The Relationship between Turgor Pressure Change and Cell Hydraulics of Midrib Parenchyma Cells in the Leaves of Zea mays

Leaf dehydration decreases water potential and cell turgor pressure. Therefore, changes in cell turgor pressure may regulate water transport across plant cell membranes. Using a cell pressure probe, the hydraulic properties of parenchyma cells in the midrib of maize (Zea mays L.) leaves were measured (half time T1/2 of water exchange in cells as a measure of hydraulic conductivity Lp). Using intact plants with root systems encased in a pressure chamber, the root systems were pressurized and the turgor pressure in leaf cells increased by increments up to 0.3 MPa. However, the increase in the cell turgor did not increase but stabilized T1/2 values. Increased water potential in leaf cells seemed to have stabilizing effects on the T1/2 probably due to enhanced water availability. When the cell turgor decreased by 0.1 MPa to 0.3 MPa with releasing the pressure in the pressure chamber, T1/2 was temporarily increased to a large degree, a factor of up to 13 within 30 min.


Introduction
Plant leaves experience various ranges of water potential in response to the changes in transpiration rate or soil water content. In response to such changes, plants need to adjust their ability to conduct water through leaves, the leaf hydraulic conductance [1]. The leaf hydraulic conductance (K leaf ) is the inverse of the hydraulic resistance R leaf , and expressed in kg H 2 O s −1 MPa −1 . Leaf dehydration has been known to decrease K leaf [2][3][4]. One of the possible causes for the K leaf decline with dehydration is the outside-xylem hydraulic conductance, which includes the cell hydraulic conductivity, Lp [5][6][7][8]. The cell Lp is a measure of how efficiently water is transported through a single cell normalized by cell area and expressed in m −1 s −1 MPa −1 . However, the cause for the K leaf decline has not been fully studied yet. The cell Lp could decline in response to the decrease of the cell turgor pressure by leaf dehydration [3,9]. Increasing evidence shows that the hydraulics in a single plant cell level are mainly regulated by water channels, aquaporins (AQPs) [10][11][12][13][14][15][16]16,18]. In response to biotic or/and abiotic stress, AQPs can either increase or decrease the cell Lp by either opening or closing ('gating') in a short-term response. On the other hand, in a long-term response, the de novo expression of AQPs can increase cell Lp or the development of the apoplastic barrier can decrease cell Lp [14,15,[19][20][21].
Turgor pressure has been suspected to be a signal of gating AQPs [22,23]. A previous study showed that change in the turgor pressure or mechanical stimuli affected the cell Lp [24]. Moreover, the cell Lp change has been shown to be attributed to the action on AQPs [9,[24][25][26]. Wan et al. [24] reported that both positive and negative pressure pulses decreased the cell Lp and that the action of AQPs was involved. They suggested a model in which the mechanical stimuli (pressure pulses) induced water flux and closed the AQPs. Kim and Steudle [9] investigated the change in the cell Lp in response to illumination, which reduced the turgor pressure because of the increase in leaf transpiration. They reported that the cell Lp was first increased by light and then decreased as the turgor pressure decreased. In this case, the light and turgor pressure changed together, so the effects caused by light and turgor coexisted and separation of the effects by light and turgor was difficult. When Kim and Steudle [9] maintained the turgor constant during illumination to eliminate the turgor effect, the change in light increased the cell Lp. This result indirectly demonstrated that the decrease in turgor pressure led to the decrease in the cell Lp. However, it was not a direct measurement of the effect of turgor pressure.
In the present study, we aimed to examine the effect of turgor pressure change on the water permeability of parenchyma cells in the midrib of a maize leaf using a cell pressure probe [27,28]. We investigated the effects of the cell turgor pressure on the water permeability of parenchyma cells in the midrib of a maize leaf. We varied the turgor pressure by applying pressure to the root system encased in a pressure chamber to manipulate the root pressure and the leaf cell turgor. To increase the cell turgor, the pressure chamber pressurized the root system, and then the pressure in the pressure chamber was released to the atmospheric pressure. After changing the pressure, cell Lp values were continuously measured. This measurement result showed the kinetics of cell Lp and allowed the discussion in terms of the gating of AQPs.

