Biotechnological interventions for improving the seed longevity in cereal crops: progress and prospects.

Seed longevity is a measure of the viability of seeds during long-term storage and is crucial for germplasm conservation and crop improvement programs. Also, longevity is an important trait for ensuring food and nutritional security. Thus, a better understanding of various factors regulating seed longevity is requisite to improve this trait and to minimize the genetic drift during the regeneration of germplasm. In particular, seed deterioration of cereal crops during storage adversely affects agricultural productivity and food security. The irreversible process of seed deterioration involves a complex interplay between different genes and regulatory pathways leading to: loss of DNA integrity, membrane damage, inactivation of storage enzymes and mitochondrial dysfunction. Identifying the genetic determinants of seed longevity and manipulating them using biotechnological tools hold the key to ensuring prolonged seed storage. Genetics and genomics approaches had identified several genomic regions regulating the longevity trait in major cereals such as: rice, wheat, maize and barley. However, very few studies are available in other Poaceae members, including millets. Deploying omics tools, including genomics, proteomics, metabolomics, and phenomics, and integrating the datasets will pinpoint the precise molecular determinants affecting the survivability of seeds. Given this, the present review enumerates the genetic factors regulating longevity and demonstrates the importance of integrated omics strategies to dissect the molecular machinery underlying seed deterioration. Further, the review provides a roadmap for deploying biotechnological approaches to manipulate the genes and genomic regions to develop improved cultivars with prolonged storage potential.

Multi-omics intervention in Setaria to dissect climate-resilient traits: Progress and prospects.

Millets constitute a significant proportion of underutilized grasses and are well known for their climate resilience as well as excellent nutritional profiles. Among millets, foxtail millet (Setaria italica) and its wild relative green foxtail (S. viridis) are collectively regarded as models for studying broad-spectrum traits, including abiotic stress tolerance, C4 photosynthesis, biofuel, and nutritional traits. Since the genome sequence release, the crop has seen an exponential increase in omics studies to dissect agronomic, nutritional, biofuel, and climate-resilience traits. These studies have provided first-hand information on the structure, organization, evolution, and expression of several genes; however, knowledge of the precise roles of such genes and their products remains elusive. Several open-access databases have also been instituted to enable advanced scientific research on these important crops. In this context, the current review enumerates the contemporary trend of research on understanding the climate resilience and other essential traits in Setaria, the knowledge gap, and how the information could be translated for the crop improvement of related millets, biofuel crops, and cereals. Also, the review provides a roadmap for studying other underutilized crop species using Setaria as a model.

Genome Editing and Designer Crops for the Future.

Domestication spanning over thousands of years led to the evolution of crops that are being cultivated in recent times. Later, selective breeding methods were practiced by human to produce improved cultivars/germplasm. Classical breeding was further transformed into molecular- and genomics-assisted breeding strategies, however, these approaches are labor-intensive and time-consuming. The advent of omics technologies has facilitated the identification of genes and genetic determinants that regulate particular traits allowing the direct manipulation of target genes and genomic regions to achieve desirable phenotype. Recently, genome editing technologies such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-Associated protein 9 (Cas9) have gained popularity for precise editing of genes to develop crop varieties with superior agronomic, physiological, climate-resilient, and nutritional traits. Owing to the efficiency and precision, genome editing approaches have been widely used to design the crops that can survive the challenges posed by changing climate, and also cater the food and nutritional requirements for ever-growing population. Here, we briefly review different genome editing technologies deployed for crop improvement, and the fundamental differences between GE technology and transgene-based approach. We also summarize the recent advances in genome editing and how this radical expansion can complement the previously established technologies along with breeding for creating designer crops.

De novo transcriptome analysis identifies key genes involved in dehydration stress response in kodo millet (Paspalum scrobiculatum L.).

Kodo millet (Paspalum scrobiculatum L.) is a small millet species known for its excellent nutritional and climate-resilient traits. To understand the genes and pathways underlying dehydration stress tolerance of kodo millet, the transcriptome of cultivar 'CO3' subjected to dehydration stress (0 h, 3 h, and 6 h) was sequenced. The study generated 239.1 million clean reads that identified 9201, 9814, and 2346 differentially expressed genes (DEGs) in 0 h vs. 3 h, 0 h vs. 6 h, and 3 h vs. 6 h libraries, respectively. The DEGs were found to be associated with vital molecular pathways, including hormone metabolism and signaling, antioxidant scavenging, photosynthesis, and cellular metabolism, and were validated using qRT-PCR. Also, a higher abundance of uncharacterized genes expressed during stress warrants further studies to characterize this class of genes to understand their role in dehydration stress response. Altogether, the study provides insights into the transcriptomic response of kodo millet during dehydration stress.

Hormonal crosstalk in regulating salinity stress tolerance in graminaceous crops.

