seasonal changes of plasma membrane h -atpase and ... · accumulated in cells of the cambial...

Seasonal Changes of Plasma Membrane H�-ATPase andEndogenous Ion Current during Cambial Growth inPoplar Plants1

Matthias Arend*, Manfred H. Weisenseel, Maria Brummer, Wolfgang Osswald, and Jorg H. Fromm

Fachgebiet Angewandte Holzbiologie (M.A., J.H.F.) and Fachgebiet Pathologie der Waldbaume (M.B., W.O.),Technische Universitat Munchen, Munich, Germany; and Botanisches Institut, Universitat Karlsruhe,Karlsruhe, Germany (M.H.W.)

The plasma membrane H�-ATPase (PM H�-ATPase), potassium ions, and endogenous ion currents might play a funda-mental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs of Populus nigraand Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H�-ATPase, x-ray analysis for K� localizationand a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants duringautumn and winter, only a slight immunoreactivity against the PM H�-ATPase was found in cross sections and tissuehomogenates, K� was distributed evenly, and the density of endogenous current was low. In spring during cambial growth,strong immunoreactivity against a PM H�-ATPase was observed in cambial cells and expanding xylem cells using themonoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K�

accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immaturexylem zone. Addition of auxin to dormant twigs induced the formation of this PM H�-ATPase in the dormant cambial regionwithin a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase inPM H�-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity ofan H�-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H�-ATPase generates the necessaryH�-gradient (proton-motive force) for the uptake of K� and nutrients into cambial and expanding xylem cells.

The plasma membrane H�-ATPase (PM H�-ATPase) is a key enzyme of plants and fungi, and itplays a central role in nutrient uptake and growth ofhigher plants (Serrano, 1989; Palmgren, 2001). Theenzyme generates the proton-motive force that drivesthe uptake of nutrients such as sugars and ions acrossthe plasma membrane of growing plant cells. Espe-cially the uptake of potassium ions through specifictransport proteins has been related to the activity ofthe PM H�-ATPase (Hoth et al., 1997; Maathuis et al.,1997). This uptake is essential for osmotic regulationand cell enlargement in differentiating tissues (Mac-Robbie, 1977; Hsiao and Lauchli, 1986). Most of theinformation concerning the PM H�-ATPase has beenobtained from biochemical and histochemical studieson herbaceous plant species, where the enzyme wasfound predominantly in vascular tissue, in compan-ion cells of the phloem, in epidermal cells of roots,and in guard cells of leaves (compare with Palmgren,1998). The results revealed an important role of theenzyme in phloem loading, uptake of minerals byroots, and turgor regulation in guard cells.

In contrast to herbaceous plants, very little isknown about the occurrence of this vital enzyme inwoody plants or about its distribution and functionin individual tissues. Some work has been done re-garding enzyme activities of vessel-associated cells inwalnut trees (Alves et al., 2001) or the variation ofdifferent PM H�-ATPase transcripts in buds of peach(Prunus persica) trees during breaking of dormancy(Gevaudant et al., 2001). Because of the strong de-mand for minerals by woody plants during the timeof cambial growth, an involvement of the PM H�-ATPase in the process of wood formation is expected.The cambial region acts as a strong axial sink thatcompetes for minerals and assimilates with othersinks such as young leaves and roots during the timeof wood formation (Dunisch and Bauch, 1994; Krabel,2000). The energy for the uptake of the solutes intogrowing cambial cells is most likely provided by theproton-motive force generated by the PM H�-ATPase (Michelet and Boutry, 1995). Especially thesupply and uptake of potassium ions might affect theprocess of wood formation due to the significance ofturgor-driven cell enlargement of cambial cellderivatives.

The acid growth hypothesis holds that susceptiblecells exposed to auxin secrete protons into the apo-plast, which causes hyperpolarization of the cells, adecrease in apoplastic pH, and an increase in thegrowth rate (Rayle and Cleland, 1992). This hyper-

1 This work was supported by the Deutsche Forschungsgemein-schaft (grant no. FR 955/3–1,2).

* Corresponding author; e-mail [email protected];fax 49–89–2180–6429.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.003905.

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polarization and the apoplastic acidification drive theuptake of nutrients into cells via secondary activetransport and cause the opening of ion channels, forinstance K� channels (Michelet and Boutry, 1995;Hoth et al., 1997; Maathuis et al., 1997). With respectto trees, numerous studies have shown that auxin isan important regulator of cambial growth (Savidgeand Wareing, 1981; Little and Savidge, 1987; Lachaud,1989; Little and Pharis, 1995; Uggla et al., 1998; Sund-berg et al., 2000). However, our understanding of thecellular events is still poor, and no effect of auxin onthe PM H�-ATPase in cambial cells has been demon-strated so far.

In poplar (Populus spp.) plants, with their fast-growing trunks, shoots, and twigs, a PM H�-ATPasemay have a vital role in the uptake of nutrients bycambial and other growing cells. Suc has been shownto be the major carbohydrate of cambial metabolism(Krabel, 2000). During the period of rapid cell divi-sions and cell growth in spring and summer, thedemand for Suc reaches its peak, and the number andactivity of a PM H�-ATPase might be high to ener-gize the uptake of Suc into cells. Some support for aninvolvement of a PM H�-ATPase during cambialgrowth comes from investigations showing differ-ences in electric membrane potential among the var-ious cell types involved in assimilate flux from sievetubes to cambial cells (van Bel and Ehlers, 2000).Moreover, transport of assimilates to the cambiumhas been demonstrated directly in willow (Salix vimi-nalis), i.e. close relatives to poplar plants, using la-beled sugars and microautoradiography (Fromm,1997). Circumstantial evidence indicating the cambialcells as the major sink for nutrients is derived fromthe large number of plasmodesmata in tangentialwalls of ray cells (Sauter and Kloth, 1986).

