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Soluble and bound hydroxyproline-rich glycoproteins in control and wounded oat and barley primary leaves
Phytochemistry, Vol. 29, No. 9, pp. 2811-2813, 1990 Printedin Great Britain.
0
SOLUBLE AND BOUND HYDROXYPROLINE-RICH CONTROL AND WOUNDED OAT AND BARLEY ZHEN-CHANG
Department
of Botany, (Received in
Key Word
Index-hena
satiua; Hordeum
LI
and
JERRY W.
Miami University,
003 l-9422/90 $3.00 + 0.00 1990 PergamonPressplc
GLYCOPROTEINS IN PRIMARY LEAVES
MCCLURE*
Oxford,
Ohio, U.S.A.
revisedform13 March 1990)
uulgare; Gramineae; leaves; apoplast; symplast; hydroxyproline-rich
glycoprotein; localization; peroxidase.
Abstract-The partitioning of hydroxyproline-rich glycoprotein (HRGP) was determined between the apoplast and symplast of control and wounded six-day-old barley (Hordeurn uulgare) and oat (Arena satiua) primary leaves. In controls, from 5% (barley) to 9% (oat) of the total HRGP was soluble or weakly ionically bound within the apoplast, 32% (barley) to 35% (oats) was symplastic and soluble in 50 mM K-Pi buffer, no HRGP was released by extracting washed cell wall preparations with 1 M NaCl, and about 60% of the HRGP was covalently bound to the cell wall in either species. Wounding dicot seedlings normally causes marked HRGP induction; in contrast, wounding barley or oat leaves 24 hr before sampling had no appreciable effect on HRGP levels or localization, but apoplastic peroxidase activity increased markedly. These results suggest that HRGP may play an important role in dicots, but not in cereals such as oats and barley, as a defensive response to tissue damage.
INTRODUCTION
Extensins are 4-hydroxyproline-rich glycoproteins (HRGP) [l] which may cross-link cell walls through isodityrosine linkages [24]. Most extensins are covalently bound to cell walls and therefore not extractable by 1 M NaCl [2]. Appreciable amounts of extensins or extensin precursors can, however, be eluted from the surface of cell suspension cultures by buffers or low ionicstrength salt solutions which do not disrupt cell integrity [S, 63. As cells differentiate, extensin precursors gradually become covalently bound in the walls through reactions presumably catalysed by cell-wall bound peroxidase C3,7]. Cross-linked extensins in the cell wail may play a role in disease resistance in dicots where their accumulation is enhanced by pathogenic infection or by mechanical wounding [S, 93. Extensins have only recently been conclusively demonstrated in cereal cell walls [6,9]. Cereals have much lower levels of extensins than do dicots and cereal extensins are not significantly increased by pathogenic infection [9, lo]. In cereals, cell-wall bound phenolics may have replaced extensins as cross-linking materials [l l] induced in response to pathogen infection [ 12,131. The partitioning of HRGP between the apoplast and symplast of cereal leaves, and the effects of wounding on this distribution, has not been previously reported. We have recently developed procedures for sequentially extracting soluble, ionically bound, and covalently bound, proteins and enzyme activities from the apoplast of oat and barley primary leaves [14, 153. In the present investigation we applied these techniques to determine the localization of HRGP in oat and barley primary leaves under control and wounded conditions. Contam*Author
to whom correspondence
should
be addressed. 2811