Plant Material
Corn (Zea mays L. cv. monitor) plants were grown in plastic pots with soil in a greenhouse of Bayreuth University, Germany as described in Kim and Steudle [9]. When plants were 4 to 8 weeks old, the cell pressure probe measurements were performed on parenchyma cells in the midrib region of the leaves, which were fourth or fifth leaves counting from the oldest. The cells were located 100-200 mm behind the leaf tip. Material used in this study was the same tissue of the plants of a similar age, as in Kim and Steudle [9].

Experimental Setup Using a Cell Pressure Probe
As described earlier [9], parenchyma cells in the midrib were punctured by a microcapillary of a cell pressure probe (CPP). The capillary with a fine tip of about 6 µm in diameter was filled with silicon oil (oil type AS4 from Wacker, Munich, Germany). The measurements of the cell turgor pressure (P) using CPP are described in Kim and Steudle [9]. Hydrostatic relaxation of turgor was performed and the half time of the hydrostatic relaxation, T 1/2 , was obtained to calculate the hydraulic conductivity of cell membranes (Lp) using the Equation (1) in Kim and Steudle [9]. The volumetric elastic modulus (ε) was calculated using the pressure change and relative volume change by Equation (2) in Kim and Steudle [9]. In the calculation of Lp and ε, we used average values of cell diameter and length from Kim and Steudle [9], who used the same type of cells located in a similar position from the plants of a similar age. In most of cases, T 1/2 was used to indicate the change in Lp because ε did not change significantly during the whole measurements even though there is a change in turgor pressure (see Results). The half time T 1/2 is inversely proportional to Lp, that is, large T 1/2 means small Lp. During the hydrostatic relaxation, the size of the pressure peak was maintained at less than 0.1 MPa to Cells 2018, 7, 180 3 of 12 avoid closing aquaporins by large pressure pulses, as reported in Wan et al. [24]. After puncturing cells, T 1/2 varied in the leaf cells of intact corn plants grown in soil [9]. Less than half of the population of cells measured in this study had small T 1/2 values of approximately 1 s after a transient increase in T 1/2 caused by the cell puncture, as discussed later. For those cells having small T 1/2 values, we checked whether or not T 1/2 was affected by the change in turgor pressure. Further information on the CPP measurement is described in previous studies [29][30][31].

Pressurization Experiment
The root system of an intact corn plant was encased in a pressure chamber and light lamp (Siemens AG, Frankfurt, Germany) was installed above the plant to illuminate the whole plant. It was the same set-up used in Kim and Steudle [9]. The root system was pressurized with the increment of pressure in the range of 0.05 MPa-0.1 MPa (small), 0.11 MPa-0.2 MPa (medium), or 0.21 MPa-0.3 MPa (large). Pressurizing the root caused to increase cell turgor pressure in leaves (see Results). Hydrostatic relaxations (T 1/2 ) were evaluated before and after the root system was pressurized. After the root was pressurized, T 1/2 values of hydrostatic relaxations were evaluated only when the turgor value was rather constant to eliminate the errors in determining the T 1/2 under changing turgor. When pressure was released to the atmospheric pressure, the cell turgor pressure returned to the original level, and then T 1/2 was analyzed.

Pressure Chamber Pressure vs. Cell Turgor Pressure
For intact plants, the root pressure was modified to change leaf cell turgor. When a certain level of pressure was applied to the root system, the cell turgor pressure increased with the ratio of 1:1 (Figures 1 and 2). Before time zero, the whole plant in Figure 1 was illuminated with the light intensity of 160 µmol m −2 s −1 at the level of the measured cell and the cell turgor was about 0.1 MPa, which was lower than it was without illumination. When the pressure applied to the root was bigger than 0.3 MPa, guttation was observed. In the occurrence of guttation, the responses in the turgor were no longer 1:1 ( Figure 1).
A representative curve of turgor variation upon pressurizing the root chamber is shown in Figure 3A. When a pressure of 0.25 MPa was applied to the pressure chamber, the turgor pressure increased by the same amount (0.25MPa). The changes in turgor pressure were much slower than those of pressure in the root pressure chamber, that is, half time of the pressure change in increasing pressure: 80 s and 220 s for the root chamber pressure and turgor pressure, respectively.        The Lp calculation needed T 1/2 of hydrostatic relaxation, the volumetric elastic modulus (ε), and cell geometry. To calculate Lp for each hydrostatic relaxation in Figure 3A, T 1/2 of the relaxation curve was used and ε value at each turgor pressure was used ( Figure 3B,C). For cell geometry, it was assumed that the cell is a cylinder with a diameter of 63 µm and a length of 76 µm as in Kim and Steudle [9]. The elastic modulus did not change much along the change in turgor pressure ( Figure 3C; ε = 1.1-1.4 MPa). Therefore, using T 1/2 as a direct measure of change in Lp is justified and we investigated changes in T 1/2 upon turgor pressure alterations.

