(w/v) p-phenylenediamine, 0.2% (w/v) pyrocatechol, and. 0.09% (w/v) hydrogen peroxide in 0.1 M Tris buffer (pH 7.5). (12) for 20 min and photographed.
Received for publication September 18, 1991 Accepted February 15, 1992
Plant Physiol. (1992) 99, 879-885 0032-0889/92/99/0879/07/$01 .00/0
Peroxidase Activity II.
the Leaf Elongation Zone of Tall Fescue' in
Spatial Distribution of Apoplastic Peroxidase Activity in Genotypes Differing'in Length of the Elongation Zone Jennifer W. MacAdam*2, Robert E. Sharp, and Curtis 1. Nelson Department of Agronomy, University of Missouri, Columbia, Missouri 65211 ABSTRACT
ity extracted from homogenized tissue segments was wellcorrelated with decrease in elongation rate and cessation of elongation. Length of the elongation zone differed by approximately 25% between the two genotypes, and peroxidase activity increased similarly with respect to stage of development in each genotype rather than with distance from the ligule. Histochemical localization of peroxidase in transverse leaf blade sections also demonstrated an increase in cell wall peroxidase activity as cells were displaced through the elongation zone. Taken together, the biochemical and histochemical results suggested that secretion of peroxidase into the walls of longitudinally expanding tissues preceded cessation
Previous work suggested that cell wall peroxidase activity increased as cells were displaced through the elongation zone in leaf blades of tall fescue (Festuca arundinacea Schreb.). In this study, two genotypes that differ in length of the elongation zone were used to examine the relationship between peroxidase activity in apoplastic fluid of intact leaf blade segments and the spatial distribution of leaf growth. Apoplastic fluid was extracted by vacuum infiltration and centrifugation, and peroxidase activity was assayed spectrophotometrically. Isoelectric focusing was used to characterize the isoforms of apoplastic peroxidase within the region of elongation and in the region of secondary cell wall deposition, which is distal to the elongation zone. A striking correlation was found in each genotype between both the location and timing of increase in apoplastic peroxidase activity and the onset of growth deceleration. Only cationic isoforms of apoplastic peroxidase could be identified in the elongation zone, whereas additional anionic isoforms appeared in the region of secondary cell wall deposition. We conclude that cessation of elongation growth in tall fescue leaf blades is likely to be related to the secretion of cationic isoforms of peroxidase into the cell wall.
of elongation. Mader et al. (19) suggested that at least some ionically bound peroxidase activity may be an artifact of homogenization. Therefore, the intent of the present study was to test directly the hypothesis that an increase in cell wall peroxidase activity preceded cessation of elongation, by assaying apoplastic fluid extracted from segments of the leaf blade elongation zone. In studies of epicotyl and hypocotyl elongation, increase in activity of anionic isozymes of peroxidase has been associated with cessation of elongation (8, 10). Therefore, the peroxidase isoforms in the apoplastic fluid were also characterized. As in the previous paper (17), low-LER3 and high-LER genotypes of tall fescue, which are known to differ in length of the elongation zone, were used so that changes in peroxidase activity could be related with greater certainty to the spatial distribution of growth.
Cessation of elongation occurs rapidly as cells are displaced through the distal half of the elongation zone of tall fescue (Festuca arundinacea Schreb.) leaf blades (18). The wall tightening that is thought to decrease the rate of cell expansion may result from formation of cross-linkages among cell wall polymers. Many of these covalent bonds occur by oxidation/ reduction reactions catalyzed by peroxidase (7). In the companion paper (17), we examined the spatial and temporal relationships between ionically bound peroxidase activity, which has previously been equated with cell wall activity (8, 10, 21), and the profile of elongation growth in two genotypes of tall fescue differing in length of the elongation zone. Increase in ionically bound peroxidase activ-
MATERIALS AND METHODS Plant Material and Growth Measurements Vegetative tillers of a low-LER or high-LER genotype (13) of tall fescue (Festuca arundinacea Schreb.) were transplanted into a commercial potting soil (12 tillers per 25-cm-diameter pot) and grown in a controlled-environment chamber at a mean photon flux density of 500 ,gmol mr2 s-' PAR at canopy height, a photoperiod of 14 h, constant day/night temperature of 200C, and RH of 80%. Pots were watered with 150 mL tap water four times per day, and fertilized four times
'
Supported in part by the Food for the 21st Century Program and the Interdisciplinary Plant Group, University of Missouri, Columbia. Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 11,483. 2 Present address: Department of Plants, Soils, and Biometeorology, Utah State University, Logan, UT 84322-4820.
