Plant Physiol. (1 997) 11 5: 1499-1 503

Specific Localization of the Respiratory Alternative Oxidase in Meristematic and Xylematic Tissues from Developing

Soybean Roots and Hypocotyls'

Mirna Hilal, Atilio Castagnaro, Hortensia Moreno, and Eddy M. Massa*

Departamento Bioquímica de Ia Nutrición, Instituto Superior de lnvestigaciones Biológicas, Consejo Nacional de lnvestigaciones Cientificas y Tecnológicas-Universidad Nacional de Tucumán, and Instituto de Química

Biológica Dr. Bernabé Bloj, Chacabuco 461, San Miguel de Tucumán, (4000) Argentina

We used tissue printing and specific immunostaining to examine the localization of the alternative oxidase (AOX) protein in corre- lation with measurements of AOX capacity. Selected root and hy- pocotyl regions were analyzed during the first 14 d of growth. It i s shown that AOX protein is localized in the apical meristem and in developing xylem. The temporal pattern of expression is coincident with the evolution of AOX capacity. Data suggest that AOX expres- sion is linked to xylem differentiation. Since heat is a major product of the alternative pathway, we speculate that thermogenesis i s implicated in morphogenesis.

In higher plants the mitochondrial electron transport is branched and there are two terminal oxidases: the usual Cyt oxidase and a KCN-insensitive AOX, which is not coup\led to ATP production (Moore and Siedow, 1991; Siedow and Umbach, 1995). The capacity for AOX respira- tion has been shown to depend on the organ, developmen- tal stage, growth conditions, and treatments (Bingham and Farrar, 1989; Obenland et al., 1990; Kearns et al., 1992; Vanlerberghe and McIntosh, 1992a). This capacity for AOX respiration, measured as the O, consumption blocked by the addition of an AOX inhibitor in the presence of an inhibitor of the Cyt path, correlates with the amount of AOX protein as determined by western-blot analysis (Hiser and McIntosh, 1990; Obenland et al., 1990; Kearns et al., 1992; Rhoads and McIntosh, 1992; Vanlerberghe and McIn- tosh, 1992a, 1992b).

No information is available concerning the spatial local- ization or possible tissue specificity of AOX protein within the root or the hypocotyl. In floral buds high levels of AOX protein were found in the younger tapetal cells of fertile anthers (Conley and Hanson, 1994), and this pattern of expression was considered possible evidence supporting the hypothesis that AOX is needed for NAD+ regeneration in tissues with high biosynthetic activity.

In this study we used tissue printing and specific immu- nostaining to examine the expression and localization of AOX protein in developing soybean (GZycine mux L.) roots

This work was partially supported by the Consejo de Investi-

* Corresponding author; e-mail massaQinsibio.unt.edu.ar; fax gaciones de la Universidad Nacional de Tucumán.

54- 81-24- 8025.

and hypocotyls, in correlation with measurements of AOX capacity. Selected root and hypocotyl regions were ana- lyzed during the first 14 d of growth. To our knowledge, this is the first report of a comparison of the temporal evolution of AOX capacity in the apical meristem and the differentiation zone of primary and secondary roots, since previous studies were performed at the whole-organ level. It is shown that AOX protein is specifically expressed in meristematic and xylematic tissues. The temporal pattern of AOX expression, which coincides with the temporal evolution of AOX capacity, appears to be linked to devel- opmental processes associated with xylem differentiation. The information presented here could serve as a basis for studies aimed at unveiling the still-unknown function of this enzyme.

MATERIALS A N D METHODS

Plant Growth

Soybean (Glycine max L.) seeds of variety UFV-8 were germinated at 28°C in sterile sand moistened with tap water for 3 d. Then, the seedlings were transferred to hydroponic culture in 25% Hoagland medium and grown at 28°C under greenhouse conditions, until harvested at different developmental stages between d 3 and 14 (the day of sowing was designated d O). The nutrient medium was renewed every 3 d.

