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Leaf thickness and turgor pressure in bean during plant desiccation
Scientia Horticulturae 184 (2015) 55–62
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Leaf thickness and turgor pressure in bean during plant desiccation Hans-Dieter Seelig a,∗ , Adelheid Wolter b , Fritz-Gerald Schröder b a b
University of Applied Sciences Dresden, Department of Electrical Engineering, Friedrich-List-Platz 1, Dresden 01069, Germany University of Applied Sciences Dresden, Department of Agriculture/Landscape Management, Friedrich-List-Platz 1, Dresden 01069, Germany
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Article history: Received 10 October 2014 Received in revised form 17 December 2014 Accepted 18 December 2014 Available online 13 January 2015 Keywords: Leaf thickness Turgor pressure Leaf water content Water deficit stress Irrigation control
a b s t r a c t This study investigated a possible relationship between the relative change of leaf thickness and the relative degree of turgor pressure in leaf cells. Leaf thickness and turgor pressure were measured simultaneously during forced plant desiccation in bean plants. Although leaf thickness followed closely the steep decline of the total water potential ( ) during the desiccation process, no conclusive relationship could be observed between leaf thickness and the osmotic water potential ( s ). Thus, a potential link between the relative change of leaf thickness and the relative degree of turgor pressure could not be verified under the experimental conditions. Earlier findings, that the relative change of leaf thickness is closely related to the relative change of the total water potential of leaf cells, are corroborated. Additional observations were: (i) a potential relationship between the relative change of leaf thickness during desiccation and the totally achieved leaf thickness, and (ii) the apparent decrease of leaf thickness during desiccation in discrete levels rather than a truly continuous decline. © 2015 Elsevier B.V. All rights reserved.
1. Introduction It has been demonstrated that the thickness of leaves can fluctuate quickly in response to certain environmental factors. For example, if soil moisture content decreases below a specific threshold, leaf thickness typically collapses within one to several hours. Taking advantage of this effect, the continuous measurement of leaf thickness may be used to control irrigation (Sharon and Bravdo, 2001). This method has the potential to save enormous amounts of irrigation water (Seelig et al., 2012; Sharon and Bravdo, 2001). It has also been shown that leaf thickness can change quickly, that is within minutes, in response to fluctuations of air temperature and air humidity. Thus, it appears that leaf thickness responds in some way to changes of the water status of leaf tissue. But what is the mechanism relating these observations? Said another way: what exactly is the message of observing short term changes of leaf thickness? In order to interpret the measurements of leaf thickness fruitfully, it is essential to have a sound understanding of the underlying cause-and-effect relationship (Jones, 2007; Scoffoni et al., 2014). Malone and Alarcon have demonstrated that the thickness of leaves can change almost immediately in response to leaf wounding (Alarcon and Malone, 1994; Malone and Alarcon, 1994). These
Abbreviations: LT, leaf thickness; TP, turgor pressure. ∗ Corresponding author. Tel.: +49 3514622346; fax: +49 3514622193. E-mail address: [email protected] (H.-D. Seelig). http://dx.doi.org/10.1016/j.scienta.2014.12.025 0304-4238/© 2015 Elsevier B.V. All rights reserved.
studies have also provided convincing evidence about the cause for this quick change in leaf thickness. When a leaf is wounded, some water is being dispersed to other regions of the leaf or plant, causing leaf thickness to increase there. This principle is termed hydraulic dispersal. Other studies have documented that the thickness of leaves can change substantial within hours or minutes in response to soil moisture content, air temperature, and air humidity. When soil is well watered, or exerts only a mild water deficit stress on the plant, leaf thickness is typically kept fairly constant. On the contrary, if the moisture content of the soil falls below a certain threshold level, leaf thickness can literally collapse within one to several hours. It may increase to its nominal value rather quickly though if the plant is being watered soon (Búrquez, 1987; McBurney, 1992; Seelig et al., 2012; Tyree and Cameron, 1977). Leaf thickness also seems to respond within minutes to ambient air conditions. During day times, leaf thickness tends to increase when the relative humidity of the ambient air rises (Búrquez, 1987), and to decrease as the vapor pressure deficit of the ambient air increases (Meidner, 1952; Syvertsen and Levy, 1982). It has also been reported that daytime leaf thickness tends to decline when the temperature of the ambient air increases (Búrquez, 1987; Li et al., 2009; McBurney, 1992; Meidner, 1952; Syvertsen and Levy, 1982). Rather than seeing a true temperature effect, this observation may simply reflect changes in the demand for evapotranspiration as well. Because both, the relative humidity as well as the vapor pressure deficit, will fluctuate as the temperature of the air changes. Overall, it appears that leaf thickness
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Fig. 1. Relationships between leaf thickness and turgor pressure (A) as well as between the relative leaf thickness and the relative turgor pressure (B) in all three plants investigated.
responds to two forces: excessive water deficit stress from the soil and daytime water demand from the air. However, although these changes of leaf thickness due to soil moisture content, air temperature, and air humidity have been documented multiple times, it remains unclear what exactly causes them. While hydraulic dispersal due to leaf wounding is the only plausible explanation so far as to why leaf thickness can fluctuate within short periods of time, hydraulic dispersal does not really serve as an explanation for the changes of leaf thickness in response to environmental factors such as soil moisture content, air temperature, and air humidity. Given the fact that leaf thickness can fluctuate rather quickly due to both factors, water deficit stress from the soil as well as water demand from the air, it appears that these types of leaf thickness variations reflect changes in the water status of the leaf tissue. If so, which type or component of leaf water status would actually be the true independent variable for these quick changes of leaf thickness? Obviously, when leaves dehydrate, leaf thickness will shrink due to this loss of water. Thus, leaf thickness is a function of the absolute water content of leaf cells. The relative water content (RWC) of leaves, which is often used to describe leaf water status, will equally reflect this loss of water during plant dehydration. So, on one hand it is easy to demonstrate a relationship between leaf thickness and the RWC, or between leaf thickness and the absolute leaf water content. On the other hand, leaf thickness appears to react very sensitive when even minute amounts of water are lost from leaf cells.
Fig. 2. Dynamics of leaf thickness, water potential (A), osmotic water potential s (B), and turgor pressure p (C) in plant 1 (P1 ).
Comparing the loss of leaf thickness with the loss of RWC, leaf thickness seems to change much more drastically than the RWC during the early process of plant desiccation. What exactly is the physiological reason for this initial change of leaf thickness if neither the absolute nor the relative water content of leaves can explain such drastic changes of leaf thickness? Perhaps the turgor pressure of leaf cells? Turgor pressure would be a likely candidate. Because turgor pressure decreases substantially when only small amounts of water move out of leaf cells (Bowman, 1989; Major and Johnsen, 2001; Morgan, 1995). This idea is supported by the observation that the thickness of leaves is to a certain extend correlated with the total water potential of leaf tissue (McBurney, 1992; Syvertsen and Levy, 1982). This idea is further supported by the observation that during daytimes, turgor pressure is related to the vapor pressure deficit of the ambient air (Major and Johnsen, 2001). And, it is supported by the observation that the bulk modulus of elasticity of plant tissue is linearly related to the turgor pressure of leaf cells (Heathcote et al., 1979; Major and Johnsen, 2001).
