Journal of Experimental Botany, Vol. 52, No. 361, pp. 1673-1682,
August 1, 2001
© 2001 Oxford University Press
Original Papers |
Gibberellic acid and dwarfism effects on the growth dynamics of B73 maize (Zea mays L.) leaf blades: a transient increase in apoplastic peroxidase activity precedes cessation of cell elongation
Department of Plants, Soils, and Biometeorology, Utah State University, Logan, UT 84322-4820, USA
Received 2 February 2001; Accepted 23 April 2001
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Abstract |
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The relationship between apoplastic peroxidase (EC 1.11.1.7) activity and cessation of growth in maize (Zea mays L.) leaf blades was investigated by altering elongation zone length. Apoplastic peroxidase activity in the elongation and secondary cell wall deposition zones of elongating leaf blades of the maize inbred line B73 was used as a control and compared to leaves of the dwarf mutant D8-81127, a near-isogenic line of B73 unresponsive to gibberellins, and to leaves of B73 plants to which gibberellic acid (GA3) had been applied via root uptake. Elongation zone length was increased by treatment with GA3 through an increase in cell number as well as increased final cell length. The shorter elongation zone of dwarf leaves occurred primarily through reduced final cell length. Although elongation zone length differed among dwarf, control, and GA3-treated leaf blades, in all three treatments a transient increase in apoplastic peroxidase activity preceded a reduction in the segmental elongation rate in leaves. A peroxidase isoenzyme with pI 7.0 occurred in the leaf elongation zone during growth deceleration in all three treatments, and its activity decreased as growth displaced tissue into the region of secondary cell wall deposition. Growth cessation for all treatments coincided with the first appearance of peroxidase isozymes with pIs of 5.6 and 5.7. Based on the activity of particular isozymes relative to growth and differentiation, the pI 7.0 isoenzyme is most likely to be involved in cessation of cell elongation, while isozymes with pIs 5.6 and 5.7 are likely to be active in lignification.
Key words: Apoplastic peroxidase, B73 maize, gibberellic acid, leaf growth.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Leaves of dwarf maize (McCune and Galston, 1959
) and internodes of dwarf sorghum [Sorghum bicolor (L.) Moench] (Schertz et al., 1971) have higher peroxidase activity than the same organs in normal plants, suggesting that peroxidase inhibits elongation growth. An association between the inhibition of cell expansion and increased peroxidase activity ionically bound to cell walls of particular organs has also been demonstrated (Gardiner and Cleland, 1974; Goldberg et al., 1986; Rama Rao et al., 1982). However, most studies correlating increased peroxidase activity with decreased growth reported on the activity of peroxidase extracted following tissue homogenization. Ionically bound peroxidase from homogenized tissues has been shown to contain peroxidase isozymes relocated from the vacuole and bound to the cell wall during cell disruption (Mader et al., 1986). Therefore, extraction of apoplastic fluid from the cell walls of intact leaf blade segments was used to investigate cell wall peroxidase activity in the present study.
The existence of an inverse relationship between gibberellic acid (GA) level and peroxidase activity has also been known for many years. Gibberellic acid applied to dwarf maize leaves (Galston and McCune, 1961) or applied to pea (Pisum sativum) internodes (Bireka and Galston, 1970) promoted elongation and altered the activity of specific peroxidase isozymes, but did not change the complement of peroxidase isozymes in the tissue.
In wheat (Triticum aestivum), the basal one-fifth of the leaf blade, defined as the elongating region, contained most of the gibberellin in growing leaves (Wheeler, 1973). Baluska et al., using roots of a gibberellin-deficient maize dwarf (d5 mutant), demonstrated that the meristem and the immediately post-mitotic (proximal elongation) zone were the targets of gibberellin deficiency (Baluska et al., 1993).
