effect low root medium ph net proton release, root ... · effect of low root medium ph figure 3....

Plant Physiol. (1992) 99, 415-4210032-0889/92/99/041 5/07/$01 .00/0

Received for publication September 11, 1991Accepted December 10, 1991

Effect of Low Root Medium pH on Net Proton Release, RootRespiration, and Root Growth of Corn (Zea mays L.) and

Broad Bean (Vicia faba L.)1

Feng Yan, Sven Schubert*, and Konrad MengelInstitute of Plant Nutrition, Justus Liebig University, Suedanlage 6, D-6300 Giessen, Germany

ABSTRACT

The effect of low pH on net H+ release and root growth of corn(Zea mays L.) and broad bean (Vicia faba L.) seedlings wasinvestigated in short-term experiments at constant pH. Broadbean was more sensitive to low pH than corn: the critical values(pH values below which net H+ release and root growth ceased)were pH 4.00 (broad bean) and pH 3.50 (com) at 1 millimolar Ca2 .Both proton release and root growth were progressively inhibitedas the medium pH declined. Additional Ca2+ in the root mediumhelped to overcome the limitations of low pH for net H releaseand root growth. Potassium (for com) and abscisic acid (for broadbean) increased both net H release and root growth rate at thecritical pH value. It is concluded that poor root growth at low pHis caused by a lack of net H+ release that may decrease cyto-plasmic pH values. Inhibited net H+ release at high external H+activity is not due to a shortage of energy supply to the H+ATPase. Instead, a displacement of Ca2+ by H+ at the extemalside of the plasmalemma may enhance reentry of H+ into rootcells.

Low pH in the soil may affect plant growth indirectly, e.g.by elevated aluminum or manganese solubility and by limitedavailability of molybdenum, phosphorus, calcium, or mag-nesium (1, 9, 22). On the other hand, low pH (high H+activity) may directly inhibit plant growth (2, 15, 25, 26, 28,30), probably by adverse effects at the root plasmalemmalevel. An increase of plasmalemma permeability at high H+activity in the medium has been shown to be alleviated bythe addition of calcium (15, 17, 19, 20). Likewise, plantgrowth at low pH was improved at higher calcium concentra-tions (9, 20). Calcium is believed to have a specific functionat the external side of the plasmalemma for membrane integ-rity (5, 13).

It has recently been suggested that at low pH net H+ releaseby H+ ATPase activity is restricted, thus limiting dry matterproduction during vegetative plant growth (26, 28). Withrespect to net H+ release and growth at high H+ activity inthe root medium, broad bean seems to be particularly sensi-tive to low pH (22, 26). Although there are various reports inthe literature describing species differences in acidity tolerance(2, 12, 21), nothing is known about the role of net H' releasein acidity tolerance.

'Supported by the Deutsche Forschungsgemeinschaft.

The aim of our present investigation was to test for speciesdifferences with respect to net H+ release at low pH and totest the effect of these differences in net H+ release on rootgrowth rate. From the results by Islam et al. ( 12), it appearedthat corn was more tolerant of low pH than broad bean.Therefore, we chose corn and broad bean to evaluate theeffect of high H+ activity in the root medium on net H+release and root growth under various experimental condi-tions. Furthermore, we investigated whether at low pH in theroot medium proton pumping by H+ ATPase is limited byATP energy supply.

MATERIALS AND METHODS

Plant Cultivation

Corn (Zea mays L. cv Blizzard) and broad bean (ViciafabaL. cv Alfred) seeds were soaked in 0.5 mM CaSO4 for 1 d andthen germinated at 25C in the dark on filter paper moistenedwith 0.5 mm CaSO4. After 3 to 4 d, seedlings were transferredto a stainless steel mesh suspended over an 0.5 mM CaSO4solution in the dark. This procedure was chosen to obtain(corn) plants with one main root only. After an additional 3to 4 d, plants were used for experiments.

