knowledge on potential candidate target genes for precise breeding interventions for aflatoxin mitigation. This is the first study to demonstrate comparative proteomics analysis in Aspergillus–peanut interaction using contrasting near-isogenic lines to elucidate the underlying molecular mechanisms of resistance.

Pearl millet [Pennisetum glaucum (L) R. Br.] is an important cereal crop of the semiarid tropics, which can withstand prolonged drought and heat stress. Considering an active involvement of the aquaporin (AQP) genes in water transport and desiccation tolerance besides several basic functions, their potential role in abiotic stress tolerance was systematically characterized and functionally validated. A total of 34 AQP genes from P. glaucum were identified and categorized into four subfamilies, viz., plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs). Sequence analysis revealed that PgAQPs have conserved characters of AQP genes with a closer relationship to sorghum. The PgAQPs were expressed differentially under high vapor pressure deficit (VPD) and progressive drought stresses where the PgPIP2;6 gene showed significant expression under high VPD and drought stress. Transgenic tobacco plants were developed by heterologous expression of the PgPIP2;6 gene and functionally characterized under different abiotic stresses to further unravel their role. Transgenic tobacco plants in the T2 generations displayed restricted transpiration and low root exudation rates in low- and high-VPD conditions. Under progressive drought stress, wild-type (WT) plants showed a quick or faster decline of soil moisture than transgenics. While under heat stress, PgPIP2;6 transgenics showed better adaptation to heat (40°C) with high canopy temperature depression (CTD) and low transpiration; under low-temperature stress, they displayed lower transpiration than their non-transgenic counterparts. Cumulatively, lower transpiration rate (Tr), low root exudation rate, declined transpiration, elevated CTD, and lower transpiration indicate that PgPIP2;6 plays a role under abiotic stress tolerance. Since the PgPIP2;6 transgenic plants exhibited better adaptation against major abiotic stresses such as drought, high VPD, heat, and cold stresses by virtue of enhanced transpiration efficiency, it has the potential to engineer abiotic stress tolerance for sustained growth and productivity of crops.

Groundnut is an important global food and oil crop that underpins agriculture-dependent livelihood strategies meeting food, nutrition, and income security. Aflatoxins, pose a major challenge to increased competitiveness of groundnut limiting access to lucrative markets and affecting populations that consume it. Other drivers of low competitiveness include allergens and limited shelf life occasioned by low oleic acid profile in the oil. Thus grain off-takers such as consumers, domestic, and export markets as well as processors need solutions to increase profitability of the grain. There are some technological solutions to these challenges and this review paper highlights advances in crop improvement to enhance groundnut grain quality and nutrient profile for food, nutrition, and economic benefits. Significant advances have been made in setting the stage for marker-assisted allele pyramiding for different aflatoxin resistance mechanisms—in vitro seed colonization, pre-harvest aflatoxin contamination, and aflatoxin production—which, together with pre- and post-harvest management practices, will go a long way in mitigating the aflatoxin menace. A breakthrough in aflatoxin control is in sight with overexpression of antifungal plant defensins, and through host-induced gene silencing in the aflatoxin biosynthetic pathway. Similarly, genomic and biochemical approaches to allergen control are in good progress, with the identification of homologs of the allergen encoding genes and development of monoclonal antibody based ELISA protocol to screen for and quantify major allergens. Double mutation of the allotetraploid homeologous genes, FAD2A and FAD2B, has shown potential for achieving >75% oleic acid as demonstrated among introgression lines. Significant advances have been made in seed systems research to bridge the gap between trait discovery, deployment, and delivery through innovative partnerships and action learning.