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Fig. 3. Dynamics of leaf thickness, water potential (A), osmotic water potential s (B), and turgor pressure p (C) in plant 2 (P2 ).
Fig. 4. Dynamics of leaf thickness, water potential (A), osmotic water potential s (B), and turgor pressure p (C) in plant 3 (P3 ).
The idea that leaf thickness is partly a function of leaf cell turgor pressure is not new. Syvertsen and Levy (1982) mention a possible link between leaf turgor and leaf thickness. Sharon and Bravdo (2001) state that there is a linear and significant correlation between leaf thickness and leaf turgor potential. This study also provides an excellent explanation of the relationship between leaf water content, leaf cell turgor potential, and leaf thickness. However, the presumed relationship between leaf thickness and turgor pressure is not straightforward. At full turgor pressure, leaves may be as thin as about 150 m, or as thick as about 500 m. Thus, the absolute value of leaf thickness alone would not yield much information about the amount of turgor pressure. But if the proposed relationship between leaf thickness and turgor pressure holds true, leaf thickness should decrease as turgor pressure declines. Syvertsen and Levy (1982) investigated diurnal changes of leaf thickness, rather than total values of leaf thickness, with respect
to water deficit stress. In this study, changes of leaf thickness were reported in micrometers and termed relative leaf thickness. Considering the change of leaf thickness appeared to eliminate the dependency on absolute values of leaf thickness substantially. This study demonstrated a basic relationship between the change of leaf thickness in m and the total leaf water potential. Búrquez (1987) as well as McBurney (1992) also used the relative change of leaf thickness for estimating the water status of leaves. Búrquez normalized the measurement with respect to leaf thickness at full turgor pressure, reporting this change in percent. McBurney normalized the measurement with respect to a certain leaf water potential. Doing so removed the dependency on absolute values of leaf thickness in both studies. It therefore seems reasonable to suspect that it is the relative change of leaf thickness (with respect to maximum leaf thickness at full turgor pressure), which reflects changes of turgor pressure.
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Fig. 5. Relationships in all three plants investigated between leaf thickness and the total water potential (A), the relative change of leaf thickness and (B), and between the relative change of leaf thickness and the relative change of (C). (D) depicts the same data as in (C), except that data points with a total leaf thickness of smaller than 100 m were removed.
But what exactly is meant with “changes of turgor pressure”? Some species may exhibit a full turgor pressure of about 2 MPa (Morgan, 1995). Other species may have a full turgor pressure of just 1 MPa, or even less (Major and Johnsen, 2001). Changes of leaf thickness can therefore not be used to measure turgor pressure in absolute numbers, nor in units of pressure such as Pascal. Perhaps, relative changes of leaf thickness reflect the relative status of turgor pressure with respect to full turgor pressure. That is, a temporal value of turgor pressure divided by the value of full turgor pressure. Here, this degree of turgor pressure shall be denoted “relative turgor pressure”. “Turgescence” and “turgidity” are synonym terms. It is hypothesized that relative changes of leaf thickness reflect the relative degree of turgor pressure in leaf cells. The objective of this study was to examine this hypothesis further.
2.2. Measurements Leaf thicknesses (LT) were measured with leaf thickness sensors (SG-1000, Agrihouse Inc., Berthoud, CO, USA). Voltage outputs from these sensors were fed to a multichannel data logger (GigaLog-SGraph, Hacker+Daten Technik, Bad Breising, Germany). Before use, all leaf thickness sensors were calibrated against various thin materials, the thicknesses of which were verified with a mechanical micrometer. Given the resolution of the data logger device, resolution of leaf thickness measurements was approximately 0.25 m. One leaf thickness sensor was used per plant. The sensors were placed at leaves, which were located at approximately half of the total height of each plant. These leaves were never detached from the plants. 2.3. Experimental design
2. Materials and methods 2.1. Plant materials and growth conditions Bean plants (Phaseolus vulgaris L.) were grown from seed under controlled environmental conditions in the greenhouse. Plants were cultivated in pots containing approximately 3500 cm3 of soil (D400 with 30% Vermiculit, Stender AG, Schermbeck, Germany), including basic fertilization (Universol Blue: NPK 1811-18+2.5MgO+TE, Everris Int. B.V., Geldermalsen, Netherlands). During the growing stage, pots were irrigated daily to the drip point, and allowed to drain. Nominal value settings were: day/night temperature: 22/20 ◦ C (±2 ◦ C), relative humidity: 60% (±20%), natural lighting with a photoperiod of approximately 16 h. All plants were at least 50 days old when experiments began.
In order to investigate a possible relationship between leaf thickness and turgor pressure, plants were exposed to sudden water deficit stress by cutting the stem of the plant. Stems were cut just above the soil. As a result, plants started to loose water, and consequently turgor pressure, soon after a cut was made. During the entire process, the thickness of a selected leaf of the plant was continuously monitored with a leaf thickness sensor. Leaf thickness readings were stored once every second. Turgor pressure was determined in various time intervals before and particularly after cutting the stem. For this purpose, leaves in the vicinity of the leaf thickness sensor were collected. Water potential of those leaves ( ) was determined immediately with a Scholander pressure chamber (Model 600, PMS Instrument Company, Albany, OR, USA). The pressure of the Scholander chamber was then increased in small steps.
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Fig. 6. Magnified portions of the dynamics of leaf thickness in P1 (A) and P3 (B). In both plants, leaf thickness decreased in discrete levels as compared to a truly continuous decline, just as in P2 .
