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Plants are constantly facing abiotic and biotic stresses. To continue to thrive in their environment, they have developed many sophisticated mechanisms to perceive these stresses and provide an appropriate response. There are many ways to study these stress signals in plant, and among them, protoplasts appear to provide a unique experimental system. As plant cells devoid of cell wall, protoplasts allow observations at the individual cell level. They also offer a prime access to the plasma membrane and an original view on the inside of the cell. In this regard, protoplasts are particularly useful to address essential biological questions regarding stress response, such as protein signaling, ion fluxes, ROS production, and plasma membrane dynamics. Here, the tools associated with protoplasts to comprehend plant stress signaling are overviewed and their potential to decipher plant defense mechanisms is discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gilliard, Huby, Cordelier, Ongena, Dhondt-Cordelier and Deleu.)

A valuable strategy to improve crop yield consists in the use of plant growth-promoting rhizobacteria (PGPRs). However, the influence of PGPR colonization on plant physiology is largely unknown. PGPR Burkholderia phytofirmans strain PsJN (Bp PsJN) colonized only Arabidopsis thaliana roots after seed or soil inoculation. Foliar bacteria were detected only after leaf infiltration. Since, different bacterial times of presence and/or locations in host plant could lead to different plant physiological responses, photosynthesis, and metabolite profiles in A. thaliana leaves were thus investigated following leaf, root, or seed inoculation with Bp PsJN. Only Bp PsJN leaf colonization transiently decreased cyclic electron transport and effective quantum yield of photosystem I (PSI), and prevented a decrease in net photosynthesis and stomatal opening compared to the corresponding control. Metabolomic analysis revealed that soluble sugars, amino acids or their derivatives accumulated differently in all Bp PsJN-inoculated plants. Octanoic acid accumulated only in case of inoculated plants. Modifications in vitamin, organic acid such as tricarboxylic acid intermediates, and hormone amounts were dependent on bacterial time of presence and location. Additionally, a larger array of amino acids and hormones (auxin, cytokinin, abscisic acid) were modified by seed inoculation with Bp PsJN. Our work thereby provides evidence that relative short-term inoculation with Bp PsJN altered physiological status of A. thaliana leaves, whereas long-term bacterization triggered modifications on a larger set of metabolites. Our data highlighted the changes displayed during this plant-microbe interaction to trigger physiological and metabolic responses that could explain the increase in plant growth or stress tolerance conferred by the presence of Bp PsJN.

Several plant growth-promoting rhizobacteria (PGPR) are known to improve plant tolerance to multiple stresses, including low temperatures. However, mechanisms underlying this protection are still poorly understood. The aim of this study was to evaluate the role of the endophytic PGPR, Burkholderia phytofirmans strain PsJN (Bp PsJN), on Arabidopsis thaliana cold tolerance using photosynthesis parameters as physiological markers. Under standard conditions, our results indicated that Bp PsJN inoculation led to growth promotion of Arabidopsis plants without significant modification on photosynthesis parameters and chloroplast organization. However, bacterial colonization induced a cell wall strengthening in the mesophyll. Impact of inoculation modes (either on seeds or by soil irrigation) and their effects overnight at 0, -1, or -3°C, were investigated by following photosystem II (PSII) activity and gas exchanges. Following low temperatures stress, a decrease of photosynthesis parameters was observed. In addition, during three consecutive nights or days at -1°C, PSII activity was monitored. Pigment contents, RuBisCO protein abundance, expression of several genes including RbcS, RbcL, CBF1, CBF2, CBF3, ICE1, COR15a, and COR78 were evaluated at the end of exposure. To assess the impact of the bacteria on cell ultrastructure under low temperatures, microscopic observations were achieved. Results indicated that freezing treatment induced significant changes in PSII activity as early as the first cold day, whereas the same impact on PSII activity was observed only during the third cold night. The significant effects conferred by PsJN were differential accumulation of pigments, and reduced expression of RbcL and COR78. Microscopical observations showed an alteration/disorganization in A. thaliana leaf mesophyll cells independently of the freezing treatments. The presence of bacteria during the three successive nights or days did not significantly improved A. thaliana responses but prevented the plasmalemma disruption under freezing stress.