Selection of the Root Regions Studied

Three different root regions were studied during the growth period between d 3 and 14: the differentiation zone of the primary root, the differentiation zone of secondary roots, and the root apical meristem. In the differentiation zone of the primary root, a segment about 4 mm long was selected at d 3 of growth, and the same segment was then studied in the older seedlings. This was checked by label- ing gently with a fine pen (Sharp et al., 1988) the root segment in 3-d-old seedlings, grown in parallel, and ob- serving the localization of the labeled zone during the following days of development. It was found that the 4-

Abbreviations: AOX, alternative oxidase; SHAM, salicylhydrox- amic acid.

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mm-long segment remained almost in the middle of theprimary root during the growth period studied. Since theorganization of cells in lateral roots is similar to that of theprimary root (Dolan et al., 1993), the differentiation zone ofthe secondary roots was studied by selecting a 2- to 3-mm-long segment in the middle. The apical meristem of pri-mary and secondary roots was identified by the Feulgen'sstain (Johausen, 1940) in seedlings grown in parallel withthose used for each determination. The meristem occupiedabout 0.5 to 1 mm in the root apice.

Determination of AOX Capacity

In this study AOX "capacity" was measured, as definedby Vanlerberghe and Mclntosh (1996): SHAM-sensitive O2uptake in the presence of KCN. This measurement does notreflect the actual AOX activity during respiration in theuninhibited condition, because the addition of inhibitorscan modify the partitioning of electron flux over the Cytpathway and the alternative pathway (Millar et al., 1995;Day et al., 1996). AOX capacity shows only the activityunder the given set of conditions used (Wagner and Krab,1995).

For the assay of AOX capacity, each selected root zonewas cross-sliced (about 1 mm thick). For each determina-tion, samples from several seedlings were pooled to obtainapproximately 30 mg of the apical meristem or 50 to 150mg of the differentiation zone. During this procedure carewas taken to maintain the temperature at about 28°C (equalto the growth temperature) and to avoid tissue drying bycovering the sample with a layer of Hoagland medium.

The O2 consumption of fresh slices from each root zone,suspended in 2 mL of filtered Hoagland medium at 28°C,was measured polarographically with a Clark electrode(Yellow Springs Instrument Co., Yellow Springs, OH) andrecorded in a Gilson oxygraph (Gilson Medical Electronics,Inc., Middleton, WI), using 1 mM KCN as the inhibitor ofthe Cyt pathway and 3 mM SHAM as the inhibitor of thealternative oxidase. These concentrations of KCN and

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V 0.3cE 0.2

TOTAL

AOX

RESIDUAL

Figure 1. Soybean seedlings at d 3 (left) and d 8 (right) of growth.X0.3.

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0.4

0.3

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0.02 4 6 8 10 12

DAYS

Figure 2. Temporal evolution of total O2 uptake (top), AOX capacity(middle), and residual respiration (bottom) in different zones of de-veloping roots. •, Differentiation zone of the primary root; O, dif-ferentiation zone of secondary roots; and A, apical meristem fromprimary and secondary roots. Each value is the mean ± so of three tofive separate measurements. Fw, Fresh weight.

SHAM had no apparent side effect in our system andproduced maximal inhibition, as indicated by titrationcurves using different concentrations of each inhibitor inthe presence and absence of the other. Steady rates ofrespiratory O, consumption were determined after about20 min under the assay conditions. AOX capacity wasdetermined by subtracting the rate of O2 uptake in thepresence of both KCN and SHAM from the rate of O2uptake in the presence of only KCN. Data are expressed ona tissue fresh weight basis.

Tissue-Print Immunoblots

Tissue printing was done as described by Cassab andVarner (1987). The nitrocellulose paper was previouslysoaked in 0.2 M CaCl2 for 15 min and air dried. The selectedsegments from primary roots and hypocotyls were in-cluded in 3% agarose and then sliced with a razor blade.Slices (1-2 mm thick) were pressed onto the nitrocellulosepaper for about 5 s. The tissue prints were air dried andtreated for amido black staining of total protein or for

Tissue-Specific Localization of Alternative Oxidase 1501

detection of AOX protein. The procedure described byVarner and Ye (1995) to detect proteins in tissue prints withspecific antibodies was followed, using a 1:100 dilution ofprimary monoclonal antibody against AOX (Elthon et a\.,1989) and a 1:1000 dilution of goat anti-mouse polyvalentsecondary antibody conjugated to alkaline phosphatase(Sigma). For each root or hypocotyl region studied, a set offour pieces of the nitrocellulose paper, each one containingsix tissue prints, was processed together with another iden-tical set used as a negative control for which incubationwith the anti-AOX antibody was omitted. This immuno-staining was performed at least three times, using samplesfrom different lots of seedlings. In all of the immunoblotsanalyzed, the localization of AOX protein was as in Figures3 to 5, and the negative controls presented no staining, asshown in Figure 4C.

