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Cell wall composition of near isogenic wheat lines carrying genes for resistance to Erysiphe graminis f. sp. tritici
Plant Science Letters, 30 (1983) 339--346 Elsevier Scientific Publishers Ireland Ltd.
339
CELL WALL COMPOSITION OF NEAR ISOGENIC WHEAT LINES C A R R Y I N G GENES F O R RESISTANCE TO ER YSIPHE GRAMINIS f. sp. TRITICI
J O Y C E A. C L A R K E a.*, N O R B E R T O D E R E K T.A. L A M P O R T a
L I S K E R b,**, A L B E R T H. E L L I N G B O E b,*** and
a M S U - D O E Plant Research Laboratory and b Department of Botany and Plant Pathology Michigan State University,East Lansing, M I 48824 (U.S.A.) (Received August 23rd, 1982) (Revision received December 7th, 1982) (Accepted December 8th, 1982)
SUMMARY
Cell wall composition of near isogenic wheat (Triticum aestivum L. em Thell) lines were compared during the initial growth period of 5--9 days. The lines could n o t be distinguished in terms of hydroxyproline arabinoside (Hyp-ara) profiles, amino acids and neutral sugars composition and levels. This tends to support the idea that the host's resistance genes are n o t expressed as major constituents of the cell wall.
Key words: Resistance -- Isogenic lines -- Hydroxyproline aralinoside -Amino acids -- Neutral sugars -- Monocotyledons
INTRODUCTION
The process of primary infection of wheat by Erysiphe graminis f. sp. tritici has been reported [ 1,2]. In the appropriate environmental conditions, this parasite will proceed through a series of morphological stages with a high degree of synchrony during the first 30 h after inoculation [1,2]. This and
*Present address: Department of Botany and Plant Sciences, University of California, Riverside, CA 29521, U.S.A. **To whom correspondence should be sent at: Stored Products Division, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel. ***Present address: International Plant Research Institute, 853 Industrial Road, San Carlos, CA 94070, U.S.A. Abbreviations: Hyp-ara, hydroxyproline arabinoside; TFA, trifluoracetic acid. 03044211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
340 it's high infection efficiency and gene availability affecting primary infection, make this disease most suitable for studies associated with biochemical changes at the early stages of the infection process [1--3]. The interactions between host and parasite are controlled by corresponding genes in host and parasite [1]. The host genes are designated Pmla, Pm2a, Pm3a, for allela A a t the Pro1 locus, allele a at the Pro2 locus, etc. [4]. The corresponding parasite genes are designated Pla, P2a, etc. [1]. All but one of these genes have measurable effects on the early interactions between host and parasite [ 1--3 ]. The aim of this work was to compare the cell wall composition in noninoculated isogenic wheat lines carrying or without, single dominant Pm genes. These findings should serve in evaluating changes in the host cell wall caused during the infection process. MATERIALS AND METHODS
Plant material Wheat (Triticum aestivum L. em Thell.) lines which are highly isogenic except for single Pm genes were available. The cv. Chancellor has no known Pm genes. It has the recemive genes pro1, pro2, pro3 and pro4. The other six host lines each contained one of the following dominant Pm genes: Pmla, Pm2a, Pm3a, Pm3b, Pm3c and Pm4a. Each of these genes has been put into the Chancellor tmckground by eight backcrosses to Chancellor [4]. Chancellor is completely susceptible to E. gram/n/s f. sp. tritici (isolate MS-l). In line 3c, the first juvenile leaf is susceptible and all other lines are resistant [4 ]. Twenty seeds of each wheat line were planted in 9.5 X 50 cm pots. The pots were maintained under uniform conditions of 20°C, 65 ± 5% relative humidity and 260 ft. candies light intensity. Ten grams of leaves were harvested 5- 9 days after planting by cutting the plants about I cm above soil surface. This period was chosen because the plant response to E. graminis is first evaluated during this interval [4].
Isolation of cell walls After they were weighed, the leaves were immediately frozen in nitrogen. The frozen leaves were finely ground in a mortar and the resulting powder suspended in 150 ml cold 1 M NaCI. The suspension was sonicated twice for 3-min periods in a Biosonik III sonicator (Bronwill Scientific, Rochester, NY 14603) at 105 W. Examination of the sonicate under a light microscope showed that at least 95% of the ceils were broken. Salt end residual chlorophyll were removed_from the cell wall by the following washes: twice with 15 vol. of distilled water; three times with 15 vol.'of acetone and twice with 15 vol. of distilled water. After each wash, the preparation was centrig~ged at full speed in a clinical centrifuge. The final white cell wall pellet was lyophilized for 24 h.