Finger millet (Eleusine coracana L.) is an annual herbaceous self-pollinating C4 cereal crop of the arid and semi-arid regions of the world. Finger millet is a food security crop proven to have resilience to changing climate and scores very high in nutrition. In the current study, we have assessed sixteen candidate reference genes for their appropriateness for the normalization studies in finger millet subjected to experimental regimes and treatments. Ten candidate reference genes (GAPDH, β-TUB, CYP, EIF4α, TIP41, UBC, G6PD, S24, MACP and MDH) were cloned and six (ACT, ELF1α, PP2A, PT, S21 and TFIID) were mined from the NCBI database as well as from the literature. Expression stability ranking of the finger millet reference genes was validated using four different statistical tools i.e., geNorm, NormFinder, BestKeeper, ΔCt and RefFinder. From the study, we endorse MACP, CYP, EIF4α to be most stable candidate reference genes in all 'tissues', whereas PT, TFIID, MACP ranked high across genotypes, β-TUB, CYP, ELF1α were found to be best under abiotic stresses and 'all samples set'. The study recommends using minimum of two reference genes for RT-qPCR data normalizations in finger millet. All in all, CYP, β-TUB, and EF1α, in combination were found to be best for robust normalizations under most experimental conditions. The best and the least stable genes were validated for confirmation by assessing their appropriateness for normalization studies using EcNAC1 gene. The report provides the first comprehensive list of suitable stable candidate reference genes for nutritional rich cereal finger millet that will be advantageous to gene expression studies in this crop.

High temperature is one of the biggest abiotic stress challenges for agriculture. While, Nitric oxide (NO) is gaining increasing attention from plant science community due to its involvement in resistance to various plant stress conditions, its implications on heat stress tolerance is still unclear. Several lines of evidence indicate NO as a key signaling molecule in mediating various plant responses such as photosynthesis, oxidative defense, osmolyte accumulation, gene expression, and protein modifications under heat stress. Furthermore, the interactions of NO with other signaling molecules and phytohormones to attain heat tolerance have also been building up in recent years. Nevertheless, deep insights into the functional intermediaries or signal transduction components associated with NO-mediated heat stress signaling are imperative to uncover their involvement in plant hormone induced feed-back regulations, ROS/NO balance, and stress induced gene transcription. Although, progress is underway, much work remains to define the functional relevance of this molecule in plant heat tolerance. This review provides an overview on current status and discuss knowledge gaps in exploiting NO, thereby enhancing our understanding of the role of NO in plant heat tolerance.

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.

Water deficit is a major yield-limiting factor for many crops, and improving the root system has been proposed as a promising breeding strategy, although not in groundnut ( Arachis hypogaea L.). The present work was carried out mainly to assess how root traits are influenced under water stress in groundnut, whether transgenics can alter root traits, and whether putative changes lead to water extraction differences. Several transgenic events, transformed with DREB1A driven by the rd29 promoter, along with wild-type JL24, were tested in a lysimeter system that mimics field conditions under both water stress (WS) and well-watered (WW) conditions. The WS treatment increased the maximum rooting depth, although the increase was limited to about 20% in JL24, compared to 50% in RD11. The root dry weight followed a similar trend. Consequently, the root dry weight and length density of transgenics was higher in layers below 100-cm depth (Exp. 1) and below 30 cm (Exp. 2). The root diameter was unchanged under WS treatment, except a slight increase in the 60-90-cm layer. The root diameter increased below 60 cm in both treatments. In the WW treatment, total water extraction of RD33 was higher than in JL24 and other transgenic events, and somewhat lower in RD11 than in JL24. In the WS treatment, water extraction of RD2, RD11 and RD33 was higher than in JL24. These water extraction differences were mostly apparent in the initial 21 days after treatment imposition and were well related to root length density in the 30-60-cm layer (R2 = 0.68), but not to average root length density. In conclusion, water stress promotes rooting growth more strongly in transgenic events than in the wild type, especially in deep soil layers, and this leads to increased water extraction. This opens an avenue for tapping these characteristics toward the improvement of drought adaptation in deep soil conditions, and toward a better understanding of genes involved in rooting in groundnut. [ABSTRACT FROM AUTHOR]