3Abbreviations: LER, leaf elongation rate(s); MDH, malate dehydrogenase. 879
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per week with 100 mL 26 mm ammonium nitrate, 2 mM dibasic potassium phosphate, 3 mm monobasic potassium phosphate, and 1 mm potassium sulfate. Plants were acclimated to chamber conditions for a minimum of 6 weeks before sampling began. LER were calculated from the mean change in length of three leaves per pot measured daily for 4 d. Elongation zone length and spatial and temporal patterns of relative elemental elongation rate within the elongation zone were calculated from Formvar replicas of epidermal cell length profiles as previously described (17). Mean cell lengths were calculated from lengths of 10 (low-LER) or 20 (high-LER) cells at 5-mm intervals from two (low-LER) or three (high-LER) leaves. Other details of plant culture and growth analysis were as described by MacAdam et al. (17).
Apoplastic Peroxidase Activity
the infiltration periods were increased to 30 min. Peroxidase activity did not decrease with successive extractions at any of the infiltration times, however. This suggested that the cell walls were only partially extracted with each infiltration. The fact that the peroxidase activity extracted with three 30-min infiltrations was no higher than that extracted with three 15min infiltrations argues against the possibility that active secretion of peroxidase occurred during the longer infiltration period. The summed MDH activity of the three successive extractions was similar at all three infiltration times (Table I), and was only 4 to 7% of the activity extracted by homogenization following extraction. This indicates that infiltrations of at least 30-min duration did not cause leakage from the protoplast. Because apoplastic peroxidase activity was no greater with 30-min than with 15-min infiltrations, 15 min was chosen as the optimal duration of infiltration for extraction of apoplastic fluid from the elongation zone.
Effect of Infiltration Time
Apoplastic fluid was extracted by vacuum infiltration and low-speed centrifugation of leaf blade segments using a modification of the method described by Terry and Bonner (24); details of extraction procedures are described in the following section. The duration of vacuum infiltration used to extract apoplastic fluid from tissue segments has commonly ranged from 30 s to 4 min (4, 14). However, Li et al. (15) reported that in primary leaves of barley seedlings, peroxidase and other cell wall enzymes were continually released during 36 min of infiltration before a significant increase in activity of the cytoplasmic marker glucose-6-phosphate dehydrogenase was seen. Therefore, three infiltration times were compared to determine an optimal duration of infiltration. Leaf blades of the low-LER genotype were used, in which the elongation zone was located in the basal 25 to 30 mm (see 'Results'). Fifteen 7-mm-long segments located at 5 to 12 mm above the ligule were vacuum infiltrated for 5, 15, or 30 min. Segments were infiltrated for a further 5 min without vacuum to allow maximal influx of buffer solution following removal of intercellular air, then centrifuged for 15 min. Infiltration and centrifugation were repeated twice, and the experiment had three replications. Each successive extraction of apoplastic fluid was assayed for peroxidase and MDH activities as described below; data are shown in Table 1 as the sum of the three successive extractions. More peroxidase activity was extracted when segments were infiltrated for 15-min periods compared with 5-min periods, but no additional increase in activity was seen when
Extraction of Apoplastic Fluid
To determine the spatial distribution of apoplastic peroxidase activity, five sequential 5-mm-long segments from 0 through 25 mm above the ligule, plus additional 5-mm-long segments located at 35 to 40 mm, 50 to 55 mm, and 65 to 70 mm (in the region of secondary cell wall deposition) were collected from 15 (low-LER) or 7 (high-LER) elongating leaf blades. Segments were oriented vertically in filter baskets of tared microfilterfuge tubes (Rainin Instrument Co., Wobum, MA) from which the filter membrane had been removed. After determination of fresh weight, the baskets and leaf segments were placed in a scintillation vial where they were held in place by a stainless steel wire screen molded to fit inside the vial above the basket. Five milliliters of ice-cold, degassed 0.01 M Mes buffer (pH 5.5) containing 0.2 M KCI was added to the vial, covering the tissue, and segments were vacuum infiltrated for 15 min and for a further 5 min without vacuum. The ionic strength of the infiltration buffer was chosen to prevent plasmolysis of the cells. The osmotic potential of the leaf elongation zone in tall fescue is approximately equivalent to that of 0.2 M KCl (W.G. Spollen, C.J. Nelson, unpublished data), and this ionic strength is in the range recommended for extraction of ionically bound compounds from intact tissues by infiltration and centrifugation (7). Low temperature and a potassium salt were used to avoid stimulation of active secretion of peroxidase into the apoplast, which has been shown to require extracellular Ca2" (23). Filter baskets were removed from the vials, drained, and
Table I. Effect of Varying Infiltration Time on Apoplastic Peroxidase and MDH Activities from Low-LER Leaf Blades For each infiltration time, 7-mm-long segments located at 5 to 12 mm above the ligule were extracted three times by vacuum infiltration and centrifugation. Apoplastic enzyme activities are the sum of the three successive extractions. Data are means ± SD of three replicate experiments.
