Climate change has increased the frequency of extreme weather events, leading to the widespread occurrence of various abiotic stresses such as drought, salinity and temperature. Abiotic challenges often coincide with biotic stresses including pathogen and pest infestations. The frequent occurrence of such stresses, either individually or in combination, hinders crop growth, development, yield and quality. Plants have evolved diverse physiological and molecular adaptations to safeguard themselves against various stresses. However, plant responses to combined biotic and abiotic stresses are more complex and variable than responses to individual stresses, due to the intricate interactions among signaling networks and defense pathways. A clear understanding of how abiotic stresses influence pest and disease incidence as well as their severity is essential for developing strategies to mitigate the effect of combined stresses. Despite progress in individual stresses, there is a lack of comprehensive studies on the alterations in physiological, biochemical and molecular mechanisms of plants under combined stress conditions. This review aims to provide insights into plant responses to combined abiotic and biotic stress interactions and highlights the key morpho-physiological, biochemical, and molecular mechanisms, and presents recent case studies illustrating plant responses and effects under such combined stresses. In addition, this review highlights the integration of mechanistic insights with modern biotechnological and breeding strategies for enhancing plant tolerance to combined abiotic and biotic stresses. By providing a multi-dimensional framework that connects physiological, molecular, and computational analyses, it enables the identification of tolerant genotypes and serves as a comprehensive resource for plant breeders, molecular biologists, and agronomists to develop targeted strategies for improving crop resilience under combined stress conditions
Seed germination is a critical physiological process that transforms a quiescent seed into a metabolically active seedling and is also a crucial factor in determining maximum crop production. This transition is influenced by various intrinsic and extrinsic factors. Interestingly, reactive oxygen species (ROS) plays an important role in breaking seed dormancy by oxidation of biomolecules, weakening of the testa and degradation of endosperm. Similarly, molecular internal oxygen is also considered vital for the transition of dormancy to seed germination. However, it is essential to establish a correlation between the internal oxygen and the generation of ROS during seed germination. This chapter details protocols for imaging internal oxygen concentrations using VisiSens and fluorescent detection of ROS using H2DCFDA in chickpea seeds, complemented by qPCR analysis of key ROS-related genes (RBOH, AOX 1, UCP 1, and NADH dehydrogenase). These findings from these methods help advance our understanding of the inverse relationship between molecular oxygen and ROS dynamics during seed germination.
Chitinases can contribute to plant defence against fungal pathogens and insect herbivores, but their family organization, inducible deployment, and putative ligand-recognition behaviour remain poorly resolved in chickpea. We combined genome-wide identification, field expression profiling under controlled Helicoverpa armigera infestation, hormone treatments, and structure-guided comparison of representative proteins to prioritize defence-associated chickpea chitinases. We identified 28 chickpea chitinase loci (Car_Chits), comprising 22 glycosyl hydrolase family 18 (GH18) genes and 6 GH19 genes. Local duplication, especially tandem duplication within GH18, was the main contributor to family expansion, and interpretable duplicate pairs were retained mainly under purifying selection. Promoter scans indicated broad enrichment of defence- and hormone-associated cis-elements. Field quantitative real-time PCR (qRT-PCR) profiling of 11 candidate genes in field-grown plants subjected to controlled H. armigera infestation and hormone treatments showed treatment-specific temporal regulation. Car_Chit-4 (GH19) was strongly induced by salicylic acid (7.81-fold at 0.5 h; q < 0.05) but transiently repressed shortly after H. armigera feeding (0.15-fold at 0.5 h; q = 0.030). Car_Chit-19 (GH18) was the clearest herbivory-responsive gene, with late induction at 8 h (1.62-fold; q = 0.050) and 48 h (1.85-fold; q = 0.050). Jasmonic acid caused broad early repression across several genes, followed by delayed induction of Car_Chit-4 at 24 h. Seven Car_Chit-(GlcNAc)₄ complexes were modelled, docked, and simulated for 100 ns. GH18 proteins generally showed more favourable predicted MM-PBSA binding energies than GH19 proteins, but the structural metrics were interpreted as relative ligand-recognition indicators rather than direct evidence of anti-herbivore function. Car_Chit-17 had the most favourable predicted binding energy (ΔG_bind = − 18.51 ± 6.75 kcal/mol), whereas Car_Chit-14 and Car_Chit-27 retained the most stable ligand poses and Car_Chit-19 displayed the most stable protein scaffold. Chickpea chitinases show differentiated temporal responses to herbivory and hormone signalling. The study supports a working model in which GH19 Car_Chit-4 marks a rapid salicylic-acid-responsive arm, whereas GH18 Car_Chit-19 marks a delayed herbivory-responsive arm. A tiered prioritization framework separates expression-deployed candidates from structure-guided biochemical candidates, explaining why different genes emerge from qRT-PCR and molecular modelling analyses. The structural analyses provide complementary prioritization of Car_Chit-17, Car_Chit-14, and Car_Chit-27 for biochemical characterization. Together, these results provide a resource for dissecting chitinase-mediated defence in chickpea and for selecting candidates for functional validation.
Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is a powerful technique for amplifying genomic regions flanking Tnt1-retortransposon insertions in plants. Here, we present a TAIL-PCR protocol for amplifying Tnt1-flanking genomic sequences in chickpea using Tnt1-transformed hairy roots as the starting material. The amplified products can be cloned and sequenced for the precise mapping of Tnt1-integration sites in the chickpea genome. This method enables the functional characterization of chickpea genes governing root-specific traits and can be easily adapted for flanking sequence tag recovery in chickpea Tnt1-mutant populations.
Light is a central environmental signal that coordinates plant development, metabolism, and stress responses. By integrating external cues with internal programs, plants balance growth and resource allocation to adapt to fluctuating environments. Light-regulated signalling cascades drive morpho-physiological adaptations for optimized light capture, tuning photosynthetic efficiency, and source-sink dynamics. Key transcriptional hubs, regulated by master transcription factors such as ELONGATED HYPOCOTYL 5 (HY5), PHYTOCHROME INTERACTING FACTORS (PIFs), and GOLDEN 2-LIKE transcription factors (GLKs) coordinate photosynthesis, growth, and defence responses, driving adjustments and imposing balances for adaptation to shade, variable light, and stress. In addition to photosynthetic energy production, light signalling pathways influence carbon and nitrogen metabolism, leading to sugar allocation, starch turnover, and nutrient utilization-balancing trade-offs between growth, energy storage, and stress responses. Emerging evidence shows that light signalling pathways intersect with energy-monitoring cascades regulated by TARGET OF RAPAMYCIN (TOR), trehalose-6-phosphate (T6P), and SUCROSE NON-FERMENTING-RELATED PROTEIN KINASE 1 (SnRK1), linking the plant's energy status to growth, immunity, and stress resilience. Sugar concentration and light signals can also jointly regulate responses to both biotic and abiotic stress, with TOR acting as a central integration hub for environmental and metabolic signals, including transcriptional and translational mechanisms. This review synthesizes our current understanding on the interplay between light and TOR-mediated networks in energy production and allocation, while highlighting knowledge gaps that limit translational potential for improving plant productivity and resilience.
