Limitation of Cell Elongation in Barley (Hordeum vulgare L.) Leaves Through Mechanical and Tissue-Hydraulic Properties Mostefa Touati1, Thorsten Knipfer2,4, Tama´s Visnovitz2,5, Abdelkrim Kameli3 and Wieland Fricke2,* 1
*Corresponding author: E-mail, [email protected]; Fax, +353-1-7161153. (Received December 15, 2014; Accepted April 1, 2015)
The aim of the present study was to assess the mechanical and hydraulic limitation of growth in leaf epidermal cells of barley (Hordeum vulgare L.) in response to agents which affect cellular water (mercuric chloride, HgCl2) and potassium (cesium chloride, CsCl; tetraethylammonium, TEA) transport, pump activity of plasma membrane H+-ATPase and wall acidification (fusicoccin, FC). Cell turgor (P) was measured with the cell pressure probe, and cell osmotic pressure (p) was analyzed through picoliter osmometry of single-cell extracts. A wall extensibility coefficient (M) and tissue hydraulic conductance coefficient (L) were derived using the Lockhart equation. There was a significant positive linear relationship between relative elemental growth rate and P, which fit all treatments, with an overall apparent yield threshold of 0.368 MPa. Differences in growth between treatments could be explained through differences in P. A comparison of L and M showed that growth in all except the FC treatment was co-limited through hydraulic and mechanical properties, though to various extents. This was accompanied by significant (0.17–0.24 MPa) differences in water potential ( ) between xylem and epidermal cells in the leaf elongation zone. In contrast, FC-treated leaves showed close to zero and a 10-fold increase in L. Keywords: Barley (Hordeum vulgare L.) Fusicoccin Hydraulic conductivity Leaf cell elongation Lockhart equation Turgor. Abbreviations: Acell, cell surface; EmBL, emerged-blade portion of leaf; Epi, epidermis; EZ, elongation zone; e, cell volumetric elastic modulus; FC, fusicoccin; h, cell length; PM-H+-ATPase, plasma membrane H+-ATPase; L, tissue hydraulic conductance coefficient; LER, leaf elongation rate; Lpcell, cell hydraulic conductivity; M, wall extensibility coefficient; P, cell turgor; p, osmotic pressure; c, water potential; , water potential difference; Epi-EZ, water potential of epidermal cell in EZ; cEpi-EmBL, water potential of epidermal cell in EmBL; Medium, water potential of root medium; Xylem-EZ, water potential of xylem in EZ; Xylem-EmBL, water potential of xylem in EmBL; Xylem-Root, water potential of xylem in root; r, radius; REGR, relative elemental growth rate; s, solute reflection coefficient; TEA,
tetraethylammonium; T1/2, half-time of water exchange; Vcell, cell volume; Y, yield threshold of wall.
Introduction The quantitative relationship between cell expansive growth and the hydraulic and mechanical properties which affect cell expansion is expressed through Equations (1) and (2), respectively (Schopfer 2006): REGR ¼ L
ð1Þ
REGR ¼ MðP YÞ
ð2Þ
Equation 1 expresses the relative elemental growth rate (REGR, s1) of a cell as the product of a hydraulic conductance coefficient (L, MPa1 s1) and the water potential difference, (MPa), between a water source and target cell. For an epidermal cell in the elongation zone (EZ) of a grass leaf, the water source is the xylem in the EZ. Equation 2 expresses REGR as a product of a wall extensibility coefficient M (MPa1 s1) and the growth-effective turgor (P – Y, MPa). The latter is the amount of cell turgor (P) which exceeds the yielding threshold of the wall (Y). Equation 2 assumes that the yielding of the cell wall depends in a linear fashion on the physical wall stress in excess of a minimum yield stress. It should be noted, though, that wall stress is a complex function, which depends on the geometry of the cell and the thickness of the wall, and is difficult to measure (Cosgrove 1987). Therefore, wall properties are expressed as P and minimum P required for growth (Y) as these sizes are easier to measure than wall stress. Turgor as used in Equation 2 is the cause of the unmeasured wall stress. Lockhart (1965) combined the two equations into what is known as the ‘Lockhart equation’. Growth is viewed as a combined mechano-hydraulic process that can be limited through either or both wall mechanical and cell hydraulic properties: REGR ¼ ½ðLMÞ=ðL+MÞð +P YÞ
ð3Þ
It can be seen from Equation 3 that if M L, growth is limited by (mechanical) wall properties (M), while for L M, growth is
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055, Advance Access publication on 22 April 2015, available online at www.pcp.oxfordjournals.org ! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
Regular Paper
Department of Biology, Faculty of Nature and Life Sciences, University Ziane Achour, Djelfa, Algeria University College Dublin, School of Biology and Environmental Science, Science Centre West, Belfield, Dublin 4, Ireland 3 Laboratoire d’Eco-Physiologie Ve´ge´tale, De´partement des Sciences Naturelles, Ecole Normale Supe´rieure de Kouba, 16050, Alger, Algeria 4 Present address: Department of Viticulture and Enology, University of California, Davis, CA 95616-5270, USA. 5 Present address: Research, Chemical Works of Gedeon Richter Plc., H-1103 Budapest, Gyo¨mro00 i u´t 19-21, Hungary. 2
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
limited by hydraulic properties (L; Lockhart 1965, Boyer et al. 1985, Schopfer 2006). The Lockhart equation applies to the steady state of growth. Most of the early work on the biophysical limitation of plant cell expansive growth has been carried out on giant algae cells and, particularly, on stems of dicotyledonous plants, which were grown typically in the dark; comparatively few studies have been carried out on grasses, and under transpiring conditions. Together, these studies showed that growth can be limited more through hydraulic or mechanical properties or be co-limited through both (for reviews, see Boyer et al. 1985, Cosgrove 1999, Fricke 2002, Schopfer 2006; see also Van-Volkenburgh and Cleland 1986, Ehlert et al. 2009). The majority of studies altered the water supply to growing tissues, or the nutritional and water status of plants to affect growth rates and to deduce Y, M and L and conclude on turgor–growth relationships (for reviews, see Cosgrove 1993, Passioura and Boyer 2003, Boyer and Silk 2004, Caldeira et al. 2014) While this approach has the advantage of not directly disturbing the growing tissue, it also has the disadvantage that the growth response to treatments represents an integrated response of all tissues in a specimen. Therefore, the aim of the present study was to apply growth-promoting and inhibiting treatments locally to growing tissues and analyze local changes in hydraulic and mechanical properties. Furthermore, while many of the previous studies measured P at the, appropriate, cell level, using the cell pressure probe, p was analyzed at bulk tissue level but not at cell level. This masks any cell-specific responses in p to growth-manipulating treatments and can cause erroneous calculations of cell water potential and . To overcome this drawback, we analyzed both P and p at cell level. The growing barley leaf has been used previously to analyze changes in cellular water relation parameters in response to nutritional and stress treatments and the local application of growth-promoting substances (Fricke et al. 1997, Fricke and Peters 2002, Fricke et al. 2006, Volkov et al. 2009, Visnovitz et al. 2013). In the present study, we exposed the EZ of the growing leaf 3 of barley to treatments which are expected to affect the transport of (i) solutes, in particular K+ [5 mM cesium chloide (CsCl); 50 mM tetraethylammonium (TEA), 5 mM fusicoccin (FC); Marre´ 1979, Tode and Lu¨then 2001, Lenaeus et al. 2005, Volkov et al. 2009, Visnovitz et al. 2013] and (ii) water (100 mM HgCl2; e.g. Tazawa et al. 1997, Besse et al. 2011) into cells and the (iii) properties of the wall (5 mM FC; Cleland 1994, Visnovitz et al. 2013). The hypotheses associated with these treatments were that (i) a reduction in solute transport in response to the application of the K+ transport/ channel inhibitors Cs+ and TEA should cause a decrease in growth which is associated with a decrease in P (and P – Y) and p, and which should also possibly cause a decrease in , while L, M and Y need not change. Conversely, simultaneous stimulation of solute transport and wall extensibility properties through the application of FC, which stimulates the plasma membrane H+-ATPase (PM-H+-ATPase) activity, would be expected to cause an increase in growth which is associated with an increase in cell p, P (and P – Y) and M,
Fig. 1 Elongation rate of leaf 3 of barley in response to test reagents which are aimed at affecting the biophysical properties of cells. Test reagents were applied externally to the elongation zone of leaf 3, at the following concentrations: CsCl, 5 mM; tetraethylammonium (TEA), 50 mM; HgCl2, 100 mM; fusicoccin (FC), 5 mM. Results are averages and SE (error bars) of 6–13 plant analyses. Statistical significance of differences between values is indicated by different letters (one-way ANOVA followed by Tukey’s test). Ctrl, control plants.
and possibly also with an increase in , while L and Y need not to change. Inhibition of water transport through aquaporins in response to Hg2+ (Tazawa et al. 1997) would be expected to reduce growth through a reduction in L and possibly also through metabolic control (Zhu and Boyer 1992) as Hg2+ has cytotoxic properties (Azevedo and Rodriguez 2012).
