Limitation of Cell Elongation in Barley (Hordeum vulgare L.)
Leaves Through Mechanical and Tissue-Hydraulic Properties
Mostefa Touati1, Thorsten Knipfer2,4, Tamás Visnovitz2,5, Abdelkrim Kameli3 and Wieland Fricke2,*
1
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 Végétale, Département des Sciences Naturelles, Ecole Normale Supérieure 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, Gyömro00 i út 19-21, Hungary.
2
*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.
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Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
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); Marré 1979, Tode and
Lüthen 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
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M. Touati et al. | Turgor–growth relationship in barley leaves
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).
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
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.
(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
(Bouchabké 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
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 (<1.0) increases the
calculated cell and diminishes .
Cosgrove (1987) argued that the significant measured in
the EZ of tissues is an artifact as it results from high concentrations of solutes in the apoplast of elongating cells.
This lowers cell , because the plasma membrane of cells has
a s close to unity, yet does not cause a significant towards
the xylem, as the s of the apoplast path between the xylem and
cell is close to zero, which renders high apoplast solute
1368
concentrations osmotically inefficient. In contrast, Nonami
and Boyer (1987) and Tang and Boyer (2002) concluded that
most of the low of growing cells in soybean hypocotyls and
maize leaves, respectively, is associated with a tension in the
apoplast, and that the measured reflects the effective
forces driving water movement. Based on the present data,
we cannot distinguish between these explanations for the
observed . However, the explanation of Cosgrove (1987)
implies that most of the radial water movement in the EZ
occurs along the apoplast path. This need not be the case.
Water may move instead along a predominantly protoplasmic
path, from cell to cell, as proposed for roots (e.g. Knipfer and
Fricke 2010) and supported through recent studies which highlight the importance of aquaporins in controlling the hydraulics
of leaves (Heinen et al. 2009, Shatil-Cohen et al. 2011, Sade et al.
2014).
The in FC-treated leaves was close to zero. Leaf growth
in FC-treated plants was not limited through hydraulic properties, and L increased >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).
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
Plant Cell Physiol. 56(7): 1364–1373 (2015) doi:10.1093/pcp/pcv055
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 (Müller 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 (Homblé and Véry
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).
Materials and Methods
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.
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
Following the 1–1.5 h incubation, the plant was removed from the growth
chamber. The moist tissue paper wrapping the base of plants was replaced
with tissue paper soaked in treatment solution. Treatment solutions consisted
of (i) distilled water (control); (ii) 100 mM HgCl2; (iii) 5 mM FC; (iv) 5 mM CsCl;
and (v) 50 mM TEA. The plant was then transferred back into the 250 ml
Erlenmeyer flask and a tiny reference mark was made with a permanent
marker close to the leaf base. The distance between that mark and the tip of
leaf 3 was measured with a ruler for subsequent determination of LER. The plant
was then allowed to grow for 2–3 h in the growth chamber. Preliminary experiments using an LVTD (linear variable differential transducer; not shown) confirmed that LER attained a stable value during this period. After the 2–3 h
growth period, the distance between the reference point and the tip of leaf 3
was measured again to determine the incremental increase in distance during
the incubation period for calculation of LER. The plant was then subjected to
biophysical analyses in an ambient laboratory environment (18–20 C; 50–70%
relative humidity; supplemental lighting of 200 mmol photons m2 s1).
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 <12% from the LER in the growth chamber,
irrespective of treatment (not shown). This justified the use of LER data obtained in the growth chamber in calculations involving the Lockhart equation.
The REGR of cells had to be known for the application of the Lockhart
equation. It was beyond the scope of the present study to establish detailed
profiles of REGR along the EZ of leaves subjected to the various treatments, for
example through pin-pricking, which affects LER and requires considerably
longer incubation periods than those used here for treatments (e.g. Fricke
et al. 1997, Fricke and Peters 2002). Instead, REGR was derived as follows: the
distribution of REGR along the EZ of barley leaves is bell-shaped, like that of
many species (Fricke et al. 1997, Fricke and Peters 2002, Hu et al. 2005).