In this study, various methods were applied toinvestigate seasonal changes and an assumed effectof auxin on the PM H�-ATPase of cambial cells: (a)Changes in potassium distribution were measured byx-ray microanalysis. (b) Specific antibodies againstthe plasma membrane H�-ATPase were used to im-munolocalize the enzyme to measure its distributionand amount in thin sections of Populus trichocarpatwigs. Either the antibody was labeled with Cy3 andlocalized by fluorescence microscopy, or it was la-beled with gold particles and visualized by transmis-sion electron microscopy. (c) Tissue homogenateswere analyzed using the western-blot technique forthe presence of PM H�-ATPase at various times ofthe year and in dormant plants after the addition ofauxin. (d) Endogenous ionic current as an indicatorof the activity of a PM H�-ATPase was measuredwith a noninvasive, vibrating electrode near the faceof stem cuttings from shoots of Populus nigra, assum-ing that the PM H�-ATPase drives H� currentthrough the plasma membrane and the adjacent apo-plast. Auxin was either applied briefly to measure

short-term effects of the hormone or for several daysto allow for gene activation.

RESULTS

Potassium Distribution

Energy-dispersive x-ray analysis in combinationwith scanning electron microscopy (SEM) providesan appropriate technique for semiquantitative detect-ing of elements in the intact tissue on the microscopiclevel. Although the physical heterogeneity of the tis-sue matrix did not permit a comparison with a cali-bration standard for absolute quantification of potas-sium concentrations, this method revealed a strongseasonal change of the relative potassium concentra-tion and distribution in twig tissue of P. trichocarpa.Especially the cambial region showed strong changesin potassium content reflecting its seasonal states ofactivity: An increased level of potassium was mea-sured in the active cambial region in May, whereasonly a low level of potassium was found in thedormant cambium and its daughter cells in January(Fig. 1). In comparison with the adjacent mature tis-sues of the phloem and xylem, the highest amount ofpotassium was found in the active cambial region asindicated by the distinct accumulation of potassium-specific x-ray signals in this part of the twigs (Fig. 2,C and D). In contrast, potassium showed an equaldistribution across twigs during the time of cambialdormancy (Fig. 2, A and B).

Western-Blot Analysis

The specificity of the monoclonal antibody 46 E5B11 F6 against a PM H�-ATPase of P. trichocarpa waschecked by western-blot analysis of twig tissue ho-mogenates. After electrophoresis of twig tissue ho-mogenate collected in May after cambial reactivation,a binding of the antibody to a polypeptide in the

Figure 1. Relative potassium concentrations in dormant (January)and active (May) cambium cells from P. trichocarpa expressed as thepeak-to-background ratio of element-specific x-ray signals. Valuesare mean � SE from measurements of 10 different tissue areas.

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100-kD range was detected on the blots (Fig. 3A). Asecond minor band was recognized by the antibodyin the 60-kD range, probably a proteolytic degrada-tion product of the enzyme with an epitope for theantibody. In contrast, no PM H�-ATPase was de-tected by the antibody on blots that were preparedfrom twig tissue in November during cambial dor-mancy (Fig. 3A). Auxin applied for 2 d to debuddedtwigs (40 �m indole-3-acetic acid [IAA]-sodium saltin water) during the period of dormancy induced anew expression of PM H�-ATPase as indicated byimmunodetectable amounts of the enzyme on theblots (Fig. 3B). A polyclonal antibody raised against aPM H�-ATPase from Nicotiana plumbaginifolia re-acted with twig tissue homogenate from poplarplants similarly to the monoclonal antibody 46 E5B11 F6 (Fig. 3C). During cambial activity a majorband was detected by the polyclonal antibody in the100-kD range and a second minor band in the 60-kDrange, whereas twig tissue prepared during cambialdormancy showed only a very slight immunoreactiv-ity in the 100-kD range.

Immunofluorescence Microscopy

Cross sections were cut from twigs in spring aftercambial reactivation and incubated with the mono-clonal antibody 46 E5 B11 F6 against PM H�-ATPaseand a Cy3-conjugated secondary antibody. A strongspecific fluorescence labeling was visible predomi-nantly in the active cambial zone and the adjacentregion of xylem cell differentiation (Fig. 4A). In themature xylem, there was some specific labeling of raycells that contacted vessel cells (Fig. 4B). In contrast,no specific labeling was observed in cross sectionstaken during cambial dormancy (Fig. 4C). After treat-ment of debudded twigs in December during cambialdormancy with 40 �m IAA for 4 d, specific labeling ofa PM H�-ATPase became visible in the cambial zoneand in some ray cells that contact vessels in themature xylem (Fig. 4, D and E). Controls showedonly an unspecific background labeling, either in theabsence of the primary antibody or after replacementof the specific antibody with nonimmune IgG (Fig.4F). For comparison, twig sections were also treatedwith the polyclonal antibody L3E against PM H�-

Figure 2. Potassium distribution in twigs of P. trichocarpa. A, SEM micrograph of a transverse section taken during cambialdormancy (November). B, Mapping of potassium-specific x-ray signals in the same twig section as shown in A. C, SEMmicrograph of a transverse twig section taken after cambial reactivation (May). D, Mapping of potassium-specific x-raysignals in the same twig section as shown in C, indicating a strong accumulation of potassium in the activated cambial zone(arrows).

Plasma Membrane H�-ATPase in Poplar

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ATPase in place of the monoclonal antibody. Sectionstaken during cambial activity showed a fluorescencepattern different from that given with the monoclo-nal antibody. A distinct specific labeling occurred incells of the inner phloem, the transition zone phlo-em/cambium, and the ray parenchyma, whereas noPM H�-ATPase was detected by the polyclonal anti-body in the differentiating xylem (Fig. 5A). In addi-tion, a weak specific fluorescence was visible in manycells of the cortex, indicating the presence of a smallamount of PM H�-ATPase in this tissue (Fig. 5B).Twig sections taken during cambial dormancyshowed a reduced intensity of the fluorescence label-ing, mostly restricted to ray cells crossing the cambialzone and the inner phloem (Fig. 5C). Controlsshowed no fluorescence, either in the absence of theprimary antibody or after replacement of the specificantibody with nonimmune IgG (Fig. 5D).