341
Analysis for starch Cell walls were tested for starch by the iodine reaction [5]. As starch was not detected by this method, cell w Alla were not treated with s-amylase prior to further analyses.
Amino acid analysis Five milligrams of cell wall were hydrolysed in 0.6 ml 5.5 N HC1 at 110°C for 18 h. The hydrolyzate was dried under nitrogen at 40°C and dissolved in 0.5 ml 0.001 N HC1. Aliquots were taken for hydroxyproline and amino acid analysis. Amino acid analyses were performed on a modified technicon Auto analyser as described by Lamport [6]. Hydroxyproline was assayed by a modification of the method of Kivirikko [7] as described by Lamport and Miller [8].
Neutral sugar analysis Five milligrams of cell wall were hydrolyzed in 2 ml trifluoracetic acid (TFA) at 121°C for 1 h. The hydrolysate was centrifuged in a clinical centrifuge at full speed for 15 min, 200/~1 of the supernatant were taken for preparation of alditol acetates. The alditol acetates were prepared by the method of Albersheim et al. [9] and inositol, added prior to hydrolysis, was used as an internal standard. Gas chromatographic separations were carried out on a Perkin Elmer 910 gas chromatograph (Perkin Elmer, Norwalk, CT 06556) fitted with a flame ionization detector and 180 X 0.2 cm i.d. glass column. The column was packed with a mixture of 0.2% polyethylene glycol adipate, 0.2% polyethylene glycol succinate and 0.4% silicone XE-115 on gas-chrom Q, 100--180 mesh (Supelco, Inc., Bellefonte, PA 16823). The flow rate of the carrier gas, helium, was 35 ml/min and the temperature program was: 1°C per min from 130°C to 180°C. Peaks were integrated by an Autolab IV module (Spectra-Physics, 655 Clyde Avenue, Mountain View, CA 94040). Aliquots of the TFA hydrolysate of cell walls were assayed for pentoses using orcinol [10].
Estimation of the Hyp.ara Hyp-ara were obtained by hydrolyzing 100 mg cell wall in 4 ml saturated 0.22 M Ba(OH)2 at 100°C for 18 h. The pH was adjusted to within the range of 7--8 with concentrated H2SO4. The supernatant was lyophilized and the lyophilisates dissolved in 200 ml deionized water and applied to the first in series of two Biogel P-2 (-400 mesh) (Biorad, 32 + Griffin, Richmond, CA 94804) columns (1 m × 1 cm) at 67°C. The columns were eluted with deionized water at a pump pressure of 250 lb/in 2 and the effluent was continuously monitored for Hyp-ara using the hydroxyproline assay described in Methods. The hydroxyproline peaks were identified by comparison of elution times with those of a sample of known composition, i.e., tomato cell wall, run under the same conditions. On the hot columns, resolution was not sufficient to separate free hydroxyproline from Hyp-ara; this resolution was achieved by running 2 columns cold under the same conditions.
20
40
.~
\
/
t t l l ~
GlucOse
*XylOSi
3C
IA
\
./
I I I I I
/\
/-
DAYS
4A
2A
o\ i.~,J
t l i : ~
o
F
Fig. 1. Neutral sugar profiles of cell walls isolated from near-isogenie wheat lines.
0 I--
Arobinose
CH
(.9 ,03 3e ...I 6 0
I1:
"~20~
!
