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Reduced Root Elongation ofLupinus angustifoliusL. by High pH is not Due to Decreased Membrane Integrity of Cortical Cells or Low Proton Production by the Roots
Annals of Botany 78 : 409–414, 1996
Reduced Root Elongation of Lupinus angustifolius L. by High pH is not Due to Decreased Membrane Integrity of Cortical Cells or Low Proton Production by the Roots C. T A N G*†, N. E. L O N G N E C K ER*, H. G R E E N W AY‡ and A. D. R O B S ON* * Centre for Legumes in Mediterranean Agriculture}Soil Science and Plant Nutrition, and ‡ Plant Sciences, The Uniersity of Western Australia, Nedlands, WA 6907, Australia Received : 24 June 1995
Accepted : 4 January 1996
Root elongation and cell expansion were decreased markedly by pH & 6±0 compared to pH 5–5±5 in Lupinus angustifolius but only slightly in Lupinus pilosus and Pisum satium. We tested whether poor root growth of L. angustifolius at high pH correlates with decreased proton extrusion or increased membrane permeability by comparing effects of pH on intact and excised roots of L. angustifolius, L. pilosus and P. satium in solution culture. Root elongation rates of L. angustifolius exposed to pH 6±5–8±0 were much decreased, yet a pH of 7±5 neither decreased the membrane potential nor increased the permeability of Na+ relative to K+ in cortical cells of either L. angustifolius or P. satium. There was no correlation between low net proton efflux and decreased rate of root elongation ; in all three species, net proton efflux by both intact and excised roots in solution was lower at pH 5±0–5±3 than at pH 6±5–6±7. Exposing shoots to light increased acidification of the external solution by the roots, but did not restore a rapid root elongation of L. angustifolius at high pH. Increasing buffer concentration in the external solution decreased the rate of root elongation more in L. angustifolius than in L. pilosus and P. satium. It is suggested that the arrested root elongation in L. angustifolius by high pH does not result from an inability to extrude protons to the external solution or an impaired membrane permeability in the cortex, but may be related to a failure to acidify the apoplast. # 1996 Annals of Botany Company Key words : Membrane permeability (α), proton extrusion, buffer concentration, Lupinus angustifolius L., root elongation, Lupinus pilosus Murr., Pisum satium L.
INTRODUCTION Root growth of Lupinus angustifolius L. is particularly sensitive to high pH (& 6) compared to Pisum satium L. and Lupinus pilosus L. (Tang et al., 1992, 1993). In solution culture, root elongation of P. satium was not affected by pH in the range from 4±5 to 8±0, whereas root elongation of L. angustifolius was halved by increasing pH from 5±5 to 6±0. High pH decreased root growth of L. angustifolius by decreasing cell elongation and not cell division, and reducing cell volume in the epidermis and the outer cortex (Tang et al., 1992, 1993). The physiological reasons for the reduction of root growth by high external pH in L. angustifolius but not in P. satium and L. pilosus are not understood. Cell wall acidification causing wall loosening is thought to be a prerequisite for cell growth (Taiz, 1984). One possibility for the inhibited cell elongation in roots of L. angustifolius is that high external pH decreases the efficiency of wall acidification. Alternatively, high pH may impair plasma membrane integrity, leading to poor cell wall formation. In a rose species, pH 8 increased the leakage of electrolytes from the roots (Zieslin and Snir, 1989). Increased plasma membrane permeability can lead to poor cell wall formation
as indicated by reduced synthesis of cellulose in cotton roots exposed to high Na+}Ca#+ ratios ; these high ratios are known to increase membrane permeability (Zhong and La$ uchli, 1988). The membrane permeability can be measured by comparing the change in membrane potential elicited by adding Na+ with that by K+ in root tissues in which respiration is inhibited by CN− (Loos and Lu$ ttge, 1984). Proton extrusion can be estimated from pH changes in bulk solution. The aim of the present study was to examine whether high pH affects membrane permeability and}or impairs proton extrusion of roots of L. angustifolius but not of P. satium or L. pilosus. In the study we added neutral buffers in solution to modify the degree of cell wall acidification of the roots. Neutral buffers have been shown to neutralize cell wall pH in roots of Brassica napus L. and inhibit Fe$+EDTA reduction in the plasma membrane (Toulon et al., 1992), the process known to be pH dependent. Neutral buffers also inhibit auxin-induced growth of scrubbed soybean hypocotyls (Rayle and Cleland, 1980). We also placed shoots in the light and showed that proton extrusion from roots of L. angustifolius was increased. The possible effect of this proton extrusion on root elongation at high pH was evaluated.
† For correspondence.