Proton Release and Root Growth Studies

Proton release and root growth were studied in a growthchamber for 1 d (broad bean) or 2 d (corn) under the followingconditions: plants were subjected to a day/night cycle of 17h/7 h with temperatures of 25 and 1 8°C, respectively, startingabout 5 h after the beginning of the day period. Fluorescentlamps (66% Osram L58 W/3 1, 34% Osram L58 W/78) gavea light intensity of approximately 80 W m-2, and relative airhumidity was 60%. Corn (36 plants per container) and broadbean (20 plants per container) were transferred to 14 L of thefollowing solution: 1 mm CaSO4, 0.25 mm Na2SO4, andK2SO4 as indicated in "Results." Sodium was added to avoideffects of NaOH titration on Na+ concentration during pHadjustment.The pH of this solution was adjusted by addition ofNaOH

or H2S04 and kept constant by continuous titration with0.5 N NaOH or H2SO4 with a Schott pH stat system. Theaddition of NaOH or H2SO4 to maintain a given pH (accu-racy, 0.02 pH units) allowed us to calculate net H+ release oruptake. Root growth rate was determined by measuring themain root length before and after the experiment with a ruler.

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Plant Physiol. Vol. 99, 1992

Oxygen Uptake

Oxygen uptake was determined by means of a Clark-typeelectrode. Roots of intact plants were preincubated in 1 mMCaSO4 at the pH to be tested for 2 d. Root tips (0.5-1 cm, 35mg) were then incubated in 5 mL of identical solution in theClark electrode for measurement of continuous 02 uptake.After a short time of adjustment (5-15 min), 02 uptake waslinear when plotted against time for at least 30 min; for thisperiod of time, 02 uptake rates were calculated.

Organic Acid and ATP Analysis

Root samples were dried at 80°C, milled, and extracted fororganic acids in 60°C water for 1 h. Malate, pyruvate, andlactate were determined using UV test kits from Boehringer(Mannheim, Germany).For the analysis of ATP, root fresh matter was rapidly

frozen in liquid N2 and analyzed enzymically as described bySchubert and Mengel (24).

Statistical Treatment

Variation is indicated by SE. Significant differences betweentreatments were calculated by using the t test.

RESULTS

Root growth of both corn and broad bean was markedlyreduced by high H' activity (low pH) in the medium (Fig. 1).However, growth of corn roots was more tolerant of high H'activity than growth ofbroad bean. Whereas at 32 /M H' (pH4.50) similar growth rates were observed for the two species,

pH

E

E£

0

__

-C

0

cn

0

0w

4.50 4.00 3.75,4

1K4

3.50r

1.0

0.6 F

0.2

100 200 300H+ Activity (,uM)

Figure 1. Effect of H+ activity in the medium on root growth rates ofcorn (0) and broad bean (0) seedlings. Root medium was 0.25 mmNa2SO4 + 25 AM K2SO4 + 1 mm CaSO4.

0.1 0

0.08

7 0.06ca

0- 0.04

-c 0.02E

E

.I,0.060

a 0.04

0

0m 0.024-

0

0

2x

- 60 -20 0 20 60

Net H' release (nmoles h-1 ptant-1 )

Figure 2. Relationship between net H+ release and root growth rateof corn (a) and broad bean (b) seedlings at various medium pH values.Experimental conditions were as described for Figure 1.

between 56 and 316 ,M H+ activity (pH 4.25 and 3.50) growthof broad bean roots was significantly more strongly inhibited(P < 0.1%) than that of corn. Above 100 jsM H+ (<pH 4.00)growth of broad bean roots may represent transient physicalcell extension because root tips died after 2 d incubationbelow pH 4.00. Corn, on the other hand, showed viable roottips even at pH 3.50, with measurable but strongly reducedgrowth rates.