5
0.012 ± 0.006
Peroxidase Activity
MDH Activity
Infiltration time (min) 15 AA470 min- mm-' 0.071 ± 0.025
Infiltration time (min) 30
0.068 ± 0.027
5
15
30
0.092 ± 0.022
AA340 min-1 mm-' 0.095 ± 0.009
0.067 ± 0.010
881
APOPLASTIC PEROXIDASE AND DISTRIBUTION OF LEAF GROWTH excess buffer was blotted away from the segments through the perforated base of the baskets. Baskets with segments were transferred to microfuge tubes and centrifuged for 15 min at 1000g. Each 5-mm-long segment yielded about 2 ,L of apoplastic fluid. All steps were carried out on ice or in a cold room at 40C. Infiltration and centrifugation were then repeated three times, and the experiment had three replications. Apoplastic fluid from the four successive extractions was pooled, and each replication was assayed for peroxidase and MDH activities as described below. Apoplastic fluid from the replications was then pooled in Centricon-10 Microconcentrators (Amicon, Danvers, MA), the extract was desalted to less than 0.01 M KCl for isoelectric focusing, and the volume was reduced to 50 ML. The desalted extract was again assayed for peroxidase activity, and soluble protein was determined using a Bradford (3) assay.
RESULTS Leaf Growth Distribution The spatial distribution of relative elemental elongation rates in leaf blades of the two genotypes (Fig. 1) was calculated from epidermal cell length profiles (Fig. 1, insets). As in the preceding paper (17), elongation zone lengths were 25 to 30 mm for the low-LER and 35 to 40 mm for the high-LER genotype. Maximal elemental elongation rates occurred in the same locations as in the previous study (17), but were lower in this study, as were overall LER (Fig. 1). These differences may have resulted from longer sheath lengths associated with higher tiller density at sampling in this study (26).
Spatial Distribution of Apoplastic Peroxidase Activity The spatial distribution of apoplastic peroxidase activity in the two genotypes is shown in Figure 2. Data are expressed
Enzyme Assays
Peroxidase activity in apoplastic fluid was assayed in a M potassium phosphate buffer (pH 6.0), 0.0167 mL of 0.2 M guaiacol, and 0.0133 mL of 0.03 M hydrogen peroxide (17). Reaction was initiated by adding 0.01 mL of apoplastic fluid, and increase in A470 min'1 was measured (4, 23). MDH activity was assayed in a mixture containing 1 mL 0.1 M potassium phosphate buffer (pH 7.5), 0.033 mL oxalacetic acid in 0.095 M potassium phosphate buffer (pH 8.3), and 0.0167 mL 1% (w/v) NADH in 1% (w/v) sodium carbonate. Reaction was initiated by adding 0.01 mL of apoplastic fluid, and decrease in A340 min-' was measured (1). Both enzymes were assayed at room temperature.
c0.2 '= 0 2 v
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0.02
20
40
60
Distance (mm)
-
0.01
.2
Low LER
i
.°
4
0.0o 0.04 B
Insoluble Dry Weight To estimate the content of cell wall material, insoluble dry weight of the segments was determined. Following infiltration and centrifugation, segments were extracted for two 15min periods in 5 to 7 mL of 95% ethanol at 800C, and for three 20-min periods in 5 to 7 mL of distilled water at 90 to 1000C. Insoluble leaf blade material was dried at 800C for approximately 2 d before weighing.