Immunogold Electron Microscopy

Because the specific fluorescence labeling given bythe monoclonal antibody appeared all over the la-beled cells, immunohistochemical experiments werecarried out with the transmission electron micro-scope for detailed localization of this PM H�-ATPase.A procedure of labeling before embedding was car-ried out using ultrasmall gold conjugates in combi-nation with silver enhancing. The silver-enhancedgold particles appeared predominantly along theplasma membrane of labeled cells, indicating that alarge number of the PM H�-ATPases was present inthis cellular domain (Fig. 6, A–F). In agreement withresults obtained from immunofluorescence micros-copy, a specific labeling of a PM H�-ATPase wasfound along the plasma membrane of cells of theactive cambium, as well as in expanding cells of theimmature xylem and in ray cells contacting vessels of

the mature xylem. In cambial cells, the intensity ofimmunolabeling along the plasma membrane wasalways higher in radial sections than in cross sectionsbecause of the much better preservation of cellularstructures, including membranes, in the former case(Fig. 6, A and B). Differentiating xylem cells, newlyformed vessels, fiber cells, and ray cells showed la-beling of a lower intensity than the cambial cellsproper (Fig. 6, C–F). A very strong immunolabelingof the plasma membrane was observed in companioncells of the inner phloem in contact with the activecambium (Fig. 7C), whereas the sieve elements at-tached to these companion cells showed no labeling.The transmission electron microscopy results alsoshow that a labeling of ray cells in mature xylem onlyoccurs when these cells contact vessels (Fig. 7, A andB). A few gold particles were bound to other cell struc-tures. The latter labeling was nonspecific, because asimilar pattern was observed in control sections incu-bated with nonimmune IgG (Fig. 7, D and E).

Endogenous Ionic Current during the Seasons

The direction and density of endogenous ionic cur-rent was measured at different sites of cross sectionsduring various times of the year. The results showthat the current pattern and current density in shootcuttings of P. nigra depended on the season: In dor-mant shoots in December, only small outward cur-rent was measured at all sites, i.e. the pith paren-chyma, secondary xylem (wood), cambium/phloemzone, and cortex parenchyma (Fig. 8). In April, aperiod of active cambial growth, strong outward cur-rent with densities of more than 6 �A cm�2 wasmeasured in the cambium/phloem zone and thegreen cortex. Outward current was also found in thepith and the mature xylem, whereas strong inwardcurrent with densities of up to 8 �A cm�2 was mea-

Figure 3. Western blots of twig tissue homoge-nates from P. trichocarpa. A, Blot incubatedwith the monoclonal antibody 46 E5 B11 F6.Samples taken from twig tissue during cambialactivity (May), respectively, cambial dormancy(November). B, Blot incubated with the mono-clonal antibody 46 E5 B11 F6. Samples takenduring cambial dormancy (November) fromtwig tissue treated with auxin for 2 d, respec-tively, without auxin. C, Blot incubated with thepolyclonal antibody L3E. Samples taken fromtwig tissue during cambial activity, respectively,cambial dormancy. Molecular mass markers areindicated on the left.

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sured in the young xylem. A similar current patternwas measured in August, but the current direction inthe cambium/phloem zone had changed to inward.The overall current density decreased from April toAugust and from August to December at all mea-surement sites.

During experiments performed in August, theionic composition of the medium was changed toobtain information about the ions involved in theendogenous current. When the pH of the mediumwas increased, the current density also increased sig-nificantly at all measurement sites, and outward cur-

Figure 4. Localization of a PM H�-ATPase in twig tissue of P. trichocarpa using the monoclonal antibody 46 E5 B11 F6. A,Section taken after cambial reactivation (April). Strong fluorescence labeling appears in the cambium zone (arrows) and theadjacent region of xylem cell differentiation. Arrowheads show newly formed vessels. B, Section taken after cambialreactivation. Fluorescence labeling of some ray cells that contact vessels in the mature xylem (arrows) can be seen. C,Section taken during cambial dormancy (December). No fluorescence labeling of a PM H�-ATPase is visible. D, Sectiontaken during cambial dormancy (December) after treatment of debudded twigs with auxin for 4 d. Fluorescence labelingappears in the cambium/phloem zone (arrows). E, Fluorescence labeling of ray cells in the mature xylem after treatment ofdebudded twigs with auxin in December (arrow). F, Control section taken after cambial reactivation (April) incubated withnonimmune IgG instead of the monoclonal antibody against a PM H�-ATPase. Xy, Xylem; Ph, phloem.




rent became dominant. Figure 9 shows a typical ex-ample of current density vectors measured across theface of cuttings, first at pH 4.5 and then at pH 7.5. Atboth sites, i.e. the cortex parenchyma (Fig. 9A) andthe cambium/phloem zone (Fig. 9B), the current den-sity increased, and the direction changed from in-ward to outward in the cambium/phloem zone. Noimmediate changes of the current density were mea-sured when the potassium or chloride concentrationof the medium was changed (data not shown). Afterincreasing the calcium concentration of the mediumto 1.0 mm, a decrease of the outward current wasobserved at the cortex and a decrease of the inwardcurrent in the cambium/phloem zone.

Because it is well known that plant hormones areinvolved in cambial growth and differentiation oftrees, we investigated the effects of two prominentphytohormones, i.e. IAA and abscisic acid (ABA), onthe endogenous ionic current of poplar shoots. Theeffect of 30 �m IAA was measured in Decemberwhen only small endogenous current was present.In accord with the IAA activation of a PM H�-

ATPase shown with immunoblot and immunofluo-rescence (compare with Figs. 3 and 4), IAA causedan increase of current density at all measurementsites already 2 h after application (Fig. 10A). Themean current density increased by 60% from 1.7 to2.8 �A cm�2.

The formation of latewood at the end of summerhas been attributed to an increase in the level of ABAin the cambial zone (Jenkins and Shephard, 1974;Wodzicki and Wodzicki, 1980). ABA was also shownto have a strong inhibitory effect on wood growth(Fromm, 1997) and magnitude of endogenous currentof willow roots (Fromm et al., 1997). During the timeof cambial activity and high-current densities inspring, application of 30 �m ABA to the mediumcaused the density of outward current to decrease byalmost 50% within 2 h, i.e. from 6.2 to 3.2 �A cm�2

(Fig. 10B). These results suggest an opposing role ofboth hormones in the regulation of endogenous cur-rent and activity of the PM H�-ATPase activity inpoplar plants.