Proton release does not always occur uniformly along theroot axis but may be restricted to certain regions, e.g. the roottip during nitrate nutrition (18). Furthermore, controversialresults exist concerning net H+ release in the root elongationzone, where net H+ release (8, 23) or net H+ uptake (29) hasbeen observed. For these reasons, and for a comparison ofroot growth rates of two species with very different rootmorphology (broad bean, corn), root weight is not an appro-priate basis for the measurement of H+ extrusion. Instead, toestablish relationships between net H+ release and root growthrate, we chose net H+ extrusion per plant as a parameter.Because of differences in root morphology and root physiol-ogy, absolute amounts of H+ release by the two species maynot be comparable. However, it is possible to assess differentialspecies behavior in H+ release as the pH varies.Between pH 6.50 and 3.25 root growth rates of corn were

linearly correlated with net H+ release (y = 0.001 x + 0.038,r = 0.92; P = 0.1%). Below pH 3.50, net H+ uptake wasobserved (Fig. 2). In contrast to corn, root growth rates ofbroad bean were linearly correlated only between pH 5.00and 3.75 (y = 0.001 x + 0.015, r = 0.98; P = 5%). Growthwas slightly reduced above pH 5.00, and net H+ uptakeoccurred below pH 4.00. Corn and broad bean differed inacidity tolerance in two respects: (a) critical pH value below

(a ) / H6.50('a) I pH4.50 p 5-50 -

p 4.00

0 pH 3.75

* pH 3.50

pH 3.25

;(b) pH 5.00pH 5.50

pH 4. pH.50 pH 6.50

pH 3.50 pH 6.50pH3.50 pH3.7 pH 4.00

416 YAN ET AL.

i

14



EFFECT OF LOW ROOT MEDIUM pH

Figure 3. Effect of low pH on root morphologyof corn (a) and broad bean (b) after 48 h treat-ment in 0.25 mm Na2SO4 + 5 mm K2SO4 + 1 mMCaSO4.

which root growth ceased (broad bean, pH 4.00; corn, pH3.50), and (b) critical pH value below which net H+ releaseceased (broad bean, pH 4.00; corn, pH 3.50). Differences inacidity tolerance of the two species may be related to thesecritical values. However, the decrease in root growth rate witha lowering of net proton release was identical for broad beanand corn (identical slopes of the regression lines). This indi-cates a similar response of root growth of the two species toan increase in H+ activity but on different levels (Fig. 2).

Figure 3 demonstrates the impairment of roots observedbelow or at the critical pH values. After 2 d treatment withpH 3.25 (corn) or pH 4.00 (broad bean), roots of both speciesappeared healthy except for the extension zones which were

constricted and looked gray. Root tips (meristems, root caps),however, appeared to be vital at this stage (they were turgid).Corn root tips changed color to typical red or violet pigmen-tation. Additionally, in contrast to broad bean, corn rootsinitiated laterals (Fig. 3).As observed earlier in long-term growth experiments (26),

the results described above suggested a close relationshipbetween root growth and the ability of plants to extrudeprotons. At low pH, a lack of net H+ release seemed to limitroot growth. To test this hypothesis, we triggered additionalH+ extrusion by the plant roots near the critical pH values byincreased Ca2' and K+ concentration as well as by ABAtreatment.

Higher Ca2" concentration (5 mm relative to 1 mM) in theroot medium significantly promoted net H+ release of bothspecies (P < 0.1%), whereas the reverse was true for lowerCa2+ concentration (0.1 mM; Table I). At the same time,higher Ca2+ concentration in the root medium increased rootgrowth rate (P < 0.1%). Again, the reverse was found forlower Ca2" concentration. The Ca2' requirement of corn at a

given pH (e.g. pH 4.1) was lower than that of broad bean.Thus, at pH 4.1, root growth rate of corn was not furtherincreased by 5 mm relative to 1 mm CaSO4 (Table I).

Testing the effect of K+ on net H+ release and root growth

rate, significant differences were observed for corn but not forbroad bean (P < 5%) (Table II). K treatment (probably bypartial depolarization of the electrical membrane potential)changed net H+ uptake to net H+ release and increased rootgrowth rate of corn. Treatment with 20 ,uM ABA increasednet H+ release by roots of corn in the fourth leaf stage (S.Schubert, unpublished data). The same treatment reduced netH+ release and growth rates of corn seedlings. On the otherhand, 20 ,uM ABA near the critical pH value stimulated netH+ release by broad bean roots and significantly promotedroot growth (P < 5%, Table III). Even though pH 4.1 causeda constriction of the root extension zone, ABA treatmenthelped to overcome the adverse effects of low pH on rootgrowth (Fig. 4).