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0.01 Isoelectric Focusing
Aliquots (5 ,L) of the desalted apoplastic fluid were applied across the center of hand-cast, nondenaturing isoelectric focusing gels containing 1% (w/v) agarose (FMC BioProducts, Rockland, ME), 12% (w/v) sorbitol, and 6.7% (v/v) carrier ampholytes, pH 3.5 to 9.5 (Pharmacia LKB, Piscataway, NJ). During focusing, which was carried out at 4 W constant power over approximately 2 h, the gels were maintained at 100C. Gels were stained for peroxidase activity with 0.1% (w/v) p-phenylenediamine, 0.2% (w/v) pyrocatechol, and 0.09% (w/v) hydrogen peroxide in 0.1 M Tris buffer (pH 7.5) (12) for 20 min and photographed.
High LER (I 0.00 0
20
40
60
80
Distance Above Ligule (mm) Figure 1. Spatial distribution of relative elemental elongation rates in leaf blades of the low-LER (A) and high-LER (B) genotypes. Data were calculated from the first derivative of an equation for displacement velocity against distance above the ligule, derived from profiles of epidermal cell lengths. LER were 17 ± 3.4 mm d-l for the low-LER and 27 ± 0.9 mm d-l for the high-LER genotype (± SD, n = 3). The insets show intercostal abaxial epidermal cell lengths in leaf blades. Error bars at each location represent ± SE of means of 10 to 20 observations from two (low-LER) or three (high-LER) leaves.
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slowly than water influx (18, 22). Beyond the elongation zone, insoluble dry weight increased with secondary cell wall deposition (18). Peroxidase activity of the desalted apoplastic fluid used for characterization of peroxidase isoforms was lower at most locations than that of the crude apoplastic extract; however, the overall patterns of activity remained similar (Fig. 2, A and B). In both genotypes, the pattern of change in soluble protein content of the desalted apoplastic fluid with distance from the ligule was similar to the profile of change in peroxidase activity (Fig. 3). Both peroxidase activity and protein content in the apoplastic fluid from the low-LER genotype were approximately 40% that of the high-LER genotype, causing the specific activity of apoplastic peroxidase to be similar in the two genotypes at the location of maximal activity per mg insoluble dry weight. Comparison of data in Figure 3 with the histochemical localization of cell wall peroxidase activity presented in the previous paper (17) argues against the possibility that the pattern of apoplastic peroxidase activity resulted from differential extractability due to variation in wall structure within the elongation zone. Staining intensity was very low near the
10
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Figure 2. Spatial distribution of apoplastic peroxidase activity in crude (solid lines) or desalted (dashed lines) extracts from leaf blades of the low-LER (A) and high-LER (B) genotypes as a function of insoluble dry weight. Five-millimeter-long segments were infiltrated for 15 min under vacuum and 5 min without vacuum, and centrifuged for 15 min at 1000g. Segments were extracted four times, and successive crude extracts were pooled before assay for peroxidase activity. The experiment had three replications; error bars for crude extracts represent ± SD. Extracts from the three replications were pooled prior to desalting. The insets show dry weight per mm of leaf blades following extraction with 95% ethanol and water. Error bars represent ± SD (n = 3).
as a function of insoluble dry weight to approximate the enzyme activity per unit of cell wall. Almost no peroxidase activity was detectable in apoplastic fluid from the proximal one-third of the elongation zone of either genotype (Fig. 2, A and B). As cells were displaced through the distal twothirds of the elongation zone, however, activity increased sharply and then decreased. Peroxidase activity in tissue displaced beyond the elongation zone continued to decrease with distance. Although spatial pattems were similar with respect to stage of development, maximal peroxidase activity in leaves of the low-LER genotype was only about 40% that of the high-LER genotype. Insoluble dry weight per mm leaf length (Fig. 2, insets) decreased with distance through the elongation zone because deposition of dry weight during elongation occurs more
3
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Figure 3. Spatial distribution of apoplastic peroxidase activity (dashed lines) and soluble protein content (solid lines) of desalted and concentrated extracts from leaf blades of the low-LER (A) and high-LER (B) genotypes. Desalted peroxidase activity data are from Figure 2. The insets show spatial distribution of apoplastic MDH activity in the same crude extracts assayed for peroxidase activity in Figure 2 (AA340 min-' mg-1 insoluble dry weight). Error bars represent ± SD.