Results Leaf elongation rate (LER) The elongation rate of leaf 3 of plants, which had a window cut at the shoot base, averaged 1.02 mm h1 (Fig. 1; control value). In comparison, leaf 3 of plants which did not have a window cut elongated at about twice the rate (2.20 mm h1; not shown; compare also Fricke and Peters 2002). Application of CsCl decreased LER significantly, by 47% to 0.54 mm h1. The K+ channel inhibitor TEA and the aquaporin inhibitor HgCl2 also decreased growth, by 26% (to 0.75 mm h1) and 34 % (to 0.67 mm h1), respectively. These decreases were not significant. Application of FC increased LER significantly by 34%, to 1.37 mm h1.
Biophysical cell analyses in the leaf EZ Treatments had a significant effect on P and p of cells [P < 0.001, analysis of variance (ANOVA) followed by Tukey’s test]. Turgor of epidermal cells in the leaf EZ averaged 0.491 MPa in control plants and decreased most in response to CsCl, to 0.426 MPa (Fig. 2A). Smaller decreases in P were observed in response to TEA (0.455 MPa) and HgCl2 1365
M. Touati et al. | Turgor–growth relationship in barley leaves
Fig. 2 (A) Epidermal cell turgor (P), (B) osmotic pressure (p) and (C) water potential ( ) in the elongation zone of leaf 3 of barley exposed to test reagents. Bulk leaf p is also shown (B). Test reagents were applied externally to the elongation zone of leaf 3 at the following concentrations: CsCl, 5 mM; tetraethylammonium (TEA), 50 mM; HgCl2, 100 mM; fusicoccin (FC), 5 mM. Results are averages and SE (error bars). Cell P was analyzed in 98 cells of control plants and 13–23 cells of treated plants, with a minimum of four plants of each treatment being analyzed. Cell p was analyzed in 15–22 cells of each treatment including the control (Ctrl), with a minimum of four plants of each treatment being analyzed. Cell was calculated as the difference between average P and p. Bulk leaf p was derived from the analysis of six leaves of each treatment and the control. Statistical significance of differences between values is indicated by different letters (one-way ANOVA followed by Tukey’s test).
(0.474 MPa). In contrast, FC treatment increased P significantly, to 0.531 MPa. The p of epidermal cells was similar in control plants and plants treated with CsCl, TEA or HgCl2 (Fig. 2B). Values ranged from 0.780 to 0.802 MPa. In contrast, FC decreased p significantly, to 0.667 MPa. Bulk leaf p was not affected significantly by treatments (Fig. 2B). Average values ranged from 0.810 to 0.844 MPa. While bulk leaf p exceeded epidermal cell p by 0.02–0.05 MPa in control plants and plants exposed to CsCl, TEA or HgCl2, it exceeded cell p in FC-treated plants by 0.15 MPa. Epidermal cell in the EZ averaged –0.289 MPa in control plants and became more negative in response to TEA (0.340 MPa), HgCl2 (–0.328 MPa) and CsCl (–0.372 MPa; Fig. 2C). In contrast, treatment with FC rendered less negative (0.136 MPa). 1366
The half-time of water exchange, T1/2, of epidermal cells of the leaf EZ was significantly shorter in control plants (0.85 s) and in plants treated with FC (0.91 s) compared with plants subjected to HgCl2 treatment (2.64 s; Fig. 3A). The cell volumetric elastic modulus differed comparatively little between these plants (Fig. 3B). This resulted in a significant decrease, by 75%, of Lpcell in HgCl2-treated plants (Fig. 3C). FC application increased Lpcell by 19%, from 2.05106 m s1 MPa1 in control plants to 2.43106 m s1 MPa1.
Water potential in the emerged-blade portion Average values of epidermal cell P and p (not shown) were used to calculate the of epidermal cells in the emerged-blade portion ( Epi-EmBL), which approximates of xylem in this leaf region ( Xylem-EmBL). Values differed little between treatments and ranged from –0.17 MPa (TEA and Hg treatment) to –0.19 MPa (control and FC treatment) and –0.24 MPa (CsCl treatment; Table 1).
Range of xylem ) and of ") driving water uptake into growing epidermal cells As outlined in the Methods section, Xylem-EZ must attain values within the range set by Medium (here: –0.04 MPa) and Epi-EmBL. For example, in control plants, Xylem-EZ was within the range – 0.19 to –0.04 MPa (Table 1). As Epi-EZ of control plants averaged –0.29 MPa, between epidermal cells and xylem in the EZ of control plants was within the range 0.249 MPa (maximum value in Table 1) to 0.09 MPa (minimum value in Table 1), with a mean (midpoint) value of 0.174 MPa. In comparison, the midpoint value of in plants treated with CsCl, TEA or HgCl2 was slightly larger (0.223–0.235 MPa). The midpoint of in FC-treated plants was the smallest (0.021 MPa) of all treatments and close to zero (Table 1).
Relationship between REGR and P and ") Across all treatments, including the control, REGR was linearly and positively related to mean cell P [r2 = 0.90, probability
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
Fig. 3 (A) Half-time of water exchange (T1/2), (B) volumetric elastic modulus (e) and (C) hydraulic conductivity (Lpcell) in epidermal cells of the elongation zone of leaf 3 of barley in response to treatment with 100 mM HgCl2 (Hg) and 5 mM fusicoccin (FC) as determined using a cell pressure probe. Results are averages and SE (error bars) of 8–16 cell analyses, with a minimum of four plants of each treatment including the control (CTRL) being analyzed. The statistical significance of differences between values is indicated by different letters (one-way ANOVA followed by Tukey’s test).
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
Table 1 Water potential ( , unit MPa) values used to calculate the maximum, minimum and mean size of the water potential difference ( ) between xylem in the leaf elongation zone and elongating epidermal (Epi) cell Treatment
)Medium
)Xylem-EmBL
)Epi-EZ
") = ()Xylem-EZ) – ()Epi-EZ) Maximum
Minimum
Mean
Control
–0.04
–0.19
–0.289
0.249
0.099
0.174
CsCl
–0.04
–0.24
–0.372
0.332
0.132
0.232
TEA
–0.04
–0.17
–0.34
0.3
0.17
0.235
HgCl2
–0.04
–0.17
–0.328
0.288
0.158
0.223
FC
–0.04
–0.19
–0.136
0.096
-0.054
0.021
Medium is the of the root medium (compare Fig. 6). Xylem-EZ was assumed to approximate either Medium (maximum values) or Xylem-EmBL (minimum values of ). ‘Mean’ is the value in the middle of the range set by the maximum and minimum.
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
(P) < 0.05; Fig. 4A]. The extrapolation of the regression line to zero REGR (intercept of x-axis), gave an apparent yield threshold, Y (compare Equation 2) of 0.365 MPa. The relationship between REGR and was also linear (r2 = 0.89; P < 0.05; Fig. 4B), yet was negative. Growth decreased with an increasing . There was no common relationship between REGR and as predicted by Equation 1. Therefore, L was derived separately for each treatment, entering values of REGR and into Equation 1. Values of L ranged from 2.6105 to 7.2104 MPa1 s1 between treatments (Fig. 4C). The highest value of L was observed for FC-treated plants. Treatment with CsCl, HgCl2 and TEA reduced L by about 50% compared with the value in control plants. Knowing all the sizes except M, the Lockhart equation was solved for M: M ¼ ðREGRLÞ=f½ð +P YÞL REGRg
ð4Þ
Values of M calculated in this way differed little between treatments and ranged from 6.83105 (HgCl2 treatment) to 9.84105 (CsCl treatment) s1 MPa1 (Fig. 4C).
