A new experimental setup to measure hydraulic conductivity of plant segments

Abstract Plant hydraulic conductivity and its decline under water stress are the focal point of current plant hydraulic research. The common methods of measuring hydraulic conductivity control a pressure gradient to push water through plant samples, submitting them to conditions far away from those that are experienced in nature where flow is suction driven and determined by the leaf water demand. In this paper, we present two methods for measuring hydraulic conductivity under closer to natural conditions, an artificial plant setup and a horizontal syringe pump setup. Both approaches use suction to pull water through a plant sample while dynamically monitoring the flow rate and pressure gradients. The syringe setup presented here allows for controlling and rapidly changing flow and pressure conditions, enabling experimental assessment of rapid plant hydraulic responses to water stress. The setup also allows quantification of dynamic changes in water storage of plant samples. Our tests demonstrate that the syringe pump setup can reproduce hydraulic conductivity values measured using the current standard method based on pushing water under above-atmospheric pressure. Surprisingly, using both the traditional and our new syringe pump setup, we found a positive correlation between changes in flow rate and hydraulic conductivity. Moreover, when flow or pressure conditions were changed rapidly, we found substantial contributions to flow by dynamic and largely reversible changes in the water storage of plant samples. Although the measurements can be performed under sub-atmospheric pressures, it is not possible to subject the samples to negative pressures due to the presence of gas bubbles near the valves and pressure sensors. Regardless, this setup allows for unprecedented insights into the interplay between pressure, flow rate, hydraulic conductivity and water storage in plant segments. This work was performed using an Open Science approach with the original data and analysis to be found at https://doi.org/10.5281/zenodo.7322605.

The limit due to this air entry of the setup was tested in the second experiment. If the water pressure falls below a certain point, air enters through the membrane's pores and stop water from flowing to the top. The pressure difference required between the air and the liquid for air to enter is referred to here as the "air entry value".
The expected air entry value for the membranes can calculated based on the pressure drop across the water-air interface in the largest pore, using a form of the Young-Laplace equation (Young et al., 1807): where σ w is the surface tension of water (N m −1 ), r p is the radius of the pores in the membrane (m), and P ae is the air entry value (Pa). Taking a surface tension of water at 20 o C of 0.07286 N m −1 and the pore radius of 2.5 × 10 −6 m reported for the membranes, the expected pressure drop would be 58288 Pa, and thus, the theoretical lowest pressure the water in the membrane can reach before air entry happens at atmospheric pressure would be 101325 − 58288 = 43037 Pa. Note that liquid pressures below this values would be expected to allow air to enter, but as long as the air entry rate is slower than the evaporation rate, liquid pressures could still decrease further below these values. This is consistent with our experimental results where a minimum pressure of 30 kPa was achieved, followed by a progressive increase in pressure and then catastrophic failure at a pressure of 35 kPa (Fig. S1.2). It is likely that air entry first occurs slowly in the form of bubbles, which accumulate at the top of the system, leading to the afore-mentioned additional pressure drop along the membrane and eventually air entry through all pores in the top part of the membrane. Figure S1.1 -Altered vertical experimental setup to determine minimum attainable pressure before air entry into membrane. Figure S1.2 -Flow and pressure measurements of the artificial plant setup. In the three trials, the valve below the pressure sensors was closed at 'a', leading to a decrease in pressure to a minimum value of 'b'. The membrane continued evaporating until air entered the membrane's pores and flow reversed at 'c'.
The measurements of conductivity, flow, and pressure of a 12.3cm twig undergoing the same bubble experiment as in Fig. 3 of the main text. Figure S2 -Flow, pressure, and conductivity measurements of 12.3 cm fagus sylvatica sample in the artificial plant setup. Air bubble was added below the lower pressure sensor 'a', and reached the sample at time 'b' where flow stopped and pressure decreased until air entered the membrane at the top and flow reversed at 'c'.

Conductivity using syringe vs measured flow
The conductivity measurements using both syringe and flowmeter values for the experiment of Fig. 5 in the main text. Figure S3 -Time series of hydraulic conductivity calculated using the syringe flow and the measured flow for a 13.5 cm fagus sylvatica twig while simulating different sorts of water stress. At 'a', a constant pull through the twig at 25 µL min −1 is applied and flow goes around the capillary. At 'b', flow is lead through a capillary upstream of the twig. At 'c', the syringe pump is stopped. At 'd', conditions are similar to 'a'. At 'e', flow is further increased to 50 µL min −1 and returned to 25 µL min −1 at 'f'. The experiment ends at 'g'.
Here we look at the change in water storage of the experimental setup without a twig and only the capillary. The capacitance of the system is used as a correction for twig storage in Fig. 6 of the main text. Figure S4 -Flow and change in water storage of the horizontal syringe method when stressing the system. At 'a', a constant pull through the twig at 25 µL min −1 is applied and flow goes around the capillary. At 'b', flow is lead through a capillary. At 'c', the syringe pump is stopped.
Here we show comparison of the system's change in water storage to that of the twig from Fig. 6 in the main text, presenting the magnitude difference between the storage values. Figure S5 -Change in water storage of the system when creating stress condition with and without twig sample. The changes in water storage of the twig are represented with and without taking the leak into consideration in the calculations.