RESULTS

Figure 1 shows the appearance of 3- and 8-d-old soybeanseedlings. The differentiation zone and the apical meristemwere selected (see "Materials and Methods") in both theprimary and secondary roots, whereas in the hypocotylsthe middle region was studied.

Temporal and Spatial Patterns of AOX Capacity inDeveloping Roots

The temporal evolution of AOX capacity in different rootzones is shown in the middle panel of Figure 2. In thedifferentiation zone of primary roots AOX capacity wasmaximal at d 3 to 4 of development; thereafter, AOX ca-pacity gradually decreased to reach negligible levels at d 8of plant growth. Since the secondary roots began to emergein 4-d-old seedlings, and subsequently new secondary rootscontinuously appeared, a large population of lateral roots in

different developmental stages was present in each seedlingduring the period following d 4 of growth (Fig. 1). The olderand youngest secondary roots from seedlings at a given agehad similar levels of AOX capacity (not shown), and tem-poral evolution was coincident with that in the primary root(Fig. 2). At d 4, AOX capacity in the secondary roots waslower than in the primary root. In the root apical meristemAOX capacity was higher, on a fresh weight basis, than inthe root differentiation zone (Fig. 2), and although it de-creased over the period studied, an appreciable level waspresent between d 8 and 12 of growth when AOX capacitywas already negligible in the maturation region.

Total (uninhibited) O2 uptake and residual (in the pres-ence of KCN plus SHAM) respiration at each age areshown in the top and bottom panels, respectively, of Figure2. The difference between total respiration and residualrespiration plus AOX capacity in Figure 2 is the KCN-sensitive O2 uptake. As can be seen, total O2 uptake andresidual respiration in the apical meristem were consider-ably higher than in the differentiation zone of primary andsecondary roots. The proportion of AOX capacity relativeto total respiration at each age was not the same in thedifferent root zones (Fig. 2). In the differentiation zone ofthe primary root, at d 4 AOX capacity was about 84% of thetotal O2 uptake, whereas after d 8 AOX capacity was neg-ligible and only a low total respiration sensitive to KCNwas observed. On the other hand, in the apical meristemAOX capacity remained about 25% of the total O2 uptakeduring the period from d 3 to 12.

Localization of AOX in Developing Roots byTissue-Print Immunoblots

To determine both the expression and the tissue distri-bution of AOX in young roots, tissue prints were preparedand either stained for total protein or immunostained tolocalize AOX protein. In Figure 3, A and C, the anatomy of

Figure 3. Tissue prints of cross-sections fromthe differentiation zone of the primary root at d3 (A and B) and d 8 (C and D) of growth. A andC, Amido black stains of total protein; B and D,immunostains specific for AOX. x, Xylem; p,phloem; and c, cortex. Bars = 250 ju,m.

1502 Hilal et al. Plant Physiol. Vol. 115, 1997

staining (not shown). As is shown in Figure 3B, at d 3 ofgrowth the anti-AOX monoclonal antibody strongly re-acted with the xylematic tissue, whereas none of the othertissues immunostained above the background. At d 8, how-ever, the tissue prints did not show any immunostaining(Fig. 3D). Therefore, in the root differentiation zone AOXshows xylem-specific expression with a temporal patternthat correlates well with the evolution of AOX capacityshown in Figure 2.