Discussion Relationship between REGR and P The present data show a positive and linear relationship between REGR and P. Changes in growth rate in response to treatments, which were applied locally to the leaf EZ, were mediated through changes in P. This relationship included treatments which targeted solute and water uptake, wall properties and the energization of plasma membrane. The relationship between REGR and P need not indicate constant wall properties as it may reflect an effect of P on wall-loosening processes as concluded for leaves of Begonia (Begonia argenteo-guttata, Serpe and Matthews 1992). Values of P were in the range of values reported previously for growing plant tissues (e.g. Boyer et al. 1985, Schmalstig and Cosgrove, 1988, Nonami and Boyer 1987, Serpe and Matthews 1992, Fricke et al. 1997, Martre et al. 1999). A positive relationship between growth rate and P has also been reported for leaves of maize using dynamic approaches, where plants were subjected to changes in root water
Fig. 4 Relative elemental growth rate (REGR) as a function of (A) cell turgor (P) and (B) water potential difference between xylem and an expanding epidermal cell ( = Xylem-EZ – Epi-EZ) in the elongation zone of the developing leaf 3 of barley. Each point represents a pair (x/ y) of mean values of (A) P/REGR and (B) /REGR for a treatment including the control. For , the midpoint of the range shown in Table 1 was used. The error bars for REGR and P are SEMs; the error bars for give the range of lowest and highest possible value (compare ‘maximum’ and ‘minimum’ values in Table 1). Data were subjected to linear regression and correlation analyses. Regression coefficients (r2) were 0.90 in (A) and 0.89 in (B), at P < 0.05. (C) A tissue hydraulic conductance coefficient ‘L’ was calculated for each treatment, by entering values of REGR and into Equation 1. This value was used together with values of REGR, P, Y and to calculate a wall extensibility coefficient ‘M’ using the Lockhart equation.
availability, root hydraulics and shoot evaporational demand (Bouchabke´ et al. 2006, Ehlert et al. 2009, Caldeira et al. 2014). The authors explained the observed changes in P through hydraulic processes, as increased plant water loss and reduced 1367
M. Touati et al. | Turgor–growth relationship in barley leaves
root water uptake rates caused reductions (more negative) in xylem in the EZ of leaves and a subsequent decrease in water supply rates to peripherally expanding cells. The reductions in xylem paralleled reductions in growth and P. Although the nature of underlying hydraulic processes is not known (Ehlert et al. 2009), a more negative xylem will diminish . According to Equation 1, this reduces growth, and L need not change. In the present study, treatments targeted specifically the leaf EZ. Cells did not grow at reduced rates because of decreased driving less water towards cells. Rather, L of the hydraulic path between xylem and epidermal cells changed, decreasing by 50% in response to CsCl, HgCl2 and TEA.
") in the EZ
1368
The mechanism of changes in P and n in response to inhibitor treatments The test agents used here can cause non-specific effects. This has to be remembered when interpreting data. For example, Cramer (1992) studied the involvement of ethylene in the growth response of maize leaves to salt stress. Silver thiosulfate was applied, with the aim of inhibiting ethylene action. The inhibitor treatment (applied to the root system of plants) decreased growth through an increase in Y, yet ethylene was found not to be involved in this response. The inhibitors used here are likely to affect more processes in cells than the targeted function. While this does not affect the
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
The existence of a significant (>0.1 MPa) , being indicative of some hydraulic (co-)limitation of cell expansion, in growing plant tissues has been discussed intensely in the literature, with data supporting and questioning the idea of a hydraulic limitation of growth (e.g. for reviews, see Boyer et al. 1985, Cosgrove 1993, Fricke 2002, Boyer and Silk 2004). In the present study, a significant was observed in all except the FC treatment. This coincided with M:L ratios ranging from 1.4 (control) to 2.4 (HgCl2) and 2.5 (TEA) to 5.1 (CsCl treatment). Growth in these treatments was co-limited by hydraulic and mechanical properties, and treatments which reduced growth (HgCl2, CsCl and TEA) increased the hydraulic limitation compared with the mechanical one (M:L >>1.0; compare Equation 3). These treatments also showed the largest . In FC-treated leaves, the M:L ratio was 0.3 and was close to zero. This suggests that growth in these leaves was limited more through mechanical than through hydraulic properties. The present data are consistent with previous studies, in that they show that growth can be co-limited through hydraulic and mechanical properties (Radin and Boyer 1982, Boyer et al. 1985, Cramer 1992; for a review, see Fricke 2002) or that mechanical (Cosgrove 1981, Cosgrove et al. 1984, Shackel et al. 1987, Cosgrove and Sovonick-Dunford 1989, Park and Cosgrove 2012) or hydraulic properties (Nonami et al. 1997, Fricke and Flowers 1998, Martre et al. 1999, Tang and Boyer 2002, Tang and Boyer 2008) limit growth more. In contrast to studies which do not support the idea of significant in growing plant tissues, in the present study of growing cells was determined through the combined analysis of both P and p at the cell level—and a significant resulted for the highest and lowest possible estimates of xylem in the EZ. The only element of uncertainty in deriving cell from P and p is the value of the solute reflection coefficient, s, of the plasma membrane of epidermal cells. Any significant deviation of a from unity (10-fold. Work by Tang and Boyer (2002) and Passioura and Boyer (2003) suggests that the hydraulic limitation, which causes to establish, occurs upstream along the supply route of water from xylem to peripheral (leaf) epidermal cells, rather than at the plasma membrane of epidermal cells itself. In the present study, this is supported through calculations (see Supplementary Data File S1) which show that across the plasma membrane of growing epidermal cells approximated 3.65105 MPa and was almost 1,000 times smaller than the smallest (2.1102 MPa) observed for FC-treated plants. Nonami et al. (1997) and Tang and Boyer (2002, 2008) proposed for soybean hypocotyls and maize leaves, respectively, that the hydraulic limitation resulted from the cumulative hydraulic resistance of many smaller, xylem parenchyma cells which were located next to xylem vessels (for a review, see Boyer and Silk 2004). The barley leaves studied here show comparatively few xylem parenchyma cells (Fricke 2002). The hydraulic limitation and the changes in L in response to treatments may reside instead in the mestome and parenchymateous sheaths of vascular bundles (Fricke 2002, Heinen et al. 2009, Besse et al. 2011). These structures are suited to fulfill a transport-controlling role similar to that of the endodermis in roots (O’Brien and Kuo 1975, Lersten 1997, Steudle and Peterson 1998, Steudle 2000, Fricke 2002, Heinen et al. 2009, Besse et al. 2011).
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
data as such, it does affect the interpretation of data. Cesium, which targets K+ transport, and Hg2+, which targets water transport, also have more general (cyto)toxic properties (Hampton et al. 2004, Azevedo and Rodriguez 2012), and Hg2+ may affect plant hormonal status and cell expansion through inducing oxidative stress (Montero-Palmero et al. 2014); TEA, which targets K+ transport, can also inhibit water channel activity (Mu¨ller et al. 2008, Yool et al. 2009). Similarly, Cs+ may have impacted on L through co-regulation of K+ and water transport (Gazzarini et al. 2006, Liu et al. 2006) or through coupling of water flows through K+ channels (Homble´ and Ve´ry 1992). FC has a comparatively specific molecular target, the PMH+-ATPase, yet changes in the activity of PM-H+-ATPase are bound to feed back on the physiology of cell. There exist alternative inhibitors, in particular for water transport through aquaporins. Anoxia, acid load and H2O2 are suitable alternatives when aiming to reduce root hydraulics to study distant changes in the REGR/P relations of elongating leaf cells (Ehlert et al. 2009). However, these inhibitors are not suitable when being applied directly to the leaf EZ in biophysical studies as they impact directly on apoplast pH (acid load, anoxia), cause oxidative stress (H2O2) and perturb cytosolic pH (acid load, anoxia), a key parameter of cell metabolism. Therefore, we consider the present choice of inhibitors as the best possible compromise between targeting a specific cellular process which facilitates cell expansion and applying the inhibitor locally to the EZ. The observed reduction in Lpcell in response to Hg2+ is consistent with the aquaporin-inhibiting properties of Hg2+ (e.g. Tazawa et al. 1997, Tazawa et al. 2001). The three treatments (CsCl, TEA and HgCl2) which reduced P did not decrease cell p. We do not know whether s decreased and rendered cell p less osmotically efficient. A likely explanation of the data is that the decrease in cell P was facilitated through an increase in apoplast p. The transport of K+ across the plasma membrane is inhibited by TEA and, in particular, CsCl (e.g. Lenaeus et al. 2005, Volkov et al. 2009). As growing barley leaf cells take up K+ to maintain their K+ concentration during growth (Volkov et al. 2009), an inhibition of K+ uptake will lead to an increase in apoplast K+. Similarly, Hg2+ may affect uptake of K+ through some non-specific cytotoxic effects; the latter could explain why the REGR of Hg2+-treated cells was lower than predicted from the common REGR–P relationship of treatments (regression line in Fig. 4A). Cell p need not to change, as any reduced K+ and solute accumulation rates in leaves treated with CsCl, TEA or HgCl2 were accompanied by reduced expansion and dilution rates of cell contents (see Fig. 5). Turgor increased significantly in FC-treated cells. These cells display increased PM-H+-ATPase activity and wall acidification (Visnovitz et al. 2012, Visnovitz et al. 2013). Both processes facilitate the increased uptake of solutes, in particular K+. This could lead to a lowering of apoplast solute concentration and p, which facilitates the increase in P. Increased rates of solute accumulation should also lead to an increase in cell p. However, p of epidermal cells decreased. The data can be explained best through growth dilution. FC-treated leaves grew at 34% higher rates than leaves of control plants. If FC-treated cells
Fig. 5 Model to explain the changes in turgor (P) in growing leaf epidermal cells of barley in response to treatments (CsCl; TEA, tetraethylammonium; HgCl2; FC, fusicoccin; CTRL, control) through solute movement from the cell exterior (apoplast) to the interior (protoplast) and accompanying growth dilution of cell contents. Values of P (unit: MPa), the net rate of solute accumulation (‘Transport’, unit: mmol l1 s1) and relative elemental growth rates (REGR, unit: s1) as determined/derived in this study are also shown. The size of arrows symbolizes the magnitude of the processes involved; filled arrows symbolize net import of solutes into cells; dashed arrows symbolize the relative rate at which cells expand and cell contents are diluted. Treatments (CsCl, TEA and HgCl2) which decrease REGR do so primarily through decreasing P; this is the result of decreased uptake rates of solutes from the cell apoplast, which in turn leads to higher solute concentrations and osmotic pressure (p) in the apoplast. In the protoplasm, solute concentrations hardly change as reduced solute uptake rates are accompanied by reduced cell expansion rates (REGRs). In FC-treated leaves, the solute uptake rate increases. This leads to lower solute concentrations and p in the apoplast and minimizes the decrease in solute concentration and p in the protoplasm during cell volume expansion. Together, this raises P in FC-treated cells above the value in control cells; neither rheological wall properties nor the solute reflection coefficient of the plasma membrane need to change in this model. Apoplast and protoplast solutes are shown in different colors, for easier distinction, even though the chemical identity of solutes may be the same.
