Chickpea chitinases responsive to Helicoverpa herbivory and phytohormone signaling: genome-wide identification, field expression profiling, and structure-guided prioritization

Abstract

Background: 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.

Results: 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.

Conclusions: 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.