Reduction of net H+ release at low pH in the medium maybe due to a lack of active H+ pumping or, alternatively, to

Table I. Effect of Ca Concentration on Net H+ Release and RootGrowth Rate of Corn and Broad Bean Seedlings

Root medium was 0.25 mm Na2SO4 + 0.5 mm K2SO4 + CaSO4.Treatment Net H+ Release Root Growth Rate

,umol g-l h-' mm h-

Corn (pH 4.1)0.1 mm Ca +0.10 1.15 (±0.03)a1.0 mm Ca +0.48 1.73 (±0.03)5.0 mm Ca +0.69 1.56 (±0.04)

Corn (pH 3.5)0.1 mm Ca -1.14 0.02 (±0.01)1.0 mm Ca -0.27 0.39 (±0.01)5.0 mm Ca +0.13 0.95 (±0.03)

Broad bean (pH 4.1)0.1 mm Ca -0.85 0.02 (±0.01)1.0 mm Ca +0.05 0.08 (±0.02)5.0 mm Ca +0.51 0.70 (±0.03)a Mean (± SE).

417




Table II. Effect of 5 mM K2S04 on Net H+ Release and Root GrowthRate of Corn and Broad Bean Seedlings

Root medium was 0.25 mm Na2SO4 + 5 mm K2SO4 + 1 mm CaSO4.Treatment Net H+ Release Root Growth Rate

mrnol g'h-' mm h-'

Corn (pH 3.5)-K -0.63 0.48 (±0.02)a+K +0.68 0.58 (±0.03)

Broad bean (pH 4.1)-K 0.00 0.03 (±0.05)+K 0.00 0.04 (±0.10)8 Mean (± SE).

increased reentry of protons back into root cells because ofhigher membrane permeability. To test whether ATP supplylimited net H+ release by H+ ATPase activity, we simultane-ously measured net H+ release, ATP concentrations, and 02uptake of corn roots after transferring roots from pH 7.0 to4.0 (Fig. 5). At this moderately low pH, net H+ release rateswere (at least transiently) changed to net H+ uptake (Fig. 5a),ATP concentrations were first slightly reduced but signifi-cantly increased after 45 min (Fig. 5b), and 02 uptake ratesincreased at pH 4.0 relative to pH 7.0 (Fig. 5c). These resultsindicate that, at moderately low pH, corn may cope with highH+ activity in the root medium by increasing root respirationand ATP concentrations, which in turn increase net H+ release(Fig. Sa). In the long run, corn is capable of net H+ release atpH 4 (Fig. 2a), whereas broad bean is not (Fig. 2b, Table III).At the critical pH values (corn, 3.5; broad bean, 4.1), root

respiration of both species was significantly reduced relativeto pH 7.0 (Fig. 6). However, as demonstrated for corn (Fig.7b) even extremely low pH values of the root medium didnot reduce but rather increased ATP levels. Also, when roottips of corn were analyzed for ATP, no decrease of ATPconcentrations was detected at pH 3.25 relative to pH 7.00(data not shown).

Contrary to ATP, malate concentrations in roots of corngradually decreased to a minimum value of about 25 mmol/g root dry weight within 8 h (Fig. 7a) when treated with pH3.25. In root tips, malate concentrations under these condi-

Table Ill. Effect of 20 AM ABA on Net H+ Release and Root GrowthRate of Corn and Broad Bean Seedlings

Root medium was 0.25 mm Na2SO4 + 5 mm K2SO4 + 1 mm CaSO4± 20 AM ABA.

Treatment Net H+ Release Root Growth Rate

pimol g-' h-1 mm h-'

Corn (pH 4.0)-ABA +0.727 1.67 (±0.16)8+ABA +0.307 0.68 (±0.15)

Broad bean (pH 4.1)-ABA -0.001 0.29 (±0.04)+ABA +0.001 0.42 (±0.04)a Mean (± SE).

Figure 4. Effect of 20 Mm ABA on root growth of broad beanseedlings at pH 4.1. Left, +ABA; right, -ABA. Root medium was0.25 mm Na2SO4 + 0.5 mm K2SO4 + 1 mm CaSO4 ±20 Mm ABA. Theexperimental period was 2 d.