At each pressure step, sap exiting the leaf’s petiole was collected with a cotton-wick, which was housed in a sealable glass-tube. After collecting expressed sap, each glass-tube was weighted with a precision scale (PS-20, Conrad, Hirschau, Germany). Expressed sap volume could subsequently be calculated by subtracting the empty-weight of each glass-tube, and by taking the density of water into account. Using these measurements, pressure–volume curves were constructed, from which the osmotic water potential of the leaves ( s ) could be derived (Kirkham, 2004; Turner, 1988; Tyree and Hammel, 1972). Turgor pressure ( p or TP) was then calculated using the relationship: TP = p = − s
(1)
In total, 3 plants were investigated (P1 , P2 , P3 ) under greenhouse conditions. Relative leaf thickness was calculated as LT divided by the maximum LT of each leaf investigated. Relative turgor pressure was calculated as TP divided by the maximum TP occurring in this plant. Relative leaf thickness was also plotted against the measurement of the total water potential and against the relative total water potential. Relative total water potential was calculated as divided by the maximum (absolute) value of occurring in this plant. 3. Results After inducing sudden water deficit stress by cutting the stem, leaf thickness in all three plants decreased quickly. Leaf cell turgor pressure also declined soon after the cut was made in all three plants. Fig. 1A shows the plot of leaf thickness versus turgor pressure, and Fig. 1B depicts the relative leaf thickness versus relative turgor pressure for all three plants. Although leaf thickness and
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turgor pressure decreased overall in all three plants during the desiccation process, this relationship turned out to be quite different for the individual plants. While in P1 a possible correlation between relative leaf thickness and relative turgor pressure seemed quite obvious, such a relationship was less pronounced in P2 , and almost non-existent in P3 . The apparent general correlation between relative leaf thickness and relative turgor pressure is supported by the regression of a linear fit to the data of all three plants (n = 23). This regression is statistically significant at p = 0.001. However, the coefficient of determination (R2 = 0.60) of this fit is far from satisfactory. Thus, a generally valid correlation between relative leaf thickness and relative turgor pressure cannot be supported by these data. In order to understand this non-anticipated result better, the development of leaf thickness during the desiccation process was plotted versus time. It was then compared to the dynamics of all mainly involved components of leaf water potential separately: turgor pressure p , the osmotic water potential s , and the total water potential . The data of each plant was plotted separately in order to comment these dynamics individually. In all three plants, leaf thickness followed the pattern of the total water potential of the plants closely. After cutting the stems of the plants, decreased swiftly and substantially, and so did leaf thickness (Figs. 2A, 3A and 4A). In contrast, the observed relationship between leaf thickness and the osmotic water potential s was not as consistent. In plant one (P1 ), s appeared to be very constant during the first part of the desiccation process, only to increase suddenly, followed by a steep decrease (Fig. 2B). In plant two (P2 ), s decreased during the first part, but increased clearly during the later part (Fig. 3B). In plant three (P3 ), s appeared to be very inconsistent during the entire desiccation process, lacking any observable trend (Fig. 4B). Fig. 5 shows the relationship between leaf thickness and total water potential of all three plants combined. In Fig. 5A, total leaf thickness is plotted versus . Although a clear relation appears to be present for each individual plant, the difference in total leaf thickness obviously does not allow to generalize these relationships. In Fig. 5B, relative leaf thickness is plotted versus . Normalizing leaf thickness to the maximum thickness of each leaf investigated removed the dependency on totally achieved leaf thickness. It also seems to demonstrate a more generalized connection between leaf thickness and , at least in plant 1 and plant 3. Fig. 5A and B also displays linear fit functions for P1 and P2 . These are for comparison with Fig. 5D. In Fig. 5C, relative leaf thickness is plotted versus the relative total water potential. Normalizing the measurements of to the maximum water potential of each leaf investigated appeared to reinforce the relationship between leaf thickness and water potential. In this plot, all data points with a total leaf thickness of greater than 100 m seem to fall along a straight line. On the contrary, all data points with a total leaf thickness of less than 100 m do not seem to follow this linear correlation. Fig. 5D depicts the same relationship as in Fig. 5C, except that data points with a total leaf thickness of less than 100 m were removed. That is, all data points of P2 , and some data points of P3 . Fig. 5D also displays a linear fit to these data points (n = 14). The regression of this fit is statistically significant at p = 0.001. The coefficient of determination (R2 ) of this fit equals 0.95. Similar significant fit functions can be observed in Fig. 5A and B, at least for P1 and P2 . However, leaves can achieve various degrees of overall thickness. It therefore is important to normalize leaf thickness. And to consider the relative changes of leaf thickness rather than absolute values. Likewise, leaves can exhibit various degrees of total water potential, even for the very same turgor pressure, depending on the current osmotic water potential. It therefore is equally important to normalize the water potential. And to
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Fig. 7. Pressure–volume curves of four selected data points from Fig. 2. The pressure–volume curves in (A–D) correspond to the data points marked in Fig. 2A as #1, #3, #5, and #8, respectively. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
consider relative changes of water potential rather than absolute values. In addition to the primary objective of this study, two other observations could be made from the results shown. One is the magnitude of the relative change of leaf thickness at the end of the desiccation process, compared to the maximum leaf thickness. In P2 , LT decreased from 116 m to 70 m, which equals a relative change of LT of about 40%. In P1 , LT decreased from 415 m to 200 m, which equals a relative change of LT of almost 60%. In P3 , LT decreased from 520 m to 100 m, which equals a relative change of LT of about 80%. The second incidental observation is the stepwise decrease of LT in P2 , as compared to the seemingly continuous decline of LT in P1 and P3 . Obviously, LT decreased in discrete steps of about 5 m per step in P2 . However, upon closer investigation, the decline of LT in P1 and P3 was also not as continuous as it seemed at first glance. When small portions of the LT data in P1 and P3 were magnified, this decline of LT in discrete steps of about 5 m per step was observed in P1 and P3 as well (Fig. 6). 4. Discussion The apparent lack of support for the suspected relationship between the relative changes of leaf thickness and the relative degree of turgor pressure was disappointing. More insight into this non-anticipated outcome brought the comparison of the dynamics of leaf thickness with the mainly involved components of leaf water potential. Comparison of the dynamics of leaf thickness with the total water potential seemed to support the suspected link between
leaf thickness and turgor pressure at first glance. Upon the sudden deprivation of water supply, leaf cell turgor pressure was expected to be lost fairly quickly. Assuming the osmotic water potential to stay rather constant, a decline of turgor pressure would be manifested by a corresponding decrease of the total water potential. Thus, the rapid decrease of the total water potential observed in Figs. 2A, 3A and 4A appears to indicate an equally quick decline of turgor pressure, and hence a strong relationship between turgor pressure and leaf thickness. This conclusion was further supported by plotting leaf thickness versus the total water potential (Fig. 5). The osmotic water potential s was not expected to change at all within this short period of time, or only slightly due to the loss of leaf cell water content. If the osmotic water potential changed, it was expected to change consistently in all three plants. However, the observed dynamics of the osmotic water potential did not meet this expectation. Perhaps, the apparent lack of evidence is due to improper determination of the osmotic water potential. The dynamics of s in all three plants were very inconsistent, raising questions about the validity of these particular data. In fact, the data reveal a fundamental discrepancy in this study concerning the true value of s and its proper determination. Considering Fig. 2A and the time course of the total water potential in plant 1: when the stem of a plant is being cut as in this example, the plant will dehydrate rapidly. That means, turgor pressure will decline very quickly. In Fig. 2A it appears that this decline of turgor pressure can be observed in the first four data points of the time course of . More importantly, turgor pressure seems to be completely lost for the last four data points of . If so, than would equal s in these last four data points. Thus, s could be
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Fig. 8. Relationships between leaf thickness and a hypothetical turgor pressure*, which is denoted with an asterisk, (A), as well as between relative leaf thickness and the hypothetical relative turgor pressure* (B) in all three plants investigated.
determined from these last four data points of the time course of : s = −0.95 MPa in plant 1. This value, however, is much lower (in absolute number) compared to the s -values determined with the pressure–volume method in plant 1, ranging between −1.5 MPa and −1.9 MPa. Which values are right? In order to investigate this apparent discrepancy further, Fig. 7 depicts the pressure–volume curves of four selected data points from Fig. 2. The data points are #1, #3, #5, and #8, as marked in Fig. 2A. Assuming for a moment that the s value obtained from Fig. 2, i.e. −0.95 MPa, were the true s : this value would correspond to an inverse balance-pressure of (−1)·(1/−0.95 MPa) = 1.05 MPa−1 , which is marked in all four p–v curves in Fig. 7 with a red circle. For an expressed sap-volume of 0.0 ml, all four p–v curves were consistent with this s number. Fig. 7A would show some turgor pressure, Fig. 7B would indicate turgor pressure to be almost lost, and Fig. 7C and D would signify turgor pressure to be completely lost. As more sap was expressed from the petioles, the p–v curves turned into straight lines, as expected. The last two data points of each p–v curve were used to calculate the slopes of these straight lines. However, when extending the straight lines to an expressed volume of 0.0 ml, they did not meet the assumed −1/ s value of 1.05 MPa−1 . Instead, these lines consistently met the ordinates of these plots at about 0.55 MPa−1 , which corresponds to s values of about −1.82 MPa. Those numbers were used in Fig. 2B for the purpose of plotting the dynamics of s in plant 1.