Gibberellic acid can stimulate growth by increasing cell elongation in some plant species, and by increasing both cell elongation and cell division in others (Metraux, 1987; Jupe et al., 1988). Fry, working with spinach (Spinach oleracea L.) cell cultures, suggested that gibberellic acid stimulated cell expansion by suppressing peroxidase secretion into cell walls (Fry, 1979). This could function in part to lower the rate of the peroxidase-catalysed formation of intermolecular bridges formed through dimerization of ferulic acid residues of hemicellulose molecules, allowing cell wall expansion and therefore growth to continue.
In previous work with B73 maize leaf blades (Souza and MacAdam, 1998) and with tall fescue leaf blades (MacAdam et al., 1992), a transient increase in apoplastic (extracellular) peroxidase activity at the distal end of the elongation zone preceded a rapid decrease in the segmental elongation rate of leaves. In maize leaf blades, peroxidase activity in the elongation zone was due to isozymes with neutral or basic pIs (Souza and MacAdam, 1998), while in the zone of secondary cell wall deposition and lignification, two isozymes with pI 5.6 and 5.7 appeared. In tall fescue leaf blades, this increase in peroxidase activity appeared to be due to increased activity of several cationic isozymes, while subsequent secondary cell wall deposition and lignification were accompanied by an increase in anionic peroxidase isozymes.
The objective of the present study was to determine whether the appearance of peroxidase in the apoplast and the subsequent cessation of leaf elongation would occur in the same way when elongation zone length was reduced by mutation or increased through application of exogenous GA. Zymograms of apoplastic peroxidase isozymes were also examined to determine whether particular isozymes were consistently related to growth deceleration.
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Materials and methods |
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Plant growth conditions
Seeds of maize inbred line B73 were surface-sterilized by soaking for 1 min in a sodium hypochlorite solution with 1.1% (w/v) available chlorine, then rinsed several times with tap water before being sown in 12 cm deep by 15 cm diameter pots in Terra-Lite Redi-Earth Peat-Lite potting mix (Scotts-Sierra Horticultural Products Co., Marysville, OH, USA). Constant light with an irradiance of 400 µmol m-2 s-1 at seedling level was provided by Sylvania 45 W cool white high-output lamps, temperature was maintained at 25 °C, and relative humidity was 40%.
Treatments
The three treatments were the B73 maize inbred line, which served as the control, the dwarf D8-81127, a near-isogenic line of B73, and the B73 inbred line treated with GA3 solution. The dwarf line used is maintained as a heterozygote and segregates as one heterozygous dwarf to one wild-type plant (Winkler and Freeling, 1994). D8 is a dominant dwarfing mutation in which the mutants are unresponsive to gibberellins (Harberd and Freeling, 1989). Dwarf plants were easily distinguished from the wild-type plant at the seedling stage due to slow growth and broad, short leaves.
Gibberellic acid-A3 (GA3) (Sigma, St Louis, MO, USA) was used in an aqueous solution at a concentration of 3.6x10-5 M applied directly to the potting medium. Before planting, the potting medium was saturated either with the GA3 solution for the GA3-treated plants, or with tap water alone for the control and dwarf treatments. Pots of the control and dwarf treatments were irrigated with 200 ml tap water per pot every other day and the GA3-treated B73 inbred line was irrigated in the same way with the GA3 solution.
Epidermal cell number, length and width
All experimentation was done using the expanding second true maize leaf blade at a stage before growth of the leaf sheath, which is seen as displacement of the ligule from the leaf base. Seven leaf blades from each of the three treatments were collected by excising the enclosing first leaf sheath from the stem at the point of its attachment and discarding the first leaf. A 4% (w/v) solution of polyvinylformaldehyde in chloroform (Schnyder et al., 1990) was spread with a small-tipped paintbrush over the abaxial surface of the leaf. When the chloroform had evaporated, a strip of clear cellophane tape was applied to the leaf and the replica was lifted off and applied to a glass microscope slide. Cell measurements were taken from the replicas on 10 intercostal cells at 2 mm increments from the ligule to 40 mm distal to the ligule using a Zeiss Axioskop microscope equipped with an eyepiece micrometer.