ination of apoplastic fractions by symplastic proteins was monitored by assaying for the cytoplasmic marker glucose-6-phosphate dehydrogenase (G6PD). Apoplastic peroxidase induction was used as an indicator of wounding. RESULTS AND DISCUSSION
The soluble or weakly ionically bound apoplastic fraction extracted by vacuum-infiltrating control leaves with buffered 200 mM NaCl contained little (barley) or no detectable (oat) G6PD activity and about 1% of the total protein of the leaf. However, this intercellular washing solution (IWS) had from 5% (barley) to 9% (oat) of the total HRGP, and from 8% to 23% of the total leaf peroxidase activity (Table 1). When leaves were wounded by peeling away the lower epidermis 24 hr before fractionation, the IWS contained no G6PD, slightly less protein or HRGP than did this fraction in control leaves, but peroxidase activity in the IWS was increased from six(oat) to 18-fold (barley) (Table 1). After the IWS was eluted, soluble symplastic constituents were determined in the supernatant of leaves homogenized and centrifuged in 50 mM K-Pi. In control leaves this fraction had essentially all of the G6PD activity, about 95% of the total soluble protein, from 63% (barley) to 69% (oat) of the peroxidase activity, and about 32% (oat) and 35% (barley) of the HRGP (Table 1). Wounding decreased total soluble protein in the symplastic fraction by about 20% in barley and about 35% in oats. Wounding increased symplastic peroxidase activity about two-fold in oats and almost seven-fold in barley (Table 1). Strongly ionically bound cell wall constituents were obtained by extracting thoroughly washed cell wall pellets with buffered 1 M NaCl. In control leaves this
Table 1. Partitioning of hydroxyproline-rich glycoprotein, peroxidase activity, glucose-hphosphate dehydrogenase activity, and total protein, between apoplastic fractions and the symplast of control and wounded six-day-old barley and oat primary leaves -Symplastic
Apoplastic 200mM IWS fraction Hydroxyproline-rich
NaCl
glycoprotein
I M NaCl soluble fraction
Covalently bound fraction ___-
(pg 4-hydroxyprohne/lO
-
Homogenate after IWS &ted
segments)
Barley Control
0.72
Wounded
(4.6) 0.41
nd* nd
to.1
9.2 f 0.2 (63.8) 9.5+0.1 (67.5)
4.55
6.1 kO.3 (56.9) 7.420.2 (67.8)
3.710.3 (34.5) 3.210.1 (29.1)
1.5+0.1 (12.5) 1.9+_0.1 (2.5)
7.620.4 (63.3) 53.0k0.4 (69.7)
11.8+0.6 (68.6) 23.05 3.1 (42.4)
(31.6) 4.2iO.l (29.5)
Oats Control
0.9+0.1
nd
Wounded
(8.6) 0.3+
nd
(3.0) Peroxidase
@kat tetraguaiacol/lO
segments) Barley
Control Wounded
l.O&O.l (8.0) 19.0+2.2 (25.0)
1.9kO.2 (15.8) 2.110.1 (2.8) Oats
Control Wounded
4.0+0.1 (23.3) 27.7+ 1.5 (51.1)
Glucose-6-phosphate
dehydrogenase
0.1 f < 0.1
1.3+0.1
(0.6) 1.4fO.2
(7.6) 2.1 FO.2
(2.6)
(3.9)
(pkat NADPHjsegment) Barley
Control
2.3 & 2.7
2.7* 1.5
nd*
Wounded
(0.3) nd
(0.4) nd
nd
676.1 + 38.3 (99.3) 673.5 153.3 t 100)
Oats Control
nd
nd
nd
496.3 _+18.8
Wounded
nd
nd
nd
(100) 259.3 I 6.3 (100)
Total protein
(pg/segment) Barley
Control
7.6 ~0.2
14.7 22.0
na#
Wounded
(1.2) 4.4 k 0.6
(2.3) 15.3kO.8 (3.0)
na
(0.9)
609.2 + I 1.9 (96.5) 483.0 & 12.0 (96.1)
Oats Control Wounded
12.2+ 1.0
12.8F0.6
(2.3) 4.0*0.1
(2.5) 10.1 kO.4
(1.2)
(3.0)
na
499.0 & 2 1.3 (95.2) 324.7 + 24.3 (95.8)
Peeled leaves were fractionated as described in the Experimental. Values are means If-se. Per cent of peeled leaf enzyme activity or soluble protein in each fraction 1s shown in parentheses. *nd = Not detected; G6PD less than 0.6 pkat NADPH per segment and HRG P less than 0. I /lg 4-hydroxyprohne per 10 segments. # na =Cannot be assayed by these techniques.