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# 1996 Annals of Botany Company
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Tang et al.—Root Elongation at High pH MATERIALS AND METHODS
Seeds of L. angustifolius cv. Yandee, L. pilosus P23030 or P. satium cv. Dundale were germinated for 2–4 d on a stainless-steel screen suspended over aerated unbuffered solution containing 1 m CaSO and 5 µ H BO at % $ $ pH 5±0–5±5 in the dark at 20–22 °C. Experiments on membrane permeability Measurements were made using roots of L. angustifolius and P. satium. Basal experimental solution (solution A) contained 2 µ H BO and a mixture of 1 m MES [2-(N$ $ morpholino)ethane-sulphonic acid] and 1 m TES [Ntris(hydroxymethyl)-methyl-2-aminoethane-sulphonic acid] at pH 7±5 or 5±2. The pH 7±5 was achieved by adding Ca(OH) , while pH 5±2 was obtained by adding Ca(OH) to # # bring pH up to 7±5 and then adding H SO , so that the # % calcium concentrations were maintained constant in all treatments. To measure root elongation, 20 3-d-old seedlings (ten of each species) supported in a lid were transferred into 5 l plastic pots containing solution A at pH 5±2 or 7±5. The elongation rate was determined by measuring length of taproots (no laterals at this stage) before and after 24 h of treatment. Membrane potentials in the cortex were measured with glass microelectrodes (0±5–1 µm tip) as described by UllrichEberius, Novacky and Ball (1983). Briefly, an excised root tip was mounted in a plexiglass chamber of 3 ml volume, which was perfused by solution A at a rate of 6 ml min−" at room temperature. The solution flowed through the chamber by gravity and could be readily changed. In preliminary experiments, the depolarization of root cortical cells of L. angustifolius and P. satium was very similar between treatments of 0±5 m CN−, 1 m CN− and 1 m CN−0±5 m SHAM (salicylhydroxamic acid). The electrical potential of roots treated for 30–60 min was rapidly restored to the original membrane potential following withdrawal of these respiratory inhibitors. Addition of 0±2 m NaN gave the lowest electrical potential, which $
repolarized very slowly after withdrawal of the inhibitor. Therefore, 0±5 m CN− was used in subsequent experiments for the reduction of energy production by the tissue. The cyanide was added as the Na+ or K+ salt. Na SO or K SO # % # % was added to the basal solution to obtain final concentrations of Na+ or K+ at 2 m. Root tissues exposed to CN− are referred to as ‘ non-energized ’ and those in media without the inhibitor are designated as ‘ energized ’. The relative Na+ : K+ permeability α (α ¯ PNa+}PK+) was calculated using an equation derived from the Goldman equation : α ¯ e∆(∆E)F/RT (where F is Faraday, R is the universal gas constant and T is the absolute temperature), ∆(∆E) is the difference between the membrane depolarization in mV resulting from addition of K+ and that of Na+. The equation is based on the assumption that Na+ and K+ are the major permeating ions and the internal concentrations are constant (Loos and Lu$ ttge, 1984). Experiments on proton efflux To measure how exposure of shoots to light affected proton efflux in relation to root elongation at high pH, seeds were germinated for 3 d and then 25 seedlings were grown for 4 d in 5 l solution B at pH 5±0 or 6±5 with or without natural light in a phytotron at 15}20 °C in April. Solution B contained nutrients of the following composition (µ) : NH NO , 100 ; Ca(NO ) , 400 ; KH PO , 20 ; K SO , 600 ; % $ $# # % # % MgSO , 200 ; CaCl , 600 ; FeEDDHA, 10 ; H BO , 5 ; % # $ $ Na MoO , 0±03 ; ZnSO , 0±75 ; MnSO , 1±0 and CuSO 0±2 # % % % % (Tang et al.,1992). In the dark treatment, the entire plants were covered with thick black plastic. Solutions were buffered with 1 m MES. Root elongation was recorded daily until day 4. For measurements of proton efflux, the roots of three intact seedlings were transferred from 5 l solution B buffered with 1 m MES into a single vial containing 120 ml solution B buffered with 0±1 m MES and with pH 5±0 or 6±5. All other experiments were conducted in a dark room at 20–22 °C. For the measurement of root elongation rate, ten seedlings of each species were grown in solution B with pH 5±0, 6±5 or 8±0. The solution was buffered with a mixture
T 1. Details of experimental design for studies of proton efflux and root elongation Experiment no. and purpose
pH treatments Buffer system Excision
Light}dark
Glucose (5 m) Plant species
1. Root elongation 5±0, 6±5, 8±0
1 m MES Intact 1 m TES
Dark
®
2. Proton efflux
5±3, 6±7
0±1 m MES
Dark
³
3. Proton efflux
5±3, 6±7
0±1 m MES
Dark
Light, dark
®
Light, dark
®
4. Root elongation 5±0, 6±5
1 m MES
Intact and excised entire roots Excised roots (3-cm tips) Intact
5. Proton efflux
0±1 m MES
Intact
5±0, 6±5
Age of seedling (d)
L. L. P. L. P.
angustifolius 3 pilosus satium angustifolius 3 satium 4
L. L. L. P. L. P.
angustifolius 3–4 pilosus angustifolius 3 satium angustifolius 3, 4, 5, 7 satium
Volume of Experimental solution duration (ml) (h) 5000
24, 48
120
6
20
4
5000 120
24, 48, 72, 96 6
Tang et al.—Root Elongation at High pH
411
T 2. Membrane potential of cortical cells in roots of L. angustifolius and P. sativum and depolarization following addition of 1 mM K+ at different external pH. Values are means³s.e. and number of experiments is shown in parentheses Solution pH during measurement 5±2 Species
Pretreatment
L. angustifolius
None
P. satium
None
L. angustifolius
Intact plants in buffered solution at pH 5±2 for 24–30 h Intact plants in buffered solution at pH 7±5 for 24–30 h
7±5
0 m K+ (Em)
1 m K+ (∆Em)
0 m K+ (Em)
1 m K+ (∆Em)
®191³7 (5) ®206³7 (5) ®199³8 (6)
49³7 (5) 67³3 (5) 44³2 (6)
®218³3 (4) ®218³5 (4)
58³7 (4) 67³7 (4)
®216³5 (4)
48³2 (4)
Measurements were taken in the regions with the smallest root hairs for pH pretreated roots of L. angustifolius (cells were the same age in both pH treatments) and in the regions of 5–8 mm from root apex for unpretreated roots of L. angustifolius and P. satium. Excised root tips were placed in treatment solution for about 2 h before measurement.
of 1 m MES and 1 m TES. For the determination of proton efflux, intact or excised roots were placed in 120 ml or 20 ml solution B with an initial pH of either 5±3 or 6±7, with or without 5 m glucose. The solution was buffered with 0±1 m MES ; pH changes in the solution were recorded and the rate of proton efflux was calculated from a titration curve. The details of the above experiments are listed in Table 1.
T 3. Relatie Na+ : K+ permeability α (PNa+}PK+) for membranes of root cortical cells of L. angustifolius and P. sativum in non®energized state at pH 5±2 and 7±5. Values are means³s.e. and number of experiments is shown in parentheses
Effect of buffer concentration This experiment was carried out in the dark at 20–22 °C. Fifteen 4-d-old seedlings were grown in solutions of pH 5±0 or 7±0 and containing basal nutrients identical to solution B except that CaCl was added at 1200 µ to prevent adverse # effects of high K+. A mixture of MES and TES was added to the solution at four different concentrations. Target pH values were obtained by titrating the solution with KOH. Potassium concentration (final concentration about 16 m) was maintained constant in all treatments by the addition of K SO . # % RESULTS Experiments on membrane permeability Membrane potential in the energized state. Exposure of either L. angustifolius or P. satium to pH 7±5 did not decrease membrane potentials. Neither did the pretreatment of L. angustifolius roots at pH 7±5 for over 24 h decrease membrane potential (Table 2). Membrane depolarization following addition of 1 m K+ was not significantly affected by pH in either non-pretreated or pH-pretreated lupin roots (Table 2). Membrane potential in the non-energized state. Potential changes elicited by transitions from Na+ to K+ solutions in the presence of 0±5 m CN− allowed the assessment of the
Species
pH
L. angustifolius
5±2 7±5 5±2
P. satium
7±5
α in nonenergized state 0±66³0±01 (8) 0±65³0±02 (8) 0±54³0±03 (9) 0±54³0±02 (9)
Excised roots were placed in solution either at pH 5±2 or pH 7±5 for 2®3 h. Potential changes [∆(∆E)] were measured in the root elongating zone (5®8 mm from tips) by transitions from 2 m Na+ to 2 m K+. α was calculated as α ¯ e∆(∆E)F/RT (see text).