"E4 0
60 e
3A
o
F
/
343
RESULTS AND DISCUSSION The similarity of neutral sugar composition of the host lines is shown in Fig. 1. The major neutral sugars are xylose, arabinose and glucose. The glucose is not derived from starch as it was shown that the cell walls are starch-free and cellulose is resistant to hydrolysis with 2 N TFA under the conditions used [11]. Low levels of rhamnose, 1.2 mol%; galactose, 2.4 mol%; galacturonic acid, 1.4 mol%; traces of mannose are present in all lines. The neutral sugar composition of wheat cell wall is similar in the high level of xylose as that reported for other monocotyledons but differs in the ratio of xylose/arabinose of 3 : 1 rather than unity, the low level of galactose, and the higher levels of non-cellulosic glucose [ 12]. Relative amounts of xylose and non-cellulosic glucose vary with age, with the increase in xylose parallelling the decrease in glucose. The decrease in glucose as the leaf elongates from 5 to 9 days is in agreement with the observation of a decrease in cell wall glucan level in Arena during auxinstimulated expansion and may implicate non-cellulosic glucose in cell wall loosening [13]. The levels of neutral sugars were similar in all cell walls and decreased from 40 weight% 5 days after planting to 25 weight% 9 days after planting. Table I shows the amino acid profile of all lines and of the dicotyledon, cotton [14]. The data for each day are not presented as there were no significant differences between days, as is shown by the low standard deviation for each value. Cell walls of all lines have similar amino acid profiles. The amino acid composition of the cell walls of wheat is similar to that of the cotton cell wall. The similarity of cell wall protein composition of a monocotyledon and that of a dicotyledon suggests that the protein is highly conserved. The Hyp-ara profile, which has been shown to change upon infection in the CoUetotrichum.melon system [ 15] is similar for all lines. Also the levels of hydroxyproline which enhance in the successful development of Erysiphe graminis on wheat [3] did not change during the experiment (Table II). The Hyp-ara profiles are characteristic for monocotyledons in that most of the hydroxyproline is non-glycosylated. The level of each Hyp-ara differs slightly from those reported for Zea mays [8] and this difference may reflect a source difference. That the lines cannot be distinguished in terms of Hyp-ara profiles, neutral sugar composition and levels, and amino acid composition suggests that the host's resistance genes are not expressed as major constituents of the cell wall and support the conclusion of Albersheim and Anderson-Prouty [ 16] that varietal specificity in the host-pathogen interaction is not determined by the constitutive components of the cell wall.
9.8 +- 0.5 5.5 ± 0.5 6.2 +- 0.7 9.7 +- 0.3 11.8 + 1.3 10.7 + 0.7 7.0 ± 0.5 Tr 4 . 3 + 0.4 1 0 . 0 + 0.4 2.4 + 0.5 4 . 9 + 0.5 6.9 ± 0.7 2.1 + 0.2 4.9 ± 0.4
8 4 . 0 ± 6.0
76.0 ± 9.0
Pm3c
9.9 -+ 0.7 5.9 ± 0.3 6.4 ± 0.4 9 . 6 ± 0.7 12.5 ± 0.4 1 0 . 9 ± 0.3 7.2 ± 0.5 Tr c 4.4 -+ 0.4 1 0 . 2 ± 0.7 2.6 ± 0.7 4.9 ± 0 . 3 6.3 + 0.4 2.3 ± 0.4 4.2 +- 0.4