Effect of Turgor Pressure Alterations on T 1/2
Turgor pressure was increased by the increment of 0.05 to 0.1 MPa (small), 0.11 to 0.2 MPa (medium), or 0.21 to 0.3 MPa (large), and then it was decreased back to the original value. A representative case in Figure 3B shows that T 1/2 initially varied after puncturing of a cell and reduced to a small value around 1 s at 40 min. The increase in turgor pressure of the cell by the increment of 0.25 MPa (large range) stabilized T 1/2 . However, when the turgor of the cell decreased by 0.25 MPa and returned to the original turgor pressure in the beginning of the measurement, there was an increase in T 1/2 by a factor of bigger than 10. The T 1/2 s were finally recovered to small values within 30 min. The transient increase in T 1/2 by the large turgor decrease had some analogy to the effect of pressure pulses [24].

Effect of Turgor Pressure Increase on T 1/2
The experiments for the case of turgor pressure increase were conducted with varying increments in pressure increase and the results are summarized in Figures 4 and 5. Single cells were employed to read T 1/2 values before and after increasing the turgor pressure. After the cell puncture, T 1/2 data were varied in the leaf cells of intact corn plants in a stochastic manner [9]. Less than half of the population of cells measured in this study had small T 1/2 values of approximately 1 s after a transient increase in T 1/2 caused by the cell puncture, as illustrated in Figure 3B. For those cells having small T 1/2 values after the lapse of some time, we checked whether or not T 1/2 values were affected by the change in turgor pressure. The increments of turgor pressure increase were classified into three ranges; small (0.05 to 0.1 MPa), medium (0.11 to 0.2 MPa), and large (0.21 to 0.3 MPa). We compared the T 1/2 value just before the turgor pressure increased and the maximum T 1/2 after the turgor increased to a stable value (T max ). The increases in turgor pressure by small, medium, or large increments did not cause a temporary increase in the T 1/2 (Figure 4; n = 6-10 cells, paired t-tests for each range of turgor pressure increase, p < 0.05). Aside from the temporary effect, the increases in turgor pressure by small, medium, or large increments tended to decrease the T 1/2 , however, the decreases were not so statistically significant ( Figure 5; n = 6-10 cells, paired t-tests for each range of turgor increment, p < 0.05). For statistical analysis, we compared the mean values of T 1/2 s for 10 min before the turgor increased and those for 20 min after the turgor pressure increased to a stable saturated value. The half times might further decrease in response to a longer period of high turgor pressure (see Section 4).    The increases of the turgor by the increments in small, medium, or large ranges did not increase T 1/2 . Mean values ± SD were plotted. The significance in differences was tested and noted with different alphabets (n = 6-10 cells, paired t-tests for each range of turgor pressure increase, p < 0.05). / 1 T . Mean values ± SD were plotted. The significance in differences was tested and noted with different alphabets (n = 6-10 cells, paired t-tests for each range of turgor pressure increase, p < 0.05).  Figure 3B. However, the decrease was not so statistically significant for repeated cells. Mean values ± SD were plotted. The significance in differences was tested and noted with different alphabets (n = 6-10 cells, paired t-tests for each range of turgor pressure increase, p < 0.05).

Effect of Decreases in Turgor Pressure Back to the Original Value on
The experiments for the case of turgor pressure decrease as seen in Figure 3 were conducted for varying pressure decreases and summarized in Figure 6. Following a single cell, we compared the   Figure 3B. However, the decrease was not so statistically significant for repeated cells. Mean values ± SD were plotted. The significance in differences was tested and noted with different alphabets (n = 6-10 cells, paired t-tests for each range of turgor pressure increase, p < 0.05).