Y 1

+-1',- 1

0EIL:Q - 3

61.2a-c-<0.8(AU-

E 0.4

7 16cm

° 80E

-10 0 10 20 30 40Time (minutes)

Figure 5. Effect of medium pH on net H+ release (a), ATP concentra-tion (b), and respiration (c) of corn roots. Seedlings were grown for 2d on 1 mm CaSO4 pH 7.0 and treated with 1 mm CaSO4, pH 7.0 (0)or pH 4.0 (@) at time "0." Negative values denote H+ uptake. Signifi-cant differences at *P = 5% or ***P = 0.1% level. RFW, Root freshweight.

418 YAN ET AL.




8

6

4

2IL

Lfl

c-

0E

8

6

4

2

5 15 25Time (minutes)

Figure 6. Effect of medium pH on the respiration rate of corn (a) andbroad bean (b) root tips. Seedlings were grown at pH 7.0 (0) or 3.5and 4.1 (0) (corn and broad bean, respectively) in 1 mm CaSO4 for 2d, and root tips were then incubated in identical medium for 02 uptakemeasurement as described in "Matenals and Methods." Significantdifferences at *P = 5%, **P = 1%, or ***P = 0.1% level. RFW, Rootfresh weight.

tions declined to about 30 ,umol/g root dry weight (data notshown).As observed for corn roots, malate concentrations decreased

in broad bean roots when plants were kept at pH 4.1 (TableIV). Pyruvate, on the other hand, was not changed by lowpH, and lactate only slightly increased in broad bean rootswhen roots kept at pH 7.0 were compared with those at pH4.1 (Table IV).

DISCUSSION

Comparison of Corn and Broad Bean

Our results have shown that acidification of the root me-dium (increase in H+ activity) reduced net H+ release androot growth of both corn and broad bean (Fig. 2, Table I).Root growth and net H+ release were linearly correlatedbetween pH 3.25 and 6.50 (corn) and pH 3.75 and 5.00(broad bean), suggesting a close relationship between the twoparameters (Fig. 2). Also, the slopes of the regression lineswere identical for the two species. This indicates that, accord-ing to the hypothesis that net H+ release limits growth at lowpH (26, 28), a given decrease in net H+ release reduced rootgrowth of broad bean and corn to the same extent. Neverthe-less, corn was more tolerant of high H+ activity in the rootmedium than broad bean (Fig. 1). This is manifested by the

fact that at 1 mm CaSO4 net H+ release and root growthceased below pH 3.50 (corn) or below pH 4.00 (broad bean;Fig. 2: intersections of the regression lines with the y axis).Even though corn does not rank among the most acid-tolerantplant species (12), it is appreciably more tolerant of high H+activity than broad bean.

Additional supply of calcium to the root medium triggerednet H+ release and stimulated root growth, whereas the reversewas found when calcium supply was reduced (Table I). Thesame applies to K treatment of corn and ABA treatment ofbroad bean: in both cases an increase in net H+ release at thecritical pH stimulated root growth (Tables II and III). Theseresults support the hypothesis that net H+ release by H+ATPase activity is a basic necessity of root growth and limitsgrowth at low pH (26, 28).

Does H+ ATPase Activity Limit Net H+ Release at LowpH?

Even though we did not measure H+ ATPase activity, twolines of evidence indicate that it was not active H+ pumpingthat limited net H+ release but rather that reentry of H+ backinto root cells was enhanced at low medium pH.

(a) High Ca2" concentrations in the root medium changednet H+ uptake to net H+ release or increased net H+ release(Table I). It has been shown (5, 13) that high Na+ activities inthe external medium displace Ca2" from external plasma-lemma-binding sites and thereby increase Na+ influx into rootcells (6, 14). High Ca2" concentrations may counteract thiseffect. Calcium displacement from external plasmalemma-

> 3'0C

E 31In

02

-

E 3

11LY

cn

In

0

EQ,

1.2

1.0

0 4 8 12 16Time (hours)

Figure 7. Effect of low pH on the time course of malate and ATPconcentrations of corn roots. At time "O," plants were transferredfrom 0.25 mm Na2O4 + 25 Mm K2SO4 + 1 mm CaSO4, pH 7.00, to anidentical solution, pH 3.25, as indicated by arrow. RFW, Root freshweight.