APOPLASTIC PEROXIDASE AND DISTRIBUTION OF LEAF GROWTH
ligule in both genotypes, increased to its highest level at 15 and 25 mm in the low-LER and high-LER genotypes, respectively, and decreased in succeeding sections. Thus, very similar pattems of change in cell wall peroxidase activity were obtained using the two techniques. The spatial distribution of apoplastic MDH activity (Fig. 3, insets) was dissimilar to the patterns of peroxidase activity and soluble protein content (Fig. 3). In both genotypes, MDH activity was highest at the base of the elongation zone and decreased with distance above the ligule. Therefore, it is also unlikely that the increased peroxidase activity in the distal two-thirds of the elongation zone resulted from differential protoplast leakage in this region. Comparison of the spatial distributions of growth in Figure 1 and apoplastic peroxidase activity in Figure 2 suggests that cessation of growth was related to increase in cell wall peroxidase activity, but the rapidity with which cessation of cell elongation occurs (18) makes an evaluation of the temporal relationship of increase in apoplastic peroxidase activity and decline in elongation rate crucial to our hypothesis. Therefore, data for crude apoplastic peroxidase activity and relative elemental elongation rate for each genotype were replotted as a function of time before growth cessation (Fig. 4, A and B). Elongation rate began to decline in both genotypes just after apoplastic peroxidase activity began to increase. Comparison of Figure 3 from the previous paper (17) with Figure 4 from this paper indicates that the increases in activity of ionically bound and apoplastic peroxidase as cells are displaced through the elongation zone are closely related.
883
both genotypes, even though elongation zone lengths differed by 10 mm. On a temporal basis, increase in both ionically bound (17) and apoplastic peroxidase activities began immediately prior to the onset of growth deceleration in each genotype. Goldberg et al. (11), working with etiolated mung bean hypocotyls, also compared ionically bound peroxidase activity from homogenized tissue with apoplastic peroxidase activity extracted by vacuum infiltration and centrifugation. Although ionically bound activity increased along the growth gradient with distance from the hook, apoplastic peroxidase activity extracted with a buffered salt solution decreased, in contrast with our results. The only apoplastic peroxidase fraction from mung bean hypocotyls for which isozyme content was illustrated was that extracted with a low-ionicstrength buffer, and the only change was in a slow-moving anionic band that also decreased in activity with distance from the hook. In tall fescue leaf blades, cationic isoforms of peroxidase appeared in the distal half of the elongation zone as the growth rate decelerated, whether extracted with low or high
0
U)
a
E m
Apoplastic Peroxidase Isoforms
E 0 m
as
In both genotypes, only cationic isoforms of apoplastic peroxidase were detected in the region corresponding to increase in total apoplastic activity (Fig. 5, A and B). Five isoforms were present in each genotype, with four of the five being common to both. In apoplastic fluid from segments in the region of secondary cell wall deposition, beyond the elongation zone, two (high-LER) or three (low-LER) anionic isoforms of peroxidase were also detected. Because apoplastic fluid from each location was concentrated to a constant volume and an aliquot of equal volume was applied to each lane of the isoelectric focusing gels, activities of isoforms in Figure 5 are comparable on the basis of activity per segment. Spatial patterns are not directly comparable to those for apoplastic activity per mg insoluble dry weight (Figs. 2 and 3) because insoluble dry weight per segment varied with distance above the ligule (Fig. 2, insets).
.c4
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0.4 45
c
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DISCUSSION
0 0 3( -60 -30 Time Before Growth Cessation (h)
The spatial distribution of ionically bound peroxidase activity in the leaf elongation zone of tall fescue reported in the previous paper (17) suggested that an increase in cell wall peroxidase activity preceded cessation of cell elongation. Results from the present study confirm that hypothesis. Apoplastic peroxidase activity increased sharply in the region where the relative elemental elongation rate peaked and began to decrease. The same pattem of increase in peroxidase activity followed by cessation of cell elongation occurred in
Figure 4. Relative elemental elongation rates (dashed lines, from Fig. 1) and apoplastic peroxidase activity in crude extracts (solid lines, from Fig. 2) from leaf blades of the low-LER (A) and high-LER (B) genotypes. Data were replotted as a function of time before growth cessation, and represent values associated with a particular tissue element as it was displaced through the elongation zone. In both genotypes, increase in apoplastic peroxidase activity immediately preceded deceleration and cessation of cell elongation. Data for the low-LER genotype are plotted on a longer time scale than those for the high-LER genotype.
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MACADAM ET AL.