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The question remains: which values of s are right? Both possible values of s for this plant, −0.95 MPa and about −1.82 MPa, appear to be reasonable. If, for a brief moment, it is being assumed that s could be obtained from the steady state value of at the end of the desiccation process, as described above, s would equal about −0.95 MPa in P1 . For P2 and P3 , the lowest values of were chosen. Hence, s would equal about −1.30 MPa in P2 and about −1.35 MPa in P3 . If it is further assumed that s would stay rather constant during this desiccation process, turgor pressure would essentially follow the total water potential . This hypothetical turgor pressure shall be denoted with an asterisk. Fig. 8A shows the plot of leaf thickness versus this hypothetical turgor pressure*, and Fig. 8B depicts relative leaf thickness versus the relative hypothetical turgor pressure* for all three plants. As expected, there appears to be a linear correlation for P1 and P2 , and also for P3 . Somewhat unexpectedly the slope and offset was markedly different in P3 compared to P1 and P2 . Apparently, this difference in slope and offset was caused by the magnitude of the relative change of leaf thickness during the desiccation process. Thus, the extent of the relative change of leaf thickness needs to be taken into account. However, the relationship depicted in Fig. 8 stands on very shaky ground, because it is based on several assumptions. First, it was assumed that turgor pressure was completely lost at the end of the desiccation process. Although this assumption seems reasonable, it is not known for a fact. Secondly, it was assumed that s would stay rather constant during the quick desiccation process. Although it seems unlikely that s changes drastically within minutes, there is no verification for this assumption either. As a matter of fact, when leaf cells dehydrate, the ratio between leaf cell water and leaf cell solutes will shift in favor of the solutes, and s should actually increase slightly. Thus, the second assumption being made is very weak. As a consequence, the relation depicted in Fig. 8 should remain a hypothetical speculation. The method of determining s from the steady state value of was therefore dismissed, although it was a tempting trial. Instead, the established method of determining s from p–v curves was being trusted. Interesting are the two additional observations, which were not directly related to the primary objective of this study. It appears that the relative change of LT depends on the absolute amount of LT. The thicker the leaf was before the desiccation process began, the greater was the relative change of LT in percent. When leaf thickness sensors are used for irrigation control, for example, such information would be important in order to know what relative change of LT can be expected for a certain leaf thickness given. More experimental work is needed to verify such a relationship properly, or to disprove it. Somewhat puzzling is the observed decrease of LT in discrete steps, instead of a truly continuous decline. These discrete steps cannot be due to low resolution of the data acquisition system, since the resolution of the data acquisition system was about 0.25 m. In fact, it can be seen that sometimes the leaf thickness sensor measured several data points in between two apparently discrete levels of LT. Are those discrete levels of LT during desiccation perhaps governed by leaf anatomy/morphology? If so, what exactly causes them?
5. Conclusions The original idea of a suspected relationship between relative changes of leaf thickness and the relative degree of turgor pressure could not be verified with this study. However, this
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non-anticipated result might be due to an improper determination of the osmotic water potential s . The results of this study do therefore not necessarily weaken the hypothesis itself, or call for alternative hypotheses. Rather, in this case the measurement of s should be revisited and possibly enhanced. Perhaps, for the measurement of turgor pressure in rapid succession, as in this study, the use of pressure–volume curves for the determination of s is not an appropriate method. Possibly, when s is being measured more accurately by using other techniques, a better link between leaf thickness and turgor pressure can be observed. The idea of this suspected relationship remains therefore twofold: intriguing on one hand, yet challenging to proof on the other hand. Still, the data of the conducted experiment may help to understand the mechanism between leaf water content and leaf thickness better. And to interpret short term fluctuations of leaf thickness in response to environmental factors fruitfully. The data of this study may be one piece of the overall puzzle. The observed close relationship between leaf thickness and the total water potential corroborates the findings of Syvertsen and Levy (1982) as well as McBurney (1992). The continuous measurement of leaf thickness might therefore be used to indirectly observe changes of with high temporal resolution. This application could prove useful to plant physiology. Because diurnal fluctuations of may happen quite instantaneously, i.e. within minutes, and sensitive to the environment (Jones, 2007). Such instantaneous fluctuations of could be missed with low time resolving measurements. Acknowledgement We thank the anonymous reviewer for his or her valuable comments. References Alarcon, J.J., Malone, M., 1994. Substantial hydraulic signals are triggered by leafbiting insects in tomato. J. Exp. Bot. 45, 953–957.