Leaf blade width and thickness
Width of 10 leaf blades of each treatment was measured from margin to margin at 10 mm intervals from zero to 60 mm distal to the ligule. To determine leaf blade thickness, entire control, dwarf and GA3-treated plants were excised at the mesocotyl, fixed for 1 h in 3 : 1 ethanol-acetic acid, and transferred to 70% ethanol (Jensen, 1962). The basal 60 mm of the expanding leaf blade was sectioned at 5 mm intervals using a hand microtome. The thickness of transverse sections of leaf blades was determined at small veins for three leaves of each treatment.
Fresh weight and insoluble dry weight
Ten expanding leaf blades from the control and from GA3-treated plants were sectioned at 6 mm intervals from the ligule to 60 mm. All segments from the same location of a given treatment were combined and the fresh weight determined. Cell soluble material and membranes were extracted from these segments by boiling successively in ethanol and water (MacAdam et al., 1992) to determine the insoluble (cell wall) dry weight of segments. This experiment was repeated three times. Fresh weight and dry weight of dwarf plants were not determined due to limited seed availability, but the relationship of increase in fresh weight with cell expansion and of increase in dry weight with secondary cell wall deposition were similar for control and GA3-treated plants.
Growth measurements
For determination of leaf elongation rate (LER), increase in length of the second leaf of 30 seedlings from each treatment was measured daily for 3 d, beginning when the tip of the leaf emerged from the enclosing leaf sheath, and finishing on the day of harvest. The distance from the base of the sheath of the first leaf to the tip of the enclosed younger second leaf was measured daily to determine change in leaf length. Displacement velocities within the elongation zone were calculated using the equation
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; Carmona and Cuadrado, 1986; Silk et al., 1989). Segmental elongation rate (mm mm-1 h-1) was obtained by differentiating a cubic equation fitted to displacement velocity as a function of distance above the ligule. To express elongation rate as a function of duration of displacement from the ligule, displacement velocity at each location was divided by 2 mm, the length of the interval at which cell length measurements were taken along the leaf, and time intervals antecedent to a specific location were summed. Duration of elongation, or the displacement time required for an element to move from the base of the leaf blade to a distal position inside the elongation zone, was the sum of the antecedent time intervals.
Apoplastic fluid extraction
Apoplastic fluid was extracted from 30 expanding leaf blades sectioned into 6 mm segments between the ligule and 60 mm distal to the ligule. Baskets from microfilterfuge tubes (Rainin Instrument Co., Woburn, MA, USA), with the filter membrane removed, were filled with segments from a given location. At the seedling stage, the basal leaf blade segments are fragile, so to minimize handling, segments were rolled in a small strip of plastic before being inserted into infiltration baskets. Leaf blade segments were rinsed three times with cold, distilled, deionized water for 5 min to remove contaminants from the cut ends of the sections. Leaf sections were submerged in cold 10 mM phosphate buffer, pH 6.0, containing 0.2 M KCl, and vacuum-infiltrated at -70 kP for 10 min.
After infiltration, baskets were blotted with lint-free tissues to remove excess buffer, transferred to microcentrifuge tubes and centrifuged at 1000 g for 15 min at 4 °C (Terry and Bonner, 1980). Infiltration and centrifugation were repeated three times with a typical yield of about 2 µl apoplastic fluid per segment per infiltration. Apoplastic fluid from successive infiltrations was pooled, then desalted and concentrated to 30 µl using Amicon Microcon-10 microconcentrators (Millipore, Bedford, MA, USA). After apoplastic fluid extraction, insoluble (cell wall) dry weight was determined as described above.
Enzyme assays
The activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49), a cytoplasm marker, was assayed in apoplastic fluid at 30 °C in a 1 ml reaction volume at a wavelength of 340 nm (Li et al., 1989). Ten µl of apoplastic fluid was added to a reaction mixture containing 68 mM Tricine adjusted to pH 8.0 with NaOH, 6 mM glucose-6-phosphate, 0.1 mM MgCl2, and 0.6 mM NADP. The functionality of this assay was first tested with purified enzyme and also with homogenate. No detectable G6PDH activity was found in apoplastic fluid extracts using this procedure.