Chancellor
N e a r i s o g e n i c w h e a t lines
a Each value is an average o f results o f 5 - - 9 b M. Meinhert and D.P. D e l m e r [ 14 ]. c Traces.
Leu Tyr Phe Lys His Arg Hyp/100 mg cell w a l l
Ileu
Asp Thr Ser GIu Gly Ala Val Met
(tool%)
A m i n o acids
0.2 0.4 0.6 1.2 1.0 0.4 0.4
0.5 0.2 0.2 0.2 0.3 0.5 0.5
days.
8 9 . 0 ± 7.0
10.4 ± 5.9 ± 6.7 • 10.2 ~ 12.5 ± 11.0 ± 7.9 ± Tr 4.8 ± 10.7 ± 2.4 ± 4.9 ± 7.0 ± 2.3 + 5.2 +
Pm3b
0.5 0.8 0.5 0.9 0.8 1.6 0.9
1.2 0.9 0.5 0.5 1.4 0.4 0.8
87.0 ± 11.0
9.8 ± 5.3 ± 6.2 ± 10.2 + 1 1 . 4 -+ 10.7 + 6.5 + Tr 4.2 + 10.1 + 2.6 -+ 5.4 + 6.0 + 3.5 + 4.7 +
Pm3a
AMINO ACID COMPOSITION OF NEAR ISOGENIC WHEAT LINES a
TABLE I
0.6 0.6 0.4 1.7 1.2 0.3 1.2
0.7 0.3 0.5 0.7 0.9 1.3 0.8
83.0 ± 9.0
9.8 + 5.8 ± 6.5 -+ 10.0-+ 12.8 + 10.4 ± 7.4 + Tr 4.7 + 10.8 ± 2.9 + 5.5 ± 6.4 + 2.3 ± 5.1 ±
Pm2a
0.4 0.7 0.5 0.9 0.9 1.6 1.3
0.7 0.3 0.5 0.4 0.8 1.0 0.6
98.0 ± 12.0
9.7 ± 5.4 ± 6.2 ± 1 0 . 2 -+ 11.4 + 10.7 ± 7.1 ± Tr 4.3 + 7.6 + 2.6 + 5.3 -+ 7.0 ± 2.9 ± 5.6 ±
Pmla
0.5 0.3 0.5 0.9 0.6 0.9 0.5
0.8 0.3 0.5 1.4 0.9 1.2 0.8
79.0 ± 9.0
9.5 + 5.5 ~ 6.4 ± 9.0 + 13.3 ± 10.2 ± 6.9 + Tr 4.5 + 10.6 ± 3.2 + 5.8 ± 6.2 ± 2.2 + 5.4 ±
Pm4a 10.8 + 6.0 + 8.4 ± 11.7-+ 11.8 ± 10.0 ± 7.0 ± 0.7 ± 4.9 + 9.7 + 2.0 ± 4.5 + 6.0 ± 2.2 + 4.2 +
0.8 0.4 1.2 1.0 1.3 1.7 0.7 0.9 0.4 0.6 0.7 0.3 1.6 0.6 0.4
Cotton b
345 TABLE II THE Hyp-ara PROFILE IN NEAR-ISOGENIC WHEAT LINES Near isogenic wheat lines
Age of plant a
Hyp-arab (%)
Hyp-ara~ (%)
Hyp-ara: (%)
Hyp-ara (%)
Unsubstituted Hyp (%)
5.9 6.6 6.2 7.1 6.6 6.0 6.1 5.2 5.2 4.6 4.0
21.8 21.9 21.7 23.4 24.0 22.7 15.2 21.8 19.8 23.1 13.0
8.9 6.9 7.9 8.1 8.3 7.8 6.3 8.2 7.1 6.6 2.0
14.4 48.9 17.5 46.9 16.5 47.6 14.7 46.6 13.6 47.6 15.0 48.4 23.4 48.9 16.0 48.7 12.9 55.0 20.6 45.0 15.0 66.0 Hyp-Ara + Hyp d
6.7 7.3 5.3 6.5 7.4
18.6 22.7 23.3 18.7 22.7
7.7 7.9 7.7 8.1 8.7
(1) Separated on cold columns
Chancellor
Pmla Pm2a Pm2a Pm3a Pm3a Zea mays (pericarp) c
5 6 7 8 9 9 6 9 6 9
(2) Separated on hot columns Pm2a 6 Pm3b 9 Pm3c 9 Pm4a 6 Pm4a 9
66.9 62.0 63.6 66.6 61.1
a Numeral refers to days after planting seed. Hyp-ara, is hydroxyproline tetra-arabinoside. c From D.T.A. Lamport and D.H. Miller [8 ]. d Hydroxyproline and Hyp-ara fused when the sample was run on hot columns. b
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t b y G r a n t s GB 4 1 2 1 4 , PCM 7 7 - 0 5 3 4 3 , a n d PCM 7 6 - 0 2 5 4 9 f r o m the N a t i o n a l Science F o u n d a t i o n a n d b y G r a n t O R D - 2 3 6 2 7 f r o m the U n i t e d States D e p a r h n e n t o f Agriculture. REFERENCES 1 A.H. Ellingboe, Phytopathology, 62 (1972) 401. 2 A.H. Ellingboe, Genetics of host-parasite interactions, in: R. Heitefus and P.H. Williams (Eds.), Encyclopedia of Plant Physiology, Vol. 4, Springer-Verlag, Heidelberg, 1976, p. 761. 3 J.A. Clarke, N. Lisker, D.T.A. Lamport and A.H. Ellingboe, Plant Physiol., 67 (1981) 188. 4 L.W. Briggle, Crop Sci., 9 (1969) 70. 5 J.E. Varner and R.M. Mense, Plant Physiol., 49 (1972) 187. 6 D.T.A. Lamport, Biochemistry, 8 (1969) 1155. 7 K.I. Kivirikko, Acta Physiol. Scand. (Suppl.), 219 (1963) 1.