Bowman, W.D., 1989. The relationship between leaf water status, gas exchange, and spectral reflectance in cotton leaves. Remote Sens. Environ. 30, 249–255. Búrquez, A., 1987. Leaf thickness and water deficit in plants: a tool for field studies. J. Exp. Bot. 38, 109–114. Scoffoni, C., Vuong, C., Diep, S., Cochard, H., Sack, L., 2014. Leaf shrinkage with dehydration: coordination with hydraulic vulnerability and drought tolerance. Plant Physiol. 164 (4), 1772–1788. Heathcote, D.G., Etherington, J.R., Woodward, F.I., 1979. An instrument for nondestructive measurement of the pressure potential (turgor) of leaf cells. J. Exp. Bot. 30, 811–816. Jones, H.G., 2007. Monitoring plant and soil water status: established and novel methods revisited and their relevance to studies of drought tolerance. J. Exp. Bot. 58 (2), 119–130. Kirkham, M.B., 2004. Principles of Soil and Plant Water Relations. Elsevier Academic Press, Burlington, MA, pp. 295–297. Li, J., Yang, J., Fei, P., Song, J., Li, D., Ge, C., Chen, W., 2009. Responses of rice leaf thickness, SPAD readings and chlorophyll a/b ratios to different nitrogen supply rates in paddy field. Field Crop Res. 114, 426–432. Major, J.E., Johnsen, K.H., 2001. Shoot water relations of mature black spruce families displaying a genotype × environment interaction in growth rate. III. Diurnal patterns as influenced by vapor pressure deficit and internal water status. Tree Physiol. 21, 579–587. Malone, M., Alarcon, J.J., 1994. Only xylem-borne factors can account for systemic wound signaling in the tomato plant. Planta 196, 740–746. McBurney, T., 1992. The relationship between leaf thickness and plant water potential. J. Exp. Bot. 43 (248), 327–335. Meidner, H., 1952. An instrument for the continuous determination of leaf thickness changes in the field. J. Exp. Bot. 3, 319–325. Morgan, J.M., 1995. Growth and yield of wheat lines with differing osmoregulative capacity at high soil water deficit in seasons of varying evaporative demand. Field Crop Res. 40, 143–152. Seelig, H.D., Stoner, R.J., Linden, J.C., 2012. Irrigation control of cowpea plants using the measurement of leaf thickness under greenhouse conditions. Irrig. Sci. 30, 247–257. Sharon, Y., Bravdo, B., 2001. A fully-automated orchard irrigation system based on continuous monitoring of turgor potential with a leaf sensor. Acta Hortic. 562, 55–61. Syvertsen, J., Levy, Y., 1982. Diurnal changes in citrus leaf thickness, leaf water potential, and leaf to air temperature difference. J. Exp. Bot. 33, 783–789. Turner, N.C., 1988. Measurement of plant water status by the pressure chamber technique. Irrig. Sci. 9, 289–308. Tyree, M.T., Cameron, S.I., 1977. A new technique for measuring oscillatory and diurnal changes in leaf thickness. Can. J. For. Res. 7, 540–544. Tyree, M.T., Hammel, H.T., 1972. The measurement of the turgor pressure and the water relations of plants by the pressure-bomb technique. J. Exp. Bot. 23, 267–282.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Leaf thickness and turgor pressure in bean during plant desiccation Hans-Dieter Seelig a,∗ , Adelheid Wolter b , Fritz-Gerald Schröder b a b
University of Applied Sciences Dresden, Department of Electrical Engineering, Friedrich-List-Platz 1, Dresden 01069, Germany University of Applied Sciences Dresden, Department of Agriculture/Landscape Management, Friedrich-List-Platz 1, Dresden 01069, Germany
a r t i c l e
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Article history: Received 10 October 2014 Received in revised form 17 December 2014 Accepted 18 December 2014 Available online 13 January 2015 Keywords: Leaf thickness Turgor pressure Leaf water content Water deficit stress Irrigation control
a b s t r a c t This study investigated a possible relationship between the relative change of leaf thickness and the relative degree of turgor pressure in leaf cells. Leaf thickness and turgor pressure were measured simultaneously during forced plant desiccation in bean plants. Although leaf thickness followed closely the steep decline of the total water potential ( ) during the desiccation process, no conclusive relationship could be observed between leaf thickness and the osmotic water potential ( s ). Thus, a potential link between the relative change of leaf thickness and the relative degree of turgor pressure could not be verified under the experimental conditions. Earlier findings, that the relative change of leaf thickness is closely related to the relative change of the total water potential of leaf cells, are corroborated. Additional observations were: (i) a potential relationship between the relative change of leaf thickness during desiccation and the totally achieved leaf thickness, and (ii) the apparent decrease of leaf thickness during desiccation in discrete levels rather than a truly continuous decline. © 2015 Elsevier B.V. All rights reserved.
1. Introduction It has been demonstrated that the thickness of leaves can fluctuate quickly in response to certain environmental factors. For example, if soil moisture content decreases below a specific threshold, leaf thickness typically collapses within one to several hours. Taking advantage of this effect, the continuous measurement of leaf thickness may be used to control irrigation (Sharon and Bravdo, 2001). This method has the potential to save enormous amounts of irrigation water (Seelig et al., 2012; Sharon and Bravdo, 2001). It has also been shown that leaf thickness can change quickly, that is within minutes, in response to fluctuations of air temperature and air humidity. Thus, it appears that leaf thickness responds in some way to changes of the water status of leaf tissue. But what is the mechanism relating these observations? Said another way: what exactly is the message of observing short term changes of leaf thickness? In order to interpret the measurements of leaf thickness fruitfully, it is essential to have a sound understanding of the underlying cause-and-effect relationship (Jones, 2007; Scoffoni et al., 2014). Malone and Alarcon have demonstrated that the thickness of leaves can change almost immediately in response to leaf wounding (Alarcon and Malone, 1994; Malone and Alarcon, 1994). These
Abbreviations: LT, leaf thickness; TP, turgor pressure. ∗ Corresponding author. Tel.: +49 3514622346; fax: +49 3514622193. E-mail address: [email protected] (H.-D. Seelig). http://dx.doi.org/10.1016/j.scienta.2014.12.025 0304-4238/© 2015 Elsevier B.V. All rights reserved.
studies have also provided convincing evidence about the cause for this quick change in leaf thickness. When a leaf is wounded, some water is being dispersed to other regions of the leaf or plant, causing leaf thickness to increase there. This principle is termed hydraulic dispersal. Other studies have documented that the thickness of leaves can change substantial within hours or minutes in response to soil moisture content, air temperature, and air humidity. When soil is well watered, or exerts only a mild water deficit stress on the plant, leaf thickness is typically kept fairly constant. On the contrary, if the moisture content of the soil falls below a certain threshold level, leaf thickness can literally collapse within one to several hours. It may increase to its nominal value rather quickly though if the plant is being watered soon (Búrquez, 1987; McBurney, 1992; Seelig et al., 2012; Tyree and Cameron, 1977). Leaf thickness also seems to respond within minutes to ambient air conditions. During day times, leaf thickness tends to increase when the relative humidity of the ambient air rises (Búrquez, 1987), and to decrease as the vapor pressure deficit of the ambient air increases (Meidner, 1952; Syvertsen and Levy, 1982). It has also been reported that daytime leaf thickness tends to decline when the temperature of the ambient air increases (Búrquez, 1987; Li et al., 2009; McBurney, 1992; Meidner, 1952; Syvertsen and Levy, 1982). Rather than seeing a true temperature effect, this observation may simply reflect changes in the demand for evapotranspiration as well. Because both, the relative humidity as well as the vapor pressure deficit, will fluctuate as the temperature of the air changes. Overall, it appears that leaf thickness
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Fig. 1. Relationships between leaf thickness and turgor pressure (A) as well as between the relative leaf thickness and the relative turgor pressure (B) in all three plants investigated.
responds to two forces: excessive water deficit stress from the soil and daytime water demand from the air. However, although these changes of leaf thickness due to soil moisture content, air temperature, and air humidity have been documented multiple times, it remains unclear what exactly causes them. While hydraulic dispersal due to leaf wounding is the only plausible explanation so far as to why leaf thickness can fluctuate within short periods of time, hydraulic dispersal does not really serve as an explanation for the changes of leaf thickness in response to environmental factors such as soil moisture content, air temperature, and air humidity. Given the fact that leaf thickness can fluctuate rather quickly due to both factors, water deficit stress from the soil as well as water demand from the air, it appears that these types of leaf thickness variations reflect changes in the water status of the leaf tissue. If so, which type or component of leaf water status would actually be the true independent variable for these quick changes of leaf thickness? Obviously, when leaves dehydrate, leaf thickness will shrink due to this loss of water. Thus, leaf thickness is a function of the absolute water content of leaf cells. The relative water content (RWC) of leaves, which is often used to describe leaf water status, will equally reflect this loss of water during plant dehydration. So, on one hand it is easy to demonstrate a relationship between leaf thickness and the RWC, or between leaf thickness and the absolute leaf water content. On the other hand, leaf thickness appears to react very sensitive when even minute amounts of water are lost from leaf cells.