T 4. The rate of root elongation (mm h®", an aerage of 24 h) of L. angustifolius, L. pilosus and P. sativum grown under different pH. Values are means³s.e. of three replicates (Expt 1 as shown in Table 1)
Day 1 Day 2
Solution pH
L. angustifolius
L. pilosus
P. satium
5±0 6±5 8±0 5±0 6±5 8±0
1±37³0±03 0±67³0±04 0±52³0±01 1±59³0±02 0±96³0±03 0±81³0±02
1±41³0±06 1±33³0±06 1±16³0±06 1±68³0±05 1±67³0±07 1±56³0±04
1±55³0±02 1±61³0±06 1±47³0±10 1±64³0±06 1±62³0±06 1±62³0±10
412
Tang et al.—Root Elongation at High pH
T 5. Net proton efflux (µmol g®"f.wt\h®") in intact or excised roots of L. angustifolius, P. sativum and L. pilosus under initial pH of 5±3 and 6±7. At the end of treatments, solution pH had dropped by 0±2 to 0±6 units. Values are means³s.e. of two replicates Experiment no. (as shown in Table 1)
L. angustifolius Initial pH
2
5±3 6±7
3
5±3 6±7
P. satium
L. pilosus
Excision
®glucose
glucose
®glucose
glucose
Intact Excised Intact Excised Excised Excised
0±64³0±01 0±32³0±02 1±16³0±01 0±60³0±03
0±79³0±01 0±67³0±01 1±71³0±06 1±32³0±06 0±97³0±09 2±21³0±02
0±88³0±06 0±28³0±04 1±02³0±09 0±42³0±06
1±26³0±05 0±96³0±17 1±65³0±01 1±08³0±09
glucose
1±47³0±14 2±03³0±08
Proton efflux (µmoles g–1 f. wt h–1)
3 A
B
C
D
2
1
0
Root elongation rate (mm h–1)
1.6
1.2
0.8
0.4
0.0
0
1
2
3
4 0 Days after treatment
1
2
3
4
F. 1. Net proton efflux (A and B) and root elongation rate in roots (C and D) of L. angustifolius (A and C) and P. satium (B and D) grown at pH 5±0 and 6±5 with and without light exposure on shoots in a phytotron. (D, pH 5±0, light ; E, pH 5±0, dark ; *, pH 6±5, light ; +, pH 6±5, dark). Effects of pH are significant for (A), (B) and (C) (P ! 0±05) ; effects of light significant for (A) (P ! 0±05) ; interaction between pH and light not significant for any of (A) to (D) (P " 0±05) (Expts 4 and 5 as in Table 1). Bars indicate l.s.d. at P ¯ 0±05.
relative Na+ : K+-permeability α (PNa+}PK+). pH treatments did not affect the relative Na+ : K+ permeability in either roots of L. angustifolius or P. satium (Table 3). Experiments on proton efflux Root elongation rates of L. angustifolius exposed to pH 6±5 and 8±0 were markedly decreased compared with those exposed to pH 5±0. This reduction was greater during
the first 24 h period than during the second 24 h period. However, increasing pH from 5±0 to 6±5 or 8±0 only slightly decreased elongation rate of roots of L. pilosus during 0–24 h and not during 24–48 h and did not decrease the elongation rate of P. satium roots in either period (Table 4). Rate of net proton efflux from intact and excised L. angustifolius roots was higher at pH 6±7 than at pH 5±3
Tang et al.—Root Elongation at High pH
413
Root elongation rate (% of the rate at 0.1 mM buffer)
120
100
80
60
40
20
0
A 0.1
B 1
4
16 0.1 1 4 Buffer concentration in solution (mM MES + mM TES)
16
F. 2. Effect of buffer concentration on root elongation rate of L. angustifolius (D), L. pilosus (*) and P. satium (^) grown at pH 7±0 during the first 24 h (A) and the second 24 h of treatment (B). The data are expressed as % of the rate at 0±1 m buffer. Root elongation rates of L. angustifolius, L. pilosus and P. satium grown at pH 7±0 with 0±1 m buffers were 0±91³0±01, 1±36³0±09 and 1±55³0±04, respectively, during the first and 1±26³0±05, 1±72³0±01 and 1±72³0±00 mm h−", respectively, during the second 24 h treatment, and were taken as 100 %. Vertical bars are the standard error of means of two replicates.