346 8 I).T.A. Lamport and D.H. Miller, Plant Physiol., 48 (1971) 454. 9 P. Albersheim, D.J. Nevins, P.D. English and A. Karl Carbohydr. Res., 5 (1967) 340. 10 Z. Dische, Color reactions of pentoses, in: R.L. Whistler and M.W. Wolfrom (Eds.), Methods in Carbohydrate Chemistry, Vol. 1, Academic Press, New York, 1962, p. 485. 11 K. Talmadge, K. Keegstra, W.D. Bauer and P. Albersheim, Plant Physiol., 51 (1973) 158. 12 D. Burke, P. Kaufman, M. McNeil and P. Albersheim, Plant Physiol., 54 (1974) 109. 13 W. Loescher and D.J. Nevins, Plant Physiol., 50 (1972) 556. 14 M. Meinhert and D. Delmer, Plant Physiol., 59 (1977) 1088. 15 M.T. Esquerre-Tugaye, C. Lafitte, D. Mazau, A. Toppan and A. Touze, Plant Physiol., 64 (1979) 320. 16 P. Albersheim and A.J. Anderson-Prouty, Annu. Rev. Plant Physiol., 26 (1975) 31.
339
CELL WALL COMPOSITION OF NEAR ISOGENIC WHEAT LINES C A R R Y I N G GENES F O R RESISTANCE TO ER YSIPHE GRAMINIS f. sp. TRITICI
J O Y C E A. C L A R K E a.*, N O R B E R T O D E R E K T.A. L A M P O R T a
L I S K E R b,**, A L B E R T H. E L L I N G B O E b,*** and
a M S U - D O E Plant Research Laboratory and b Department of Botany and Plant Pathology Michigan State University,East Lansing, M I 48824 (U.S.A.) (Received August 23rd, 1982) (Revision received December 7th, 1982) (Accepted December 8th, 1982)
SUMMARY
Cell wall composition of near isogenic wheat (Triticum aestivum L. em Thell) lines were compared during the initial growth period of 5--9 days. The lines could n o t be distinguished in terms of hydroxyproline arabinoside (Hyp-ara) profiles, amino acids and neutral sugars composition and levels. This tends to support the idea that the host's resistance genes are n o t expressed as major constituents of the cell wall.
Key words: Resistance -- Isogenic lines -- Hydroxyproline aralinoside -Amino acids -- Neutral sugars -- Monocotyledons
INTRODUCTION
The process of primary infection of wheat by Erysiphe graminis f. sp. tritici has been reported [ 1,2]. In the appropriate environmental conditions, this parasite will proceed through a series of morphological stages with a high degree of synchrony during the first 30 h after inoculation [1,2]. This and
*Present address: Department of Botany and Plant Sciences, University of California, Riverside, CA 29521, U.S.A. **To whom correspondence should be sent at: Stored Products Division, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel. ***Present address: International Plant Research Institute, 853 Industrial Road, San Carlos, CA 94070, U.S.A. Abbreviations: Hyp-ara, hydroxyproline arabinoside; TFA, trifluoracetic acid. 03044211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
340 it's high infection efficiency and gene availability affecting primary infection, make this disease most suitable for studies associated with biochemical changes at the early stages of the infection process [1--3]. The interactions between host and parasite are controlled by corresponding genes in host and parasite [1]. The host genes are designated Pmla, Pm2a, Pm3a, for allela A a t the Pro1 locus, allele a at the Pro2 locus, etc. [4]. The corresponding parasite genes are designated Pla, P2a, etc. [1]. All but one of these genes have measurable effects on the early interactions between host and parasite [ 1--3 ]. The aim of this work was to compare the cell wall composition in noninoculated isogenic wheat lines carrying or without, single dominant Pm genes. These findings should serve in evaluating changes in the host cell wall caused during the infection process. MATERIALS AND METHODS
Plant material Wheat (Triticum aestivum L. em Thell.) lines which are highly isogenic except for single Pm genes were available. The cv. Chancellor has no known Pm genes. It has the recemive genes pro1, pro2, pro3 and pro4. The other six host lines each contained one of the following dominant Pm genes: Pmla, Pm2a, Pm3a, Pm3b, Pm3c and Pm4a. Each of these genes has been put into the Chancellor tmckground by eight backcrosses to Chancellor [4]. Chancellor is completely susceptible to E. gram/n/s f. sp. tritici (isolate MS-l). In line 3c, the first juvenile leaf is susceptible and all other lines are resistant [4 ]. Twenty seeds of each wheat line were planted in 9.5 X 50 cm pots. The pots were maintained under uniform conditions of 20°C, 65 ± 5% relative humidity and 260 ft. candies light intensity. Ten grams of leaves were harvested 5- 9 days after planting by cutting the plants about I cm above soil surface. This period was chosen because the plant response to E. graminis is first evaluated during this interval [4].