Figure 5. Localization of the PM H�-ATPase in twig tissue of P. trichocarpa using the polyclonal antibody L3E. A, Sectiontaken after cambial reactivation. Fluorescence labeling appears in the inner phloem and the transition zone phloem/cambium (arrows). No specific labeling occurs in the differentiating xylem (arrowheads). B, Slight fluorescence labeling ofcortex cells in a section taken after cambial reactivation (arrows). C, Section taken during cambial dormancy, with slightfluorescence labeling of cells in the inner phloem. Arrows indicate ray cells crossing the cambium and the inner phloem.D, Control section taken after cambial reactivation and incubated with nonimmune IgG instead of the polyclonal antibodyagainst a PM H�-ATPase. Xy, Xylem; Ph, phloem; Scl, sclerenchyma.

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Figure 6. Cellular fine localization of a PM H�-ATPase in the active cambium zone and the region of xylem celldifferentiation using the monoclonal antibody 46 E5 B11 F6. Samples were taken in May. A and B, Radial section of an activecambial cell with strong labeling along the plasma membrane (arrows). C and D, Cross section of newly formed xylem cells.Labeling of the plasma membrane appears in a ray cell and a fiber cell (arrows). E and F, Radial section of an enlarging vesselcell with labeling of the plasma membrane (arrows). V, Vacuole; CW, cell wall; VC, vessel; RC, ray cell; FC, fiber cell.




Figure 7. Cellular fine localization of a PM H�-ATPase in the innerphloem and in ray cells of mature xylem. Samples were taken in May.A and B, Cross section of mature xylem. Labeling appears along theplasma membrane of a ray cell that contacts a mature vessel cell(arrows). C, Cross section of a sieve element/companion cell com-plex of the inner phloem. Strong labeling occurs along the plasmamembrane of the companion cell. D and E, Controls. Cross sectionsof a ray cell and a cambial cell. VC, Vessel; RC, ray cell; CA, cambialcell; SE, sieve element; CC, companion cell.

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DISCUSSION

In this study, the PM H�-ATPase, K�-ion contentand endogenous ionic currents were investigated inpoplar plants using specific antibodies, x-ray analy-sis, and a noninvasive vibrating probe. A PM H�-ATPase was visualized by the monoclonal antibody46 E5 B11 F6 in a very sensitive manner in physio-logically active poplar plants during cambial growth,but not in dormant ones. In contrast to herbaceousspecies, this PM H�-ATPase was localized in thecambium zone, in differentiating xylem cells and inray cells surrounding vessels in the mature xylem.Auxin applied to dormant plants induced the forma-tion of this PM H�-ATPase in the cambial region aswell as in ray cells. Experiments carried out addition-ally with the electron microscope showed that thePM H�-ATPase was localized in the plasma mem-brane of the labeled cells. No immunoreactivity wasobtained with this antibody in the cortex paren-chyma and in pith cells. This result came as a surprisebecause these tissues produced ionic currents withsimilar direction and density as the cambial regionduring periods of high-growth activity. Using a poly-clonal antibody, further PM H�-ATPases were local-ized in the inner phloem and in cells of the cortexparenchyma. Surprisingly, no immunoreactivity wasfound using the polyclonal antibody in the differen-tiating xylem and in pith cells. Only a slight labelingwas observed during cambial dormancy.

Taken together, a clear seasonal variation in theamount of the PM H�-ATPase was shown in poplar

twigs using different specific antibodies and corre-lated to cambial growth activity. Similar to this ob-servation, a seasonal variation of different PM H�-ATPase transcripts was demonstrated recently in thebud tissue of peach trees (Gevaudant et al., 2001). Thedifferent labeling pattern given by monoclonal andpolyclonal antibodies indicated that different iso-forms of the enzyme are present in poplar tissues, ofwhich one group is mainly found in the cambialtissue and the differentiating xylem. Molecular stud-ies have shown that in plants, the PM H�-ATPase isencoded by a multigene family, and PM H�-ATPaseisoforms exhibit a distinct pattern of tissue-specificexpression (compare with Palmgren, 1998; Palmgren,2001). For castor bean (Ricinus communis) plants, itwas recently demonstrated that the monoclonal an-tibody 46 E5 B11 F6 discriminates between differentPM H�-ATPase isoforms in a cell-specific manner(Langhans et al., 2001). The complete lack of specificlabeling for a PM H�-ATPase in pith cells remains anopen question, because this tissue drove ionic cur-rents during periods of high-growth activity. Besidesthe occurrence of further PM H�-ATPase isoforms,an alternative explanation for the outward current atthe pith might be an efflux of K� and Na� or aninflux of Cl� ions. K� and Na�, however, are veryunlikely to carry the outward current. A massive andprolonged leakage of K� from the small number ofinjured cells at the cutting plane does not seem fea-sible, and a Na�/K� ATPase that would transportNa� to the medium is absent in plant cells. Net Cl�

uptake, on the other hand, might be a real possibilityfor the outward current. Such uptake has been re-ported from bean leaves during photosynthesis (Sha-bala and Newman, 1999), and pith cells showed adistinct fluorescence of chlorophyll when twig sec-tions from poplar plants were inspected with thefluorescence microscope.

During all seasons, the cortex parenchyma, the ma-ture xylem, and the pith parenchyma drove currentfrom the protoplasts to the apoplast. The cambialzone showed outward current in the spring, but in-ward current in the late summer. Young wood cellswere a current sink during the entire growing season.The current densities were highest in spring, de-clined in late summer, and became rather small inwinter when the plants were dormant.

Combining the data from PM H�-ATPase localiza-tion and current measurements, there are severalindicators that a major component of the endogenouscurrent is H� ions: (a) The high concentration of PMH�-ATPases, particular in the cambial zone and thedifferentiating xylem, during cambial growth corre-lates positively with outward current during thesame time. The slight amount of immunodetectablePM H�-ATPases in dormant plants conversely corre-lates with low-current densities. (b) The current den-sity increased with increasing pH and addition ofauxin. A higher pH on the outside of cells favors H�

Figure 8. Pattern and densities (�SE) of endogenous ionic current ofdifferent tissues across sections of vertically oriented shoot cuttingsfrom P. nigra measured in April, August, and December. Currentdensities were measured in more than 10 different shoots collectedfrom the trunks or roots of P. nigra trees.




export by the PM H�-ATPase (Takeuchi et al., 1985;Takeshige et al., 1986), and auxin is known to stim-ulate H� excretion (Talbott et al., 1988; Luthen et al.,1990; Hager et al., 1991). (c) The current densitydecreased with increasing Ca2� concentration andaddition of ABA. Both compounds are known to turndown the activity of the PM H�-ATPase (Beffagna etal., 1995; Goh et al., 1996; Lino et al., 1998).