(Table 5). Excision decreased the rate by about 50 % in both pH treatments. Adding glucose to the solution increased proton efflux to a greater extent from excised roots than from roots of intact plants (Table 5). Net proton efflux from intact and excised roots of P. satium and excised roots of L. pilosus followed a similar pattern to L. angustifolius. However, the ratio of efflux at pH 6±7 to efflux at pH 5±3 was higher in L. angustifolius roots (approx. 2±0) than in P. satium (approx. 1±2) or in L. pilosus (approx. 1±4) ; excision decreased proton extrusion more in P. satium than in L. angustifolius (Table 5). Exposure of the shoots to light increased net proton efflux from intact roots of L. angustifolius at both pH 5±0 and 6±5. The rate was generally higher at pH 6±5 than at pH 5±0 except in the dark at day 4 (Fig. 1 A). In roots of P. satium, light did not affect net proton efflux. The rate of proton efflux was higher at pH 6±5 than at pH 5±0 at days 0 and 1 but not afterwards (Fig. 1 B). Net proton efflux in roots of both P. satium and L. angustifolius decreased over time (Fig. 1 A and B). However, light treatments did not change the response of root elongation of L. angustifolius to pH (Fig. 1 C). Regardless of light treatments, increasing solution pH from 5±0 to 6±5 markedly decreased root elongation rate in L. angustifolius during 4 d exposure (Fig. 1 C). Root elongation rate in light was slightly greater than in dark after 3 d treatment, presumably due to carbohydrate becoming limited in dark treatment (Fig. 1 C). Neither light nor pH treatments significantly affected root elongation in P. satium (Fig. 1 D). Effects of buffer concentration In the first 24 h at 0±1 m buffer, root elongation rate decreased by 46 % in L. angustifolius and 10 % in L. pilosus but increased by 10 % in P. satium when pH was increased
from 5±0 to 7±0. In the second 24 h, root elongation rate decreased by 30 % in L. angustifolius in response to increased pH, but was not affected in L. pilosus and P. satium (data not shown). Increasing buffer concentration in solution decreased root elongation rate in all species grown at pH 7±0 with a much greater decrease in L. angustifolius than in L. pilosus and P. satium (Fig. 2, data expressed as % of rate at 0±1 m buffer), but did not affect the elongation rate of any of the three species at pH 5±0 (data not shown). DISCUSSION The reduction of cell elongation in L. angustifolius by high pH is not due to impaired membrane permeability in the cortex. In sunflower roots, boron deficiency, which impairs membrane function, decreased the extent of depolarization of the cells resulting from an increase of external [K+] (Schon, Novacky and Blevins, 1990). In Lemna gibba, mercury, an inhibitor of SH-group dependent metabolic reactions, increased the ratio of passive permeability α ¯ PNa+}PK+ of cells in the non-energized state (Loos and Lu$ ttge, 1984). In this study, high pH (7±5) did not decrease membrane potential, the extent of membrane depolarization by addition of K+, nor alter the relative membrane permeability of cortical cells to Na+ and K+ in L. angustifolius or P. satium, indicating that high pH did not reduce membrane integrity. This response appears to differ from results of Zieslin and Snir (1989) who showed a greater leakage of electrolytes in rose roots exposed to pH 8 than to pH 6. Tang et al. (1993) found that high pH led to disintegration of the root surface, inhibited the formation of root hairs and decreased cell volume in the epidermis and the outer cortex but not in the inner cortex of L. angustifolius. The absence of an effect of high pH on cell volume in the inner cortex
414
Tang et al.—Root Elongation at High pH
implies that these cells had functioned normally and expanded fully, and this is supported by our finding that high pH did not decrease membrane potential in the cortex. In this study, effects of increasing pH from 5±0–5±3 to 6±5–7±5 in decreasing root elongation rate in L. angustifolius were not associated with decreased net proton efflux. Proton efflux from both intact and excised roots at high pH was generally similar between L. angustifolius, L. pilosus and P. satium. Higher net proton efflux at high pH than at low pH is probably due to more absorption of cations at high pH. In L. angustifolius, exposure of light on the shoots increased proton efflux from the roots but did not restore a rapid root elongation at high pH. This contrasts with findings in the literature using other species and organs. In Phaseolus ulgaris, light induced an expansion of leaf cells by stimulating proton excretion (van Volkenburgh and Cleland, 1980). Light-stimulated acidification of P. ulgaris leaves occurred prior to the growth, and infiltration of the tissues with a neutral buffer inhibited this light-induced growth. The ‘ acid growth ’ hypothesis has been based mainly on work with shoot tissues (e.g. excised coleoptiles and epicotyls) (Kutschera, 1994). Wall acidification has been implicated in the root growth of some species (e.g. maize, Evans, Mulkey and Vesper, 1980 ; Mulkey and Evans, 1981) but not others (e.g. barley, O’Neill and Scott, 1983). In intact plants, contradictory results on the relation of growth and acidification indicate that growth rate is also regulated by other mechanisms (Taiz, 1984). Nevertheless, our study showed that increasing concentration of neutral buffers in the pH 7±0 solution decreased the rate of root elongation more in L. angustifolius than in L. pilosus or P. satium. Thus roots of L. angustifolius relative to those of L. pilosus or P. satium might have had faster diffusion of H+ out of the apoplast or of buffer into the apoplast, less buffering capacity of cell wall, or lower threshold of apoplast pH for cell wall growth.
LITERATURE CITED Evans ML, Mulkey TJ, Vesper MJ. 1980. Auxin action on proton influx in corn Zea mays roots and its correlation with growth. Planta 148 : 510–512. Kutschera U. 1994. The current status of the acid-growth hypothesis. New Phytologist 126 : 549–569. Loos S, Lu$ ttge U. 1984. Effects of HgCl on membranes of Lemna # gibba in the energized and non-energized state. Physiologie VeU geU tale 22 : 171–179. Mulkey TJ, Evans ML. 1981. Geotropism in corn Zea mays root : Evidence for its mediation by differential acid efflux. Science 212 : 70–71. O’Neill RL, Scott TK. 1983. Proton flux and elongation in primary roots of barley (Hordeum ulgare L.). Plant Physiology 73 : 199–201. Schon MK, Novacky A, Blevins DG. 1990. Boron induces hyperpolarization of sunflower root cell membranes and increases membrane permeability to K+. Plant Physiology 93 : 566–571. Taiz L. 1984. Plant cell expansion : regulation of cell wall mechanical properties. Annual Reiew of Plant Physiology 35 : 585–657. Tang C, Kuo J, Longnecker NE, Thomson CJ, Robson AD. 1993. High pH causes disintegration of root surface in Lupinus angustifolius L. Annals of Botany 71 : 201–207. Tang C, Longnecker NE, Thomson CJ, Greenway H, Robson AD. 1992. Lupin (Lupinus angustifolius L.) and pea (Pisum satium L.) roots differ in their sensitivity to pH above 6±0. Journal of Plant Physiology 140 : 715–719. Toulon V, Sentenac H, Thibaud JB, Davidian JC, Moulineau C, Grignon C. 1992. Role of apoplast acidification by the H+ pump : Effect on the sensitivity to pH and CO of iron reduction by roots of # Brassica napus L. Planta 186 : 212–218. Ullrich-Eberius C, Novacky A, Ball E. 1983. Effect of cyanide in dark and light on the membrane potential and ATP level of young and mature green tissues of higher plants. Plant Physiology 72 : 7–15. van Volkenburgh E, Cleland RE. 1980. Proton excretion and cell expansion in bean leaves. Planta 148 : 273–278. Zhong H, La$ uchli A. 1988. Incorporation of ["%C] glucose into cell wall polysaccharides of cotton roots : effects of NaCl and CaCl . Plant # Physiology 88 : 511–514. Zieslin N, Snir P. 1989. Responses of rose plants cultivar ‘ Sonia ’ and Rosa indica major to changes in pH and aeration of the root environment in hydroponic culture. Scientia Horticulturae 37 : 339–349.