Isolation of cell walls After they were weighed, the leaves were immediately frozen in nitrogen. The frozen leaves were finely ground in a mortar and the resulting powder suspended in 150 ml cold 1 M NaCI. The suspension was sonicated twice for 3-min periods in a Biosonik III sonicator (Bronwill Scientific, Rochester, NY 14603) at 105 W. Examination of the sonicate under a light microscope showed that at least 95% of the ceils were broken. Salt end residual chlorophyll were removed_from the cell wall by the following washes: twice with 15 vol. of distilled water; three times with 15 vol.'of acetone and twice with 15 vol. of distilled water. After each wash, the preparation was centrig~ged at full speed in a clinical centrifuge. The final white cell wall pellet was lyophilized for 24 h.
341
Analysis for starch Cell walls were tested for starch by the iodine reaction [5]. As starch was not detected by this method, cell w Alla were not treated with s-amylase prior to further analyses.
Amino acid analysis Five milligrams of cell wall were hydrolysed in 0.6 ml 5.5 N HC1 at 110°C for 18 h. The hydrolyzate was dried under nitrogen at 40°C and dissolved in 0.5 ml 0.001 N HC1. Aliquots were taken for hydroxyproline and amino acid analysis. Amino acid analyses were performed on a modified technicon Auto analyser as described by Lamport [6]. Hydroxyproline was assayed by a modification of the method of Kivirikko [7] as described by Lamport and Miller [8].
Neutral sugar analysis Five milligrams of cell wall were hydrolyzed in 2 ml trifluoracetic acid (TFA) at 121°C for 1 h. The hydrolysate was centrifuged in a clinical centrifuge at full speed for 15 min, 200/~1 of the supernatant were taken for preparation of alditol acetates. The alditol acetates were prepared by the method of Albersheim et al. [9] and inositol, added prior to hydrolysis, was used as an internal standard. Gas chromatographic separations were carried out on a Perkin Elmer 910 gas chromatograph (Perkin Elmer, Norwalk, CT 06556) fitted with a flame ionization detector and 180 X 0.2 cm i.d. glass column. The column was packed with a mixture of 0.2% polyethylene glycol adipate, 0.2% polyethylene glycol succinate and 0.4% silicone XE-115 on gas-chrom Q, 100--180 mesh (Supelco, Inc., Bellefonte, PA 16823). The flow rate of the carrier gas, helium, was 35 ml/min and the temperature program was: 1°C per min from 130°C to 180°C. Peaks were integrated by an Autolab IV module (Spectra-Physics, 655 Clyde Avenue, Mountain View, CA 94040). Aliquots of the TFA hydrolysate of cell walls were assayed for pentoses using orcinol [10].
Estimation of the Hyp.ara Hyp-ara were obtained by hydrolyzing 100 mg cell wall in 4 ml saturated 0.22 M Ba(OH)2 at 100°C for 18 h. The pH was adjusted to within the range of 7--8 with concentrated H2SO4. The supernatant was lyophilized and the lyophilisates dissolved in 200 ml deionized water and applied to the first in series of two Biogel P-2 (-400 mesh) (Biorad, 32 + Griffin, Richmond, CA 94804) columns (1 m × 1 cm) at 67°C. The columns were eluted with deionized water at a pump pressure of 250 lb/in 2 and the effluent was continuously monitored for Hyp-ara using the hydroxyproline assay described in Methods. The hydroxyproline peaks were identified by comparison of elution times with those of a sample of known composition, i.e., tomato cell wall, run under the same conditions. On the hot columns, resolution was not sufficient to separate free hydroxyproline from Hyp-ara; this resolution was achieved by running 2 columns cold under the same conditions.
20
40
.~
\
/
t t l l ~
GlucOse
*XylOSi
3C
IA
\
./
I I I I I
/\
/-
DAYS
4A
2A
o\ i.~,J
t l i : ~
o
F
Fig. 1. Neutral sugar profiles of cell walls isolated from near-isogenie wheat lines.
0 I--
Arobinose
CH
(.9 ,03 3e ...I 6 0
I1:
"~20~
!