Orf147, a cytotoxic peptide, has been found to cause cytoplasmic male sterility (CMS) in Cajanus cajanifolius (pigeonpea). In our study, Orf147 was introduced into self-pollinating Cicer arietinum (chickpea) using Agrobacterium-mediated transformation for induction of CMS. The stable integration and expression of the transgene has been assessed through PCR and qRT-PCR analysis. In addition, phenotypic sterility analysis has been performed, considering developmental parameters like flower development, pod formation and flower drop. Transgene inheritance analysis demonstrates that out of the five PCR positive events in the T 0 generation, two events have segregated according to the Mendelian segregation ratio (3:1) in the T 2 generation. Further, pollen viability test using microscopic analysis confirms the induction of partial CMS in transgenic chickpea. The study holds significant value regarding the heterosis of self-pollinating legumes like chickpea. As a part of the prospect, exploring inducible promoters of species-specific or related legumes would be the next step to developing a two-line hybrid system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Aflatoxins are immunosuppressive and carcinogenic secondary metabolites, produced by the filamentous ascomycete Aspergillus flavus , that are hazardous to animal and human health. In this study, we show that multiplexed host-induced gene silencing (HIGS) of Aspergillus flavus genes essential for fungal sporulation and aflatoxin production ( nsdC , veA , aflR , and aflM) confers enhanced resistance to Aspergillus infection and aflatoxin contamination in groundnut (<20 ppb). Comparative proteomic analysis of contrasting groundnut genotypes (WT and near-isogenic HIGS lines) supported a better understanding of the molecular processes underlying the induced resistance and identified several groundnut metabolites that might play a significant role in resistance to Aspergillus infection and aflatoxin contamination. Fungal differentiation and pathogenicity proteins, including calmodulin, transcriptional activator-HacA, kynurenine 3-monooxygenase 2, VeA, VelC, and several aflatoxin pathway biosynthetic enzymes, were downregulated in Aspergillus infecting the HIGS lines. Additionally, in the resistant HIGS lines, a number of host resistance proteins associated with fatty acid metabolism were strongly induced, including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol Δ-7 desaturase, ceramide kinase-related protein, sphingolipid Δ-8 desaturase, and phospholipase-D. Combined, this knowledge can be used for groundnut pre-breeding and breeding programs to provide a safe and secure food supply.

Advances in biocontrol potentials and fungicide resistance are highly desirable for Trichoderma. Thus, it is profitable to use mutagenic agents to develop superior strains with enhanced biocontrol properties and fungicide tolerance in Trichoderma. This study investigates the N-methyl-n-nitro-N-nitrosoguanidine (NTG) (100 mg/L) induced mutants of Trichoderma asperellum. Six NTG (3 each from 1st & 2nd round) induced mutants were developed and evaluated their biocontrol activities and carbendazim tolerance. Among the mutant N2-3, N2-1, N1 and N2-2 gave the best antagonistic and volatile metabolite activities on inhibition of chickpea F. oxysporum f. sp. ciceri, B. cinerea and R. bataticola mycelium under in vitro condition. Mutant N2-2 (5626.40 μg/ml) showed the highest EC50 value against carbendazim followed by N2-3 (206.36 μg/ml) and N2-1 (16.41 μg/ml); and succeeded to sporulate even at 2000 μg/ml of carbendazim. The biocontrol activity of N2-2 and N2 with half-dose of carbendazim was evaluated on chickpea dry root rot under controlled environment. Disease reduction and progress of the dry root rot was extremely low in T7 (N2-2 + with half-dose of carbendazim) treatment. Further, carbendazim resistant mutants demonstrated mutation in tub2 gene of β-tubulin family which was suggested through the 37 and 183 residue changes in the superimposed protein structures encoded by tub2 gene in N2 and N2-2 with WT respectively. This study conclusively implies that the enhanced carbendazim tolerance in N2-2 mutant did not affect the mycoparasitism and plant growth activity of Trichoderma. These mutants were as good as the wild-type with respect to all inherent attributes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 G. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Technologies and innovations are critical for addressing the future food system needs where genetic resources are an essential component of the change process. Advanced breeding tools like "genome editing" are vital for modernizing crop breeding to provide game-changing solutions to some of the "must needed" traits in agriculture. CRISPR/Cas-based tools have been rapidly repurposed for editing applications based on their improved efficiency, specificity and reduced off-target effects. Additionally, precise gene-editing tools such as base editing, prime editing, and multiplexing provide precision in stacking of multiple traits in an elite variety, and facilitating specific and targeted crop improvement. This has helped in advancing research and delivery of products in a short time span, thereby enhancing the rate of genetic gains. A special focus has been on food security in the drylands through crops including millets, teff, fonio, quinoa, Bambara groundnut, pigeonpea and cassava. While these crops contribute significantly to the agricultural economy and resilience of the dryland, improvement of several traits including increased stress tolerance, nutritional value, and yields are urgently required. Although CRISPR has potential to deliver disruptive innovations, prioritization of traits should consider breeding product profiles and market segments for designing and accelerating delivery of locally adapted and preferred crop varieties for the drylands. In this context, the scope of regulatory environment has been stated, implying the dire impacts of unreasonable scrutiny of genome-edited plants on the evolution and progress of much-needed technological advances., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sharma, Palakolanu, Bhattacharya, Shankhapal and Bhatnagar-Mathur.)