Reduced Root Elongation of Lupinus angustifolius L. by High pH is not Due to Decreased Membrane Integrity of Cortical Cells or Low Proton Production by the Roots C. T A N G*†, N. E. L O N G N E C K ER*, H. G R E E N W AY‡ and A. D. R O B S ON* * Centre for Legumes in Mediterranean Agriculture}Soil Science and Plant Nutrition, and ‡ Plant Sciences, The Uniersity of Western Australia, Nedlands, WA 6907, Australia Received : 24 June 1995
Accepted : 4 January 1996
Root elongation and cell expansion were decreased markedly by pH & 6±0 compared to pH 5–5±5 in Lupinus angustifolius but only slightly in Lupinus pilosus and Pisum satium. We tested whether poor root growth of L. angustifolius at high pH correlates with decreased proton extrusion or increased membrane permeability by comparing effects of pH on intact and excised roots of L. angustifolius, L. pilosus and P. satium in solution culture. Root elongation rates of L. angustifolius exposed to pH 6±5–8±0 were much decreased, yet a pH of 7±5 neither decreased the membrane potential nor increased the permeability of Na+ relative to K+ in cortical cells of either L. angustifolius or P. satium. There was no correlation between low net proton efflux and decreased rate of root elongation ; in all three species, net proton efflux by both intact and excised roots in solution was lower at pH 5±0–5±3 than at pH 6±5–6±7. Exposing shoots to light increased acidification of the external solution by the roots, but did not restore a rapid root elongation of L. angustifolius at high pH. Increasing buffer concentration in the external solution decreased the rate of root elongation more in L. angustifolius than in L. pilosus and P. satium. It is suggested that the arrested root elongation in L. angustifolius by high pH does not result from an inability to extrude protons to the external solution or an impaired membrane permeability in the cortex, but may be related to a failure to acidify the apoplast. # 1996 Annals of Botany Company Key words : Membrane permeability (α), proton extrusion, buffer concentration, Lupinus angustifolius L., root elongation, Lupinus pilosus Murr., Pisum satium L.
INTRODUCTION Root growth of Lupinus angustifolius L. is particularly sensitive to high pH (& 6) compared to Pisum satium L. and Lupinus pilosus L. (Tang et al., 1992, 1993). In solution culture, root elongation of P. satium was not affected by pH in the range from 4±5 to 8±0, whereas root elongation of L. angustifolius was halved by increasing pH from 5±5 to 6±0. High pH decreased root growth of L. angustifolius by decreasing cell elongation and not cell division, and reducing cell volume in the epidermis and the outer cortex (Tang et al., 1992, 1993). The physiological reasons for the reduction of root growth by high external pH in L. angustifolius but not in P. satium and L. pilosus are not understood. Cell wall acidification causing wall loosening is thought to be a prerequisite for cell growth (Taiz, 1984). One possibility for the inhibited cell elongation in roots of L. angustifolius is that high external pH decreases the efficiency of wall acidification. Alternatively, high pH may impair plasma membrane integrity, leading to poor cell wall formation. In a rose species, pH 8 increased the leakage of electrolytes from the roots (Zieslin and Snir, 1989). Increased plasma membrane permeability can lead to poor cell wall formation
as indicated by reduced synthesis of cellulose in cotton roots exposed to high Na+}Ca#+ ratios ; these high ratios are known to increase membrane permeability (Zhong and La$ uchli, 1988). The membrane permeability can be measured by comparing the change in membrane potential elicited by adding Na+ with that by K+ in root tissues in which respiration is inhibited by CN− (Loos and Lu$ ttge, 1984). Proton extrusion can be estimated from pH changes in bulk solution. The aim of the present study was to examine whether high pH affects membrane permeability and}or impairs proton extrusion of roots of L. angustifolius but not of P. satium or L. pilosus. In the study we added neutral buffers in solution to modify the degree of cell wall acidification of the roots. Neutral buffers have been shown to neutralize cell wall pH in roots of Brassica napus L. and inhibit Fe$+EDTA reduction in the plasma membrane (Toulon et al., 1992), the process known to be pH dependent. Neutral buffers also inhibit auxin-induced growth of scrubbed soybean hypocotyls (Rayle and Cleland, 1980). We also placed shoots in the light and showed that proton extrusion from roots of L. angustifolius was increased. The possible effect of this proton extrusion on root elongation at high pH was evaluated.
† For correspondence.
0305-7364}96}10040906 $18.00}0
# 1996 Annals of Botany Company
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Tang et al.—Root Elongation at High pH MATERIALS AND METHODS
Seeds of L. angustifolius cv. Yandee, L. pilosus P23030 or P. satium cv. Dundale were germinated for 2–4 d on a stainless-steel screen suspended over aerated unbuffered solution containing 1 m CaSO and 5 µ H BO at % $ $ pH 5±0–5±5 in the dark at 20–22 °C. Experiments on membrane permeability Measurements were made using roots of L. angustifolius and P. satium. Basal experimental solution (solution A) contained 2 µ H BO and a mixture of 1 m MES [2-(N$ $ morpholino)ethane-sulphonic acid] and 1 m TES [Ntris(hydroxymethyl)-methyl-2-aminoethane-sulphonic acid] at pH 7±5 or 5±2. The pH 7±5 was achieved by adding Ca(OH) , while pH 5±2 was obtained by adding Ca(OH) to # # bring pH up to 7±5 and then adding H SO , so that the # % calcium concentrations were maintained constant in all treatments. To measure root elongation, 20 3-d-old seedlings (ten of each species) supported in a lid were transferred into 5 l plastic pots containing solution A at pH 5±2 or 7±5. The elongation rate was determined by measuring length of taproots (no laterals at this stage) before and after 24 h of treatment. Membrane potentials in the cortex were measured with glass microelectrodes (0±5–1 µm tip) as described by UllrichEberius, Novacky and Ball (1983). Briefly, an excised root tip was mounted in a plexiglass chamber of 3 ml volume, which was perfused by solution A at a rate of 6 ml min−" at room temperature. The solution flowed through the chamber by gravity and could be readily changed. In preliminary experiments, the depolarization of root cortical cells of L. angustifolius and P. satium was very similar between treatments of 0±5 m CN−, 1 m CN− and 1 m CN−0±5 m SHAM (salicylhydroxamic acid). The electrical potential of roots treated for 30–60 min was rapidly restored to the original membrane potential following withdrawal of these respiratory inhibitors. Addition of 0±2 m NaN gave the lowest electrical potential, which $