"E4 0
60 e
3A
o
F
/
343
RESULTS AND DISCUSSION The similarity of neutral sugar composition of the host lines is shown in Fig. 1. The major neutral sugars are xylose, arabinose and glucose. The glucose is not derived from starch as it was shown that the cell walls are starch-free and cellulose is resistant to hydrolysis with 2 N TFA under the conditions used [11]. Low levels of rhamnose, 1.2 mol%; galactose, 2.4 mol%; galacturonic acid, 1.4 mol%; traces of mannose are present in all lines. The neutral sugar composition of wheat cell wall is similar in the high level of xylose as that reported for other monocotyledons but differs in the ratio of xylose/arabinose of 3 : 1 rather than unity, the low level of galactose, and the higher levels of non-cellulosic glucose [ 12]. Relative amounts of xylose and non-cellulosic glucose vary with age, with the increase in xylose parallelling the decrease in glucose. The decrease in glucose as the leaf elongates from 5 to 9 days is in agreement with the observation of a decrease in cell wall glucan level in Arena during auxinstimulated expansion and may implicate non-cellulosic glucose in cell wall loosening [13]. The levels of neutral sugars were similar in all cell walls and decreased from 40 weight% 5 days after planting to 25 weight% 9 days after planting. Table I shows the amino acid profile of all lines and of the dicotyledon, cotton [14]. The data for each day are not presented as there were no significant differences between days, as is shown by the low standard deviation for each value. Cell walls of all lines have similar amino acid profiles. The amino acid composition of the cell walls of wheat is similar to that of the cotton cell wall. The similarity of cell wall protein composition of a monocotyledon and that of a dicotyledon suggests that the protein is highly conserved. The Hyp-ara profile, which has been shown to change upon infection in the CoUetotrichum.melon system [ 15] is similar for all lines. Also the levels of hydroxyproline which enhance in the successful development of Erysiphe graminis on wheat [3] did not change during the experiment (Table II). The Hyp-ara profiles are characteristic for monocotyledons in that most of the hydroxyproline is non-glycosylated. The level of each Hyp-ara differs slightly from those reported for Zea mays [8] and this difference may reflect a source difference. That the lines cannot be distinguished in terms of Hyp-ara profiles, neutral sugar composition and levels, and amino acid composition suggests that the host's resistance genes are not expressed as major constituents of the cell wall and support the conclusion of Albersheim and Anderson-Prouty [ 16] that varietal specificity in the host-pathogen interaction is not determined by the constitutive components of the cell wall.
9.8 +- 0.5 5.5 ± 0.5 6.2 +- 0.7 9.7 +- 0.3 11.8 + 1.3 10.7 + 0.7 7.0 ± 0.5 Tr 4 . 3 + 0.4 1 0 . 0 + 0.4 2.4 + 0.5 4 . 9 + 0.5 6.9 ± 0.7 2.1 + 0.2 4.9 ± 0.4
8 4 . 0 ± 6.0
76.0 ± 9.0
Pm3c
9.9 -+ 0.7 5.9 ± 0.3 6.4 ± 0.4 9 . 6 ± 0.7 12.5 ± 0.4 1 0 . 9 ± 0.3 7.2 ± 0.5 Tr c 4.4 -+ 0.4 1 0 . 2 ± 0.7 2.6 ± 0.7 4.9 ± 0 . 3 6.3 + 0.4 2.3 ± 0.4 4.2 +- 0.4
Chancellor
N e a r i s o g e n i c w h e a t lines
a Each value is an average o f results o f 5 - - 9 b M. Meinhert and D.P. D e l m e r [ 14 ]. c Traces.
Leu Tyr Phe Lys His Arg Hyp/100 mg cell w a l l
Ileu
Asp Thr Ser GIu Gly Ala Val Met
(tool%)
A m i n o acids
0.2 0.4 0.6 1.2 1.0 0.4 0.4
0.5 0.2 0.2 0.2 0.3 0.5 0.5
days.