Peroxidase activity was assayed at 30 °C in a reaction mixture containing 1 ml of 0.1 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. The reaction was initiated by adding 10 µl apoplastic fluid, and increase min-1 in absorption of guaiacol was measured at 470 nm.
Isoelectric focusing gel
The isoelectric focusing gel was prepared using 1% (w/v) Isogel agarose (BioWhittaker Molecular Applications, Rockland ME, USA) and 12% sorbitol as recommended by BMA, with ampholytes pH 3 to 10 (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Gels were cast by hand onto Gelbond film (BMA).
Each lane of the isoelectric focusing gel was loaded with 15 µl of sample. The gel was prefocused for 1 h and focused for 1.5 h. Both prefocusing and focusing were done at constant power and 1500 V; prefocusing was done at 1 W, and focusing at 8 W. Gels were run at 10 °C and stained for peroxidase activity using the PPD-PC method (Imberty et al., 1984). To determine pI, markers ranging from 3.6 to 10.2 (BMA) were run on each gel.
Statistical analysis
All analyses were carried out using the SAS (SAS Institute Inc., Cary, NC, USA) mixed model analysis of variance procedure PROC MIXED.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Peroxidase is involved in a number of reactions that can inhibit cell wall expansion, including the intracellular cross-linking of extensin (Cooper and Varner, 1983
; Lamport and Epstein, 1983), the cross-linking of ferulic acids residues of cell wall components (Fry, 1979) and the oxidation of lignin precursors (Northcote, 1989). No lignin could be detected with phloroglucinol staining of epidermal or fibre cells at the location of cessation of elongation in these maize leaf blades; only xylem cell walls stained for lignin within the elongation zone. Therefore, it is proposed that the transient increase in peroxidase activity within the elongation zone is related to the subsequent cessation of elongation rather than to lignification of secondary cell walls. To aid in accurately relating change in peroxidase activity to successive stages of growth and development, the location of cell division and elongation was documented, with particular attention to the site where elongation of leaf blade tissue stops. Secondary cell wall deposition and lignification also begin at this location, based on studies in tall fescue (MacAdam and Nelson, 1987). Figure 1 illustrates the relative locations and lengths of these zones for the B73 maize inbred line under the growth conditions of this study.
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Effects on the dynamics of leaf growth
The elongation zone was defined as the region where epidermal cell lengths increased with location distal to the ligule (or toward the leaf tip; Fig. 2A
). Average maximum cell lengths at the distal end of the elongation zone were 179 µm, 216 µm, and 247 µm, respectively, for the dwarf, control and GA3-treated plants. The length of the leaf blade elongation zone averaged 18 mm for the dwarf, 22 mm for the control and 30 mm for GA3-treated plants. Mean leaf blade lengths from base to tip were 95±3.4 mm (±SD, n=10), 140±9.9 mm, and 220±12 mm, respectively, for the dwarf, control and GA3-treated plants. In Brassica rapa genotypes, epidermal cells of dwarf plants were approximately one-third the length of normal plants. The exogenous application of GA3 to normal B. rapa plants increased cell number, cell length, and altered leaf morphology to a more elongated shape (Rood et al., 1990b).
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Comparing the ratio of leaf blade lengths to final cell lengths for all three treatments, it can be concluded that cell division was increased by exogenous GA. GA3 applied to tomato (Lycopersicon esculentum Mill.) plants caused an increase in internode length, and in the length and number of cells in the outer cell layers of internodes (Jupe et al., 1988
). Based on a calculation of the total leaf area of dwarf and GA3-treated plants, there was a decrease in the leaf area of dwarf plants and an increase in the leaf area of GA3-treated plants compared to the control, not just a redistribution of leaf blade tissue area. In other studies using maize, total leaf area of dwarf plants was also reduced and GA treatment of normal maize plants resulted in increased leaf area (Rood et al., 1990a).