Hydroxyproline-rich glycoproteins in oat and barley primary leaves fraction contained no HRGP, no G6PD activity, from 2 to 3% of the total leaf protein, and less than 1% of the peroxidase activity of oat but 16% of the peroxidase activity of barley (Table 1). This fraction from wounded leaves had no detectable HRGP and no activity for G6PD in either species. In barley, wounding had no appreciable effect on strongly ionically bound peroxidase activity. In oats, however, wounding increased strongly ionically bound peroxidase activity more than 13-fold (Table 1). The covalently bound fraction left in the wall of control leaves after 1 M NaCl extraction retained from 57% (oat) to 64% (barley) of the total HRGP, no detectable G6PD activity, and from 7% (oat) to 12% (barley) of the peroxidase activity. Wounding had no appreciable effect on HRGP levels in this fraction but slightly increased peroxidase activity (Table 1). The total amount of HRGP in the leaves of either species was not appreciably altered by wounding, and the decrease in soluble apoplastic HRGP in response to wounding was roughly proportional to increased amounts of HRGP covalently bound to cell walls. Since wounding markedly increased apoplastic peroxidase activity (Table 1) and peroxidase is presumed to cross-link extensin [3,7], we suggest that wounding caused a fraction of the soluble or weakly ionically bound apoplastic HRGP to become covalently bound into cell walls of the barley and oat primary leaves. When cucumber or carrot plants are wounded or infected with pathogens they accumulate more HRGP [8, 9, 161 and it has been suggested that this may play a role in dicot disease resistance [Z]. Mazau and Esquerrb Tugayt reported that pathogenic infection caused marked induction of HRGP in all dicots that they examined but had no significant effect on HRGP levels in the cereals barley, wheat or rice. Our results demonstrate that wounding has no appreciable effect on HRGP accumulation, or on its partitioning between the apoplast and symplast, in barley and oat primary leaves. In contrast, wounding markedly enhances apoplastic peroxidase activity (Table 1) which is associated with cross-linking of cell wall phenolics [3,7]. Cereals have far less HRGP than do dicots [2] and cereal cell walls commonly respond to wounding or infection by enhanced accumulation of various cell wall phenolics [ 12, 131. Our results are consistent with the view [ 1l] that in cereal cell walls, ferulic acid and related phenolics may be more important than 4-hydroxyproline-rich glycoproteins as adaptive responses to tissue damage. EXPERIMENTAL
Barley (Hordeum oulgare L.) and oat (Aoena satiua L.) seedlings were grown and harvested as previously described [14]. Extraction of apoplastic and symplastic constituents. Apoplastic and symplastic fractions were prepared as previously described [14, 151. Briefly, the lower epidermis was peeled from 10 leaves and the peeled segments vacuum-infiltrated with 5 ml of 5 mM K-Pi buffer, pH 6.5, containing 200 mM NaCI, for 36 min, to elute an intercellular washing solution (IWS) containing soluble and weakly ionically bound apoplastic
2813
proteins and enzymes. Segments were next homogenized in 5 ml of 50 mM K-Pi buffer, pH 6.5, centrifuged, and the supernatant collected as the soluble symplastic fraction. After washing with 1% (v/v) Triton-X100 and buffer to remove cytoplasmic contamination, the pellet was stirred in buffered 1 M NaCl to liberate strongly ionically bound apoplastic fractions. The final pellet was assayed for covalently bound apoplastic proteins and enzymes. Mechanical wounding. Wounding was accomplished by peeling away the lower epidermis and incubating leaf segments on Hz0 at 30” in the dark for 24 hr [12]. At the end of incubation, segments were processed as described above to obtain symplastic and apoplastic protein and enzyme fractions. HRGP determination. Fractions were adjusted to 6 M HCI and hydrolysed at 116” in the dark for 18 hr in sealed tubes [17]. After hydrolysis the samples were evaporated to dryness at 65” under N,, the residue dissolved in H,O, and HRGP determined as 4-hydroxyproline by the method of Richard [17] using 4hydroxy-L-proline (Sigma Biochemical, St Louis, U.S.A.) as a standard. Enzyme assays. Peroxidase and glucose-6-phosphate dehydrogenase activities were assayed as before [14]. Total protein was determined by Bradford’s dye binding technique [18].