had net accumulated solutes at the same rate as in control plants, cell p should have decreased to (1/1.34) 75% of the control value. The observed cell p was 86% of the control value. This implies that FC stimulated the net rate of solute accumulation by [(0.861.34) – (1)] 15% (see also Fig. 5). Based on the above, it is proposed that FC stimulated the rate of solute accumulation in cells and caused an accompanying decrease in apoplast p. This allowed P and P – Y to increase beyond the level in control cells. Such an interpretation of data does not require an alteration in wall rheological properties, in contrast to previous studies which showed that FC affects M (Lado et al. 1973, Van Volkenburgh and Cleland 1986, Keller and Van Volkenburgh 1998, Park and Cosgrove 2012; for reviews, see Cosgrove 1999, Schopfer 2006). Katou and Furumoto (1986) reached a similar conclusion for concurrent increases in P and growth in response to auxin, although they did not actually measure p at the cell level; neither did studies 1369
M. Touati et al. | Turgor–growth relationship in barley leaves
which do not support the present interpretation of FC data (for a review, see Cosgrove 1993). The present study highlights the need to measure P and p at the cell level when deriving the of a cell (and also ) from values of P and p and when concluding on changes in cellular p. A bulk leaf analysis of p would have masked the epidermal cellspecific decrease in p in response to FC (see Fig. 2B). The epidermis-specific response in p may govern the leaf response to FC, as the epidermis is thought to limit mechanically the growth of plant organs (Brummell and Hall 1980, Van Volkenburgh and Cleland 1986, Schopfer 2006).
Plant material Barley (Hordeum vulgare L. cv. Jersey) was grown hydroponically in a growth chamber (Snyders Microclimate MC1000) under a 16 h day/8 h night cycle, at 20 C day and 18 C night temperature, 70–80% relative humidity and a photosynthetically active radiation of 250 mmol photons m2 s1 (compare Knipfer and Fricke 2010). Nutrient solutions were continuously aerated. Plants were analyzed when they were 14–16 d old, 3–9 h into the photoperiod. At this developmental stage, leaves 1 and 2 were fully expanded and the main growing leaf was leaf 3. The base 40 mm of leaf 3 contained the leaf EZ and was enclosed by the sheaths of older leaves 1 and 2 (Fricke and Flowers 1998, Fricke and Peters 2002). The transpiring portion of leaf 3 which had emerged from the sheath of leaf 2 and which was exposed to ambient environmental conditions (light, wind, air humidity) was the ‘emerged blade’ (EmBL).
Outline of approach to determine the components of the Lockhart equation The approach was as follows:
(i) Expose the growth zone of leaf 3 of 2- to 3-week-old barley plants to treatments which affect cellular water transport (100 mM HgCl2), solute transport (5 mM CsCl; 50 mM TEA; 5 mM FC) and wall properties (5 mM FC). Determine LER and deduce REGRs for treatments. (ii) Measure P in epidermal cells of the EZ and emerged-blade portion (EmBL) of the growing leaf using the cell pressure probe, and determine cell p using picoliter osmometry of extracted cell sap. Use these data to calculate cell-EZ and cell-EmBL. (iii) Using the above values of cell and a value of root-medium, determine the range of possible values of xylem in the EZ ( xylemEZ). The difference between cell-EZ and xylem-EZ [( cell-EZ) – ( xylem-EZ)] is the difference in ( ) between the xylem in the EZ and expanding, epidermal cell (see Fig. 6). (iv) Construct plots of (a) P vs. REGR and (b) vs. REGR. This will show whether all treatments fit one general relationship as predicted by Equations (1) and (2); if so, a value of apparent yield threshold can be derived from the REGR vs. P relationship (Y equals P for extrapolated zero REGR). (v) Derive L from Equation 1 using values of REGR and . Solve the Lockhart equation for M, and calculate M using measured (P) and derived (Y) values. (vi) Using the information provided through (iv) and (v), conclude on the relative limitation of cell expansion through mechanical and hydraulic properties and whether changes in REGR in response to treatments can be explained through changes in P.
1370
Fig. 6 Estimation of the water potential ( ) of xylem in the elongation zone (EZ) ( Xylem-EZ) of leaf 3 of barley. The picture on the left shows a 16-day-old barley plant as analyzed in the present study; the pictures on the right show cross-sections of the root and of leaf regions. The EZ of the leaf is located at the base, with the root being located upstream and the emerged-blade portion (EmBL) of the growing leaf being located downstream of the transpiration stream which supplies water to the EZ. Most of the water moving axially through roots and the EmBL will move along metaxylem vessels, while most of the water moving axially through the EZ will move through protoxylem at proximal regions and through metaxylem at more distal regions (towards the leaf tip). Metaxylem vessels (root: early metaxylem, peripheral location; late metaxylem, central location) are indicated by blue dots; protoxylem vessels in leaf regions are indicated by red dots. The of xylem in the EZ ( Xylem-EZ) could not be determined directly and an indirect approach was taken to derive its range of values. Water flow through the root medium–plant–air continuum is driven by a difference in . The least negative is in the root medium ( Medium); the most negative within the plant is close to the exit point of water from the transpiring portion of blade (EmBL for leaf 3) and should approach the of leaf epidermal cells in this leaf region ( Epi-EmBL). The value of Xylem-EZ will be within the range set by these two ‘extremes’. Scale bars are 20 mm for EZ and EmBL and 100 mm for root.
Preparation of the leaf EZ for the application of test reagents The cuticle in the EZ of leaf 3 of barley is not fully developed and has a permeance which is much higher than that in transpiring leaf tissue (Richardson et al. 2005, Richardson et al. 2007). This makes it possible to apply test reagents externally to the EZ (Visnovitz et al. 2013). A plant to be analyzed was removed from the growth chamber and the shoot was placed under a stereomicroscope, with the root system remaining in nutrient solution. The sheath of leaf 1 was partially peeled back and a window ( 2 mm wide, 5 mm long) was cut into the sheath of leaf 2 at 15–25 mm from the base of the shoot to expose a small portion of the EZ of leaf 3 located beneath. At this location, REGRs are highest in the expanding leaf 3 (e.g. Fricke et al. 1997, Fricke and Peters 2002). Only leaves with no visible injury to the EZ were used for experiments. The portion of the EZ from about 10–30 mm from the base was wrapped in tissue paper which had been moistened with distilled water. The plant was then transferred into a 250 ml Erlenmeyer flask containing nutrient solution, with the shoot base being supported by a foam piece. The plant was moved back into the growth chamber for between 1 and 1.5 h to acclimatize to the new situation of having a window cut. The root medium was aerated throughout.