8 9 . 0 ± 7.0
10.4 ± 5.9 ± 6.7 • 10.2 ~ 12.5 ± 11.0 ± 7.9 ± Tr 4.8 ± 10.7 ± 2.4 ± 4.9 ± 7.0 ± 2.3 + 5.2 +
Pm3b
0.5 0.8 0.5 0.9 0.8 1.6 0.9
1.2 0.9 0.5 0.5 1.4 0.4 0.8
87.0 ± 11.0
9.8 ± 5.3 ± 6.2 ± 10.2 + 1 1 . 4 -+ 10.7 + 6.5 + Tr 4.2 + 10.1 + 2.6 -+ 5.4 + 6.0 + 3.5 + 4.7 +
Pm3a
AMINO ACID COMPOSITION OF NEAR ISOGENIC WHEAT LINES a
TABLE I
0.6 0.6 0.4 1.7 1.2 0.3 1.2
0.7 0.3 0.5 0.7 0.9 1.3 0.8
83.0 ± 9.0
9.8 + 5.8 ± 6.5 -+ 10.0-+ 12.8 + 10.4 ± 7.4 + Tr 4.7 + 10.8 ± 2.9 + 5.5 ± 6.4 + 2.3 ± 5.1 ±
Pm2a
0.4 0.7 0.5 0.9 0.9 1.6 1.3
0.7 0.3 0.5 0.4 0.8 1.0 0.6
98.0 ± 12.0
9.7 ± 5.4 ± 6.2 ± 1 0 . 2 -+ 11.4 + 10.7 ± 7.1 ± Tr 4.3 + 7.6 + 2.6 + 5.3 -+ 7.0 ± 2.9 ± 5.6 ±
Pmla
0.5 0.3 0.5 0.9 0.6 0.9 0.5
0.8 0.3 0.5 1.4 0.9 1.2 0.8
79.0 ± 9.0
9.5 + 5.5 ~ 6.4 ± 9.0 + 13.3 ± 10.2 ± 6.9 + Tr 4.5 + 10.6 ± 3.2 + 5.8 ± 6.2 ± 2.2 + 5.4 ±
Pm4a 10.8 + 6.0 + 8.4 ± 11.7-+ 11.8 ± 10.0 ± 7.0 ± 0.7 ± 4.9 + 9.7 + 2.0 ± 4.5 + 6.0 ± 2.2 + 4.2 +
0.8 0.4 1.2 1.0 1.3 1.7 0.7 0.9 0.4 0.6 0.7 0.3 1.6 0.6 0.4
Cotton b
345 TABLE II THE Hyp-ara PROFILE IN NEAR-ISOGENIC WHEAT LINES Near isogenic wheat lines
Age of plant a
Hyp-arab (%)
Hyp-ara~ (%)
Hyp-ara: (%)
Hyp-ara (%)
Unsubstituted Hyp (%)
5.9 6.6 6.2 7.1 6.6 6.0 6.1 5.2 5.2 4.6 4.0
21.8 21.9 21.7 23.4 24.0 22.7 15.2 21.8 19.8 23.1 13.0
8.9 6.9 7.9 8.1 8.3 7.8 6.3 8.2 7.1 6.6 2.0
14.4 48.9 17.5 46.9 16.5 47.6 14.7 46.6 13.6 47.6 15.0 48.4 23.4 48.9 16.0 48.7 12.9 55.0 20.6 45.0 15.0 66.0 Hyp-Ara + Hyp d
6.7 7.3 5.3 6.5 7.4
18.6 22.7 23.3 18.7 22.7
7.7 7.9 7.7 8.1 8.7
(1) Separated on cold columns
Chancellor
Pmla Pm2a Pm2a Pm3a Pm3a Zea mays (pericarp) c
5 6 7 8 9 9 6 9 6 9
(2) Separated on hot columns Pm2a 6 Pm3b 9 Pm3c 9 Pm4a 6 Pm4a 9
66.9 62.0 63.6 66.6 61.1
a Numeral refers to days after planting seed. Hyp-ara, is hydroxyproline tetra-arabinoside. c From D.T.A. Lamport and D.H. Miller [8 ]. d Hydroxyproline and Hyp-ara fused when the sample was run on hot columns. b
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t b y G r a n t s GB 4 1 2 1 4 , PCM 7 7 - 0 5 3 4 3 , a n d PCM 7 6 - 0 2 5 4 9 f r o m the N a t i o n a l Science F o u n d a t i o n a n d b y G r a n t O R D - 2 3 6 2 7 f r o m the U n i t e d States D e p a r h n e n t o f Agriculture. REFERENCES 1 A.H. Ellingboe, Phytopathology, 62 (1972) 401. 2 A.H. Ellingboe, Genetics of host-parasite interactions, in: R. Heitefus and P.H. Williams (Eds.), Encyclopedia of Plant Physiology, Vol. 4, Springer-Verlag, Heidelberg, 1976, p. 761. 3 J.A. Clarke, N. Lisker, D.T.A. Lamport and A.H. Ellingboe, Plant Physiol., 67 (1981) 188. 4 L.W. Briggle, Crop Sci., 9 (1969) 70. 5 J.E. Varner and R.M. Mense, Plant Physiol., 49 (1972) 187. 6 D.T.A. Lamport, Biochemistry, 8 (1969) 1155. 7 K.I. Kivirikko, Acta Physiol. Scand. (Suppl.), 219 (1963) 1.
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