repolarized very slowly after withdrawal of the inhibitor. Therefore, 0±5 m CN− was used in subsequent experiments for the reduction of energy production by the tissue. The cyanide was added as the Na+ or K+ salt. Na SO or K SO # % # % was added to the basal solution to obtain final concentrations of Na+ or K+ at 2 m. Root tissues exposed to CN− are referred to as ‘ non-energized ’ and those in media without the inhibitor are designated as ‘ energized ’. The relative Na+ : K+ permeability α (α ¯ PNa+}PK+) was calculated using an equation derived from the Goldman equation : α ¯ e∆(∆E)F/RT (where F is Faraday, R is the universal gas constant and T is the absolute temperature), ∆(∆E) is the difference between the membrane depolarization in mV resulting from addition of K+ and that of Na+. The equation is based on the assumption that Na+ and K+ are the major permeating ions and the internal concentrations are constant (Loos and Lu$ ttge, 1984). Experiments on proton efflux To measure how exposure of shoots to light affected proton efflux in relation to root elongation at high pH, seeds were germinated for 3 d and then 25 seedlings were grown for 4 d in 5 l solution B at pH 5±0 or 6±5 with or without natural light in a phytotron at 15}20 °C in April. Solution B contained nutrients of the following composition (µ) : NH NO , 100 ; Ca(NO ) , 400 ; KH PO , 20 ; K SO , 600 ; % $ $# # % # % MgSO , 200 ; CaCl , 600 ; FeEDDHA, 10 ; H BO , 5 ; % # $ $ Na MoO , 0±03 ; ZnSO , 0±75 ; MnSO , 1±0 and CuSO 0±2 # % % % % (Tang et al.,1992). In the dark treatment, the entire plants were covered with thick black plastic. Solutions were buffered with 1 m MES. Root elongation was recorded daily until day 4. For measurements of proton efflux, the roots of three intact seedlings were transferred from 5 l solution B buffered with 1 m MES into a single vial containing 120 ml solution B buffered with 0±1 m MES and with pH 5±0 or 6±5. All other experiments were conducted in a dark room at 20–22 °C. For the measurement of root elongation rate, ten seedlings of each species were grown in solution B with pH 5±0, 6±5 or 8±0. The solution was buffered with a mixture
T 1. Details of experimental design for studies of proton efflux and root elongation Experiment no. and purpose
pH treatments Buffer system Excision
Light}dark
Glucose (5 m) Plant species
1. Root elongation 5±0, 6±5, 8±0
1 m MES Intact 1 m TES
Dark
®
2. Proton efflux
5±3, 6±7
0±1 m MES
Dark
³
3. Proton efflux
5±3, 6±7
0±1 m MES
Dark
Light, dark
®
Light, dark
®
4. Root elongation 5±0, 6±5
1 m MES
Intact and excised entire roots Excised roots (3-cm tips) Intact
5. Proton efflux
0±1 m MES
Intact
5±0, 6±5
Age of seedling (d)
L. L. P. L. P.
angustifolius 3 pilosus satium angustifolius 3 satium 4
L. L. L. P. L. P.
angustifolius 3–4 pilosus angustifolius 3 satium angustifolius 3, 4, 5, 7 satium
Volume of Experimental solution duration (ml) (h) 5000
24, 48
120
6
20
4
5000 120
24, 48, 72, 96 6
Tang et al.—Root Elongation at High pH
411
T 2. Membrane potential of cortical cells in roots of L. angustifolius and P. sativum and depolarization following addition of 1 mM K+ at different external pH. Values are means³s.e. and number of experiments is shown in parentheses Solution pH during measurement 5±2 Species
Pretreatment
L. angustifolius
None
P. satium
None
L. angustifolius
Intact plants in buffered solution at pH 5±2 for 24–30 h Intact plants in buffered solution at pH 7±5 for 24–30 h
7±5
0 m K+ (Em)
1 m K+ (∆Em)
0 m K+ (Em)
1 m K+ (∆Em)
®191³7 (5) ®206³7 (5) ®199³8 (6)
49³7 (5) 67³3 (5) 44³2 (6)
®218³3 (4) ®218³5 (4)
58³7 (4) 67³7 (4)
®216³5 (4)
48³2 (4)
Measurements were taken in the regions with the smallest root hairs for pH pretreated roots of L. angustifolius (cells were the same age in both pH treatments) and in the regions of 5–8 mm from root apex for unpretreated roots of L. angustifolius and P. satium. Excised root tips were placed in treatment solution for about 2 h before measurement.
of 1 m MES and 1 m TES. For the determination of proton efflux, intact or excised roots were placed in 120 ml or 20 ml solution B with an initial pH of either 5±3 or 6±7, with or without 5 m glucose. The solution was buffered with 0±1 m MES ; pH changes in the solution were recorded and the rate of proton efflux was calculated from a titration curve. The details of the above experiments are listed in Table 1.
T 3. Relatie Na+ : K+ permeability α (PNa+}PK+) for membranes of root cortical cells of L. angustifolius and P. sativum in non®energized state at pH 5±2 and 7±5. Values are means³s.e. and number of experiments is shown in parentheses
Effect of buffer concentration This experiment was carried out in the dark at 20–22 °C. Fifteen 4-d-old seedlings were grown in solutions of pH 5±0 or 7±0 and containing basal nutrients identical to solution B except that CaCl was added at 1200 µ to prevent adverse # effects of high K+. A mixture of MES and TES was added to the solution at four different concentrations. Target pH values were obtained by titrating the solution with KOH. Potassium concentration (final concentration about 16 m) was maintained constant in all treatments by the addition of K SO . # % RESULTS Experiments on membrane permeability Membrane potential in the energized state. Exposure of either L. angustifolius or P. satium to pH 7±5 did not decrease membrane potentials. Neither did the pretreatment of L. angustifolius roots at pH 7±5 for over 24 h decrease membrane potential (Table 2). Membrane depolarization following addition of 1 m K+ was not significantly affected by pH in either non-pretreated or pH-pretreated lupin roots (Table 2). Membrane potential in the non-energized state. Potential changes elicited by transitions from Na+ to K+ solutions in the presence of 0±5 m CN− allowed the assessment of the
Species
pH
L. angustifolius
5±2 7±5 5±2
P. satium
7±5
α in nonenergized state 0±66³0±01 (8) 0±65³0±02 (8) 0±54³0±03 (9) 0±54³0±02 (9)
Excised roots were placed in solution either at pH 5±2 or pH 7±5 for 2®3 h. Potential changes [∆(∆E)] were measured in the root elongating zone (5®8 mm from tips) by transitions from 2 m Na+ to 2 m K+. α was calculated as α ¯ e∆(∆E)F/RT (see text).