Cell width, like cell length, increased through the end of the elongation zone (Fig. 2B
). Cell width of the dwarf increased from 12.5 µm at the leaf base to 25.7 µm at the end of the elongation zone, control cell width increased from 11.3 to 23.5 µm, and cell width of GA3-treated plants increased from 10.2 to 22.8 µm. This trend is in general agreement with cell width differences of Brassica rapa genotypes, which were 48 µm for dwarf, 41 µm for normal, and 24 µm for GA3-treated plants (Rood et al., 1990b). At the location where elongation stopped, the increased cell length in GA3-treated plants was compensated somewhat by decreased cell width, and in dwarf plants increased cell width was compensated by reduced cell length.
Leaf width was greatest for dwarf plants (Fig. 3A). While cell width only increased within the elongation zone, leaf blade width of all plants continued to increase beyond the location at which epidermal cells had ceased to elongate: to 30 mm, 50 mm and beyond 60 mm distal to the ligule for the dwarf, control and GA3-treated plants, respectively. Since mesophyll cell division is confined to the basal half of the elongation zone (Souza and MacAdam, 1998), the continued increase in leaf width after cessation of cell elongation may be related to continued differentiation of fibre or bulliform cells, as noted previously (Maurice et al., 1997).
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Leaf thickness was also greatest for dwarf maize and least for leaves of GA3-treated plants (Fig. 3B
). Like leaf width, the increase in leaf thickness continued beyond the point where epidermal cell expansion in both length and width ceased, to about 40, 45, and 50 mm distal to the ligule for the dwarf, control and GA3-treated plants, respectively. Gibberellic acid applied to sunflower also caused a decreased leaf thickness in leaves that were not fully expanded (Starman et al., 1990).
GA3-treated leaf blades had lower fresh and insoluble dry weights than control leaf blades within the basal 60 mm distal to the ligule (Fig. 4A, B). Fresh weight increased in these treatments from the ligule through the distal end of the elongation zone, in the region where water uptake occurs to support cell expansion. In GA3-treated plants, where cells reached a greater final mature length, water uptake, represented by an increase in fresh weight, occurred over a greater distance. In both treatments, dry weights decreased from the ligule through the distal end of the elongation zone as the number of cell end walls per mm decreased with cell elongation. Dry weight increased as tissue was displaced beyond the elongation zone. The increase in dry weight was most likely due to the thickening of secondary cell walls in fibre cells, as observed in tall fescue leaf blades (MacAdam and Nelson, 1987).
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The ratio of fresh weight to insoluble dry weight (FW/IDW), indicative of water content per unit cell wall, was similar in GA3-treated plants to that of control plants, although the pattern of change was shifted to a position more distal to the ligule when exogenous GA3 increased elongation zone length (Fig. 4C
). This similarity in FW/IDW ratios suggests that water uptake and cell wall synthesis are co-regulated within a narrow range regardless of final cell dimensions.
The difference among treatments in the number of cells in a horizontal file of epidermal cells was confined to the basal 20 mm of the leaf blade (Fig. 5A). GA3-treated plants had 30–60% more cells in this region compared to the control.
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The rate of elongation of epidermal cells as they were displaced through the elongation zone can be calculated by plotting cell length as a function of time. Cell length increase is exponential with time, and fits the function
x
where y is the cell length at a given position within the elongation zone, y0 is the initial cell length, r is the rate constant or relative growth rate (Erickson, 1976), and t is time (Fig. 5B).
The increase in cell number in GA3-treated plants was offset somewhat by a reduced rate constant for cell elongation; rate constants were 0.25, 0.34, and 0.29 for dwarf, control and GA3-treated leaf blades, respectively. The duration of cell elongation was greater for both dwarf (46 h) and GA3-treated plants (45 h) than for the control (34 h) (Fig. 5B). This is in apparent contrast to the conclusions of Metraux (Metraux, 1987) and Jupe et al. (Jupe et al., 1988) who state that duration of elongation is not increased by GA. However, the studies cited in these papers evaluated the overall response time rather than examining the duration of cell growth as in the present study (Fig. 5B). The shorter cells of dwarf maize leaf blades were caused by a lower cell elongation rate compared to control plants (Fig. 5B).