REFERENCES
1. Tierney, M. L. and Varner, J. E. (1987) Plant Physiol. 84, 1. 2. Lamport, D. T. A. and Catt, J. W. (1981) in Encyclopaedia of Plant Physiology (Tanner, W. and Holmes, F. A., eds), 13B, p. 133. Springer. 3. McNeil, M., Darvill, A. G., Fry, S. C. and Albersheim, P. (1984) Ann. Reo. Biochem. 53,625. 4. Fry, S. C. (1986) Ann. Rev. Plant Physiol. 37, 165. 5. Smith, J. J., Muldoon, E. P. and Lamport, D. T. A. (1984) Phytochemistry 23, 1233. 6. Kieliszewski, M. and Lamport, D. T. A. (1987) Plant Physiol. 85, 823.
7. Everdeen, D. S., Kiefer, S., Willard, J. J., Muldoon, E. P., Dey, P. M., Li, X. and Lamport, D. T. A. (1988) Plant Physiol. 87, 616.
8. Hammerschmidt, R., Lamport, D. T. A. and Muldoon, E. P. (1984) Physiol. Plant Pathol. 24, 43. 9. Mazau, D. and EsquerrQ-TugayQ M. T. (1986) Physiol. Mol. Plant Pathol. 29, 147.
10. Clarke, J. A., Lisker, N., Ellingboe, A. H. and Lamport, D. T. A. (1983) Plant Sci. Letters 30, 339. 11. Carpita, N. C. and Kanabus, J. (1988) Plant Physiol. 88,671. 12. Geballe, G. T. and Galston, A. W. (1982) Plant Physiol. 70, 781.
13. Reisener, H. J., Tiburzy, R., Kogel, K. H., Moerschbacher, B. and Keck, B. (1986) in Biology and Molecular Biology of Plant-Pathogen Interactions (Bailey, J., ed.), Vol. Hl, p. 133. NATO AST Series. 14. Li, Z.-C., McClure, J. W. and Hagerman, A. E. (1989) Plant Physiol. 90, 185.
15. 16. 17. 18.
Li, Z.-C. and McClure, J. W. (1989) Phytochemistry 28,2255. Tierney, M. L. (1986) Plant Physiol. 80, S-70. Richard, A. B. (1982) Meth. Enzymol. 82, 372. Bradford, M. (1976) Anal. Biochem. 72,248.
0
SOLUBLE AND BOUND HYDROXYPROLINE-RICH CONTROL AND WOUNDED OAT AND BARLEY ZHEN-CHANG
Department
of Botany, (Received in
Key Word
Index-hena
satiua; Hordeum
LI
and
JERRY W.
Miami University,
003 l-9422/90 $3.00 + 0.00 1990 PergamonPressplc
GLYCOPROTEINS IN PRIMARY LEAVES
MCCLURE*
Oxford,
Ohio, U.S.A.
revisedform13 March 1990)
uulgare; Gramineae; leaves; apoplast; symplast; hydroxyproline-rich
glycoprotein; localization; peroxidase.
Abstract-The partitioning of hydroxyproline-rich glycoprotein (HRGP) was determined between the apoplast and symplast of control and wounded six-day-old barley (Hordeurn uulgare) and oat (Arena satiua) primary leaves. In controls, from 5% (barley) to 9% (oat) of the total HRGP was soluble or weakly ionically bound within the apoplast, 32% (barley) to 35% (oats) was symplastic and soluble in 50 mM K-Pi buffer, no HRGP was released by extracting washed cell wall preparations with 1 M NaCl, and about 60% of the HRGP was covalently bound to the cell wall in either species. Wounding dicot seedlings normally causes marked HRGP induction; in contrast, wounding barley or oat leaves 24 hr before sampling had no appreciable effect on HRGP levels or localization, but apoplastic peroxidase activity increased markedly. These results suggest that HRGP may play an important role in dicots, but not in cereals such as oats and barley, as a defensive response to tissue damage.
