Endosymbiotic Methylobacterium oryzae mitigates the impact of limited water availability in lentil (Lens culinaris Medik.) by increasing plant cytokinin levels

Abstract

Phytohormones regulate multiple processes of plant physiology, including responses to biotic and abiotic stresses. Among phytohormones, cytokinins (CKs) are an important group of growth regulators responsible for cell division, nutrient allocation and photosynthetic performance. In this work, the effect of an increased pool of CKs in lentil (Lens culinaris, cv. CDC Maxim) exposed to complete water withdrawal during its growth in an environmental chamber was investigated. The elevated phytohormone levels were achieved by inoculating plants with growth-promoting Methylobacterium, a beneficial plant endophyte capable of synthesizing high levels of CKs, specifically the most active forms of trans- (tZ) and cis-Zeatin (cZ). The M. oryzae strain (LMG23582(T)) was selected based on its high CK production and its tolerance to drought stress tested through in vitro culture exposure to PEG6000. To investigate the ability of Methylobacterium to alleviate the effects of drought stress, the following agronomical parameters were investigated: seed germination, seedling growth rate, biometrical characteristics (shoot and root length and weight), physiological traits (photosynthesis, transpiration, water use efficiency, chlorophyll level, and electrolyte conductivity), plant water management (relative water level and leaf water content), phytohormone levels (CK and ABA) and yield-associated traits (number and weight of seeds, Harvest Index). The presence of Methylobacterium significantly enhanced the performance of lentils exposed to drought by: 1) stimulating early growth of shoots and roots (32% and 51% increase, respectively), 2) increasing photosynthetic rates (from 6.21 μmolCO2/m2/s to 7.75 μmolCO2/m2/s for well-watered, and from 3.85 μmolCO2/m2/s, to 4.87 μmolCO2/m2/s for drought-stressed lentils), and 3) improving Harvest Index (7-fold for well-watered and 4-fold for drought-stressed plants). The stress mitigating effect of the bacterial symbiont, M. oryzae, was attributed, at least partially to the microbial CKs delivered to plants. This conclusion was supported by the considerably higher levels of CKs detected by HPLC-MS/MS in the leaves of drought-stressed, inoculated plants (279.5 pmol/gFW of total CKs, compared to 62.1 pmol/gFW in non-inoculated individuals). Furthermore, the most active free base CKs were only found in the inoculated plants.