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
Materials and Methods
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
Application of test reagents and determination of LER in the growth chamber
LER on the cell pressure probe stage, and derivation of REGR Throughout experiments, LER was determined for plants in the growth chamber, yet biophysical cell parameters were analyzed on the cell pressure probe stage in the laboratory. To test whether LER in the growth chamber was comparable with that on the cell pressure probe stage, one additional set of experiments was carried out in which LER for three plants per treatment was first determined in the growth chamber and then on the cell pressure probe stage. The latter was achieved by pointing the microcapillary tip of the cell pressure probe at a reference point on the emerged-blade portion of leaf 3 and measuring with a graticule the displacement of the reference point over a 25–30 min period—a time typically required for cell pressure probe analyses. The LER determined in this way differed by
*Corresponding author: E-mail, [email protected]; Fax, +353-1-7161153. (Received December 15, 2014; Accepted April 1, 2015)
The aim of the present study was to assess the mechanical and hydraulic limitation of growth in leaf epidermal cells of barley (Hordeum vulgare L.) in response to agents which affect cellular water (mercuric chloride, HgCl2) and potassium (cesium chloride, CsCl; tetraethylammonium, TEA) transport, pump activity of plasma membrane H+-ATPase and wall acidification (fusicoccin, FC). Cell turgor (P) was measured with the cell pressure probe, and cell osmotic pressure (p) was analyzed through picoliter osmometry of single-cell extracts. A wall extensibility coefficient (M) and tissue hydraulic conductance coefficient (L) were derived using the Lockhart equation. There was a significant positive linear relationship between relative elemental growth rate and P, which fit all treatments, with an overall apparent yield threshold of 0.368 MPa. Differences in growth between treatments could be explained through differences in P. A comparison of L and M showed that growth in all except the FC treatment was co-limited through hydraulic and mechanical properties, though to various extents. This was accompanied by significant (0.17–0.24 MPa) differences in water potential ( ) between xylem and epidermal cells in the leaf elongation zone. In contrast, FC-treated leaves showed close to zero and a 10-fold increase in L. Keywords: Barley (Hordeum vulgare L.) Fusicoccin Hydraulic conductivity Leaf cell elongation Lockhart equation Turgor. Abbreviations: Acell, cell surface; EmBL, emerged-blade portion of leaf; Epi, epidermis; EZ, elongation zone; e, cell volumetric elastic modulus; FC, fusicoccin; h, cell length; PM-H+-ATPase, plasma membrane H+-ATPase; L, tissue hydraulic conductance coefficient; LER, leaf elongation rate; Lpcell, cell hydraulic conductivity; M, wall extensibility coefficient; P, cell turgor; p, osmotic pressure; c, water potential; , water potential difference; Epi-EZ, water potential of epidermal cell in EZ; cEpi-EmBL, water potential of epidermal cell in EmBL; Medium, water potential of root medium; Xylem-EZ, water potential of xylem in EZ; Xylem-EmBL, water potential of xylem in EmBL; Xylem-Root, water potential of xylem in root; r, radius; REGR, relative elemental growth rate; s, solute reflection coefficient; TEA,
tetraethylammonium; T1/2, half-time of water exchange; Vcell, cell volume; Y, yield threshold of wall.
Introduction The quantitative relationship between cell expansive growth and the hydraulic and mechanical properties which affect cell expansion is expressed through Equations (1) and (2), respectively (Schopfer 2006): REGR ¼ L
ð1Þ
REGR ¼ MðP YÞ
ð2Þ
Equation 1 expresses the relative elemental growth rate (REGR, s1) of a cell as the product of a hydraulic conductance coefficient (L, MPa1 s1) and the water potential difference, (MPa), between a water source and target cell. For an epidermal cell in the elongation zone (EZ) of a grass leaf, the water source is the xylem in the EZ. Equation 2 expresses REGR as a product of a wall extensibility coefficient M (MPa1 s1) and the growth-effective turgor (P – Y, MPa). The latter is the amount of cell turgor (P) which exceeds the yielding threshold of the wall (Y). Equation 2 assumes that the yielding of the cell wall depends in a linear fashion on the physical wall stress in excess of a minimum yield stress. It should be noted, though, that wall stress is a complex function, which depends on the geometry of the cell and the thickness of the wall, and is difficult to measure (Cosgrove 1987). Therefore, wall properties are expressed as P and minimum P required for growth (Y) as these sizes are easier to measure than wall stress. Turgor as used in Equation 2 is the cause of the unmeasured wall stress. Lockhart (1965) combined the two equations into what is known as the ‘Lockhart equation’. Growth is viewed as a combined mechano-hydraulic process that can be limited through either or both wall mechanical and cell hydraulic properties: REGR ¼ ½ðLMÞ=ðL+MÞð +P YÞ
ð3Þ
It can be seen from Equation 3 that if M L, growth is limited by (mechanical) wall properties (M), while for L M, growth is
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055, Advance Access publication on 22 April 2015, available online at www.pcp.oxfordjournals.org ! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
Regular Paper
Department of Biology, Faculty of Nature and Life Sciences, University Ziane Achour, Djelfa, Algeria University College Dublin, School of Biology and Environmental Science, Science Centre West, Belfield, Dublin 4, Ireland 3 Laboratoire d’Eco-Physiologie Ve´ge´tale, De´partement des Sciences Naturelles, Ecole Normale Supe´rieure de Kouba, 16050, Alger, Algeria 4 Present address: Department of Viticulture and Enology, University of California, Davis, CA 95616-5270, USA. 5 Present address: Research, Chemical Works of Gedeon Richter Plc., H-1103 Budapest, Gyo¨mro00 i u´t 19-21, Hungary. 2
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
limited by hydraulic properties (L; Lockhart 1965, Boyer et al. 1985, Schopfer 2006). The Lockhart equation applies to the steady state of growth. Most of the early work on the biophysical limitation of plant cell expansive growth has been carried out on giant algae cells and, particularly, on stems of dicotyledonous plants, which were grown typically in the dark; comparatively few studies have been carried out on grasses, and under transpiring conditions. Together, these studies showed that growth can be limited more through hydraulic or mechanical properties or be co-limited through both (for reviews, see Boyer et al. 1985, Cosgrove 1999, Fricke 2002, Schopfer 2006; see also Van-Volkenburgh and Cleland 1986, Ehlert et al. 2009). The majority of studies altered the water supply to growing tissues, or the nutritional and water status of plants to affect growth rates and to deduce Y, M and L and conclude on turgor–growth relationships (for reviews, see Cosgrove 1993, Passioura and Boyer 2003, Boyer and Silk 2004, Caldeira et al. 2014) While this approach has the advantage of not directly disturbing the growing tissue, it also has the disadvantage that the growth response to treatments represents an integrated response of all tissues in a specimen. Therefore, the aim of the present study was to apply growth-promoting and inhibiting treatments locally to growing tissues and analyze local changes in hydraulic and mechanical properties. Furthermore, while many of the previous studies measured P at the, appropriate, cell level, using the cell pressure probe, p was analyzed at bulk tissue level but not at cell level. This masks any cell-specific responses in p to growth-manipulating treatments and can cause erroneous calculations of cell water potential and . To overcome this drawback, we analyzed both P and p at cell level. The growing barley leaf has been used previously to analyze changes in cellular water relation parameters in response to nutritional and stress treatments and the local application of growth-promoting substances (Fricke et al. 1997, Fricke and Peters 2002, Fricke et al. 2006, Volkov et al. 2009, Visnovitz et al. 2013). In the present study, we exposed the EZ of the growing leaf 3 of barley to treatments which are expected to affect the transport of (i) solutes, in particular K+ [5 mM cesium chloide (CsCl); 50 mM tetraethylammonium (TEA), 5 mM fusicoccin (FC); Marre´ 1979, Tode and Lu¨then 2001, Lenaeus et al. 2005, Volkov et al. 2009, Visnovitz et al. 2013] and (ii) water (100 mM HgCl2; e.g. Tazawa et al. 1997, Besse et al. 2011) into cells and the (iii) properties of the wall (5 mM FC; Cleland 1994, Visnovitz et al. 2013). The hypotheses associated with these treatments were that (i) a reduction in solute transport in response to the application of the K+ transport/ channel inhibitors Cs+ and TEA should cause a decrease in growth which is associated with a decrease in P (and P – Y) and p, and which should also possibly cause a decrease in , while L, M and Y need not change. Conversely, simultaneous stimulation of solute transport and wall extensibility properties through the application of FC, which stimulates the plasma membrane H+-ATPase (PM-H+-ATPase) activity, would be expected to cause an increase in growth which is associated with an increase in cell p, P (and P – Y) and M,
Fig. 1 Elongation rate of leaf 3 of barley in response to test reagents which are aimed at affecting the biophysical properties of cells. Test reagents were applied externally to the elongation zone of leaf 3, at the following concentrations: CsCl, 5 mM; tetraethylammonium (TEA), 50 mM; HgCl2, 100 mM; fusicoccin (FC), 5 mM. Results are averages and SE (error bars) of 6–13 plant analyses. Statistical significance of differences between values is indicated by different letters (one-way ANOVA followed by Tukey’s test). Ctrl, control plants.
and possibly also with an increase in , while L and Y need not to change. Inhibition of water transport through aquaporins in response to Hg2+ (Tazawa et al. 1997) would be expected to reduce growth through a reduction in L and possibly also through metabolic control (Zhu and Boyer 1992) as Hg2+ has cytotoxic properties (Azevedo and Rodriguez 2012).