Pearl millet is an important cereal crop of semi-arid regions since it is highly nutritious and climate resilient. However, pearl millet is underutilized commercially due to the rapid onset of hydrolytic rancidity of seed lipids post-milling. We investigated the underlying biochemical and molecular mechanisms of rancidity development in the flour from contrasting inbred lines under accelerated aging conditions. The breakdown of storage lipids (triacylglycerols; TAG) was accompanied by free fatty acid accumulation over the time course for all lines. The high rancidity lines had the highest amount of FFA by day 21, suggesting that TAG lipases may be the cause of rancidity. Additionally, the high rancidity lines manifested substantial amounts of volatile aldehyde compounds, which are characteristic products of lipid oxidation. Lipases with expression in seed post-milling were sequenced from low and high rancidity lines. Polymorphisms were identified in two TAG lipase genes ( PgTAGLip1 and PgTAGLip2 ) from the low rancidity line. Expression in a yeast model system confirmed these mutants were non-functional. We provide a direct mechanism to alleviate rancidity in pearl millet flour by identifying mutations in key TAG lipase genes that are associated with low rancidity. These genetic variations can be exploited through molecular breeding or precision genome technologies to develop elite pearl millet cultivars with improved flour shelf life., Competing Interests: KF, JE, LW, BR, and BD were employed by the company Corteva™ Agriscience. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AR declared a past co-authorship with the author SG to the handling editor., (Copyright © 2022 Aher, Reddy, Bhunia, Flyckt, Shankhapal, Ojha, Everard, Wayne, Ruddy, Deonovic, Gupta, Sharma and Bhatnagar-Mathur.)

Pearl millet [ Pennisetum glaucum (L) R. Br.] is an important cereal crop of the semiarid tropics, which can withstand prolonged drought and heat stress. Considering an active involvement of the aquaporin (AQP) genes in water transport and desiccation tolerance besides several basic functions, their potential role in abiotic stress tolerance was systematically characterized and functionally validated. A total of 34 AQP genes from P. glaucum were identified and categorized into four subfamilies, viz., plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs). Sequence analysis revealed that PgAQPs have conserved characters of AQP genes with a closer relationship to sorghum. The PgAQPs were expressed differentially under high vapor pressure deficit (VPD) and progressive drought stresses where the PgPIP2;6 gene showed significant expression under high VPD and drought stress. Transgenic tobacco plants were developed by heterologous expression of the PgPIP2;6 gene and functionally characterized under different abiotic stresses to further unravel their role. Transgenic tobacco plants in the T 2 generations displayed restricted transpiration and low root exudation rates in low- and high-VPD conditions. Under progressive drought stress, wild-type (WT) plants showed a quick or faster decline of soil moisture than transgenics. While under heat stress, PgPIP2;6 transgenics showed better adaptation to heat (40°C) with high canopy temperature depression (CTD) and low transpiration; under low-temperature stress, they displayed lower transpiration than their non-transgenic counterparts. Cumulatively, lower transpiration rate (Tr), low root exudation rate, declined transpiration, elevated CTD, and lower transpiration indicate that PgPIP2;6 plays a role under abiotic stress tolerance. Since the PgPIP2;6 transgenic plants exhibited better adaptation against major abiotic stresses such as drought, high VPD, heat, and cold stresses by virtue of enhanced transpiration efficiency, it has the potential to engineer abiotic stress tolerance for sustained growth and productivity of crops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Reddy, Dhaware, Sivasakthi, Divya, Nagaraju, Sri Cindhuri, Kavi Kishor, Bhatnagar-Mathur, Vadez and Sharma.)