Plant Physiol. Vol. 99, 1992
Figure 5. Spatial distribution of apoplastic peroxidase isozymes in leaf blades of the low-LER (A) and high-LER (B) genotypes. Apoplastic fluid from 5-mm-long leaf blade segments was desalted and concentrated to 50 AL, and 5 ,uL was applied to each lane of the isoelectric focusing gel. Peroxidase activity was stained using pphenylenediamine and pyrocatechol. Cell elongation occurred over the basal 25 to 30 mm and 35 to 40 mm of the leaf blade for the low-LER and high-LER genotypes, respectively (Fig. 2), and was followed by secondary (20) cell wall deposition.
isdiyrsie because. a,,peoiase isozymewith a isolcn pon ofi 4.6 fro cutue tomt cells formed
to. for. B.,:
ionic strength buffers (data not shown). Anionic isoforms only appeared later, in the region where secondary cell wall deposition and signification occur (16). A temporal sequence of peroxidase secretion, similar to that observed in this study, occurs in response to developmental phenomena such as root formation or flower initiation, and as a response to mechanical stress or wounding (9). In those cases, cationic isoperoxidases are secreted rapidly, and are followed more slowly by secretion of anionic isoperoxidases. Decrease in cell wall extensibility via formation of covalent bonds among wall polymers would likely occur before cessation of elongation and, therefore, may be associated with the cationic peroxidase isoforms detected in apoplastic fluid from the distal half of the elongation zone. Positively charged cationic isozymes could be attracted to the net negatively charged cell wall and function in the oxidation of ferulic acid, leading to formation of diferulic acid and covalent binding of hemicelluloses and pectins into the cell wall (5, 6). Cationic peroxidases are also thought to catabolize IAA in the cell wall compartment, decreasing cell expansion, and have been reported to act as 1-aminocyclopropane-1-carboxylic acid oxidases (2). In elongating oat and maize coleoptiles, ethylene inhibits cell elongation and changes the orientation of cell expansion (20); ethylene generated by oxidation of 1-aminocyclopropane-1-carboxylic acid could have a similar function in tall fescue leaf blades, which continue to increase in width after cell elongation ceases (22). In each tall fescue genotype, activity of both cationic and anionic isoforms of apoplastic peroxidase was detected in the region of secondary cell wall deposition, where signification occurs. Although increase in the activity of anionic isozymes of peroxidase was previously associated with inhibition of cell elongation (8, 10), the enhanced spatial resolution of our data demonstrates that, at least in tall fescue leaf blades, anionic isozymes appeared only when elongation had stopped and increased in activity as signification began (16). Anionic peroxidase isozymes may also be responsible for oxidation and dimerization of tyrosine residues of extensin
to form isodityrosine, because a peroxidase isozyme with an isoelectric point of 4.6 from cultured tomato cells formed isodityrosine in extension from several species (25).
CONCLUSION A striking correlation between isfrms on apoplastic peroxidase activity and deceleration of elongation confirms the increase in cell wall-associated ioniearlier observe at cally bound peroxidase activity preceded growth cessation. This conclusion is strengthened by the consistent results obtained for two genotypes that differed in length of the elongation zone. Only cationic isoforms of peroxidase were observed within the elongation zone, whereas anionic isoforms appeared later, as cells were displaced through the region of secondary cell wall deposition. Future studies will investigate further the tissue specificity and the physiological roles of the peroxidase isozymes secreted in the region of cessation of elongation. ACKNOWLEDGMENTS We thank Dr. Doug Randall, University of Missouri, and Dr. Joe Vamer, Washington University, for useful discussions, and Nancy David for assistance with isoelectric focusing. LITERATURE CITED 1. Bergmeyer HU, Bernt E (1974) Malate dehydrogenase. In HU Bergmeyer, ed, Methods of Enzymatic Analysis. Academic Press, New York, pp 613-617 2. Boyer N, De Jaegher G (1986) Direct or indirect role of peroxidases in ethylene biosynthesis? In H Greppin, C Penel, T