(b)

ii~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

419

-




0.25 mM Na2SO4 + 1 mM CaSO4 + 0.5 mM

Treatment Malate Pyruvate Lactate

mmol g-' root dry wt

CornpH 7.0 128 (±2)a 8 (±0) 15 (±2)pH 3.5 33 (±1) 8 (±0) 13 (±1)

Broad beanpH 7.0 415 (±13) 5 (±0) 42 (±3)pH 4.1 81 (±1) 5 (±0) 62 (±2)a Mean (± SE).

binding sites at high H+ activity is a reasonable assumptionbecause low pH increases membrane permeability, particu-larly at low Ca2" concentrations (16, 17, 19, 20).

(b) Root ATP concentration never decreased at low pH butrather increased (Figs. Sb and 7b). Even transfer of corn rootsfrom pH 7.00 to the critical pH 3.25 enhanced ATP concen-

trations in the root tissue (Fig. 7b), indicating that ATP energy

supply to the H+ ATPase did not limit net H+ release.

How Does Net H+ Release Limit Root Growth at Low pH?

According to Serrano (27), net H+ release by H+ ATPaseactivity is essential for (a) nutrient uptake, (b) turgor genera-

tion, (c) external acidification for cell wall loosening (acidgrowth theory), and (d) cytoplasmic pH regulation. It isunlikely that root growth was inhibited because of a lack ofnutrient uptake (3, 26). Also, addition of potassium, whoseuptake is closely related to H+ extrusion (4, 1 1), only slightlyincreased net H' release and root growth of corn but failed inthe case of broad bean (Table II). For the same reason, turgorgeneration probably also was not limiting root growth. Finally,because the external medium was acidified by the pH statsystem, H+ activity in low pH treatments should have beensufficient for cell wall loosening.

Therefore, we propose that cytoplasmic pH regulation was

disturbed at low pH in the medium. Using the 31P NMRtechnique, Gerendas et al. (10) found a temporary decreaseof 0.3 unit of cytoplasmic pH at pH 4 of the external solutionin corn roots relative to pH 6. Because corn root growth andnet H+ release still occurred at pH 4 (Fig. 2), a more distinctcytoplasmic pH decrease may be expected at the critical valuepH 3.50.

Incomplete cytoplasmic pH regulation may also be inferredfrom a drastic decrease of malate concentrations in the roottissues of both species at low pH (Table IV, Fig. 7a). Contraryto malate, pyruvate and lactate were not reduced (Table IV).Decarboxylation of malate has been postulated to be part ofa biochemical pH stat system that helps to stabilize cyto-plasmic pH when acidification occurs (7). Because in root tips(small degree of vacuolation) malate was almost completelyexhausted (data not shown), it is likely that at critical mediumpH not only the biophysical (H+ ATPase) but also the bio-chemical pH stat system failed to control cytoplasmic pH.

Apparently, corn roots were capable of adapting to pH 4

because respiration and ATP concentrations increased,thereby reducing net H+ uptake within several minutes (Fig.5). Within hours at pH 4, corn changed net H+ uptake to netH+ release (Fig. 2). In contrast, critical pH values reducedrespiration rates of both species (Fig. 6) without decreasingATP concentrations (Fig. 7). This may indicate lower ATPdemand under conditions of inhibited growth processes.

It is interesting that root growth at high H+ activity wasprimarily affected not in the meristematic tissue but in theextension zone (Fig. 3). Inhibition of extension growth ofbroad bean at low pH was overcome by external applicationof ABA (Fig. 4) which increases ATP levels in roots (S.Schubert, unpublished data). Further work is focused on thequestion of how cytosolic acidification controls extensiongrowth.

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Table IV. Effect of Root Medium pH on Malate, Pyruvate, andLactate Concentrations of Corn and Broad Bean Roots

Root medium was

K2SO4.

420YAN ET AL.




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effect low root medium ph net proton release, root ... · effect of low root medium ph figure 3....

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