Fig. 2. Dynamics of leaf thickness, water potential (A), osmotic water potential s (B), and turgor pressure p (C) in plant 1 (P1 ).
Comparing the loss of leaf thickness with the loss of RWC, leaf thickness seems to change much more drastically than the RWC during the early process of plant desiccation. What exactly is the physiological reason for this initial change of leaf thickness if neither the absolute nor the relative water content of leaves can explain such drastic changes of leaf thickness? Perhaps the turgor pressure of leaf cells? Turgor pressure would be a likely candidate. Because turgor pressure decreases substantially when only small amounts of water move out of leaf cells (Bowman, 1989; Major and Johnsen, 2001; Morgan, 1995). This idea is supported by the observation that the thickness of leaves is to a certain extend correlated with the total water potential of leaf tissue (McBurney, 1992; Syvertsen and Levy, 1982). This idea is further supported by the observation that during daytimes, turgor pressure is related to the vapor pressure deficit of the ambient air (Major and Johnsen, 2001). And, it is supported by the observation that the bulk modulus of elasticity of plant tissue is linearly related to the turgor pressure of leaf cells (Heathcote et al., 1979; Major and Johnsen, 2001).
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Fig. 3. Dynamics of leaf thickness, water potential (A), osmotic water potential s (B), and turgor pressure p (C) in plant 2 (P2 ).
Fig. 4. Dynamics of leaf thickness, water potential (A), osmotic water potential s (B), and turgor pressure p (C) in plant 3 (P3 ).
The idea that leaf thickness is partly a function of leaf cell turgor pressure is not new. Syvertsen and Levy (1982) mention a possible link between leaf turgor and leaf thickness. Sharon and Bravdo (2001) state that there is a linear and significant correlation between leaf thickness and leaf turgor potential. This study also provides an excellent explanation of the relationship between leaf water content, leaf cell turgor potential, and leaf thickness. However, the presumed relationship between leaf thickness and turgor pressure is not straightforward. At full turgor pressure, leaves may be as thin as about 150 m, or as thick as about 500 m. Thus, the absolute value of leaf thickness alone would not yield much information about the amount of turgor pressure. But if the proposed relationship between leaf thickness and turgor pressure holds true, leaf thickness should decrease as turgor pressure declines. Syvertsen and Levy (1982) investigated diurnal changes of leaf thickness, rather than total values of leaf thickness, with respect
to water deficit stress. In this study, changes of leaf thickness were reported in micrometers and termed relative leaf thickness. Considering the change of leaf thickness appeared to eliminate the dependency on absolute values of leaf thickness substantially. This study demonstrated a basic relationship between the change of leaf thickness in m and the total leaf water potential. Búrquez (1987) as well as McBurney (1992) also used the relative change of leaf thickness for estimating the water status of leaves. Búrquez normalized the measurement with respect to leaf thickness at full turgor pressure, reporting this change in percent. McBurney normalized the measurement with respect to a certain leaf water potential. Doing so removed the dependency on absolute values of leaf thickness in both studies. It therefore seems reasonable to suspect that it is the relative change of leaf thickness (with respect to maximum leaf thickness at full turgor pressure), which reflects changes of turgor pressure.
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Fig. 5. Relationships in all three plants investigated between leaf thickness and the total water potential (A), the relative change of leaf thickness and (B), and between the relative change of leaf thickness and the relative change of (C). (D) depicts the same data as in (C), except that data points with a total leaf thickness of smaller than 100 m were removed.
But what exactly is meant with “changes of turgor pressure”? Some species may exhibit a full turgor pressure of about 2 MPa (Morgan, 1995). Other species may have a full turgor pressure of just 1 MPa, or even less (Major and Johnsen, 2001). Changes of leaf thickness can therefore not be used to measure turgor pressure in absolute numbers, nor in units of pressure such as Pascal. Perhaps, relative changes of leaf thickness reflect the relative status of turgor pressure with respect to full turgor pressure. That is, a temporal value of turgor pressure divided by the value of full turgor pressure. Here, this degree of turgor pressure shall be denoted “relative turgor pressure”. “Turgescence” and “turgidity” are synonym terms. It is hypothesized that relative changes of leaf thickness reflect the relative degree of turgor pressure in leaf cells. The objective of this study was to examine this hypothesis further.
2.2. Measurements Leaf thicknesses (LT) were measured with leaf thickness sensors (SG-1000, Agrihouse Inc., Berthoud, CO, USA). Voltage outputs from these sensors were fed to a multichannel data logger (GigaLog-SGraph, Hacker+Daten Technik, Bad Breising, Germany). Before use, all leaf thickness sensors were calibrated against various thin materials, the thicknesses of which were verified with a mechanical micrometer. Given the resolution of the data logger device, resolution of leaf thickness measurements was approximately 0.25 m. One leaf thickness sensor was used per plant. The sensors were placed at leaves, which were located at approximately half of the total height of each plant. These leaves were never detached from the plants. 2.3. Experimental design
2. Materials and methods 2.1. Plant materials and growth conditions Bean plants (Phaseolus vulgaris L.) were grown from seed under controlled environmental conditions in the greenhouse. Plants were cultivated in pots containing approximately 3500 cm3 of soil (D400 with 30% Vermiculit, Stender AG, Schermbeck, Germany), including basic fertilization (Universol Blue: NPK 1811-18+2.5MgO+TE, Everris Int. B.V., Geldermalsen, Netherlands). During the growing stage, pots were irrigated daily to the drip point, and allowed to drain. Nominal value settings were: day/night temperature: 22/20 ◦ C (±2 ◦ C), relative humidity: 60% (±20%), natural lighting with a photoperiod of approximately 16 h. All plants were at least 50 days old when experiments began.
In order to investigate a possible relationship between leaf thickness and turgor pressure, plants were exposed to sudden water deficit stress by cutting the stem of the plant. Stems were cut just above the soil. As a result, plants started to loose water, and consequently turgor pressure, soon after a cut was made. During the entire process, the thickness of a selected leaf of the plant was continuously monitored with a leaf thickness sensor. Leaf thickness readings were stored once every second. Turgor pressure was determined in various time intervals before and particularly after cutting the stem. For this purpose, leaves in the vicinity of the leaf thickness sensor were collected. Water potential of those leaves ( ) was determined immediately with a Scholander pressure chamber (Model 600, PMS Instrument Company, Albany, OR, USA). The pressure of the Scholander chamber was then increased in small steps.