Could it be that the PM H�-ATPases, the endoge-nous current, and auxin have a role in growth anddifferentiation of the cambial region? We think theyhave. During growth and differentiation of cambialcells, K� ions are taken up, the surface area of thecells expands, and cell wall polysaccharides/proteinsare secreted (Savidge, 1996). In spring, high concen-trations of endogenous auxin are present in youngshoots and twigs (Little and Savidge, 1987), and apromotion of cambial activity by auxin that was ap-plied to debudded stems has been demonstrated(Wareing et al., 1964). This auxin was transported tothe cambium and its young derivatives (Lachaud andBonnemain, 1984; Tuominen et al., 1997; Uggla et al.,

1998). Auxin has also been shown to mediate anincrease in H�-ATPase transcripts in the plasmamembrane of maize coleoptiles (Hager et al., 1991;Frias et al., 1996), and small “auxin up RNA’s” and“auxin genes” have been found during cell elonga-tion (Abel et al., 1994; Abel and Theologis, 1996).Similar effects of auxin are likely to occur in thecambial region of poplar plants because the numberof PM H�-ATPases and the ionic current densityincreased with the addition of auxin. The increase incurrent density indicates more H� ions in the apo-plast and hyperpolarization of the membrane poten-tial in cells of the cambial zone. An elevated protongradient and a higher membrane voltage favor theuptake of sugars via symport with protons into therapidly growing cambial cells and an influx of K�

ions due to opening of ion channels in the plasmamembrane of expanding young vessel cells (Briskinand Gawienowski, 1996). We were recently able todemonstrate that the formation of fewer and largervacuoles during cambial reactivation is caused byincreased K� uptake, probably modulated by the

Figure 10. Typical examples of short-term ef-fects of IAA and ABA on the endogenous ioniccurrent of shoot cuttings from P. nigra. A, IAA(30 �M) was added for 2 h to the artificial pondwater (APW) bathing a dormant shoot cutting inDecember. B, ABA (30 �M) was added for 2 h tothe APW bath of a shoot cutting during highcambial activity in April. Absolute current den-sities are given and projected onto the crosssections of the shoots as columns of inward oroutward current.

Figure 9. Typical examples of the changes incurrent density and direction after increasing thepH of the medium from pH 4.5 to 7.5. A, En-dogenous current at the cortex parenchyma of apoplar shoot cutting. B, Endogenous current atthe cambium/phloem zone. Both examples weremeasured in August in shoots from P. nigra trees.

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activity of a PM H�-ATPase (Arend and Fromm,2000). Thus, the auxin-induced up-regulation andactivation of a PM H�-ATPase in poplar plant cellsseems to be a prerequisite for nutrient uptake andgrowth of cambial and young xylem cells. This issimilar to roots, where inward H�-current is corre-lated with nutrient uptake and plasmatic growth atthe root apex, and outward current with uptake ofminerals and cell enlargement in the elongation zone(Weisenseel et al., 1992). A change in current direc-tion during the season, as for instance measured inthe cambial zone, may, therefore, be an indicator of ashift in the mode of growth. ABA, in contrast toauxin, strongly reduced the endogenous current ofpoplar plants. This correlates positively with thestrong inhibitory effect of ABA on wood growth(Fromm, 1997). In late summer, the formation of late-wood has also been attributed to an increase in thelevel of endogenous ABA in the cambial zone, effect-ing cessation of cambial activity (Jenkins and Shep-hard, 1974; Wodzicki and Wodzicki, 1980). Ourpresent results indicate a role for auxin in hardwoodformation of poplar trees via activation of a PMH�-ATPase in the cambial region.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Poplar (Populus nigra var italica L. and Populus trichocarpa Torr. et Gray.)plants were selected for the experiments because of the well-known advan-tages of the genus Populus as a model for hardwood secondary vasculargrowth (Chaffey, 1999). For electron microscopic investigations (SEM-energy-dispersive x-ray analysis), immunoblot analysis, and immunocyto-chemical procedures, twig tissue samples were collected from mature pop-lar (P. trichocarpa) trees grown in a forest near the campus of the TechnicalUniversity of Munich. For measurements of endogenous current, stemcuttings with a length of approximately 50 mm and a diameter of 5 to 7 mmwere taken from 1- to 2-year-old upright shoots sprouting from the trunk orroots of poplar trees (P. nigra) growing on the campus of the University ofKarlsruhe.

SEM and X-Ray Microanalysis

Small sections of twig tissue were cut with a razor blade and immediatelyshock-frozen in liquid isopentane at its melting point. After freeze-drying,the samples were coated with chromium and examined in a scanningelectron microscope (AMR 1200, Leitz, Wetzlar, Germany) fitted with aKEVEX 4000 x-ray analyzer. Element-specific x-ray spectra were obtainedfrom a reduced scan raster area at 1,000� magnification. Relative potassiumconcentrations were expressed as peak to background ratio from 10 re-corded spectra. For visualizing the distribution of potassium in the twigtissue, potassium-specific x-ray signals were recorded using the element-specific scan modus of the microscope.

Antibodies against Plasma Membrane H�-ATPase

The monoclonal antibody 46 E5 B11 was chosen for western blotting andimmunolocalization, because of its strong immunoreactivity against PMH�-ATPase (Lutzelschwab, 1990; Villalba et al., 1991; Baur et al., 1996;Langhans et al., 2001) The hybridoma clone 46 E5 B11 was used to produceantibodies against a PM H�-ATPase of maize (Zea mays; Lutzelschwab,1990; Villalba, et al., 1991). For the experiments described here, subclone 46E5 B11 F6 was employed according to Baur et al. (1996). The specificity ofthis antibody against a PM H�-ATPase of poplar plants was tested bywestern-blot analysis (see below). For comparison, the polyclonal antibody

L3E raised against a polypeptide corresponding to the PMA 2 isogene(plasma membrane H�-ATPase 2) of Nicotiana plumbaginifolia were used forwestern blotting and immunolocalization (Morsomme et al., 1998).