INTRODUCTION
Extensins are 4-hydroxyproline-rich glycoproteins (HRGP) [l] which may cross-link cell walls through isodityrosine linkages [24]. Most extensins are covalently bound to cell walls and therefore not extractable by 1 M NaCl [2]. Appreciable amounts of extensins or extensin precursors can, however, be eluted from the surface of cell suspension cultures by buffers or low ionicstrength salt solutions which do not disrupt cell integrity [S, 63. As cells differentiate, extensin precursors gradually become covalently bound in the walls through reactions presumably catalysed by cell-wall bound peroxidase C3,7]. Cross-linked extensins in the cell wail may play a role in disease resistance in dicots where their accumulation is enhanced by pathogenic infection or by mechanical wounding [S, 93. Extensins have only recently been conclusively demonstrated in cereal cell walls [6,9]. Cereals have much lower levels of extensins than do dicots and cereal extensins are not significantly increased by pathogenic infection [9, lo]. In cereals, cell-wall bound phenolics may have replaced extensins as cross-linking materials [l l] induced in response to pathogen infection [ 12,131. The partitioning of HRGP between the apoplast and symplast of cereal leaves, and the effects of wounding on this distribution, has not been previously reported. We have recently developed procedures for sequentially extracting soluble, ionically bound, and covalently bound, proteins and enzyme activities from the apoplast of oat and barley primary leaves [14, 153. In the present investigation we applied these techniques to determine the localization of HRGP in oat and barley primary leaves under control and wounded conditions. Contam*Author
to whom correspondence
should
be addressed. 2811
ination of apoplastic fractions by symplastic proteins was monitored by assaying for the cytoplasmic marker glucose-6-phosphate dehydrogenase (G6PD). Apoplastic peroxidase induction was used as an indicator of wounding. RESULTS AND DISCUSSION
The soluble or weakly ionically bound apoplastic fraction extracted by vacuum-infiltrating control leaves with buffered 200 mM NaCl contained little (barley) or no detectable (oat) G6PD activity and about 1% of the total protein of the leaf. However, this intercellular washing solution (IWS) had from 5% (barley) to 9% (oat) of the total HRGP, and from 8% to 23% of the total leaf peroxidase activity (Table 1). When leaves were wounded by peeling away the lower epidermis 24 hr before fractionation, the IWS contained no G6PD, slightly less protein or HRGP than did this fraction in control leaves, but peroxidase activity in the IWS was increased from six(oat) to 18-fold (barley) (Table 1). After the IWS was eluted, soluble symplastic constituents were determined in the supernatant of leaves homogenized and centrifuged in 50 mM K-Pi. In control leaves this fraction had essentially all of the G6PD activity, about 95% of the total soluble protein, from 63% (barley) to 69% (oat) of the peroxidase activity, and about 32% (oat) and 35% (barley) of the HRGP (Table 1). Wounding decreased total soluble protein in the symplastic fraction by about 20% in barley and about 35% in oats. Wounding increased symplastic peroxidase activity about two-fold in oats and almost seven-fold in barley (Table 1). Strongly ionically bound cell wall constituents were obtained by extracting thoroughly washed cell wall pellets with buffered 1 M NaCl. In control leaves this
Table 1. Partitioning of hydroxyproline-rich glycoprotein, peroxidase activity, glucose-hphosphate dehydrogenase activity, and total protein, between apoplastic fractions and the symplast of control and wounded six-day-old barley and oat primary leaves -Symplastic
Apoplastic 200mM IWS fraction Hydroxyproline-rich
