A method to experimentally clamp leaf water content to defined values to assess its effects on apoplastic pH

Background Leaf hydration is controlled by feedback mechanisms, e.g. stomatal responses, adjustments of osmotic potential and hydraulic conductivity. Leaf water content thus is an input into related feedback-loops controlling the balance of water uptake and loss. Apoplastic alkalisation upon leaf dehydration is hypothesized to be involved in water stress related signaling on tissue level. When studying these mechanisms and their intermediate signaling steps, an experimenter has only poor means to actually control the central experimental variable, leaf water content (LWC), because it is not only dependent on external variables (e.g. air humidity), which are under experimental control, but is also governed by the biological influences controlling transpiration and water uptake. Those are often unknown in their magnitude, unpredictable and fluctuating throughout an experiment and will prevent true repetitions of an experiment. The goal of the method presented here is to experimentally control and manipulate leaf water content (LWC) of attached intact leaves enclosed in a cuvette. Results An experimental setup was developed where LWC is measured by a sensor based on IR-transmission and its signal processed to control a pump which circulates air from the cuvette through a cold trap. Hereby a feedback-loop is formed, which by adjusting vapour pressure deficit (VPD) and consequently leaf transpiration can precisely control LWC. This technique is demonstrated here in a combination with microscopic fluorescence imaging of apoplastic pH (pH apo ) as indicated by the excitation ratio of the pH sensitive dye OregonGreen. Initial results indicate that pH apo of the adaxial epidermis of Vicia faba is linearly related to reductions in LWC. Conclusions Using this setup, constant LWC levels, step changes or ramps can be experimentally applied while simultaneously measuring physiological responses. The example experiments demonstrate that bringing LWC under experimental control in this way allows better controlled and more repeatable experiments to probe quantitative relationships between LWC and signaling and regulatory processes.

Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging

Building a proton gradient across a biological membrane and between different tissues is a matter of great importance for plant development and nutrition. To gain a better understanding of proton distribution in the plant root apoplast as well as across the plasma membrane, we generated Arabidopsis plants expressing stable membrane-anchored ratiometric fluorescent sensors based on pHluorin. These sensors enabled noninvasive pH-specific measurements in mature root cells from the medium–epidermis interface up to the inner cell layers that lie beyond the Casparian strip. The membrane-associated apoplastic pH was much more alkaline than the overall apoplastic space pH. Proton concentration associated with the plasma membrane was very stable, even when the growth medium pH was altered. This is in apparent contradiction with the direct connection between root intercellular space and the external medium. The plasma membrane-associated pH in the stele was the most preserved and displayed the lowest apoplastic pH (6.0 to 6.1) and the highest transmembrane delta pH (1.5 to 2.2). Both pH values also correlated well with optimal activities of channels and transporters involved in ion uptake and redistribution from the root to the aerial part. In growth medium where ionic content is minimized, the root plasma membrane-associated pH was more affected by environmental proton changes, especially for the most external cell layers. Calcium concentration appears to play a major role in apoplastic pH under these restrictive conditions, supporting a role for the cell wall in pH homeostasis of the unstirred surface layer of plasma membrane in mature roots.

Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana

Plant cells are embedded within cell walls, which provide structural integrity, but also spatially constrain cells, and must therefore be modified to allow cellular expansion. The long-standing acid growth theory postulates that auxin triggers apoplast acidification, thereby activating cell wall-loosening enzymes that enable cell expansion in shoots. Interestingly, this model remains heavily debated in roots, because of both the complex role of auxin in plant development as well as technical limitations in investigating apoplastic pH at cellular resolution. Here, we introduce 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as a suitable fluorescent pH indicator for assessing apoplastic pH, and thus acid growth, at a cellular resolution in Arabidopsis thaliana roots. Using HPTS, we demonstrate that cell wall acidification triggers cellular expansion, which is correlated with a preceding increase of auxin signaling. Reduction in auxin levels, perception, or signaling abolishes both the extracellular acidification and cellular expansion. These findings jointly suggest that endogenous auxin controls apoplastic acidification and the onset of cellular elongation in roots. In contrast, an endogenous or exogenous increase in auxin levels induces a transient alkalinization of the extracellular matrix, reducing cellular elongation. The receptor-like kinase FERONIA is required for this physiological process, which affects cellular root expansion during the gravitropic response. These findings pinpoint a complex, presumably concentration-dependent role for auxin in apoplastic pH regulation, steering the rate of root cell expansion and gravitropic response.