Comparative phytochemical analysis and gene expression profiling of leaf galls on selected medicinal plants

Abstract

Leaf galls are abnormal plant structures that are often caused by gall-inducing organisms like insects, mites, fungi or bacteria. These structures are the result of complex physiological and biochemical interactions between the gall inducer and the host plant. Traditionally viewed as detrimental, galls are increasingly recognized as sites of complex metabolic reprogramming, with the potential to modify the production of bioactive secondary metabolites in host plants. The purpose of this study is to investigate the molecular, phytochemical and structural characteristics of leaf galls in selected medicinal plants: Alstonia scholaris (L.) R. Br. and Ficus racemosa L., in which galls are induced by insects, Pauropsylla tuberculata and Pauropsylla depressa, respectively. The study aims to elucidate how gall formation influences secondary metabolism and address a gap in understanding the biochemical and molecular responses of medicinal plants to biotic stress. The methodology combines morphological, anatomical, phytochemical and molecular techniques. Microscopy was used to undertake morphological and anatomical investigations of gall-affected tissues. Phytochemical profiling entails both qualitative and quantitative investigations utilizing techniques such as Gas Chromatography-Mass Spectrometry (GC-MS) and Orbitrap High-Resolution Liquid Chromatography-Mass Spectrometry (OHR-LC-MS). Extracts from normal leaves, galled leaves and galls were evaluated for bioactivity analysis. The molecular approach used Quantitative Real-Time PCR (qRT-PCR) to characterize the expression of six essential genes involved in the shikimic acid and phenylpropanoid pathways that regulate phenolic compound biosynthesis. Morphological analysis revealed that galls are distinct, with specialized structures like larval chambers and nutritive tissues. The key findings show that gall formation profoundly changes plant physiology. Phytochemical analysis leads to the finding that galls and galled leaves contain more secondary metabolites, particularly phenolics, flavonoids and tannins, than normal leaves, indicating stronger defense responses against inducers. GC-MS and OHR- LC-MS revealed a diverse range of secondary metabolites with numerous compounds elevated or expressed only in gall tissues. These results suggest that gall enhances the phytochemical diversity and potency of the plant. When compared to normal leaves, 8bioactivity analysis revealed that ethanolic extracts from galls and galled leaves exhibited superior antioxidant, antibacterial, antifungal and cytotoxic properties. The highest bioactivity was detected in F. racemosa gall leaf extracts. According to qRT-PCR analysis, all the selected genes were significantly upregulated in galls. F. racemosa had higher fold gene expression than A. scholaris, suggesting a more robust metabolic response to P. depressa. Nonetheless, there was little gene upregulation in gall leaves, suggesting metabolites were being translocated to gall leaves from galls. The findings show that gall induction enhances secondary metabolism, boosting the production of bioactive compounds with therapeutic potential. These gall induced changes offer valuable applications in phytopharmacology, from improving herbal formulations to supporting drug discovery. Overall, the study provides a useful model for understanding inducible secondary metabolism and its relevance to agriculture and medicine. Future studies should compare other gall-forming species, evaluate ecological functions and investigate post- transcriptional regulations.