Gaspar, eds, Molecular and Physiological Aspects of Plant Peroxidases. University of Geneva, Geneva, pp 47-60 3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 4. Castillo FJ, Penel C, Greppin H (1984) Peroxidase release induced by ozone in Sedum album leaves. Involvement of Ca2". Plant Physiol 74: 846-851 5. Fry SC (1979) Phenolic components of the primary cell wall and
APOPLASTIC PEROXIDASE AND DISTRIBUTION OF LEAF GROWTH
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their possible role in the hormonal regulation of growth. Planta 146: 343-351 Fry SC (1980) Gibberellin-controlled pectinic acid and protein secretion in growing cells. Phytochemistry 19: 735-740 Fry SC (1988) The Growing Plant Cell Wall: Chemical and Metabolic Analysis. Longman, Harlow, UK Gardiner MG, Cleland R (1974) Peroxidase changes during the cessation of elongation in Pisum sativum stems. Phytochemistry 13: 1095-1098 Gaspar T, Penel C, Castillo FJ, Greppin H (1985) A two-step control of basic and acidic peroxidases and its significance for growth and development. Physiol Plant 64: 418-423 Goldberg R, Imberty A, Chu-Ba J (1986) Development of isoperoxidases along the growth gradient in the mung bean hypocotyl. Phytochemistry 25: 1271-1274 Goldberg R, Kevers C, Gaspar T (1989) Guaiacol and ascorbate peroxidase compartmentation and gradient along the growing mung bean hypocotyl. Biochem Physiol Pflanzen 184: 155-161 Imberty A, Goldberg R, Catesson AM (1984) Tetramethylbenzidine and p-phenylenediamine-pyrocatechol for peroxidase histochemistry and biochemistry: two new, non-carcinogenic chromogens for investigating lignification process. Plant Sci Lett 35: 103-108 Jones RJ, Nelson CJ, Sleper DA (1979) Seedling selection for morphological characters associated with yield of tall fescue. Crop Sci 19: 631-634 Kim SH, Terry ME, Hoops P, Dauwalder M, Roux SJ (1988) Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings. Plant Physiol 88: 1446-1453 Li ZC, McClure JW, Hagerman AE (1989) Soluble and bound apoplastic activity for peroxidase ,B-D-glucosidase, malate dehydrogenase, and nonspecific arylesterase, in barley (Hordeum vulgare L.) and oat (Avena sativa L.) primary leaves. Plant Physiol 90: 185-190 MacAdam JW (1988) Cellular dynamics, peroxidase activity,
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and secondary cell wall deposition during tall fescue leaf blade development. PhD thesis. University of Missouri, Columbia MacAdam JW, Nelson CJ, Sharp RE (1992) Peroxidase activity in the leaf elongation zone of tall fescue. I. Spatial distribution of ionically bound peroxidase activity in genotypes differing in length of the elongation zone. Plant Physiol 99: 872-878 MacAdam JW, Volenec JJ, Nelson CJ (1989) Effects of nitrogen on mesophyll cell division and epidermal cell elongation in tall fescue leaf blades. Plant Physiol 89: 549-556 Mader M, Nessel A, Schloss P (1986) Cell compartmentation and specific roles of isoenzymes. In H Greppin, C Penel, T Gaspar, eds, Molecular and Physiological Aspects of Plant Peroxidases. University of Geneva, Geneva, pp 247-260 Osborne DJ (1990) Ethylene formation, cell types and differentiation. In HE Flores, RN Arteca, JC Shannon, eds, Polyamines and Ethylene: Biochemistry, Physiology, and Interactions. American Society of Plant Physiologists, Rockville, MD, pp
203-215 21. Rama Rao N, Naithani SC, Jasdanwala RT, Singh YD (1982) Changes in indoleacetic acid oxidase and peroxidase activities during cotton fibre development. Z Pflanzenphysiol 106: 157-165 22. Schnyder H, Nelson CJ (1989) Growth rates and assimilate partitioning in the elongation zone of tall fescue leaf blades at high and low irradiance. Plant Physiol 90: 1201-1206 23. Sticher L, Penel C, Greppin H (1981) Calcium requirement for the secretion of peroxidases by plant cell suspensions. J Cell Sci 48: 345-353 24. Terry ME, Bonner BA (1980) An examination of centrifugation as a method of extracting an extracellular solution from peas, and its use for the study of indoleacetic acid-induced growth. Plant Physiol 66: 321-325 25. Upham BL, Alizadeh H, Ryan KJ, Lamport DTA (1991) A pl 4.6 peroxidase that specifically crosslinks extensin precursors (abstract No. 558). Plant Physiol 96: S-88 26. Wilson RE, Laidlaw AS (1985) The role of the sheath tube in the development of expanding leaves in perennial ryegrass. Ann Appl Biol 106: 385-391