SDS-Page and Immunoblot Analysis

Poplar twigs were collected in spring during cambial activity and inautumn during cambial dormancy. From each sample, 0.5 g of fresh tissuewas homogenized in an ice-cooled mortar with 5 mL of 10 mm Tris-HCl, 5mm EDTA, and 1% (v/v) plant protease inhibitor cocktail (Sigma, St. Louis),pH 6.8, and filtered through a layer of gauze. The filtrate was centrifuged at20,000g for 10 min at 4°C, and the resulting pellet was resuspended in 2 mLof solubilization buffer containing 50 mm Tris-HCl, 2% (w/v) SDS, and 3%(v/v) �-mercaptoethanol, pH 6.8. After incubation for 1 h at 25°C, thesolution was centrifuged for 1 min at 8,000g, and a sample of the superna-tant was loaded without heating onto an 8% (v/v) SDS-polyacrylamideminigel. Protein standards of known Mr (prestained broad range, Bio-Rad,Hercules, CA) were run on the same gel. Proteins were transferred to apolyvinylidene difluoride membrane using a semidry transfer unit (TRANS-BLOT SD, Bio-Rad). The membrane was washed in Tris-buffered saline(TBS), blocked for 2 h with 1% (w/v) bovine serum albumin (BSA) in TBS,incubated for 1 h with the monoclonal mouse antibody 46 E5 B11 F6, diluted1:500 or with the polyclonal rabbit antibody L3E, and diluted 1:1,000 both inTBS containing 1% (w/v) BSA. After washing in TBS containing 0.05% (v/v)Tween 20, the membrane was incubated for 1 h with 1-nm gold-conjugatedgoat anti-mouse antibody or anti-rabbit antibody (British Biocell Interna-tional, Cardiff, UK), diluted 1:400 in TBS containing 1% (w/v) BSA. Thelabeling of the membrane was developed with a silver enhancing Kit (BritishBiocell International) according to the manufacturer’s instructions.

Fluorescence Microscopy Immunolabeling

Samples of 10 to 20 mm in length were cut from 1- or 2-year-old twigsand fixed with 3% (w/v) formaldehyde (freshly prepared from paraformal-dehyde) and 3 mm EGTA in 50 mm PIPES, pH 6.4, for 1 h at roomtemperature. After washing in PIPES solution, cross sections of 50 �mthickness were cut with a microtome and rinsed in PBS, pH 7.2. To reduceunspecific labeling, the sections were blocked with 100 mm glycine in PBS-T(PBS containing 0.2% [w/v] Tween 20) and 3% (w/v) BSA in PBS-T, both for30 min. For immunolocalization, sections were incubated for 2 h at 37°Cwith the monoclonal mouse antibody 46 E5 B11 F6 diluted 1:500 or with thepolyclonal rabbit antibody L3E diluted 1:1,000, both in PBS containing 0.5%(w/v) BSA. After washing in PBS, the sections were incubated for 1 h at37°C with Cy3 labeled anti-mouse antibody, respectively, anti-rabbit anti-body (Cy3 conjugates, Dianova, Hamburg, Germany) diluted 1:250 in PBScontaining 0.5% (w/v) BSA. After the incubation period, the sections wererinsed in PBS-T and then viewed using an axiophot microscope (Zeiss, Jena,Germany) equipped with the filter combination 546-nm exciter and 590-nmemitter. Photographs were taken with Kodak Ektrachrome 320 T film (East-man Kodak, Rochester, NY). Incubations in medium containing Cy3-conjugated secondary antibody without primary antibody or in mediumwith mouse nonimmune IgG diluted 1:200 instead of primary antibodyserved as controls.

Electron Microscopy Immunolabeling

Small cuttings from poplar twigs were collected in spring after cambialreactivation and fixed for 1 h at room temperature with 3% (w/v) formal-dehyde (freshly prepared from paraformaldehyde) in PBS, pH 7.2, contain-ing 1% (w/v) saponin for permeabilization of cells. After washing in PBS-T,unspecific binding sites were blocked for 30 min with 100 mm glycin and foranother 30 min with 5% (v/v) goat normal serum and 1% (w/v) BSA, bothin PBS-T. For immunolabeling, samples were incubated overnight at 4°Cwith the antibody 46 E5 B11 F6 diluted 1:500 in PBS containing 0.5% (w/v)BSA, then washed in PBS-T and incubated for 2 h at room temperature with1 nm gold-conjugated goat anti-mouse antibody (British Biocell Interna-tional) diluted 1:400 in PBS containing 0.5% (w/v) BSA. Controls wereincubated with nonimmune mouse IgG in place of the specific primaryantibody. After washing in PBS-T, the samples were post-fixed for 30 minwith 2% (v/v) glutaraldehyde in PBS and washed in distilled water. Forvisualizing the 1-nm gold particles at the electron microscope level, silver




enhancement was carried out with the silver-enhancing kit from BritishBiocell International according to the manufacturer’s instructions beforethe samples were dehydrated and embedded in Spurr’s epoxy resin. Thisunusual procedure greatly improved the detection of a PM H�-ATPase.Ultrathin sections from the peripheral tissue layers were cut with a dia-mond knife on an ultramicrotome (LKB, Uppsala), transferred onto Form-var coated copper grids, and stained with lead citrate. Sections wereexamined using a Zeiss EM 10 transmission electron microscope operatedat 80 kV.

