Hormonal involvement in boosting the vegetative vigour underlying caffeine-related improvements in lentil production

Previous studies have shown that caffeine (1,3,7-trimethylxanthine) has some potential for its use as a biostimulant ingredient for boosting lentil production at suboptimal temperatures. However, some limitations to its use include its potential side effects as an emerging contaminant and the current lack of knowledge of its mechanism of action. Here, we aimed to study the mechanisms underlying improved lentil production upon caffeine application. Greenhouse-grown plants treated with caffeine (at 10^-5 M, 10^-4 M, and 10^-3 M) were compared to an untreated, control treatment, and both reproductive and vegetative vigour were evaluated in parallel with endogenous foliar concentrations of phytohormones, including both stress and growth-related hormones. Results showed an enhanced lentil production at the highest caffeine concentration (10^-3 M) which might be attributed, at least in part, to a greater vegetative vigour. The hormonal profiling revealed a dual effect. Firstly, there was a specific increase in jasmonoyl-isoleucine (JA-Ile) in the short term, which may provide a priming effect. Secondly, abscisic acid (ABA) content kept at low levels and the active cytokinin (CK) isopentenyl adenine (2-iP) increased and persisted at high levels throughout the reproductive stage. Cytokinin-mediated effects on growth, and more specifically the high CK/ABA ratios in leaves, appeared to mediate caffeine-related effects in boosting vegetative vigour. In conclusion, caffeine emerges as a compelling alkaloid for integration into biostimulant formulations due to its favorable effect in boosting lentil production through an improvement of vegetative vigour. These outcomes appear to be modulated by phytohormones, most notably jasmonates, priming plants for improved performance under suboptimal temperatures, and cytokinins, alongside ABA and its associated ratios, collectively enhancing plant growth and reproductive vigour in challenging conditions.