Results Leaf elongation rate (LER) The elongation rate of leaf 3 of plants, which had a window cut at the shoot base, averaged 1.02 mm h1 (Fig. 1; control value). In comparison, leaf 3 of plants which did not have a window cut elongated at about twice the rate (2.20 mm h1; not shown; compare also Fricke and Peters 2002). Application of CsCl decreased LER significantly, by 47% to 0.54 mm h1. The K+ channel inhibitor TEA and the aquaporin inhibitor HgCl2 also decreased growth, by 26% (to 0.75 mm h1) and 34 % (to 0.67 mm h1), respectively. These decreases were not significant. Application of FC increased LER significantly by 34%, to 1.37 mm h1.
Biophysical cell analyses in the leaf EZ Treatments had a significant effect on P and p of cells [P < 0.001, analysis of variance (ANOVA) followed by Tukey’s test]. Turgor of epidermal cells in the leaf EZ averaged 0.491 MPa in control plants and decreased most in response to CsCl, to 0.426 MPa (Fig. 2A). Smaller decreases in P were observed in response to TEA (0.455 MPa) and HgCl2 1365
M. Touati et al. | Turgor–growth relationship in barley leaves
Fig. 2 (A) Epidermal cell turgor (P), (B) osmotic pressure (p) and (C) water potential ( ) in the elongation zone of leaf 3 of barley exposed to test reagents. Bulk leaf p is also shown (B). Test reagents were applied externally to the elongation zone of leaf 3 at the following concentrations: CsCl, 5 mM; tetraethylammonium (TEA), 50 mM; HgCl2, 100 mM; fusicoccin (FC), 5 mM. Results are averages and SE (error bars). Cell P was analyzed in 98 cells of control plants and 13–23 cells of treated plants, with a minimum of four plants of each treatment being analyzed. Cell p was analyzed in 15–22 cells of each treatment including the control (Ctrl), with a minimum of four plants of each treatment being analyzed. Cell was calculated as the difference between average P and p. Bulk leaf p was derived from the analysis of six leaves of each treatment and the control. Statistical significance of differences between values is indicated by different letters (one-way ANOVA followed by Tukey’s test).
(0.474 MPa). In contrast, FC treatment increased P significantly, to 0.531 MPa. The p of epidermal cells was similar in control plants and plants treated with CsCl, TEA or HgCl2 (Fig. 2B). Values ranged from 0.780 to 0.802 MPa. In contrast, FC decreased p significantly, to 0.667 MPa. Bulk leaf p was not affected significantly by treatments (Fig. 2B). Average values ranged from 0.810 to 0.844 MPa. While bulk leaf p exceeded epidermal cell p by 0.02–0.05 MPa in control plants and plants exposed to CsCl, TEA or HgCl2, it exceeded cell p in FC-treated plants by 0.15 MPa. Epidermal cell in the EZ averaged –0.289 MPa in control plants and became more negative in response to TEA (0.340 MPa), HgCl2 (–0.328 MPa) and CsCl (–0.372 MPa; Fig. 2C). In contrast, treatment with FC rendered less negative (0.136 MPa). 1366
The half-time of water exchange, T1/2, of epidermal cells of the leaf EZ was significantly shorter in control plants (0.85 s) and in plants treated with FC (0.91 s) compared with plants subjected to HgCl2 treatment (2.64 s; Fig. 3A). The cell volumetric elastic modulus differed comparatively little between these plants (Fig. 3B). This resulted in a significant decrease, by 75%, of Lpcell in HgCl2-treated plants (Fig. 3C). FC application increased Lpcell by 19%, from 2.05106 m s1 MPa1 in control plants to 2.43106 m s1 MPa1.
Water potential in the emerged-blade portion Average values of epidermal cell P and p (not shown) were used to calculate the of epidermal cells in the emerged-blade portion ( Epi-EmBL), which approximates of xylem in this leaf region ( Xylem-EmBL). Values differed little between treatments and ranged from –0.17 MPa (TEA and Hg treatment) to –0.19 MPa (control and FC treatment) and –0.24 MPa (CsCl treatment; Table 1).
Range of xylem ) and of ") driving water uptake into growing epidermal cells As outlined in the Methods section, Xylem-EZ must attain values within the range set by Medium (here: –0.04 MPa) and Epi-EmBL. For example, in control plants, Xylem-EZ was within the range – 0.19 to –0.04 MPa (Table 1). As Epi-EZ of control plants averaged –0.29 MPa, between epidermal cells and xylem in the EZ of control plants was within the range 0.249 MPa (maximum value in Table 1) to 0.09 MPa (minimum value in Table 1), with a mean (midpoint) value of 0.174 MPa. In comparison, the midpoint value of in plants treated with CsCl, TEA or HgCl2 was slightly larger (0.223–0.235 MPa). The midpoint of in FC-treated plants was the smallest (0.021 MPa) of all treatments and close to zero (Table 1).
Relationship between REGR and P and ") Across all treatments, including the control, REGR was linearly and positively related to mean cell P [r2 = 0.90, probability
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
Fig. 3 (A) Half-time of water exchange (T1/2), (B) volumetric elastic modulus (e) and (C) hydraulic conductivity (Lpcell) in epidermal cells of the elongation zone of leaf 3 of barley in response to treatment with 100 mM HgCl2 (Hg) and 5 mM fusicoccin (FC) as determined using a cell pressure probe. Results are averages and SE (error bars) of 8–16 cell analyses, with a minimum of four plants of each treatment including the control (CTRL) being analyzed. The statistical significance of differences between values is indicated by different letters (one-way ANOVA followed by Tukey’s test).
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
Table 1 Water potential ( , unit MPa) values used to calculate the maximum, minimum and mean size of the water potential difference ( ) between xylem in the leaf elongation zone and elongating epidermal (Epi) cell Treatment
)Medium
)Xylem-EmBL
)Epi-EZ
") = ()Xylem-EZ) – ()Epi-EZ) Maximum
Minimum
Mean
Control
–0.04
–0.19
–0.289
0.249
0.099
0.174
CsCl
–0.04
–0.24
–0.372
0.332
0.132
0.232
TEA
–0.04
–0.17
–0.34
0.3
0.17
0.235
HgCl2
–0.04
–0.17
–0.328
0.288
0.158
0.223
FC
–0.04
–0.19
–0.136
0.096
-0.054
0.021
Medium is the of the root medium (compare Fig. 6). Xylem-EZ was assumed to approximate either Medium (maximum values) or Xylem-EmBL (minimum values of ). ‘Mean’ is the value in the middle of the range set by the maximum and minimum.
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
(P) < 0.05; Fig. 4A]. The extrapolation of the regression line to zero REGR (intercept of x-axis), gave an apparent yield threshold, Y (compare Equation 2) of 0.365 MPa. The relationship between REGR and was also linear (r2 = 0.89; P < 0.05; Fig. 4B), yet was negative. Growth decreased with an increasing . There was no common relationship between REGR and as predicted by Equation 1. Therefore, L was derived separately for each treatment, entering values of REGR and into Equation 1. Values of L ranged from 2.6105 to 7.2104 MPa1 s1 between treatments (Fig. 4C). The highest value of L was observed for FC-treated plants. Treatment with CsCl, HgCl2 and TEA reduced L by about 50% compared with the value in control plants. Knowing all the sizes except M, the Lockhart equation was solved for M: M ¼ ðREGRLÞ=f½ð +P YÞL REGRg
ð4Þ
Values of M calculated in this way differed little between treatments and ranged from 6.83105 (HgCl2 treatment) to 9.84105 (CsCl treatment) s1 MPa1 (Fig. 4C).