Measurement of Endogenous Ionic Currents

Stem cuttings of approximately 50 mm in length from shoots weretrimmed at their apical surface to an angle of 30° and fastened vertically intosmall petri dishes with a front window of cover glass and a downwardextending tube of Plexiglas. The dishes were filled with 25 mL of APWcontaining 1.0 mm NaCl, 0.1 mm KCl, 0.1 mm CaCl2, and 1.0 mm MES,adjusted to pH 6.0 with Tris. The dishes were suspended into the stage of aninverted microscope and viewed from the front with a horizontal stereo-scope (Leitz). The cuttings were left to settle and recover from handling for30 to 60 min before the measurements. Endogenous current was measuredwith a three-dimensional vibrating-probe set-up as described by Weisenseelet al. (1992). In brief, the three-dimensional probe measures the total currentdensity and its spatial components, i.e. it measures current density vectors.For the measurements, a 50°-tilted, insulated metal electrode (SS300305A,Micro Probe, Clarksburg, MD) with a 30-�m spherical tip of platinum blackwas vibrated lengthwise and approximately 200 �m above the green cortexparenchyma, the cambial zone, young and mature xylem cells, and the pithparenchyma. Three consecutive measurements were carried out at each siteby rotating the probe, which was mounted to the bottom of a horizontallyrevolving stage, into three positions separated by 30° and 90°, respectively.The probe was vibrated with a frequency of 317 Hz, an amplitude of 35 �m,and a duration of 20 s in each position. At sites with an electric potentialdifference, originating from ionic current flow through the aqueous me-dium, this voltage was picked up by the electrode and transformed into anAC voltage by the vibration. The latter was measured using a lock-inamplifier (5210, PerkinElmer Life Sciences, Boston). The three values ofvoltage measured at each site were then used to calculate the local currentdensity vector with the aid of a custom-made algorithm. Current densitiesmay be translated into ion fluxes by equating 1 �A cm�2 with 10 pmol cm�2

s�1 of monovalent ions.

ACKNOWLEDGMENTS

We thank Dr. Martin Lutzelschwab for kindly providing the antibodyclone 46 E5 B11 F6, Dr. Marc Boutry for polyclonal antibodies, and BarbaraSchlicke for technical assistance.

Received February 8, 2002; returned for revision March 12, 2002; acceptedMay 3, 2002.

LITERATURE CITED

Abel S, Oeller PW, Theologis A (1994) Early auxin-induced genes encodeshort-lived nuclear proteins. Proc Natl Acad Sci USA 91: 326–330

Abel S, Theologis A (1996). Early genes and auxin action. Plant Physiol 111:9–17

Alves G, Sauter JJ, Julien JL, Fleurat-Lessard P, Ameglio T, Guillot A,Petel G, Lacointe A (2001) Plasma membrane H�-ATPase, succinate andisocitrate dehydrogenases activities of vessel-associated cells in walnuttrees. J Plant Physiol 158: 1263–1271

Arend M, Fromm J (2000) Seasonal variation in the K, Ca and P contentand distribution of plasma membrane H�-ATPase in the cambium ofPopulus trichocarpa. In R Savidge, J Barnett, R Napier, eds, Cell andMolecular Biology of Wood Formation. BIOS Scientific Publishers, Ox-ford, pp 67–70

Baur M, Meyer AJ, Heumann HG, Lutzelschwab M, Michalke W (1996)Distribution of plasma membrane H�-ATPase and polar current patternsin leaves and stems of Elodea canadensis. Bot Acta 109: 382–387

Beffagna N, Romani G, Gatti L (1995) Changes in chloride fluxes and cytosolicpH induced by abscisic acid in Elodea densa leaves. Bot Acta 108: 74–79

Briskin DP, Gawienowski MC (1996) Role of the plasma membrane H�-ATPase in K� transport. Plant Physiol 111: 1199–1207

Chaffey N (1999) Cambium: old challenges-new opportunities. Trees 13:138–151

Dunisch O, Bauch J (1994) Influence of mineral elements on wood forma-tion of old growth spruce (Picea abies [L.] Karst.). Holzforschung 48: 5–14

Frias I, Caldera MT, Perez-Castineira JR, Navarro-Avino JP, Culianez-Macia FA, Kuppinger O, Stransky H, Pages M, Hager A, Serano R (1996)A major isoform of the maize plasma membrane H�-ATPase: character-ization and induction by auxin in coleoptiles. Plant Cell 8: 1533–1544

Fromm J (1997) Hormonal physiology of wood growth in willow (Salixviminalis L.): effects of spermine and abscisic acid. Wood Sci Technol 31:119–130

Fromm J, Meyer AJ, Weisenseel MH (1997) Growth, membrane potentialand endogenous ion currents of willow (Salix viminalis L.) roots are allaffected by abscisic acid and spermine. Physiol Plant 99: 529–537

Gevaudant F, Petel G, Guilliot A (2001) Differential expression of fourmembers of the H� ATPase gene family during dormancy of vegetativebuds of peach trees. Planta 212: 619–626

Goh C, Kinoshita T, Oku T, Shimazaki K (1996) Inhibition of blue light-dependent H� pumping by abscisic acid in Vicia guard-cell protoplasts.Plant Physiol 111: 433–440

Hager A, Debus G, Edel HG, Stransky H, Serrano R (1991) Auxin inducesexocytosis and the rapid synthesis of a high-turnover pool of plasma-membrane proton ATPase. Planta 185: 527–537

Hoth S, Dreyer I, Dietrich P, Becker D, Muller-Rober B, Hedrich R (1997).Molecular basis of plant-specific acid activation of K� uptake channels.Proc Natl Acad Sci USA 94: 4806–4810

Hsiao TC, Lauchli A (1986) Role of potassium in plant-water relations. In BTinker, A Lauchli, eds, Advances in Plant Nutrition, Vol 2. PraegerScientific, New York, pp 281–312

Jenkins PA, Shephard KR (1974) Seasonal changes in levels of indole-aceticacid and abscisic acid in stem tissues of Pinus radiata. J Forest Sci 4:511–519

Krabel D (2000) Influence of sucrose on cambial activity. In R Savidge, JBarnett, R Napier, eds, Cell and Molecular Biology of Wood Formation.BIOS Scientific Publishers, Oxford, pp 113–125

Lachaud S (1989) Participation of auxin and abscisic acid in the regulationof seasonal variations in cambial activity and xylogenesis. Trees 3:125–137

Lachaud S, Bonnemain JL (1984) Seasonal variations in the polar-transportpathways and retention sites of [3H]indole-3-acetic acid in youngbranches of Fagus sylvatica L. Planta 161: 207–215

