Understanding the functional role of the organic acids for stomatal movements and stress responses in Arabidopsis thaliana
Authors:   , Oliveira Hellen Oliveira de 
Published: June 2023

Organic acids (OAs) are central to cellular metabolism performing several functions: beyond their role as intermediates of the tricarboxylic acid cycle and precursors for the biosynthesis of other molecules, they are also a predominant part of root exudates, modulators of redox states between cellular compartments, and key regulators in the response of guard cells to environmental stimuli. In this thesis, we present distinct aspects of the complex regulation of OAs and how their transport mediates stomatal movements in guard cells. We initially summarize the current molecular advances underlying the complex regulation of stomatal development and movements to dynamic environmental conditions. In doing so, we were able to showcase that (i) stomatal closure in response to abscisic acid (ABA) depends on the disassembly of microtubules by the degradation of a stabilizing protein during the stomatal opening, (ii) sucrose supply in short days results in the accumulation of an conserved energy sensor kinase that promotes stomatal development, and (iii) that production of a non-protein amino acid gamma-aminobutyric acid (GABA) is necessary to reduce stomatal opening and transpirational loss through down-regulation of a vacuolar anion transporter (ALMT9) of stomatal cells in response to drought. In chapter 3, we addressed the importance of OAs for plant performance in the context of mitochondrial and guard cell metabolism under abiotic stress conditions. Accordingly, we reviewed the role of OAs as root chelating agent, in biosynthesis of stress signaling and osmoregulatory solutes during stomatal movements. We conclude that plants use different mechanisms to regulate and accumulate OAs, depending on the cell organ, cell compartment, and stress condition, allowing the proper functioning of physiological and biochemical responses in plants following stress conditions. In chapter 4, to obtain a more comprehensive picture of the vacuolar transport of OAs in guard cells, we analyzed how the impaired malate accumulation impacts effects on stomatal behavior, photosynthetic capacity and primary metabolism in leaves of plants with individual and combined repression of tonoplast dicarboxylate transporter (tDT), ALMT6 and ALMT4 channels. Briefly, the results presented here provide evidence on (i) the inefficiency of stomatal opening caused by ALMT6 repression in almt6, almt6 tdt-1 and almt6 almt4 lines suggesting that, ALMT6 compensates for vacuolar malate transport in guard cells upon tDT repression with respect to the stomatal opening. However, tDT and ALMT4 are important in the proper storage of dicarboxylates in the vacuole of mesophyll cells. In addition, (ii) we also observed that almt6 almt4 plants maintained growth by increasing dark respiration and sugar accumulation, whereas in almt6 and almt4 plants this accumulation maintained respiration rates, with unchanged and impaired growth, respectively; (iii) repression of ALMT6 and ALMT4 channels led to slower stomatal kinetics and lower stomatal conductance, highlighting the importance of ALMT6 for stomatal opening, and that non-functional ALMT4 likely downregulates the activity of other ion and solute transport channels in guard cells during stomatal opening. Despite that, we did not observe effects on stomatal behavior under high CO 2 , and, as a result, all mutants were responsive to ABA during stomatal closure, indicating the possible activity of OAs and other ion channels in the guard cell plasma membrane. Future analyses are required to determine if alternative ALMT family members and/or others ions transporters are functioning at guard cell transcriptional levels. This input knowledge will be necessary for a better understanding of the mechanisms used to bypass the impaired accumulation of organic acids in these plants. Keywords: Environmental stress. Mitochondrial metabolism. Vacuolar transport. Organic acids. Respiration. Stomata.