T 4. The rate of root elongation (mm h®", an aerage of 24 h) of L. angustifolius, L. pilosus and P. sativum grown under different pH. Values are means³s.e. of three replicates (Expt 1 as shown in Table 1)
Day 1 Day 2
Solution pH
L. angustifolius
L. pilosus
P. satium
5±0 6±5 8±0 5±0 6±5 8±0
1±37³0±03 0±67³0±04 0±52³0±01 1±59³0±02 0±96³0±03 0±81³0±02
1±41³0±06 1±33³0±06 1±16³0±06 1±68³0±05 1±67³0±07 1±56³0±04
1±55³0±02 1±61³0±06 1±47³0±10 1±64³0±06 1±62³0±06 1±62³0±10
412
Tang et al.—Root Elongation at High pH
T 5. Net proton efflux (µmol g®"f.wt\h®") in intact or excised roots of L. angustifolius, P. sativum and L. pilosus under initial pH of 5±3 and 6±7. At the end of treatments, solution pH had dropped by 0±2 to 0±6 units. Values are means³s.e. of two replicates Experiment no. (as shown in Table 1)
L. angustifolius Initial pH
2
5±3 6±7
3
5±3 6±7
P. satium
L. pilosus
Excision
®glucose
glucose
®glucose
glucose
Intact Excised Intact Excised Excised Excised
0±64³0±01 0±32³0±02 1±16³0±01 0±60³0±03
0±79³0±01 0±67³0±01 1±71³0±06 1±32³0±06 0±97³0±09 2±21³0±02
0±88³0±06 0±28³0±04 1±02³0±09 0±42³0±06
1±26³0±05 0±96³0±17 1±65³0±01 1±08³0±09
glucose
1±47³0±14 2±03³0±08
Proton efflux (µmoles g–1 f. wt h–1)
3 A
B
C
D
2
1
0
Root elongation rate (mm h–1)
1.6
1.2
0.8
0.4
0.0
0
1
2
3
4 0 Days after treatment
1
2
3
4
F. 1. Net proton efflux (A and B) and root elongation rate in roots (C and D) of L. angustifolius (A and C) and P. satium (B and D) grown at pH 5±0 and 6±5 with and without light exposure on shoots in a phytotron. (D, pH 5±0, light ; E, pH 5±0, dark ; *, pH 6±5, light ; +, pH 6±5, dark). Effects of pH are significant for (A), (B) and (C) (P ! 0±05) ; effects of light significant for (A) (P ! 0±05) ; interaction between pH and light not significant for any of (A) to (D) (P " 0±05) (Expts 4 and 5 as in Table 1). Bars indicate l.s.d. at P ¯ 0±05.
relative Na+ : K+-permeability α (PNa+}PK+). pH treatments did not affect the relative Na+ : K+ permeability in either roots of L. angustifolius or P. satium (Table 3). Experiments on proton efflux Root elongation rates of L. angustifolius exposed to pH 6±5 and 8±0 were markedly decreased compared with those exposed to pH 5±0. This reduction was greater during
the first 24 h period than during the second 24 h period. However, increasing pH from 5±0 to 6±5 or 8±0 only slightly decreased elongation rate of roots of L. pilosus during 0–24 h and not during 24–48 h and did not decrease the elongation rate of P. satium roots in either period (Table 4). Rate of net proton efflux from intact and excised L. angustifolius roots was higher at pH 6±7 than at pH 5±3
Tang et al.—Root Elongation at High pH
413
Root elongation rate (% of the rate at 0.1 mM buffer)
120
100
80
60
40
20
0
A 0.1
B 1
4
16 0.1 1 4 Buffer concentration in solution (mM MES + mM TES)
16
F. 2. Effect of buffer concentration on root elongation rate of L. angustifolius (D), L. pilosus (*) and P. satium (^) grown at pH 7±0 during the first 24 h (A) and the second 24 h of treatment (B). The data are expressed as % of the rate at 0±1 m buffer. Root elongation rates of L. angustifolius, L. pilosus and P. satium grown at pH 7±0 with 0±1 m buffers were 0±91³0±01, 1±36³0±09 and 1±55³0±04, respectively, during the first and 1±26³0±05, 1±72³0±01 and 1±72³0±00 mm h−", respectively, during the second 24 h treatment, and were taken as 100 %. Vertical bars are the standard error of means of two replicates.