Discussion Relationship between REGR and P The present data show a positive and linear relationship between REGR and P. Changes in growth rate in response to treatments, which were applied locally to the leaf EZ, were mediated through changes in P. This relationship included treatments which targeted solute and water uptake, wall properties and the energization of plasma membrane. The relationship between REGR and P need not indicate constant wall properties as it may reflect an effect of P on wall-loosening processes as concluded for leaves of Begonia (Begonia argenteo-guttata, Serpe and Matthews 1992). Values of P were in the range of values reported previously for growing plant tissues (e.g. Boyer et al. 1985, Schmalstig and Cosgrove, 1988, Nonami and Boyer 1987, Serpe and Matthews 1992, Fricke et al. 1997, Martre et al. 1999). A positive relationship between growth rate and P has also been reported for leaves of maize using dynamic approaches, where plants were subjected to changes in root water
Fig. 4 Relative elemental growth rate (REGR) as a function of (A) cell turgor (P) and (B) water potential difference between xylem and an expanding epidermal cell ( = Xylem-EZ – Epi-EZ) in the elongation zone of the developing leaf 3 of barley. Each point represents a pair (x/ y) of mean values of (A) P/REGR and (B) /REGR for a treatment including the control. For , the midpoint of the range shown in Table 1 was used. The error bars for REGR and P are SEMs; the error bars for give the range of lowest and highest possible value (compare ‘maximum’ and ‘minimum’ values in Table 1). Data were subjected to linear regression and correlation analyses. Regression coefficients (r2) were 0.90 in (A) and 0.89 in (B), at P < 0.05. (C) A tissue hydraulic conductance coefficient ‘L’ was calculated for each treatment, by entering values of REGR and into Equation 1. This value was used together with values of REGR, P, Y and to calculate a wall extensibility coefficient ‘M’ using the Lockhart equation.
availability, root hydraulics and shoot evaporational demand (Bouchabke´ et al. 2006, Ehlert et al. 2009, Caldeira et al. 2014). The authors explained the observed changes in P through hydraulic processes, as increased plant water loss and reduced 1367
M. Touati et al. | Turgor–growth relationship in barley leaves
root water uptake rates caused reductions (more negative) in xylem in the EZ of leaves and a subsequent decrease in water supply rates to peripherally expanding cells. The reductions in xylem paralleled reductions in growth and P. Although the nature of underlying hydraulic processes is not known (Ehlert et al. 2009), a more negative xylem will diminish . According to Equation 1, this reduces growth, and L need not change. In the present study, treatments targeted specifically the leaf EZ. Cells did not grow at reduced rates because of decreased driving less water towards cells. Rather, L of the hydraulic path between xylem and epidermal cells changed, decreasing by 50% in response to CsCl, HgCl2 and TEA.
") in the EZ
1368
The mechanism of changes in P and n in response to inhibitor treatments The test agents used here can cause non-specific effects. This has to be remembered when interpreting data. For example, Cramer (1992) studied the involvement of ethylene in the growth response of maize leaves to salt stress. Silver thiosulfate was applied, with the aim of inhibiting ethylene action. The inhibitor treatment (applied to the root system of plants) decreased growth through an increase in Y, yet ethylene was found not to be involved in this response. The inhibitors used here are likely to affect more processes in cells than the targeted function. While this does not affect the
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
The existence of a significant (>0.1 MPa) , being indicative of some hydraulic (co-)limitation of cell expansion, in growing plant tissues has been discussed intensely in the literature, with data supporting and questioning the idea of a hydraulic limitation of growth (e.g. for reviews, see Boyer et al. 1985, Cosgrove 1993, Fricke 2002, Boyer and Silk 2004). In the present study, a significant was observed in all except the FC treatment. This coincided with M:L ratios ranging from 1.4 (control) to 2.4 (HgCl2) and 2.5 (TEA) to 5.1 (CsCl treatment). Growth in these treatments was co-limited by hydraulic and mechanical properties, and treatments which reduced growth (HgCl2, CsCl and TEA) increased the hydraulic limitation compared with the mechanical one (M:L >>1.0; compare Equation 3). These treatments also showed the largest . In FC-treated leaves, the M:L ratio was 0.3 and was close to zero. This suggests that growth in these leaves was limited more through mechanical than through hydraulic properties. The present data are consistent with previous studies, in that they show that growth can be co-limited through hydraulic and mechanical properties (Radin and Boyer 1982, Boyer et al. 1985, Cramer 1992; for a review, see Fricke 2002) or that mechanical (Cosgrove 1981, Cosgrove et al. 1984, Shackel et al. 1987, Cosgrove and Sovonick-Dunford 1989, Park and Cosgrove 2012) or hydraulic properties (Nonami et al. 1997, Fricke and Flowers 1998, Martre et al. 1999, Tang and Boyer 2002, Tang and Boyer 2008) limit growth more. In contrast to studies which do not support the idea of significant in growing plant tissues, in the present study of growing cells was determined through the combined analysis of both P and p at the cell level—and a significant resulted for the highest and lowest possible estimates of xylem in the EZ. The only element of uncertainty in deriving cell from P and p is the value of the solute reflection coefficient, s, of the plasma membrane of epidermal cells. Any significant deviation of a from unity (10-fold. Work by Tang and Boyer (2002) and Passioura and Boyer (2003) suggests that the hydraulic limitation, which causes to establish, occurs upstream along the supply route of water from xylem to peripheral (leaf) epidermal cells, rather than at the plasma membrane of epidermal cells itself. In the present study, this is supported through calculations (see Supplementary Data File S1) which show that across the plasma membrane of growing epidermal cells approximated 3.65105 MPa and was almost 1,000 times smaller than the smallest (2.1102 MPa) observed for FC-treated plants. Nonami et al. (1997) and Tang and Boyer (2002, 2008) proposed for soybean hypocotyls and maize leaves, respectively, that the hydraulic limitation resulted from the cumulative hydraulic resistance of many smaller, xylem parenchyma cells which were located next to xylem vessels (for a review, see Boyer and Silk 2004). The barley leaves studied here show comparatively few xylem parenchyma cells (Fricke 2002). The hydraulic limitation and the changes in L in response to treatments may reside instead in the mestome and parenchymateous sheaths of vascular bundles (Fricke 2002, Heinen et al. 2009, Besse et al. 2011). These structures are suited to fulfill a transport-controlling role similar to that of the endodermis in roots (O’Brien and Kuo 1975, Lersten 1997, Steudle and Peterson 1998, Steudle 2000, Fricke 2002, Heinen et al. 2009, Besse et al. 2011).
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
data as such, it does affect the interpretation of data. Cesium, which targets K+ transport, and Hg2+, which targets water transport, also have more general (cyto)toxic properties (Hampton et al. 2004, Azevedo and Rodriguez 2012), and Hg2+ may affect plant hormonal status and cell expansion through inducing oxidative stress (Montero-Palmero et al. 2014); TEA, which targets K+ transport, can also inhibit water channel activity (Mu¨ller et al. 2008, Yool et al. 2009). Similarly, Cs+ may have impacted on L through co-regulation of K+ and water transport (Gazzarini et al. 2006, Liu et al. 2006) or through coupling of water flows through K+ channels (Homble´ and Ve´ry 1992). FC has a comparatively specific molecular target, the PMH+-ATPase, yet changes in the activity of PM-H+-ATPase are bound to feed back on the physiology of cell. There exist alternative inhibitors, in particular for water transport through aquaporins. Anoxia, acid load and H2O2 are suitable alternatives when aiming to reduce root hydraulics to study distant changes in the REGR/P relations of elongating leaf cells (Ehlert et al. 2009). However, these inhibitors are not suitable when being applied directly to the leaf EZ in biophysical studies as they impact directly on apoplast pH (acid load, anoxia), cause oxidative stress (H2O2) and perturb cytosolic pH (acid load, anoxia), a key parameter of cell metabolism. Therefore, we consider the present choice of inhibitors as the best possible compromise between targeting a specific cellular process which facilitates cell expansion and applying the inhibitor locally to the EZ. The observed reduction in Lpcell in response to Hg2+ is consistent with the aquaporin-inhibiting properties of Hg2+ (e.g. Tazawa et al. 1997, Tazawa et al. 2001). The three treatments (CsCl, TEA and HgCl2) which reduced P did not decrease cell p. We do not know whether s decreased and rendered cell p less osmotically efficient. A likely explanation of the data is that the decrease in cell P was facilitated through an increase in apoplast p. The transport of K+ across the plasma membrane is inhibited by TEA and, in particular, CsCl (e.g. Lenaeus et al. 2005, Volkov et al. 2009). As growing barley leaf cells take up K+ to maintain their K+ concentration during growth (Volkov et al. 2009), an inhibition of K+ uptake will lead to an increase in apoplast K+. Similarly, Hg2+ may affect uptake of K+ through some non-specific cytotoxic effects; the latter could explain why the REGR of Hg2+-treated cells was lower than predicted from the common REGR–P relationship of treatments (regression line in Fig. 4A). Cell p need not to change, as any reduced K+ and solute accumulation rates in leaves treated with CsCl, TEA or HgCl2 were accompanied by reduced expansion and dilution rates of cell contents (see Fig. 5). Turgor increased significantly in FC-treated cells. These cells display increased PM-H+-ATPase activity and wall acidification (Visnovitz et al. 2012, Visnovitz et al. 2013). Both processes facilitate the increased uptake of solutes, in particular K+. This could lead to a lowering of apoplast solute concentration and p, which facilitates the increase in P. Increased rates of solute accumulation should also lead to an increase in cell p. However, p of epidermal cells decreased. The data can be explained best through growth dilution. FC-treated leaves grew at 34% higher rates than leaves of control plants. If FC-treated cells
Fig. 5 Model to explain the changes in turgor (P) in growing leaf epidermal cells of barley in response to treatments (CsCl; TEA, tetraethylammonium; HgCl2; FC, fusicoccin; CTRL, control) through solute movement from the cell exterior (apoplast) to the interior (protoplast) and accompanying growth dilution of cell contents. Values of P (unit: MPa), the net rate of solute accumulation (‘Transport’, unit: mmol l1 s1) and relative elemental growth rates (REGR, unit: s1) as determined/derived in this study are also shown. The size of arrows symbolizes the magnitude of the processes involved; filled arrows symbolize net import of solutes into cells; dashed arrows symbolize the relative rate at which cells expand and cell contents are diluted. Treatments (CsCl, TEA and HgCl2) which decrease REGR do so primarily through decreasing P; this is the result of decreased uptake rates of solutes from the cell apoplast, which in turn leads to higher solute concentrations and osmotic pressure (p) in the apoplast. In the protoplasm, solute concentrations hardly change as reduced solute uptake rates are accompanied by reduced cell expansion rates (REGRs). In FC-treated leaves, the solute uptake rate increases. This leads to lower solute concentrations and p in the apoplast and minimizes the decrease in solute concentration and p in the protoplasm during cell volume expansion. Together, this raises P in FC-treated cells above the value in control cells; neither rheological wall properties nor the solute reflection coefficient of the plasma membrane need to change in this model. Apoplast and protoplast solutes are shown in different colors, for easier distinction, even though the chemical identity of solutes may be the same.