For millennia, natural and artificial selection has combined favourable alleles for desirable traits in crop species. While modern plant breeding has achieved steady increases in crop yields over the last century, on the current trajectory we will simply not meet demand by 2045. Novel breeding strategies and sources of genetic variation will be required to sustainably fill predicted yield gaps and meet new consumer preferences. Here, we highlight that stepping up to meet this grand challenge will increasingly require thinking 'beyond the gene'. Significant progress has been made in understanding the contributions of both epigenetic variation and cis-regulatory variation to plant traits. This non-genic variation has great potential in future breeding, synthetic biology and biotechnology applications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pearl millet (Pennisetum glaucum [L.] R. Br.) is an important crop capable of growing in harsh and marginal environments, with the highest degree of tolerance to drought and heat stresses among cereals. Diverse germplasm of pearl millet shows a significant phenotypic variation in response to abiotic stresses, making it a unique model to study the mechanisms responsible for stress mitigation. The present study focuses on identifying the physiological response of two pearl millet high-resolution cross (HRC) genotypes, ICMR 1122 and ICMR 1152, in response to low and high vapor pressure deficit (VPD). Under high VPD conditions, ICMR 1152 exhibited a lower transpiration rate (Tr), higher transpiration efficiency, and lower root sap exudation than ICMR 1122. Further, Pg-miRNAs expressed in the contrasting genotypes under low and high VPD conditions were identified by deep sequencing analysis. A total of 116 known and 61 novel Pg-miRNAs were identified from ICMR 1152, while 26 known and six novel Pg-miRNAs were identified from ICMR 1122 genotypes, respectively. While Pg-miR165, 168, 170, and 319 families exhibited significant differential expression under low and high VPD conditions in both genotypes, ICMR 1152 showed abundant expression of Pg-miR167, Pg-miR172, Pg-miR396 Pg-miR399, Pg-miR862, Pg-miR868, Pg-miR950, Pg-miR5054, and Pg-miR7527 indicating their direct and indirect role in root physiology and abiotic stress responses. Drought responsive Pg-miRNA targets showed upregulation in response to high VPD stress, further narrowing down the miRNAs involved in regulation of drought tolerance in pearl millet., (© 2021 Scandinavian Plant Physiology Society.)