NaCl
glycoprotein
I M NaCl soluble fraction
Covalently bound fraction ___-
(pg 4-hydroxyprohne/lO
-
Homogenate after IWS &ted
segments)
Barley Control
0.72
Wounded
(4.6) 0.41
nd* nd
to.1
9.2 f 0.2 (63.8) 9.5+0.1 (67.5)
4.55
6.1 kO.3 (56.9) 7.420.2 (67.8)
3.710.3 (34.5) 3.210.1 (29.1)
1.5+0.1 (12.5) 1.9+_0.1 (2.5)
7.620.4 (63.3) 53.0k0.4 (69.7)
11.8+0.6 (68.6) 23.05 3.1 (42.4)
(31.6) 4.2iO.l (29.5)
Oats Control
0.9+0.1
nd
Wounded
(8.6) 0.3+
nd
(3.0) Peroxidase
@kat tetraguaiacol/lO
segments) Barley
Control Wounded
l.O&O.l (8.0) 19.0+2.2 (25.0)
1.9kO.2 (15.8) 2.110.1 (2.8) Oats
Control Wounded
4.0+0.1 (23.3) 27.7+ 1.5 (51.1)
Glucose-6-phosphate
dehydrogenase
0.1 f < 0.1
1.3+0.1
(0.6) 1.4fO.2
(7.6) 2.1 FO.2
(2.6)
(3.9)
(pkat NADPHjsegment) Barley
Control
2.3 & 2.7
2.7* 1.5
nd*
Wounded
(0.3) nd
(0.4) nd
nd
676.1 + 38.3 (99.3) 673.5 153.3 t 100)
Oats Control
nd
nd
nd
496.3 _+18.8
Wounded
nd
nd
nd
(100) 259.3 I 6.3 (100)
Total protein
(pg/segment) Barley
Control
7.6 ~0.2
14.7 22.0
na#
Wounded
(1.2) 4.4 k 0.6
(2.3) 15.3kO.8 (3.0)
na
(0.9)
609.2 + I 1.9 (96.5) 483.0 & 12.0 (96.1)
Oats Control Wounded
12.2+ 1.0
12.8F0.6
(2.3) 4.0*0.1
(2.5) 10.1 kO.4
(1.2)
(3.0)
na
499.0 & 2 1.3 (95.2) 324.7 + 24.3 (95.8)
Peeled leaves were fractionated as described in the Experimental. Values are means If-se. Per cent of peeled leaf enzyme activity or soluble protein in each fraction 1s shown in parentheses. *nd = Not detected; G6PD less than 0.6 pkat NADPH per segment and HRG P less than 0. I /lg 4-hydroxyprohne per 10 segments. # na =Cannot be assayed by these techniques.
Hydroxyproline-rich glycoproteins in oat and barley primary leaves fraction contained no HRGP, no G6PD activity, from 2 to 3% of the total leaf protein, and less than 1% of the peroxidase activity of oat but 16% of the peroxidase activity of barley (Table 1). This fraction from wounded leaves had no detectable HRGP and no activity for G6PD in either species. In barley, wounding had no appreciable effect on strongly ionically bound peroxidase activity. In oats, however, wounding increased strongly ionically bound peroxidase activity more than 13-fold (Table 1). The covalently bound fraction left in the wall of control leaves after 1 M NaCl extraction retained from 57% (oat) to 64% (barley) of the total HRGP, no detectable G6PD activity, and from 7% (oat) to 12% (barley) of the peroxidase activity. Wounding had no appreciable effect on HRGP levels in this fraction but slightly increased peroxidase activity (Table 1). The total amount of HRGP in the leaves of either species was not appreciably altered by wounding, and the decrease in soluble apoplastic HRGP in response to wounding was roughly proportional to increased amounts of HRGP covalently bound to cell walls. Since wounding markedly increased apoplastic peroxidase activity (Table 1) and peroxidase is presumed to cross-link extensin [3,7], we suggest that wounding caused a fraction of the soluble or weakly ionically bound apoplastic HRGP to become covalently bound into cell walls of the barley and oat primary leaves. When cucumber or carrot plants are wounded or infected with pathogens they accumulate more HRGP [8, 9, 161 and it has been suggested that this may play a role in dicot disease resistance [Z]. Mazau and Esquerrb Tugayt reported that pathogenic infection caused marked induction of HRGP in all dicots that they examined but had no significant effect on HRGP levels in the cereals barley, wheat or rice. Our results demonstrate that wounding has no appreciable effect on HRGP accumulation, or on its partitioning between the apoplast and symplast, in barley and oat primary leaves. In contrast, wounding markedly enhances apoplastic peroxidase activity (Table 1) which is associated with cross-linking of cell wall phenolics [3,7]. Cereals have far less HRGP than do dicots [2] and cereal cell walls commonly respond to wounding or infection by enhanced accumulation of various cell wall phenolics [ 12, 131. Our results are consistent with the view [ 1l] that in cereal cell walls, ferulic acid and related phenolics may be more important than 4-hydroxyproline-rich glycoproteins as adaptive responses to tissue damage. EXPERIMENTAL