had net accumulated solutes at the same rate as in control plants, cell p should have decreased to (1/1.34) 75% of the control value. The observed cell p was 86% of the control value. This implies that FC stimulated the net rate of solute accumulation by [(0.861.34) – (1)] 15% (see also Fig. 5). Based on the above, it is proposed that FC stimulated the rate of solute accumulation in cells and caused an accompanying decrease in apoplast p. This allowed P and P – Y to increase beyond the level in control cells. Such an interpretation of data does not require an alteration in wall rheological properties, in contrast to previous studies which showed that FC affects M (Lado et al. 1973, Van Volkenburgh and Cleland 1986, Keller and Van Volkenburgh 1998, Park and Cosgrove 2012; for reviews, see Cosgrove 1999, Schopfer 2006). Katou and Furumoto (1986) reached a similar conclusion for concurrent increases in P and growth in response to auxin, although they did not actually measure p at the cell level; neither did studies 1369
M. Touati et al. | Turgor–growth relationship in barley leaves
which do not support the present interpretation of FC data (for a review, see Cosgrove 1993). The present study highlights the need to measure P and p at the cell level when deriving the of a cell (and also ) from values of P and p and when concluding on changes in cellular p. A bulk leaf analysis of p would have masked the epidermal cellspecific decrease in p in response to FC (see Fig. 2B). The epidermis-specific response in p may govern the leaf response to FC, as the epidermis is thought to limit mechanically the growth of plant organs (Brummell and Hall 1980, Van Volkenburgh and Cleland 1986, Schopfer 2006).
Plant material Barley (Hordeum vulgare L. cv. Jersey) was grown hydroponically in a growth chamber (Snyders Microclimate MC1000) under a 16 h day/8 h night cycle, at 20 C day and 18 C night temperature, 70–80% relative humidity and a photosynthetically active radiation of 250 mmol photons m2 s1 (compare Knipfer and Fricke 2010). Nutrient solutions were continuously aerated. Plants were analyzed when they were 14–16 d old, 3–9 h into the photoperiod. At this developmental stage, leaves 1 and 2 were fully expanded and the main growing leaf was leaf 3. The base 40 mm of leaf 3 contained the leaf EZ and was enclosed by the sheaths of older leaves 1 and 2 (Fricke and Flowers 1998, Fricke and Peters 2002). The transpiring portion of leaf 3 which had emerged from the sheath of leaf 2 and which was exposed to ambient environmental conditions (light, wind, air humidity) was the ‘emerged blade’ (EmBL).
Outline of approach to determine the components of the Lockhart equation The approach was as follows:
(i) Expose the growth zone of leaf 3 of 2- to 3-week-old barley plants to treatments which affect cellular water transport (100 mM HgCl2), solute transport (5 mM CsCl; 50 mM TEA; 5 mM FC) and wall properties (5 mM FC). Determine LER and deduce REGRs for treatments. (ii) Measure P in epidermal cells of the EZ and emerged-blade portion (EmBL) of the growing leaf using the cell pressure probe, and determine cell p using picoliter osmometry of extracted cell sap. Use these data to calculate cell-EZ and cell-EmBL. (iii) Using the above values of cell and a value of root-medium, determine the range of possible values of xylem in the EZ ( xylemEZ). The difference between cell-EZ and xylem-EZ [( cell-EZ) – ( xylem-EZ)] is the difference in ( ) between the xylem in the EZ and expanding, epidermal cell (see Fig. 6). (iv) Construct plots of (a) P vs. REGR and (b) vs. REGR. This will show whether all treatments fit one general relationship as predicted by Equations (1) and (2); if so, a value of apparent yield threshold can be derived from the REGR vs. P relationship (Y equals P for extrapolated zero REGR). (v) Derive L from Equation 1 using values of REGR and . Solve the Lockhart equation for M, and calculate M using measured (P) and derived (Y) values. (vi) Using the information provided through (iv) and (v), conclude on the relative limitation of cell expansion through mechanical and hydraulic properties and whether changes in REGR in response to treatments can be explained through changes in P.
1370
Fig. 6 Estimation of the water potential ( ) of xylem in the elongation zone (EZ) ( Xylem-EZ) of leaf 3 of barley. The picture on the left shows a 16-day-old barley plant as analyzed in the present study; the pictures on the right show cross-sections of the root and of leaf regions. The EZ of the leaf is located at the base, with the root being located upstream and the emerged-blade portion (EmBL) of the growing leaf being located downstream of the transpiration stream which supplies water to the EZ. Most of the water moving axially through roots and the EmBL will move along metaxylem vessels, while most of the water moving axially through the EZ will move through protoxylem at proximal regions and through metaxylem at more distal regions (towards the leaf tip). Metaxylem vessels (root: early metaxylem, peripheral location; late metaxylem, central location) are indicated by blue dots; protoxylem vessels in leaf regions are indicated by red dots. The of xylem in the EZ ( Xylem-EZ) could not be determined directly and an indirect approach was taken to derive its range of values. Water flow through the root medium–plant–air continuum is driven by a difference in . The least negative is in the root medium ( Medium); the most negative within the plant is close to the exit point of water from the transpiring portion of blade (EmBL for leaf 3) and should approach the of leaf epidermal cells in this leaf region ( Epi-EmBL). The value of Xylem-EZ will be within the range set by these two ‘extremes’. Scale bars are 20 mm for EZ and EmBL and 100 mm for root.
Preparation of the leaf EZ for the application of test reagents The cuticle in the EZ of leaf 3 of barley is not fully developed and has a permeance which is much higher than that in transpiring leaf tissue (Richardson et al. 2005, Richardson et al. 2007). This makes it possible to apply test reagents externally to the EZ (Visnovitz et al. 2013). A plant to be analyzed was removed from the growth chamber and the shoot was placed under a stereomicroscope, with the root system remaining in nutrient solution. The sheath of leaf 1 was partially peeled back and a window ( 2 mm wide, 5 mm long) was cut into the sheath of leaf 2 at 15–25 mm from the base of the shoot to expose a small portion of the EZ of leaf 3 located beneath. At this location, REGRs are highest in the expanding leaf 3 (e.g. Fricke et al. 1997, Fricke and Peters 2002). Only leaves with no visible injury to the EZ were used for experiments. The portion of the EZ from about 10–30 mm from the base was wrapped in tissue paper which had been moistened with distilled water. The plant was then transferred into a 250 ml Erlenmeyer flask containing nutrient solution, with the shoot base being supported by a foam piece. The plant was moved back into the growth chamber for between 1 and 1.5 h to acclimatize to the new situation of having a window cut. The root medium was aerated throughout.
Downloaded from http://pcp.oxfordjournals.org/ at University of Prince Edward Island on July 12, 2015
Materials and Methods
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
Application of test reagents and determination of LER in the growth chamber
LER on the cell pressure probe stage, and derivation of REGR Throughout experiments, LER was determined for plants in the growth chamber, yet biophysical cell parameters were analyzed on the cell pressure probe stage in the laboratory. To test whether LER in the growth chamber was comparable with that on the cell pressure probe stage, one additional set of experiments was carried out in which LER for three plants per treatment was first determined in the growth chamber and then on the cell pressure probe stage. The latter was achieved by pointing the microcapillary tip of the cell pressure probe at a reference point on the emerged-blade portion of leaf 3 and measuring with a graticule the displacement of the reference point over a 25–30 min period—a time typically required for cell pressure probe analyses. The LER determined in this way differed by