(Table 5). Excision decreased the rate by about 50 % in both pH treatments. Adding glucose to the solution increased proton efflux to a greater extent from excised roots than from roots of intact plants (Table 5). Net proton efflux from intact and excised roots of P. satium and excised roots of L. pilosus followed a similar pattern to L. angustifolius. However, the ratio of efflux at pH 6±7 to efflux at pH 5±3 was higher in L. angustifolius roots (approx. 2±0) than in P. satium (approx. 1±2) or in L. pilosus (approx. 1±4) ; excision decreased proton extrusion more in P. satium than in L. angustifolius (Table 5). Exposure of the shoots to light increased net proton efflux from intact roots of L. angustifolius at both pH 5±0 and 6±5. The rate was generally higher at pH 6±5 than at pH 5±0 except in the dark at day 4 (Fig. 1 A). In roots of P. satium, light did not affect net proton efflux. The rate of proton efflux was higher at pH 6±5 than at pH 5±0 at days 0 and 1 but not afterwards (Fig. 1 B). Net proton efflux in roots of both P. satium and L. angustifolius decreased over time (Fig. 1 A and B). However, light treatments did not change the response of root elongation of L. angustifolius to pH (Fig. 1 C). Regardless of light treatments, increasing solution pH from 5±0 to 6±5 markedly decreased root elongation rate in L. angustifolius during 4 d exposure (Fig. 1 C). Root elongation rate in light was slightly greater than in dark after 3 d treatment, presumably due to carbohydrate becoming limited in dark treatment (Fig. 1 C). Neither light nor pH treatments significantly affected root elongation in P. satium (Fig. 1 D). Effects of buffer concentration In the first 24 h at 0±1 m buffer, root elongation rate decreased by 46 % in L. angustifolius and 10 % in L. pilosus but increased by 10 % in P. satium when pH was increased
from 5±0 to 7±0. In the second 24 h, root elongation rate decreased by 30 % in L. angustifolius in response to increased pH, but was not affected in L. pilosus and P. satium (data not shown). Increasing buffer concentration in solution decreased root elongation rate in all species grown at pH 7±0 with a much greater decrease in L. angustifolius than in L. pilosus and P. satium (Fig. 2, data expressed as % of rate at 0±1 m buffer), but did not affect the elongation rate of any of the three species at pH 5±0 (data not shown). DISCUSSION The reduction of cell elongation in L. angustifolius by high pH is not due to impaired membrane permeability in the cortex. In sunflower roots, boron deficiency, which impairs membrane function, decreased the extent of depolarization of the cells resulting from an increase of external [K+] (Schon, Novacky and Blevins, 1990). In Lemna gibba, mercury, an inhibitor of SH-group dependent metabolic reactions, increased the ratio of passive permeability α ¯ PNa+}PK+ of cells in the non-energized state (Loos and Lu$ ttge, 1984). In this study, high pH (7±5) did not decrease membrane potential, the extent of membrane depolarization by addition of K+, nor alter the relative membrane permeability of cortical cells to Na+ and K+ in L. angustifolius or P. satium, indicating that high pH did not reduce membrane integrity. This response appears to differ from results of Zieslin and Snir (1989) who showed a greater leakage of electrolytes in rose roots exposed to pH 8 than to pH 6. Tang et al. (1993) found that high pH led to disintegration of the root surface, inhibited the formation of root hairs and decreased cell volume in the epidermis and the outer cortex but not in the inner cortex of L. angustifolius. The absence of an effect of high pH on cell volume in the inner cortex
414
Tang et al.—Root Elongation at High pH
implies that these cells had functioned normally and expanded fully, and this is supported by our finding that high pH did not decrease membrane potential in the cortex. In this study, effects of increasing pH from 5±0–5±3 to 6±5–7±5 in decreasing root elongation rate in L. angustifolius were not associated with decreased net proton efflux. Proton efflux from both intact and excised roots at high pH was generally similar between L. angustifolius, L. pilosus and P. satium. Higher net proton efflux at high pH than at low pH is probably due to more absorption of cations at high pH. In L. angustifolius, exposure of light on the shoots increased proton efflux from the roots but did not restore a rapid root elongation at high pH. This contrasts with findings in the literature using other species and organs. In Phaseolus ulgaris, light induced an expansion of leaf cells by stimulating proton excretion (van Volkenburgh and Cleland, 1980). Light-stimulated acidification of P. ulgaris leaves occurred prior to the growth, and infiltration of the tissues with a neutral buffer inhibited this light-induced growth. The ‘ acid growth ’ hypothesis has been based mainly on work with shoot tissues (e.g. excised coleoptiles and epicotyls) (Kutschera, 1994). Wall acidification has been implicated in the root growth of some species (e.g. maize, Evans, Mulkey and Vesper, 1980 ; Mulkey and Evans, 1981) but not others (e.g. barley, O’Neill and Scott, 1983). In intact plants, contradictory results on the relation of growth and acidification indicate that growth rate is also regulated by other mechanisms (Taiz, 1984). Nevertheless, our study showed that increasing concentration of neutral buffers in the pH 7±0 solution decreased the rate of root elongation more in L. angustifolius than in L. pilosus or P. satium. Thus roots of L. angustifolius relative to those of L. pilosus or P. satium might have had faster diffusion of H+ out of the apoplast or of buffer into the apoplast, less buffering capacity of cell wall, or lower threshold of apoplast pH for cell wall growth.
LITERATURE CITED Evans ML, Mulkey TJ, Vesper MJ. 1980. Auxin action on proton influx in corn Zea mays roots and its correlation with growth. Planta 148 : 510–512. Kutschera U. 1994. The current status of the acid-growth hypothesis. New Phytologist 126 : 549–569. Loos S, Lu$ ttge U. 1984. Effects of HgCl on membranes of Lemna # gibba in the energized and non-energized state. Physiologie VeU geU tale 22 : 171–179. Mulkey TJ, Evans ML. 1981. Geotropism in corn Zea mays root : Evidence for its mediation by differential acid efflux. Science 212 : 70–71. O’Neill RL, Scott TK. 1983. Proton flux and elongation in primary roots of barley (Hordeum ulgare L.). Plant Physiology 73 : 199–201. Schon MK, Novacky A, Blevins DG. 1990. Boron induces hyperpolarization of sunflower root cell membranes and increases membrane permeability to K+. Plant Physiology 93 : 566–571. Taiz L. 1984. Plant cell expansion : regulation of cell wall mechanical properties. Annual Reiew of Plant Physiology 35 : 585–657. Tang C, Kuo J, Longnecker NE, Thomson CJ, Robson AD. 1993. High pH causes disintegration of root surface in Lupinus angustifolius L. Annals of Botany 71 : 201–207. Tang C, Longnecker NE, Thomson CJ, Greenway H, Robson AD. 1992. Lupin (Lupinus angustifolius L.) and pea (Pisum satium L.) roots differ in their sensitivity to pH above 6±0. Journal of Plant Physiology 140 : 715–719. Toulon V, Sentenac H, Thibaud JB, Davidian JC, Moulineau C, Grignon C. 1992. Role of apoplast acidification by the H+ pump : Effect on the sensitivity to pH and CO of iron reduction by roots of # Brassica napus L. Planta 186 : 212–218. Ullrich-Eberius C, Novacky A, Ball E. 1983. Effect of cyanide in dark and light on the membrane potential and ATP level of young and mature green tissues of higher plants. Plant Physiology 72 : 7–15. van Volkenburgh E, Cleland RE. 1980. Proton excretion and cell expansion in bean leaves. Planta 148 : 273–278. Zhong H, La$ uchli A. 1988. Incorporation of ["%C] glucose into cell wall polysaccharides of cotton roots : effects of NaCl and CaCl . Plant # Physiology 88 : 511–514. Zieslin N, Snir P. 1989. Responses of rose plants cultivar ‘ Sonia ’ and Rosa indica major to changes in pH and aeration of the root environment in hydroponic culture. Scientia Horticulturae 37 : 339–349.