In the present study, the promoter region of the pearl millet heat shock protein 10 (PgHsp10) gene was cloned and characterized. The PgHsp10 promoter (PgHsp10pro) sequence region has all the cis-motifs required for tissue and abiotic stress inducibility. The complete PgHsp10pro (PgHsp10PC) region and a series of 5' truncations of PgHsp10 (PgHsp10D1 and PgHsp10D2) and an antisense form of PgHsp10pro (PgHsp10AS) were cloned into a plant expression vector (pMDC164) through gateway cloning. All four constructs were separately transformed into tobacco through Agrobacterium-mediated genetic transformation, and PCR-confirmed transgenic plants progressed to T 1 and T 2 generations. The T 2 transgenic tobacco plants comprising all PgHsp10pro fragments were used for GUS histochemical and qRT-PCR assays in different tissues under control and abiotic stresses. The PgHsp10PC pro expression was specific to stem and seedlings under control conditions. Under different abiotic stresses, particularly heat stress, PgHsp10PCpro had relatively higher activity than PgHsp10D1pro, PgHsp10D2pro and PgHsp10ASpro. PgHsp10pro from a stress resilient crop like pearl millet responds positively to a range of abiotic stresses, in particular heat, when expressed in heterologous plant systems such as tobacco. Hence, PgHsp10pro appears to be a potential promoter candidate for developing heat and drought stress-tolerant crop plants., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Groundnut is an important global food and oil crop that underpins agriculture-dependent livelihood strategies meeting food, nutrition, and income security. Aflatoxins, pose a major challenge to increased competitiveness of groundnut limiting access to lucrative markets and affecting populations that consume it. Other drivers of low competitiveness include allergens and limited shelf life occasioned by low oleic acid profile in the oil. Thus grain off-takers such as consumers, domestic, and export markets as well as processors need solutions to increase profitability of the grain. There are some technological solutions to these challenges and this review paper highlights advances in crop improvement to enhance groundnut grain quality and nutrient profile for food, nutrition, and economic benefits. Significant advances have been made in setting the stage for marker-assisted allele pyramiding for different aflatoxin resistance mechanisms- in vitro seed colonization, pre-harvest aflatoxin contamination, and aflatoxin production-which, together with pre- and post-harvest management practices, will go a long way in mitigating the aflatoxin menace. A breakthrough in aflatoxin control is in sight with overexpression of antifungal plant defensins, and through host-induced gene silencing in the aflatoxin biosynthetic pathway. Similarly, genomic and biochemical approaches to allergen control are in good progress, with the identification of homologs of the allergen encoding genes and development of monoclonal antibody based ELISA protocol to screen for and quantify major allergens. Double mutation of the allotetraploid homeologous genes, FAD2A and FAD2B , has shown potential for achieving >75% oleic acid as demonstrated among introgression lines. Significant advances have been made in seed systems research to bridge the gap between trait discovery, deployment, and delivery through innovative partnerships and action learning., (Copyright © 2020 Ojiewo, Janila, Bhatnagar-Mathur, Pandey, Desmae, Okori, Mwololo, Ajeigbe, Njuguna-Mungai, Muricho, Akpo, Gichohi-Wainaina, Variath, Radhakrishnan, Dobariya, Bera, Rathnakumar, Manivannan, Vasanthi, Kumar and Varshney.)

Finger millet (Eleusine coracana L.) is an annual herbaceous self-pollinating C4 cereal crop of the arid and semi-arid regions of the world. Finger millet is a food security crop proven to have resilience to changing climate and scores very high in nutrition. In the current study, we have assessed sixteen candidate reference genes for their appropriateness for the normalization studies in finger millet subjected to experimental regimes and treatments. Ten candidate reference genes (GAPDH, β-TUB, CYP, EIF4α, TIP41, UBC, G6PD, S24, MACP and MDH) were cloned and six (ACT, ELF1α, PP2A, PT, S21 and TFIID) were mined from the NCBI database as well as from the literature. Expression stability ranking of the finger millet reference genes was validated using four different statistical tools i.e., geNorm, NormFinder, BestKeeper, ΔCt and RefFinder. From the study, we endorse MACP, CYP, EIF4α to be most stable candidate reference genes in all 'tissues', whereas PT, TFIID, MACP ranked high across genotypes, β-TUB, CYP, ELF1α were found to be best under abiotic stresses and 'all samples set'. The study recommends using minimum of two reference genes for RT-qPCR data normalizations in finger millet. All in all, CYP, β-TUB, and EF1α, in combination were found to be best for robust normalizations under most experimental conditions. The best and the least stable genes were validated for confirmation by assessing their appropriateness for normalization studies using EcNAC1 gene. The report provides the first comprehensive list of suitable stable candidate reference genes for nutritional rich cereal finger millet that will be advantageous to gene expression studies in this crop., Competing Interests: The authors have declared that no competing interests exist.

Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub-Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut-Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host-induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE-Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species-scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near-immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi-arid regions., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The molecular mechanisms and targets of nitric oxide (NO) are not fully known in plants. Our study reports the first large-scale quantitative proteomic analysis of NO donor responsive proteins in chickpea. Dose response studies carried out using NO donors, sodium nitroprusside (SNP), diethylamine NONOate (DETA) and S-nitrosoglutathione (GSNO) in chickpea genotype ICCV1882, revealed a dose dependent positive impact on seed germination and seedling growth. SNP at 0.1mM concentration proved to be most appropriate following confirmation using four different chickpea genotypes. while SNP treatment enhanced the percentage of germination, chlorophyll and nitrogen contents in chickpea, addition of NO scavenger, cPTIO reverted its impact under abiotic stresses. Proteome profiling revealed 172 downregulated and 76 upregulated proteins, of which majority were involved in metabolic processes (118) by virtue of their catalytic (145) and binding (106) activity. A few crucial proteins such as S-adenosylmethionine synthase, dehydroascorbate reductase, pyruvate kinase fragment, 1-aminocyclopropane-1-carboxylic acid oxidase, 1-pyrroline-5-carboxylate synthetase were less abundant whereas Bowman-Birk type protease inhibitor, non-specific lipid transfer protein, chalcone synthase, ribulose-1-5-bisphosphate carboxylase oxygenase large subunit, PSII D2 protein were highly abundant in SNP treated samples. This study highlights the protein networks for a better understanding of possible NO induced regulatory mechanisms in plants.

High temperature is one of the biggest abiotic stress challenges for agriculture. While, Nitric oxide (NO) is gaining increasing attention from plant science community due to its involvement in resistance to various plant stress conditions, its implications on heat stress tolerance is still unclear. Several lines of evidence indicate NO as a key signaling molecule in mediating various plant responses such as photosynthesis, oxidative defense, osmolyte accumulation, gene expression, and protein modifications under heat stress. Furthermore, the interactions of NO with other signaling molecules and phytohormones to attain heat tolerance have also been building up in recent years. Nevertheless, deep insights into the functional intermediaries or signal transduction components associated with NO-mediated heat stress signaling are imperative to uncover their involvement in plant hormone induced feed-back regulations, ROS/NO balance, and stress induced gene transcription. Although, progress is underway, much work remains to define the functional relevance of this molecule in plant heat tolerance. This review provides an overview on current status and discuss knowledge gaps in exploiting NO, thereby enhancing our understanding of the role of NO in plant heat tolerance.

Sorghum is a typical short-day (SD) plant and its use in grain or biomass production in temperate regions depends on its flowering time control, but the underlying molecular mechanism of floral transition in sorghum is poorly understood. Here we characterized sorghum FLOWERING LOCUS T (SbFT) genes to establish a molecular road map for mechanistic understanding. Out of 19 PEBP genes, SbFT1, SbFT8 and SbFT10 were identified as potential candidates for encoding florigens using multiple approaches. Phylogenetic analysis revealed that SbFT1 clusters with the rice Hd3a subclade, while SbFT8 and SbFT10 cluster with the maize ZCN8 subclade. These three genes are expressed in the leaf at the floral transition initiation stage, expressed early in grain sorghum genotypes but late in sweet and forage sorghum genotypes, induced by SD treatment in photoperiod-sensitive genotypes, cooperatively repressed by the classical sorghum maturity loci, interact with sorghum 14-3-3 proteins and activate flowering in transgenic Arabidopsis plants, suggesting florigenic potential in sorghum. SD induction of these three genes in sensitive genotypes is fully reversed by 1 wk of long-day treatment, and yet, some aspects of the SD treatment may still make a small contribution to flowering in long days, indicating a complex photoperiod response mediated by SbFT genes., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