Langhans M, Ratajczack R, Lutzelschwab M, Michalke W, Wachter R,Fischer-Schliebs E, Ullrich C (2001) Immunolocalization of plasma-membrane H�-ATPase and tonoplast-type pyrophosphatase in theplasma membrane of the sieve element-companion cell complex in thestem of Ricinus communis L. Planta 213: 11–19

Lino B, Baizabal-Aguirre VM, Gonzales de la Vara LE (1998) The plasma-membrane H�-ATPase from beet root is inhibited by a calcium-dependent phosphorylation. Planta 204: 352–359

Little CHA, Pharis RP (1995) Hormonal control of radial and longitudinalgrowth in the tree stem. In B Gartner, ed, Plant Stems: Physiology andFunctional Morphology. Academic Press, San Diego, pp 281–319

Little CHA, Savidge RA (1987) The role of plant growth regulators in foresttree cambial growth. Plant Growth Regul 6: 137–169

Luthen H, Bidgon M, Bottger M (1990) Reexamination of the acid growththeory of auxin action. Plant Physiol 93: 931–939

Lutzelschwab M (1990) Biochemische und immunologische Charak-terisierung von Funktionen der Plasmamembran von Cucurbita pepo L.:Evidenz fur Heterogenitat der Plasmamembran. Dissertation Albert Lud-wigs Universitat, Freiburg, Germany

Maathuis FJM, Ichida AM, Sanders D, Schroeder JI (1997) Roles of higherplant K� channels. Plant Physiol 114: 1141–1149

MacRobbie EAC (1977) Functions of ion transport in plant cells and tissues.Int Rev Biochem 13: 211–247

Michelet B, Boutry M (1995) The plasma membrane H�-ATPase: a highlyregulated enzyme with multiple physiological functions. Plant Physiol108: 1–6

Morsomme P, Dambly S, Maudoux O, Boutry M (1998) Single pointmutations distributed in 10 soluble and membrane regions of the Nico-tiana plumbaginifolia plasma membrane PMA2 H�-ATPase activate the

Arend et al.



enzyme and modify the structure of the C-terminal region. J Biol Chem273: 34837–34842

Palmgren MG (1998) Proton gradients and plant growth: role of plasmamembrane H�-ATPase. Adv Bot Res 28: 2–70

Palmgren MG (2001) Plant plasma membrane H�-ATPases: powerhousesfor nutrient uptake. Annu Rev Plant Physiol Plant Mol Biol 52: 817–845

Rayle DL, Cleland RE (1992) The acid growth theory of auxin induced cellelongation is alive and well. Plant Physiol 99: 1271–1274

Sauter JJ, Kloth S (1986) Plasmodesmatal frequency and radial translocationrates in ray cell of poplar (Populus � canadensis Moench “robusta”).Planta 168: 377–380

Savidge RA (1996) Xylogenesis, genetic and environmental regulation: areview. Int Assoc Wood Anat J 17: 269–310

Savidge RA, Wareing PF (1981) Plant growth regulators and the differen-tiation of vascular elements. In JR Barnett, ed, Xylem Cell Development.Castle House Publishing, Tunbridge Wells, UK, 192–235

Serrano R (1989) Structure and function of plasma membrane ATPase.Annu Rev Plant Physiol Plant Mol Biol 40: 61–94

Shabala S, Newman I (1999) Light-induced changes in hydrogen, calcium,potassium, and chloride ion fluxes and concentrations from the meso-phyll and epidermal tissues of bean leaves: understanding the ionic basisof light-induced bioelectrogenesis. Plant Physiol 119: 1115–1124

Sundberg B, Uggla C, Tuominnen H (2000) Cambial growth and auxingradients. In R Savidge, J Barnett, R Napier, eds, Cell Molecular Biologyof Wood Formation. BIOS Scientific Publishers, Oxford, pp 169–188

Takeshige K, Shimmen T, Tazawa M (1986) Quantitative analysis of ATP-dependent H� efflux and pump current driven by an electrogenic pumpin Nitellopsis obutsa. Plant Cell Physiol 27: 337–348

Takeuchi Y, Kishimoto U, Ohkawa T, Kamiike N (1985) A kinetic analysisof the electrogenic pump of Chara corallina: II. Dependence of the pumpactivity on external pH. J Membr Biol 86: 17–26

Talbott LD, Ray PM, Roberts JKM (1988) Effect of indole acetic acid- andfusicoccin-stimulated proton extrusion on internal pH of pea internodecells. Plant Physiol 87: 211–216

Tuominen H, Puech L, Fink S, Sundberg B (1997) A radial gradient ofindole-3-acetic acid is related to secondary xylem development in hybridaspen. Plant Physiol 115: 577–585

Uggla C, Mellerowicz EJ, Sundberg B (1998) Indole-3-acetic acid controlscambial growth in Scots pine by positional signaling. Plant Physiol 117:113–121

van Bel AJE, Ehlers K (2000) Symplasmic organization of the transportphloem and the implications for photosynthate transfer to the cambi-um.In R Savidge, J Barnett, R Napier, eds, Cell and Molecular Biology ofWood Formation. BIOS Scientific Publishers, Oxford, pp 85–99

Villalba JM, Lutzelschwab M, Serrano R (1991) Immunocytolocalization ofplasma-membrane proton-ATPase in maize coleoptiles and enclosedleaves. Planta 185: 458–461

Wareing PF, Hanney CEA, Digby J (1964) The role of endogenous hormonesin cambial activity and xylem differentiation. In MH Zimmermann, ed, TheFormation of Wood in Forest Trees. Academic Press, New York, pp 323–344

Weisenseel MH, Becker HF, Ehlgotz JG (1992) Growth, gravitropism andendogenous ion currents of cress roots (Lepidium sativum L.): measurementsusing a novel three-dimensional recording probe. Plant Physiol 100: 16–25

Wodzicki TJ, Wodzicki AB (1980) Seasonal abscisic acid accumulation in stemand cambial region of Pinus sylvestris, and its contribution to the hypothesisof a late wood control system in conifers. Physiol Plant 48: 443–447




seasonal changes of plasma membrane h -atpase and ... · accumulated in cells of the cambial...

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