Tissue prints of longitudinal sections from the root apicalmeristem at d 3 of growth were stained for total protein(Fig. 4A) to illustrate the anatomy of this region or immu-nostained to localize AOX (Fig. 4B). A blank of the immu-nostaining omitting the anti-AOX antibody is shown inFigure 4C. As expected from the results shown in Figure 2,AOX was highly expressed in the root apical meristem (Fig.4B). It can be seen that AOX protein was distributed in allof the meristematic tissue. i

Figure 4. Tissue prints of longitudinal sections from the apical mer-istem of primary roots at d 3 of development. A, Amido black stain oftotal protein. B, Immunostain specific for AOX. C, Blank of theimmunostain omitting the anti-AOX antibody. Bar = 250 ^m; allpanels are shown at the same magnification.

the root differentiation zone at d 3 and 8 of development isillustrated by cross-sections stained with amido black. Vas-cular bundles, cortex cells, and the epidermal layer areclearly visible. In Figure 3, B and D, the localization of AOXis shown by specific immunostains. Blanks of the immu-nostains omitting the anti-AOX antibody presented no

Figure 5. Tissue prints of cross-sections fromthe hypocotyl middle region at d 3 (A and B) andd 8 (C and D) of growth. A and C, Amido blackstains of total protein. B and D, Immunostainsspecific for AOX. x, Xylem; p, phloem; and c,cortex. Bars = 250 /AITI.

Expression and Localization of AOX inDeveloping Hypocotyls

Tissue prints in Figure 5 show tissue distribution of AOXin young hypocotyls. Blanks of the immunostains omittingthe anti-AOX antibody presented no staining (not shown).As shown in Figure 5B, at d 3 of development AOX proteinwas abundant in the primary xylem, whereas other tissuesdid not immunostain. At d 8 of development (Fig. 5D), nospecific immunostaining was observed in the tissue prints.Therefore, the pattern of temporal evolution and tissuelocalization of AOX in the hypocotyl is similar to that in theroot differentiation zone.

DISCUSSION

Our data show that AOX capacity is a good reporter ofthe amount of AOX protein present in the tissues. Thus,

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Tissue-Specific Localization of Alternative Oxidase 1503

this parameter is useful for routine screening of the protein in different samples, even though it is not an estimation of the degree of functioning of the alternative pathway in the uninhibited condition. The similar pattern of AOX capacity and AOX expression shows that the AOX protein transfers electrons to O, under conditions in which the Cyt pathway is impeded by KCN. In this study AOX capacity measure- ments allowed us to decide when and where to examine AOX localization by tissue print immunoblots.

The respiratory parameters at each age are not identical in the different root regions, as shown in this paper. Thus, the root should not be considered a homogeneous system in biochemical studies of the respiratory pathways. This is an aspect that has not been noticed in previous works performed with the whole root.

The temporal evolution of AOX in this and other systems (Lennon et al., 1995) indicates that this protein is respond- ing to some developmental cues, the identities of which are unknown. The specific expression of AOX in xylematic tissues and its coordinated decline during root and hypo- cotyl development, shown in the present report, suggest a role for this enzyme linked to xylem differentiation. The implication of alternative respiration in morphogenesis has been recently illustrated by the effect that inhibition of this pathway has on Dictyostelium development. In this mold, inhibition of the alternative respiration leads to alteration in cell-type proportioning correlated with induction of the expression of specific genes and simultaneous inhibition of the expression of others (Matsuyama and Maeda, 1995). This suggests that AOX may be involved in the regulation of cell-type differentiation.

Since heat is a major product of the alternative pathway (Meeuse, 1975; Ordentlich et al., 1991; Van Der Straeten et al., 1995), we speculate that localized warming at precise times might be important in the promotion and/or coor- dination of specific processes during morphogenesis. A small or moderate temperature difference between deter- mined cells with high AOX activity and the surrounding tissues or medium, rather than a high global temperature elevation, could mediate some event in developing tissues. Although our proposal is clearly hypothetical, it is a work- ing idea that we hope will stimulate further investigation.

ACKNOWLEDGMENTS

We thank Dr. Thomas E. Elthon (University of Nebraska, Lincoln) for providing the anti-AOX monoclonal antibody, Dr. A. Molina (Escuela Técnica Superior de Ingenieros Agrónomos, Madrid, Spain) for suggestions about the tissue printing technique, and G. Ponessa (Fundación Miguel Lillo, Tucumdn, Argentina) for helpful comments concerning the manuscript.

Received March 31, 1997; accepted August 26, 1997. Copyright Clearance Center: 0032-0889/97/115/1499/05.

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