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Fig. 6. Magnified portions of the dynamics of leaf thickness in P1 (A) and P3 (B). In both plants, leaf thickness decreased in discrete levels as compared to a truly continuous decline, just as in P2 .
At each pressure step, sap exiting the leaf’s petiole was collected with a cotton-wick, which was housed in a sealable glass-tube. After collecting expressed sap, each glass-tube was weighted with a precision scale (PS-20, Conrad, Hirschau, Germany). Expressed sap volume could subsequently be calculated by subtracting the empty-weight of each glass-tube, and by taking the density of water into account. Using these measurements, pressure–volume curves were constructed, from which the osmotic water potential of the leaves ( s ) could be derived (Kirkham, 2004; Turner, 1988; Tyree and Hammel, 1972). Turgor pressure ( p or TP) was then calculated using the relationship: TP = p = − s
(1)
In total, 3 plants were investigated (P1 , P2 , P3 ) under greenhouse conditions. Relative leaf thickness was calculated as LT divided by the maximum LT of each leaf investigated. Relative turgor pressure was calculated as TP divided by the maximum TP occurring in this plant. Relative leaf thickness was also plotted against the measurement of the total water potential and against the relative total water potential. Relative total water potential was calculated as divided by the maximum (absolute) value of occurring in this plant. 3. Results After inducing sudden water deficit stress by cutting the stem, leaf thickness in all three plants decreased quickly. Leaf cell turgor pressure also declined soon after the cut was made in all three plants. Fig. 1A shows the plot of leaf thickness versus turgor pressure, and Fig. 1B depicts the relative leaf thickness versus relative turgor pressure for all three plants. Although leaf thickness and
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turgor pressure decreased overall in all three plants during the desiccation process, this relationship turned out to be quite different for the individual plants. While in P1 a possible correlation between relative leaf thickness and relative turgor pressure seemed quite obvious, such a relationship was less pronounced in P2 , and almost non-existent in P3 . The apparent general correlation between relative leaf thickness and relative turgor pressure is supported by the regression of a linear fit to the data of all three plants (n = 23). This regression is statistically significant at p = 0.001. However, the coefficient of determination (R2 = 0.60) of this fit is far from satisfactory. Thus, a generally valid correlation between relative leaf thickness and relative turgor pressure cannot be supported by these data. In order to understand this non-anticipated result better, the development of leaf thickness during the desiccation process was plotted versus time. It was then compared to the dynamics of all mainly involved components of leaf water potential separately: turgor pressure p , the osmotic water potential s , and the total water potential . The data of each plant was plotted separately in order to comment these dynamics individually. In all three plants, leaf thickness followed the pattern of the total water potential of the plants closely. After cutting the stems of the plants, decreased swiftly and substantially, and so did leaf thickness (Figs. 2A, 3A and 4A). In contrast, the observed relationship between leaf thickness and the osmotic water potential s was not as consistent. In plant one (P1 ), s appeared to be very constant during the first part of the desiccation process, only to increase suddenly, followed by a steep decrease (Fig. 2B). In plant two (P2 ), s decreased during the first part, but increased clearly during the later part (Fig. 3B). In plant three (P3 ), s appeared to be very inconsistent during the entire desiccation process, lacking any observable trend (Fig. 4B). Fig. 5 shows the relationship between leaf thickness and total water potential of all three plants combined. In Fig. 5A, total leaf thickness is plotted versus . Although a clear relation appears to be present for each individual plant, the difference in total leaf thickness obviously does not allow to generalize these relationships. In Fig. 5B, relative leaf thickness is plotted versus . Normalizing leaf thickness to the maximum thickness of each leaf investigated removed the dependency on totally achieved leaf thickness. It also seems to demonstrate a more generalized connection between leaf thickness and , at least in plant 1 and plant 3. Fig. 5A and B also displays linear fit functions for P1 and P2 . These are for comparison with Fig. 5D. In Fig. 5C, relative leaf thickness is plotted versus the relative total water potential. Normalizing the measurements of to the maximum water potential of each leaf investigated appeared to reinforce the relationship between leaf thickness and water potential. In this plot, all data points with a total leaf thickness of greater than 100 m seem to fall along a straight line. On the contrary, all data points with a total leaf thickness of less than 100 m do not seem to follow this linear correlation. Fig. 5D depicts the same relationship as in Fig. 5C, except that data points with a total leaf thickness of less than 100 m were removed. That is, all data points of P2 , and some data points of P3 . Fig. 5D also displays a linear fit to these data points (n = 14). The regression of this fit is statistically significant at p = 0.001. The coefficient of determination (R2 ) of this fit equals 0.95. Similar significant fit functions can be observed in Fig. 5A and B, at least for P1 and P2 . However, leaves can achieve various degrees of overall thickness. It therefore is important to normalize leaf thickness. And to consider the relative changes of leaf thickness rather than absolute values. Likewise, leaves can exhibit various degrees of total water potential, even for the very same turgor pressure, depending on the current osmotic water potential. It therefore is equally important to normalize the water potential. And to
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Fig. 7. Pressure–volume curves of four selected data points from Fig. 2. The pressure–volume curves in (A–D) correspond to the data points marked in Fig. 2A as #1, #3, #5, and #8, respectively. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
consider relative changes of water potential rather than absolute values. In addition to the primary objective of this study, two other observations could be made from the results shown. One is the magnitude of the relative change of leaf thickness at the end of the desiccation process, compared to the maximum leaf thickness. In P2 , LT decreased from 116 m to 70 m, which equals a relative change of LT of about 40%. In P1 , LT decreased from 415 m to 200 m, which equals a relative change of LT of almost 60%. In P3 , LT decreased from 520 m to 100 m, which equals a relative change of LT of about 80%. The second incidental observation is the stepwise decrease of LT in P2 , as compared to the seemingly continuous decline of LT in P1 and P3 . Obviously, LT decreased in discrete steps of about 5 m per step in P2 . However, upon closer investigation, the decline of LT in P1 and P3 was also not as continuous as it seemed at first glance. When small portions of the LT data in P1 and P3 were magnified, this decline of LT in discrete steps of about 5 m per step was observed in P1 and P3 as well (Fig. 6). 4. Discussion The apparent lack of support for the suspected relationship between the relative changes of leaf thickness and the relative degree of turgor pressure was disappointing. More insight into this non-anticipated outcome brought the comparison of the dynamics of leaf thickness with the mainly involved components of leaf water potential. Comparison of the dynamics of leaf thickness with the total water potential seemed to support the suspected link between
leaf thickness and turgor pressure at first glance. Upon the sudden deprivation of water supply, leaf cell turgor pressure was expected to be lost fairly quickly. Assuming the osmotic water potential to stay rather constant, a decline of turgor pressure would be manifested by a corresponding decrease of the total water potential. Thus, the rapid decrease of the total water potential observed in Figs. 2A, 3A and 4A appears to indicate an equally quick decline of turgor pressure, and hence a strong relationship between turgor pressure and leaf thickness. This conclusion was further supported by plotting leaf thickness versus the total water potential (Fig. 5). The osmotic water potential s was not expected to change at all within this short period of time, or only slightly due to the loss of leaf cell water content. If the osmotic water potential changed, it was expected to change consistently in all three plants. However, the observed dynamics of the osmotic water potential did not meet this expectation. Perhaps, the apparent lack of evidence is due to improper determination of the osmotic water potential. The dynamics of s in all three plants were very inconsistent, raising questions about the validity of these particular data. In fact, the data reveal a fundamental discrepancy in this study concerning the true value of s and its proper determination. Considering Fig. 2A and the time course of the total water potential in plant 1: when the stem of a plant is being cut as in this example, the plant will dehydrate rapidly. That means, turgor pressure will decline very quickly. In Fig. 2A it appears that this decline of turgor pressure can be observed in the first four data points of the time course of . More importantly, turgor pressure seems to be completely lost for the last four data points of . If so, than would equal s in these last four data points. Thus, s could be
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Fig. 8. Relationships between leaf thickness and a hypothetical turgor pressure*, which is denoted with an asterisk, (A), as well as between relative leaf thickness and the hypothetical relative turgor pressure* (B) in all three plants investigated.