Leaf elongation rate is equivalent to displacement velocity at the distal end of the elongation zone, and results from the combination of the number of cells in the elongation zone and their rate and duration of elongation. Average LERs were 1.04±0.095 mm h-1 (±LSD, n=30), 1.68±0.24 mm h-1, and 2.35±0.41 mm h-1, respectively, for dwarf, control and GA3-treated plants (Fig. 6A). Compared to the control, this was a decrease of 62% for the dwarf and an increase of 40% for the GA3-treated plants. GA3 applied to tall fescue also caused an increase in LER along with a decrease in leaf width (Poskuta et al., 1984). The segmental elongation rate of leaves (Fig. 6B) reached maximal values of 0.15, 0.22 and 0.24 mm mm-1 h-1 at about 14, 15 and 23 mm above the ligule for the dwarf, control and GA3-treated plants, respectively.
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Effects on apoplastic peroxidase activity
Gibberellic acid could promote growth by inhibiting peroxidase secretion into the apoplast as proposed earlier (Fry, 1979), which suggested that peroxidase cross-links the cell wall by promoting the conversion of feruloyl side-chains into diferuloyl cross-links between cell wall polysaccharides, thus restricting growth. Hartley demonstrated the presence of carbohydrate esters of ferulic acid in grass cell walls (Hartley, 1973). If an increased level of GA in the meristem and the proximal part of the elongation zone shifted the peak of peroxidase activity to a more distal location along the leaf blade, this could delay the cross-linking that rigidifies the cell wall, allowing cells to continue water uptake and cell wall synthesis and delaying the cessation of cell elongation, thereby lengthening the elongation zone.
In GA3-treated maize B73 plants, apoplastic peroxidase activity peaked at about 21 mm distal to the ligule, while for dwarf and control plants apoplastic peroxidase activity peaked at about 15 mm (Fig. 7). For all treatments, this peak of apoplastic peroxidase activity occurred inside the elongation zone near the location where cells reached their maximum rate of elongation (Fig. 6B). Apoplastic peroxidase activity decreased as tissue was displaced from the location of maximal elongation to the end of the elongation zone for all three treatments. A second increase in apoplastic peroxidase activity followed cessation of elongation in the dwarf and control treatments, and probably occurred in GA3-treated plants beyond 60 mm distal to the ligule (Fig. 7). Apoplastic peroxidase activity is reported as activity mg-1 insoluble dry weight to relate the change in activity to the amount of cell wall material present.
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Segmental elongation rate, replotted as a function of time before growth cessation (Fig. 8
), increased until the transient increase in apoplastic peroxidase activity occurred, then decreased similarly in all three treatments. The peak in apoplastic peroxidase activity occurred approximately 7 h before cessation of cell elongation regardless of treatment. In an experiment with leaf blades of annual ryegrass (Lolium tenulentum L.), ionically bound peroxidase activity began to increase about 5 h before elongation ceased in both well-watered and droughted plants, and peaked about 2 h before cessation of elongation (Bacon et al., 1997). In tall fescue leaf blades, apoplastic peroxidase activity peaked about 10 h before the cessation of leaf elongation (MacAdam et al., 1992). This rapid deceleration of segmental elongation rate following an increase in apoplastic peroxidase activity in the elongation zone could result from the formation of diferulate cross-links between cell wall polysaccharides or other functions of peroxidase such as IAA catabolism (Lagrimini et al., 1997) or cross-linking of structural proteins (Lamport and Epstein, 1983), both of which would contribute to the cessation of cell wall growth.