Barley (Hordeum oulgare L.) and oat (Aoena satiua L.) seedlings were grown and harvested as previously described [14]. Extraction of apoplastic and symplastic constituents. Apoplastic and symplastic fractions were prepared as previously described [14, 151. Briefly, the lower epidermis was peeled from 10 leaves and the peeled segments vacuum-infiltrated with 5 ml of 5 mM K-Pi buffer, pH 6.5, containing 200 mM NaCI, for 36 min, to elute an intercellular washing solution (IWS) containing soluble and weakly ionically bound apoplastic
2813
proteins and enzymes. Segments were next homogenized in 5 ml of 50 mM K-Pi buffer, pH 6.5, centrifuged, and the supernatant collected as the soluble symplastic fraction. After washing with 1% (v/v) Triton-X100 and buffer to remove cytoplasmic contamination, the pellet was stirred in buffered 1 M NaCl to liberate strongly ionically bound apoplastic fractions. The final pellet was assayed for covalently bound apoplastic proteins and enzymes. Mechanical wounding. Wounding was accomplished by peeling away the lower epidermis and incubating leaf segments on Hz0 at 30” in the dark for 24 hr [12]. At the end of incubation, segments were processed as described above to obtain symplastic and apoplastic protein and enzyme fractions. HRGP determination. Fractions were adjusted to 6 M HCI and hydrolysed at 116” in the dark for 18 hr in sealed tubes [17]. After hydrolysis the samples were evaporated to dryness at 65” under N,, the residue dissolved in H,O, and HRGP determined as 4-hydroxyproline by the method of Richard [17] using 4hydroxy-L-proline (Sigma Biochemical, St Louis, U.S.A.) as a standard. Enzyme assays. Peroxidase and glucose-6-phosphate dehydrogenase activities were assayed as before [14]. Total protein was determined by Bradford’s dye binding technique [18].
REFERENCES
1. Tierney, M. L. and Varner, J. E. (1987) Plant Physiol. 84, 1. 2. Lamport, D. T. A. and Catt, J. W. (1981) in Encyclopaedia of Plant Physiology (Tanner, W. and Holmes, F. A., eds), 13B, p. 133. Springer. 3. McNeil, M., Darvill, A. G., Fry, S. C. and Albersheim, P. (1984) Ann. Reo. Biochem. 53,625. 4. Fry, S. C. (1986) Ann. Rev. Plant Physiol. 37, 165. 5. Smith, J. J., Muldoon, E. P. and Lamport, D. T. A. (1984) Phytochemistry 23, 1233. 6. Kieliszewski, M. and Lamport, D. T. A. (1987) Plant Physiol. 85, 823.
7. Everdeen, D. S., Kiefer, S., Willard, J. J., Muldoon, E. P., Dey, P. M., Li, X. and Lamport, D. T. A. (1988) Plant Physiol. 87, 616.
8. Hammerschmidt, R., Lamport, D. T. A. and Muldoon, E. P. (1984) Physiol. Plant Pathol. 24, 43. 9. Mazau, D. and EsquerrQ-TugayQ M. T. (1986) Physiol. Mol. Plant Pathol. 29, 147.
10. Clarke, J. A., Lisker, N., Ellingboe, A. H. and Lamport, D. T. A. (1983) Plant Sci. Letters 30, 339. 11. Carpita, N. C. and Kanabus, J. (1988) Plant Physiol. 88,671. 12. Geballe, G. T. and Galston, A. W. (1982) Plant Physiol. 70, 781.
13. Reisener, H. J., Tiburzy, R., Kogel, K. H., Moerschbacher, B. and Keck, B. (1986) in Biology and Molecular Biology of Plant-Pathogen Interactions (Bailey, J., ed.), Vol. Hl, p. 133. NATO AST Series. 14. Li, Z.-C., McClure, J. W. and Hagerman, A. E. (1989) Plant Physiol. 90, 185.
15. 16. 17. 18.
Li, Z.-C. and McClure, J. W. (1989) Phytochemistry 28,2255. Tierney, M. L. (1986) Plant Physiol. 80, S-70. Richard, A. B. (1982) Meth. Enzymol. 82, 372. Bradford, M. (1976) Anal. Biochem. 72,248.