The role of apoplastic reactive oxygen species metabolism in ammonium-induced growth inhibition in Arabidopis thaliana

Nitrogen is essential for development and overall wellbeing of plants. Plants primarily absorb two forms of inorganic nitrogen in their root systems: nitrates (NO3–) and ammonium ions (NH4+). However, cultivation in the presence of reduced forms of inorganic nitrogen – ammonium ions as the only source of nitrogen, affects some physiological and biochemical processes in plants, causing a set of developmental disorders, generally called the ammonium syndrome. In this study, we hypothesized that the ammonium-induced increase in ROS production, particularly in the extracellular space, significantly contributes to the inhibition of plant growth. The primary research material used was Arabidopsis thaliana grown in the presence of nitrate (control conditions) or ammonium ions as the only source of nitrogen. In study, in addition to the A. thaliana ecotype Columbia-0 (Col-0), other mutants characterized by impaired metabolism of apoplast ROS were used, including rbohd and rbohc mutants and pox33 mutants. This study demonstrated that rbohd mutants of A. thaliana are characterized by significantly reduced production of apoplast ROS under ammonium nutrition. Dysfunction of the RBOHD gene also resulted in the disturbance of the apoplastic redox system, which led to a decrease in the concentration but an increase in the reduction state of ascorbate located in the apoplast. Reduced expression of genes whose products are considered apoplast ROS sensors was detected in rbohd mutants grown in the presence of ammonium ions as a source of nitrogen. Furthermore, reduced expression of genes whose products are considered sensors of cell wall integrity was noted in rbohd mutants under ammonium nutrition. Thus, this study proved that RBOHD activity in A. thaliana is an important element of the system that determines sensitivity to ammonium stress. As part of my PhD thesis, I attempted to identify changes in apoplastic metabolism that are the result of redox changes occurring in the cell as a consequence of nitrogen assimilation and are directly activated by the availability of ammonium ions as a source of nitrogen. For this purpose, I characterized the changes in apoplast redox metabolism in A. thaliana seedlings (ecotype Columbia-0) grown in a shaking liquid culture for short periods (30 min, 2 h, and 24 h) following the addition of ammonium ions as the only source of nitrogen. I have observed that such a short treatment with ammonium ions does not cause oxidative stress in A. thaliana tissues, but induces numerous changes in apoplastic metabolism. The results of this study indicated that the redox changes in the apoplast appeared quickly after the addition of ammonium ions as a nitrogen source to A. thaliana seedlings. The results also suggest that some signaling pathways that determine sensitivity to ammonium ions are initiated by oxidoreductive changes in the apoplastic space. Additionally, I characterized the effects of ammonium nutrition on plant cell division and concluded that the observed changes in the transcript level of cell cycle-related proteins under ammonium stress are dependent on ammonium-induced changes in apoplastic ROS metabolism. Moreover, I observed that ammonium nutrition increased the endopolyploidy of nuclei isolated from cells of apical buds collected from A. thaliana plants. Based on the results, I conclude that ROS metabolism, particularly apoplastic ROS metabolism, is an important factor determining the inhibition of plant growth under ammonium nutrition. It can be concluded that plant growth inhibition induced by changes in the apoplastic redox state under ammonium stress conditions is most likely the sum of changes, including changes in cell wall characteristics, a reduction in cell volume growth potential, modifications in ROS-dependent apoplast-to-symplast signaling, and a reduction in the number of dividing cells.