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Apoplastic peroxidase activity differed significantly among treatments as tissue was displaced beyond the elongation zone into the region where secondary cell wall deposition and lignification are likely to be occurring (Fig. 7
). Using the Bonferroni method at 
=5%, dwarf plants had higher peroxidase activity than control plants between 39 and 57 mm above the ligule (indicated by *), and dwarf plants reached a higher level of peroxidase activity than GA3-treated plants between 45 mm and 57 mm above the ligule (+). Control plants had higher peroxidase activity than GA3-treated plants at 57 mm above the ligule (#). This second, more prolonged increase in apoplastic peroxidase activity following cessation of cell elongation occurred in the region where leaves of each treatment are likely to deposit secondary cell walls in the fibre cells that constitute the structural tissue associated with every vein of the leaf (Fig. 4B); lignification follows secondary cell wall deposition.
Effects on peroxidase isozymes
While it is not possible conclusively to associate the peak in apoplastic peroxidase activity that was followed by deceleration of segmental elongation rate in B73 maize with a specific peroxidase isoenzyme, in zymograms of apoplastic fluid (Fig. 9) the pI 7.0 band decreased in activity as tissue was displaced beyond the elongation zone. The increase in peroxidase activity that occurred distal to the elongation zone coincided with an increase in the activity of two anionic isoenzymes with pIs 5.6 and 5.7. The region in which these isozymes increased in activity is also the region where secondary cell wall deposition and lignification are probably occurring (Fig. 4B). These two isozymes initially appeared in the leaf blade segment where cessation of elongation occurred, at 12–18 mm, 18–24 mm or 24–30 mm above the ligule for the dwarf, control and GA3-treated plants, respectively (Fig. 6B). The coincidence of these bands with the region of increasing cell wall dry weight suggests they could play a role in cell wall lignification.
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In tall fescue leaf blades, only cationic peroxidase isozymes appeared in apoplastic fluid from cell walls of the elongation zone, whereas new anionic peroxidase isozymes appeared in apoplastic fluid as leaf blade tissue was displaced into the zone of secondary cell wall deposition and lignification (MacAdam et al., 1992
). Cessation of cell elongation involves the formation of molecular bridges within or among cell wall polymers, so enzymes attracted to the cell wall could function well in these reactions. Because cationic isozymes have an affinity for negative charges of the cell wall, they would tend to become ionically bound to the cell wall. In the present study, the most active isozyme secreted in the elongation zone of maize leaf blades prior to cessation of elongation had a pI of 7.0. The process of lignification displaces the aqueous fraction of the cell wall and this process would be accomplished most efficiently by peroxidase isozymes that remained in the residual apoplastic fluid after secretion into the wall space rather than by isozymes localized at the cell wall. As in the earlier study of tall fescue, the peroxidase isozymes active in the region of lignification in this study have an acidic pI. In both cases, the pIs of peroxidase isozymes seem appropriate to the functions ascribed to them.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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In this study, treatment of B73 maize plants with GA3 increased the length of the leaf blade elongation zone compared to the control through an increase in cell division and duration of cell elongation (Fig. 5A
, B). The dwarf plant leaf blade elongation zone was reduced compared to the control due to a lower rate of cell elongation (Fig. 5B). Treatment of normal B73 maize with GA3 resulted in the appearance or activation of peroxidase in the apoplast at a location more distal to the ligule (Fig. 7). The transient peak in apoplastic peroxidase activity in all treatments was followed by deceleration of segmental elongation rate (Fig. 8). A peroxidase isozyme with pI 7.0 had elevated activity in the elongation zone, then decreased, while the activity of peroxidase isozymes with pIs 5.5 and 5.7 increased in the region where secondary cell wall deposition and lignification occurred. Taken together, these results suggest that a transient secretion of neutral or cationic peroxidase isozymes into the cell wall precipitates the deceleration of elongation, while lignification is caused by anionic isozymes secreted into the apoplast after deposition of secondary cell walls has begun.
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Acknowledgments |
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This research was supported by the Utah Agricultural Experiment Station, Utah State University, Logan, Utah 84322-4810. Approved as journal paper no. 7363.
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Notes |
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1 Present address: CNPMS, EMBRAPA, CX Postal 151, Sete Lagoas MG 35701-970, Brazil (IRPS).
2 To whom correspondence should be addressed. Fax: +1 435 797 3376. E-mail: jenmac{at}cc.usu.edu
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References |
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