Maximum and near maximum REGRs are attained between 15 and 25 mm
from the point of leaf insertion, the region studied here. This region accounts
for about 40% of the total elongation of leaf 3 of barley (for profiles, see, for
example, Fricke et al. 1997, Fricke and Peters 2002). Therefore, for a given leaf
growth rate of, for example, 1 mm h1, 40% of this value (0.4 mm h1) can be
accounted for by the 10 mm long region at 15–25 mm from the point of leaf
insertion. This calculates to an average REGR of [(0.4 mm h1)/(10 mm)]
0.04 h1, or 4% h1 or 1.11 105 s1. Accordingly, the REGR for a particular
treatment was calculated as:
REGRTreatment ¼ ð0:4 LERTreatment Þ=ð10 mmÞ
ð5Þ
Biophysical cell analyses in the leaf EZ
Turgor was measured with the cell pressure probe, and p was determined in sap
extracted from individual cells using picoliter osmometry (Fricke and Peters
2002, Volkov et al. 2007). Cell was calculated as the difference between P
and p.
Between four and seven adaxial epidermal cells overlying ridges (Fricke
1997) were analyzed for cell P in each leaf. Cell hydraulic conductivity (Lpcell)
was calculated according to Equation 6
Lpcell ¼ ðVcell =Acell Þ ð"+OÞ1 f½ðLnð2Þ=T1=2 g
ð6Þ
With
" ¼ ðPÞ ðVcell =Vcell Þ
ð7Þ
Cell elastic modulus (e) and half-time of water exchange (T1/2) were determined with the cell pressure probe (Volkov et al. 2007, Knipfer et al. 2011); P
and Vcell are the instantaneous changes in pressure and in microcapillary cell
volume, respectively, which are imposed with the cell pressure probe to induce
pressure relaxations with half-time T1/2. As T1/2 was in the second to subsecond
range, e was corrected for fast water flow (Steudle 1993, Steudle 2000, Volkov
et al. 2007).
The volume (Vcell, m3) and surface (Acell, m2) of cells was determined by
treating cells as cylinders (Equations 8 and 9), with radius r and length h:
Vcell ¼ ðp r2 Þh
ð8Þ
Acell ¼ ½ð2p rÞ h+2 ðp r2 Þ
ð9Þ
And
The radius and length of a cell either were determined directly for a particular
cell, using a graticule fitted to the stereomicroscope of the cell pressure probe setup; or an average value obtained through analyses of several cells was used as it
sometimes proved difficult to obtain precise measures of dimension for a pressure-probed cell due to the highly reflective leaf surface of the EZ.
Cell p was obtained through analyses of a separate set of plants, yet the same type
of adaxial epidermal cells was analyzed as during P analyses. Between three and five
pooled samples, each consisting of 3–4 successively sampled cells, were harvested and
analyzed from each leaf. Cell osmolality (mosmol kg1) was converted into p (MPa)
using the relationship that 40.75 mosmol kg1 corresponds to 0.1 MPa.
To compare cell with bulk leaf p directly, the same leaf sections used for
analyses of cell p were subsequently harvested for determination of bulk leaf p
(Suku et al. 2014) using a VAPRO (Wescor Inc.) osmometer.
Biophysical cell analyses in the EmBL portion of
leaf 3
Cell P and p were determined for adaxial epidermal cells overlying ridges in the
emerged-blade portion of leaf 3 using the cell pressure probe and picoliter
osmometry, respectively. These analyses were carried out on a set of plants
separate from that analyzed in the EZ.
Calculation of the net rate of solute accumulation
in growing cells
A growing cell which expands at a certain REGR (unit: s1), while maintaining a
certain p (unit: MPa, where 1 MPa corresponds to about 407.5 mmol kg1, or
407.5 mmol l1 solutes), needs to accumulate solutes at a net rate, ‘Transport’
(unit: mmol l1 s1), which equals the product of REGR and p:
Transport ¼ REGR p
ð10Þ
Average values of REGR (see Fig. 4A) and p (see cell values in Fig. 2B) were
used to calculate the net rate of solute accumulation of treatments
Statistical analyses
Data were subjected to correlation and regression analyses, and to ANOVA
(general linear model followed by Tukey analysis) using functions in Minitab.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the IRCSET (Irish Research Council
for Science, Engineering and Technology) [studentships to T.K.
1371
M. Touati et al. | Turgor–growth relationship in barley leaves
and T.V.]; the Algerian Ministry of Higher Education and
Scientific Research [a PhD fellowship and visiting researcher
grant to M.T.].
Acknowledgments
The authors would like to thank Ádám Solti (Eötvös Loránd
University, Budapest, Hungary) for providing Jersey barley
seeds.
Disclosures
The authors have no conflicts of interest to declare.
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