Effect of Decreases in Turgor Pressure Back to the Original Value on T 1/2
The experiments for the case of turgor pressure decrease as seen in Figure 3 were conducted for varying pressure decreases and summarized in Figure 6. Following a single cell, we compared the T 1/2 just before the turgor decreased and the maximum T 1/2 after the turgor decreased to a stable value (T max ). In response to the cell turgor decreases by small ranges (0.05 to 0.1 MPa), the changes in T 1/2 were not significant. The decreases in the turgor pressure by medium (0.11 to 0.2 MPa) or large (0.21 to 0.3 MPa) ranges induced a transient increase in T 1/2 , which finally returned to the original value before decreasing the cell turgor pressure. The significances in the difference between T 1/2 just before turgor decreased and T max are shown in Figure 6 (n = 5-7 cells, paired t-tests for each range of turgor pressure decrease, p < 0.05).   T in a bigger fold than in (B). Mean values ± SD were plotted. The significance in differences was tested and noted with different alphabets (n = 5-7 cells, paired t-tests for each range of turgor pressure decrease, p < 0.05).

Discussion
We observed that the pressurized root system increased the leaf cell turgor pressure and it rather stabilized leaf cell hydraulic conductivity (Lp). However, the return of the turgor to its original value . Mean values ± SD were plotted. The significance in differences was tested and noted with different alphabets (n = 5-7 cells, paired t-tests for each range of turgor pressure decrease, p < 0.05).

Discussion
We observed that the pressurized root system increased the leaf cell turgor pressure and it rather stabilized leaf cell hydraulic conductivity (Lp). However, the return of the turgor to its original value by releasing the chamber pressure to the atmospheric pressure, except for the case of small changes up to 0.1 MPa, decreased the cell Lp temporarily. This transient pattern in cell Lp by pressure decrease was similar to what happened when pressure pulses were imposed on cortical cells of young corn roots in the study of Wan et al. [24]. These authors observed that root cell Lp decreased along both directions of the pressure pulses and they explained the phenomena by energy injection model. If the energy injection model was the only mechanism being applied here, this type of effect should have been observed after the turgor increased. However, it did not occur. Therefore, another type of co-working mechanism may play a role when the turgor pressure increases (water availability increases). Although the mechanism for the response of the cell Lp to the cell turgor pressure is still unknown, this study contributes to the revelation that the cell Lp responds to the changes in cell turgor.
The next question concerns what the signal is to change the cell Lp when the cell turgor changes. The first candidate, which is most likely to affect the cell Lp, is the change in turgor pressure, which causes water flux and a mechanical stress. The decrease of the cell turgor pressure to its original value by releasing the root pressure was found to induce a temporary decrease in the cell Lp. This effect is analogous to that of Wan et al. [24] in which pressure pulses were imposed on the cortical cells of young corn roots. They observed a similar pattern of temporary decrease in cell Lp, which was more pronounced in larger pulses. They demonstrated this effect to be related to the aquaporins by using a heavy metal known to close the AQPs. According to the energy injection model of Wan et al. [24], the change in the turgor gives rise to a substantial water flux across the AQPs, transferring energy to the AQPs and closes them (Figure 7). There could be also another way of AQP gating in response to turgor pressure change. Johannson et al. [22] proposed that the Ca 2+ channel could be mechanosensitively gated by changes in the turgor pressure. Cytoplasmic Ca 2+ concentration has been known to be responsible for the gating of AQP by phosphorylation [22,32,33]. The next candidate is the water availability. If the pressure change was only detected, we could have observed the same effect caused by the increase in the cell turgor pressure. However, we did not observe it. The increase of turgor pressure rather stabilized the cell Lp, whose values fluctuated after the puncturing of the cell by a micro capillary of the cell pressure probe. In addition, there was a tendency of increasing cell Lp within 30 min after initiation of changing pressure, despite the negligible statistical differences. Therefore, another mechanism probably coexists to override the closure of AQPs in response to the increase in the water availability by increase in cell turgor pressure. The apoplastic water content near a cell may affect the local Ca 2+ concentration or pH, and this may influence the gating of AQPs [22,[32][33][34]. In a long-term response to high turgor pressure, the expression of AQPs might be involved and the cell Lp could respond differently [19]. In the present study, when water flow was produced by reducing the water potential in the surrounding leaf tissue by decreasing the root pressure, the water flow through the AQPs might close AQPs in the same way.

Conclusions
In conclusion, this study shows for the first time that the cell Lp temporarily decreased with the decrease in the cell turgor pressure. This study did not aim to identify the mechanism of Lp response to changes in turgor pressure. We postulated the involvement of gating of AQPs, because the cell Lp variation pattern of parenchyma cells in the midrib of maize leaves in response to the decreased cell turgor pressure was similar to that of cortical cells in young maize roots in response to pressure pulses in a previous study of Wan et al. [24]. They demonstrated the gating of AQPs by pressure pulses and heavy metals. However, we did not see the temporary increase in leaf cell Lp by increase of turgor pressure on the opposite of the case of turgor pressure decrease. These results support that both the changes in cell turgor pressure related to mechanical stimuli and the water availability may coordinate the hydraulic features of leaf cells in response to pressure changes. Acknowledgments: We commemorate the late Steudle (Department of Plant Ecology, Bayreuth University) for his valuable contribution in this study. We acknowledge the Deutscher Akademischer Austauschdienst (DAAD) for a grant to Y.X.K.