Soil salinity is one of the major threats that pose challenges to global cereal productivity and food security. Cereals have evolved sophisticated mechanisms to circumvent stress at morpho-physiological, biochemical, and molecular levels. Salt stress cues are perceived by the roots, which trigger the underlying signaling pathways that involve phytohormones. Each phytohormone triggers a specific signaling pathway integrated in a complex manner to produce antagonistic, synergistic, and additive responses. Phytohormones induce salt-responsive signaling pathways to modulate various physiological and anatomical mechanisms, including cell wall repair, apoplastic pH regulation, ion homeostasis, root hair formation, chlorophyll content, and leaf morphology. Exogenous applications of phytohormones moderate the adverse effects of salinity and improve growth. Understanding the complex hormonal crosstalk in cereals under salt stress will advance the knowledge about cooperation or antagonistic mechanisms among hormones and their role in developing salt-tolerant cereals to enhance the productivity of saline agricultural land. In this context, the present review focuses on the mechanisms of hormonal crosstalk that mediate the salt stress response and adaptation in graminaceous crops.

Diverse role of phytic acid in plants and approaches to develop low-phytate grains to enhance bioavailability of micronutrients.

Natural or synthetic compounds that interfere with the bioavailability of nutrients are called antinutrients. Phytic acid (PA) is one of the major antinutrients present in the grains and acts as a chelator of micronutrients. The presence of six reactive phosphate groups in PA hinders the absorption of micronutrients in the gut of non-ruminants. Consumption of PA-rich diet leads to deficiency of minerals such as iron and zinc among human population. On the contrary, PA is a natural antioxidant, and PA-derived molecules function in various signal transduction pathways. Therefore, optimal concentration of PA needs to be maintained in plants to avoid adverse pleiotropic effects, as well as to ensure micronutrient bioavailability in the diets. Given this, the chapter enumerates the structure, biosynthesis, and accumulation of PA in food grains followed by their roles in growth, development, and stress responses. Further, the chapter elaborates on the antinutritional properties of PA and explains the conventional breeding and transgene-based approaches deployed to develop low-PA varieties. Studies have shown that conventional breeding methods could develop low-PA lines; however, the pleiotropic effects of these methods viz. reduced yield, embryo abnormalities, and poor seed quality hinder the use of breeding strategies. Overexpression of phytase in the endosperm and RNAi-mediated silencing of genes involved in myo-inositol biosynthesis overcome these constraints. Next-generation genome editing approaches, including CRISPR-Cas9 enable the manipulation of more than one gene involved in PA biosynthesis pathway through multiplex editing, and scope exists to deploy such tools in developing varieties with optimal PA levels.

Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies.

BACKGROUND: Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efficient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for Agrobacterium rhizogenes-mediated root transformation of chickpea. RESULTS: Transformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation. Expression of green fluorescent protein further confirmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efficiency. In addition, root transformation was found to be genotype-independent and the efficacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing Arabidopsis TRANSPARENT TESTA 2 (AtTT2) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum. Furthermore, the induction of transgenic roots does not affect functional studies involving infection of roots by fungal pathogen. CONCLUSIONS: Transgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3-4 weeks, providing sufficient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop.

Interplay of neuronal and non-neuronal genes regulates intestinal DAF-16-mediated immune response during Fusarium infection of Caenorhabditis elegans.

Although precisely controlled innate immune response is governed by conserved cellular events in phylogenetically diverse hosts, the underlying molecular mechanisms by which this process is regulated against a multi-host pathogen remain unknown. Fusarium oxysporum is a model multi-host pathogen, known to be associated with neuronal stress in humans and vascular wilt in plants. The interaction between innate immune and neuronal pathways is the basis of many diverse biological responses. How these processes are coordinated in response to fungal disease is not well understood. Here, we show that F. oxysporum f. sp. ciceri causes neuronal stress and intestinal disintegration, ultimately leading to the death of Caenorhabditis elegans. To explore the regulatory framework of Fusarium-associated disease, we analysed the gene expression during infection, integrated temporal gene expression, and network analysis with genetic inactivation data in Caenorhabditis elegans. We identified 1024 genes showing significant changes in expression (corrected P-values <0.05) in response to Fusarium infection. Co-expression network analysis of our data identified prognostic genes related to disease progression. These genes were dynamically expressed in various neuronal and non-neuronal tissues exhibiting diverse biological functions, including cellular homeostasis, organ patterning, stress response, and lipid metabolism. The RNA-seq analysis further identified shared and unique signalling pathways regulated by DAF-16/FOXO and SIR-2.1 linking neuronal stress, which facilitates negative regulation of intestinal innate immunity. Genetic analysis revealed that GCY-5 in ASE functions upstream of DAF-16, whereas ASI-specific SRD-1 regulates behavioural immunity. Overall, our results indicate that a ubiquitous response occurs during Fusarium infection mediated by highly conserved regulatory components and pathways, which can be exploited further for the identification of disease-responsive genes conserved among animals and plants. Finally, this study provided a novel insight into cross-species immune signalling and may facilitate the discovery of cellular therapeutic targets for Fusarium-associated disease.

Extracellular Matrix Proteome and Phosphoproteome of Potato Reveals Functionally Distinct and Diverse Canonical and Non-Canonical Proteoforms.

The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of biological functions accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions rely on post-translational modifications, phosphorylation in particular, affecting enzymatic activity, interaction, localization and stability. To investigate the ECM proteoforms, we have isolated the cell wall proteome and phosphoproteome of a tuberous crop, potato (Solanum tuberosum). LC-MS/MS analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown functions. The findings may provide a better understanding of biochemical machinery and the integrated protein and phosphoprotein network of ECM for future functional studies of different developmental pathways and guidance cues in mechanosensing and integrity signaling.