determined from these last four data points of the time course of : s = −0.95 MPa in plant 1. This value, however, is much lower (in absolute number) compared to the s -values determined with the pressure–volume method in plant 1, ranging between −1.5 MPa and −1.9 MPa. Which values are right? In order to investigate this apparent discrepancy further, Fig. 7 depicts the pressure–volume curves of four selected data points from Fig. 2. The data points are #1, #3, #5, and #8, as marked in Fig. 2A. Assuming for a moment that the s value obtained from Fig. 2, i.e. −0.95 MPa, were the true s : this value would correspond to an inverse balance-pressure of (−1)·(1/−0.95 MPa) = 1.05 MPa−1 , which is marked in all four p–v curves in Fig. 7 with a red circle. For an expressed sap-volume of 0.0 ml, all four p–v curves were consistent with this s number. Fig. 7A would show some turgor pressure, Fig. 7B would indicate turgor pressure to be almost lost, and Fig. 7C and D would signify turgor pressure to be completely lost. As more sap was expressed from the petioles, the p–v curves turned into straight lines, as expected. The last two data points of each p–v curve were used to calculate the slopes of these straight lines. However, when extending the straight lines to an expressed volume of 0.0 ml, they did not meet the assumed −1/ s value of 1.05 MPa−1 . Instead, these lines consistently met the ordinates of these plots at about 0.55 MPa−1 , which corresponds to s values of about −1.82 MPa. Those numbers were used in Fig. 2B for the purpose of plotting the dynamics of s in plant 1.
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The question remains: which values of s are right? Both possible values of s for this plant, −0.95 MPa and about −1.82 MPa, appear to be reasonable. If, for a brief moment, it is being assumed that s could be obtained from the steady state value of at the end of the desiccation process, as described above, s would equal about −0.95 MPa in P1 . For P2 and P3 , the lowest values of were chosen. Hence, s would equal about −1.30 MPa in P2 and about −1.35 MPa in P3 . If it is further assumed that s would stay rather constant during this desiccation process, turgor pressure would essentially follow the total water potential . This hypothetical turgor pressure shall be denoted with an asterisk. Fig. 8A shows the plot of leaf thickness versus this hypothetical turgor pressure*, and Fig. 8B depicts relative leaf thickness versus the relative hypothetical turgor pressure* for all three plants. As expected, there appears to be a linear correlation for P1 and P2 , and also for P3 . Somewhat unexpectedly the slope and offset was markedly different in P3 compared to P1 and P2 . Apparently, this difference in slope and offset was caused by the magnitude of the relative change of leaf thickness during the desiccation process. Thus, the extent of the relative change of leaf thickness needs to be taken into account. However, the relationship depicted in Fig. 8 stands on very shaky ground, because it is based on several assumptions. First, it was assumed that turgor pressure was completely lost at the end of the desiccation process. Although this assumption seems reasonable, it is not known for a fact. Secondly, it was assumed that s would stay rather constant during the quick desiccation process. Although it seems unlikely that s changes drastically within minutes, there is no verification for this assumption either. As a matter of fact, when leaf cells dehydrate, the ratio between leaf cell water and leaf cell solutes will shift in favor of the solutes, and s should actually increase slightly. Thus, the second assumption being made is very weak. As a consequence, the relation depicted in Fig. 8 should remain a hypothetical speculation. The method of determining s from the steady state value of was therefore dismissed, although it was a tempting trial. Instead, the established method of determining s from p–v curves was being trusted. Interesting are the two additional observations, which were not directly related to the primary objective of this study. It appears that the relative change of LT depends on the absolute amount of LT. The thicker the leaf was before the desiccation process began, the greater was the relative change of LT in percent. When leaf thickness sensors are used for irrigation control, for example, such information would be important in order to know what relative change of LT can be expected for a certain leaf thickness given. More experimental work is needed to verify such a relationship properly, or to disprove it. Somewhat puzzling is the observed decrease of LT in discrete steps, instead of a truly continuous decline. These discrete steps cannot be due to low resolution of the data acquisition system, since the resolution of the data acquisition system was about 0.25 m. In fact, it can be seen that sometimes the leaf thickness sensor measured several data points in between two apparently discrete levels of LT. Are those discrete levels of LT during desiccation perhaps governed by leaf anatomy/morphology? If so, what exactly causes them?
5. Conclusions The original idea of a suspected relationship between relative changes of leaf thickness and the relative degree of turgor pressure could not be verified with this study. However, this
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non-anticipated result might be due to an improper determination of the osmotic water potential s . The results of this study do therefore not necessarily weaken the hypothesis itself, or call for alternative hypotheses. Rather, in this case the measurement of s should be revisited and possibly enhanced. Perhaps, for the measurement of turgor pressure in rapid succession, as in this study, the use of pressure–volume curves for the determination of s is not an appropriate method. Possibly, when s is being measured more accurately by using other techniques, a better link between leaf thickness and turgor pressure can be observed. The idea of this suspected relationship remains therefore twofold: intriguing on one hand, yet challenging to proof on the other hand. Still, the data of the conducted experiment may help to understand the mechanism between leaf water content and leaf thickness better. And to interpret short term fluctuations of leaf thickness in response to environmental factors fruitfully. The data of this study may be one piece of the overall puzzle. The observed close relationship between leaf thickness and the total water potential corroborates the findings of Syvertsen and Levy (1982) as well as McBurney (1992). The continuous measurement of leaf thickness might therefore be used to indirectly observe changes of with high temporal resolution. This application could prove useful to plant physiology. Because diurnal fluctuations of may happen quite instantaneously, i.e. within minutes, and sensitive to the environment (Jones, 2007). Such instantaneous fluctuations of could be missed with low time resolving measurements. Acknowledgement We thank the anonymous reviewer for his or her valuable comments. References Alarcon, J.J., Malone, M., 1994. Substantial hydraulic signals are triggered by leafbiting insects in tomato. J. Exp. Bot. 45, 953–957.
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