Conflicts of Interest:
The authors declare no conflict of interest.  [24]. Moving the position of meniscus between sap and oil in a micro capillary of a cell pressure probe could produce a big pressure pulse and water flow through the AQPs. The water flow might inject energy to the AQP and cause a reversible mechanical deformation of the protein resulting in the closure of the channel. (B) In the present study, when water flow was produced by reducing the water potential in the surrounding leaf tissue by decreasing the root pressure, the water flow through the AQPs might close AQPs in the same way.
At the level of leaf tissues, a similar trend of the decrease in K leaf by the turgor pressure decrease was observed [23]. The authors considered the turgor pressure as a signal to decrease K leaf . They suspected the decrease in K leaf was caused by the decrease in the cell Lp, by the closure of the AQPs in the bundle sheath. This speculation is in accordance with the result of the present study that the decrease in cell turgor decreases the parenchyma cell Lp, although there could be differences among different cell types. Moreover, K leaf declines with dehydration, and the potential sources of K leaf decline are known as xylem cavitation, xylem collapse, and the decline in conductance of extra-xylem paths [1,4]. Among these factors, recently the changes in extra-xylem paths such as AQPs in a bundle sheath were considered to play an important role to explain the K leaf decline [5][6][7][8]. Especially for mild dehydration in which cells are still turgid, cell hydraulics would still have an important role in leaf tissue hydraulics. To investigate cell hydraulics, the cell pressure probe is a powerful tool to measure the activity of AQPs in a single living cell of intact plants, which encounter water deficit. However, the use of the cell pressure probe on cells having low turgor caused by water deficit is technically difficult. Either invention of a new technique or clever designs of the experiment, or both, are required to examine cell hydraulics in response to water deficit.
The transiency in the decrease of cell Lp by turgor decrease in the present experiment may sound inconsistent that dehydration decreases K leaf . However, the current experiment is not real leaf dehydration. It is a simulation of a turgor pressure decrease, which was first increased, to test the effect of water flux produced by pressure change. The increasing of a turgor implied that there could be an effect by the absolute value of turgor pressure, that is, increasing the turgor stabilized T 1/2 . If the turgor decreased compared to the original value, the effect could be different, that is, an effect of water flux and an effect of the decrease in the absolute value of turgor (water availability). This was observed by Kim and Steudle [9] by increasing the light intensity, and T 1/2 initially decreased and then increased, but this was a co-effect of both light and turgor decrease. Other experiments reducing cell turgor, for example dehydration or application of osmoticum simulating water deficit, would give direct effects of the pressure decrease into a value lower than the original value.
The pattern of changes in cell turgor pressure by increasing the root pressure was similar to Wei et al. [28], who measured the responses in the xylem pressure using a pressure probe to the pressure applied to the root systems of corn plants. Cell pressure did not increase after guttation occurred as the xylem pressure did not increase after guttation occurred. The increased xylem pressure was delivered to the leaf cells. The increasing pressure in the leaf cells was slower than that in the xylem. This slowness is closely related to the distance from the cells to the xylem vessel and the cell hydraulic conductivity, Lp [27].

Conclusions
In conclusion, this study shows for the first time that the cell Lp temporarily decreased with the decrease in the cell turgor pressure. This study did not aim to identify the mechanism of Lp response to changes in turgor pressure. We postulated the involvement of gating of AQPs, because the cell Lp variation pattern of parenchyma cells in the midrib of maize leaves in response to the decreased cell turgor pressure was similar to that of cortical cells in young maize roots in response to pressure pulses in a previous study of Wan et al. [24]. They demonstrated the gating of AQPs by pressure pulses and heavy metals. However, we did not see the temporary increase in leaf cell Lp by increase of turgor pressure on the opposite of the case of turgor pressure decrease. These results support that both the changes in cell turgor pressure related to mechanical stimuli and the water availability may coordinate the hydraulic features of leaf cells in response to pressure changes.