Nitric oxide (NO) is a versatile gaseous signaling molecule with increasing significance in plant research due to its association with various stress responses. Although, improved drought tolerance by NO is associated greatly with its ability to reduce stomatal opening and oxidative stress, it can immensely influence other physiological processes such as photosynthesis, proline accumulation and seed germination under water deficit. NO as a free radical can directly alter proteins, enzyme activities, gene transcription, and post-translational modifications that benefit functional recovery from drought. The present drought-mitigating strategies have focused on exogenous application of NO donors for exploring the associated physiological and molecular events, transgenic and mutant studies, but are inadequate. Considering the biphasic effects of NO, a cautious deployment is necessary along with a systematic approach for deciphering positively regulated responses to avoid any cytotoxic effects. Identification of NO target molecules and in-depth analysis of its effects under realistic field drought conditions should be an upmost priority. This detailed synthesis on the role of NO offers new insights on its functions, signaling, regulation, interactions and co-existence with different drought-related events providing future directions for exploiting this molecule towards improving drought tolerance in crop plants., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Aflatoxins are toxic, carcinogenic, mutagenic, teratogenic and immunosuppressive byproducts of Aspergillus spp. that contaminate a wide range of crops such as maize, peanut, and cotton. Aflatoxin not only affects crop production but renders the produce unfit for consumption and harmful to human and livestock health, with stringent threshold limits of acceptability. In many crops, breeding for resistance is not a reliable option because of the limited availability of genotypes with durable resistance to Aspergillus. Understanding the fungal/crop/environment interactions involved in aflatoxin contamination is therefore essential in designing measures for its prevention and control. For a sustainable solution to aflatoxin contamination, research must be focused on identifying and improving knowledge of host-plant resistance factors to aflatoxin accumulation. Current advances in genetic transformation, proteomics, RNAi technology, and marker-assisted selection offer great potential in minimizing pre-harvest aflatoxin contamination in cultivated crop species. Moreover, developing effective phenotyping strategies for transgenic as well as precision breeding of resistance genes into commercial varieties is critical. While appropriate storage practices can generally minimize post-harvest aflatoxin contamination in crops, the use of biotechnology to interrupt the probability of pre-harvest infection and contamination has the potential to provide sustainable solution., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Terminal drought causes major yield loss in chickpea, so it is imperative to identify genotypes with best suited adaptive traits to secure yield in terminal drought-prone environments. Here, we evaluated chickpea (At) rd29A:: (At) DREB1A transgenic events (RD2, RD7, RD9 and RD10) and their untransformed C235 genotype for growth, water use and yield under terminal water-stress (WS) and well-watered (WW) conditions. The assessment was made across three lysimetric trials conducted in contained environments in the greenhouse (2009GH and 2010GH) and the field (2010F). Results from the greenhouse trials showed genotypic variation for harvest index (HI), yield, temporal pattern of flowering and seed filling, temporal pattern of water uptake across crop cycle, and transpiration efficiency (TE) under terminal WS conditions. The mechanisms underlying the yield gain in the WS transgenic events under 2009GH trial was related to conserving water for the reproductive stage in RD7, and setting seeds early in RD10. Water conservation also led to a lower percentage of flower and pod abortion in both RD7 and RD10. Similarly, in the 2010GH trial, reduced water extraction during vegetative stage in events RD2, RD7 and RD9 was critical for better seed filling in the pods produced from late flowers in RD2, and reduced percentage of flower and pod abortion in RD2 and RD9. However, in the 2010F trial, the increased seed yield and HI in RD9 compared with C235 came along only with small changes in water uptake and podding pattern, probably not causal. Events RD2 (2010GH), RD7 (2010GH) and RD10 (2009GH) with higher seed yield also had higher TE than C235. The results suggest that DREB1A, a transcription factor involved in the regulation of several genes of abiotic stress response cascade, influenced the pattern of water uptake and flowering across the crop cycle, leading to reduction in the percentage of flower and pod abortion in the glasshouse trials.