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Hydroxyproline and proline content of cell walls of sunflower, peanut and cotton grown under salt stress
Plaxt Sc~sce, 69 (1990) 27 - 32
27
Elsevier Scientific Publishers Ireland Ltd.
H Y D R O X Y P R O L I N E A N D P R O L I N E C O N T E N T O F CELL W A L L S OF S U N F L O W E R , P E A N U T A N D COTTON G R O W N U N D E R SALT S T R E S S
A. GOLAN-GOLDHIRSH', B. HANKAMER and S.H. LIPS
Plant Adaptation Researck Unit. Tke Jacob BlauJtein Institute for Desert Researc~ Ben.Gu)~on Unit)ersity o/tke Negev. Sede Boqer C.mnp~84993(Is~eU (Received December 8th, 1989) (Revision received February 2nd. I990) (Accepted February 2nd. 1990)
Proline and hydroxyproline content of the cell walls of peanut (A ~acku bypoga~a L. cv. Shulamit), cotton (Gossypium kirsuturn L. cv. sj) and sunflower (Heliantku.s annum) L. ¢v. Saffola S0222) were determined. Proline concentration in the leaves of all plants species tested was approximately 110 nmol •mg ~ dry matter and higher than in the root and stem. Hydroxyproline concentration was highest in the root of all species tested and was between 30 and 60 nmol" mg "~ dry matter. This was 5 - 10 times higher than the concentration in the stem and leaf. There was no significant effect of salt stress on proline and hydroxy. proline concentration in "purified cell wall fraction" of sunflower. Salt stress (100 mmol • 1~ NaCI} reduced substantially plant growth of 53-day~)ld sunflowers, stem volume decreased from 21~3 ± 5.? ml to 3.8 ± 1.6 ml, dry matter content of stem decreased from 719 ± 186 mg to 88 ± 33 mg and the number of leaves per plant decreased from 18 to 13. These results suggest that inhibition of plant growth, by salt stress, was accompanied by inhibition of cell wall proteins (extensins) synthesis. Therefore, changes in the physicochemical properties of cell wall accompanying the osmotic adjustment should be sought in other posttranslatiortal modifications of extensin(s}, either glycosylation or inter, and/or intramolecular cros.q-linking in the cell wall.
Key worda: Heliantkus annuu~ L.; Aracb/m hF/>ogoea L.; GossFpium kirsutum I,.; extensin(s); hydroxyproline; proline.
Introduction A reduction in cell size has often been noted in field grown plants and in plant cell cultures, which developed under conditions of water stress [1~ Restricted cell expansion associated with osmotic adjustment has been suggested as an adaptive mechanism to counteract reduced rates of solute uptake [2]. A significantly thicker cell wall was observed in stressed plants compared to controls [3]. If osmotic stress suppresses plant growth through an effect on cell elongation, the cell wall structure may change under such conditions. Changes in the polysaccharides fraction of cell wall under water stress were indeed reported recently
• Author to whom all correspondence should be addressed.
[4,5~ The effect of salt stress on the protein fraction of cell wall has been reported scantily {6,7]. Extensin is a major plant cell wall glycopr~ tein in dicots. In many plant species examined so far, it was found to be an hydroxyproline(hyp)-rich (46 s o l % ) glycoprotein [8~ Of the total cell hyp, 95% was found in the cell wall fraction [9]. Therefore, hyp was often used as a marker for extensin. Several reports indicated that increased hyp content was correlated with cessation of growth [10]. Growth inhibiting agents increased hyp content in excised pea epicotyls, while o,a'. dipyridyl, an iron chelator, blocked hyp formation and overcame the effect of growth inhibitors [11]. The levels of cell-wall bound hyp inversely correlated with growth rates [12]. Inhibition of proline hydroxylation in the pres-
0168-9452/90/$03.50 (c~"19~) Elsevier Scientific Publishers Ireland IAd. Printed and Published in Ireland
28 ence of low POz or a,a'-dipyridyl was correlated with increased growth [13,14]. Hydroxyproline content of cell walls of Pisum satitmm increased in successive segments cut basipetally from the root tip [15] also indicating that decreased growth rate is associated with high levels of hyp. In contrast to these findings, which suggested that high hyp is associated with cessation of growth it was reported that cell walls of rapidly growing, submerged internodes of rice plants contained more hyp and had a higher hydration capacity than control internodes [16}. The significance of hyp may not be the same in monocotyledons as in dicotyledons since monocotyledons contain much less {10-20 times) hyp [17}. The aim of this work was to evaluate the changes in cell wall proteins induced by salt stress, by determination of the changes in hyp and proline concentrations. Materials and Methods
Plant materials Peanut (Arachis hypogaea L. cv. Shulamit), and cotton (Gossypium hirsutum L. cv. sj) were grown in aerated Long Ashton (LA) [18] nutrient solutions containing 4 m m o l ' l -~ nitrate, as Ca(NOs)~ in the greenhouse. The medium of salt-treated peanut and cotton contained 100 mmol" 1-1 and 200 retool. I-~ sodium chloride. Nutrient solutions were changed weekly and water loss was replenished daily [19]. Plants were harvested after 16 days, separated into root, stem and leaves and dried at 70°C for 48 h. The dry material was ground to fine powder and saved desicated at room temperature until analysed. Sunflowers (Helianthus annuus L. cv. Saffola S0222) were grown and treated as described above, except that the medium contained 15 mmol. 1-~ nitrate, the salt treatment contained 100 mmol.l -~ NaCI. Sample plants were removed during development for analysis. Cell wall extraction Cell walls of sunflower root were extracted according to Talmadge et al. [20], with the fol-
lowing modifictions: roots were washed in ice cold water, frozen in liquid nitrogen and freeze dried. The dry tissue was ground to fine powder and stored desicated at room temperature until used. Cell wall extraction was carried out on 1-g samples of dry powder. It was washed twice in 500 ml of water containing 10 retool" 1~ NaHSO 3. The residue was resuspended in 40 ml cold washing solution (10 mmol" 1-1 NaHSO s and 0.5% Nonidet). The suspension obtained was passed through a Carver Laboratory French Press Model M at 10 0 0 0 - 3 0 000 psi and the extruded emulsion passed directly into 460 ml cold washing solution. The cell wall fraction was collected on a miracloth filter, resuspended in 200 ml of washing solution and sonicated in a Labsonic 1510 for 1 rain at 140 W. The washed cell wall fraction was centifruged at 8000 x g at 4°C for 5 rain. The resulting pellet was sonicated and centrifuged as before and washed three times in water, followed by 5 washes in 40 ml methanol/chlorform (1:1) mixture, then washed 3 times in acetone and dried at 75°C for 2 h. Completion of cell breakdown was verified microscopically. The resulting pellet referred to as the "purified cell wall fraction" was used for hydrolysis.
Acid hydrolysis of dry matter and purified cell waU fraction A sample of plant dry material or purified cell wall fraction (25-50 rag) was suspended in HCI 6 N (2 ml), in screw-capped pyrex tubes and heated at ll0°C for 20 h. In preliminary experiments it was found that the release of proline and hyp was complete after 8 h and the amino acids were stable for at least 30 h under the conditions of hydrolysis. Hydrolysates were cooled to room temperature, their pH adjusted to 6 - 7 and their volume made-up to 10 ml with water. This solution was filtered through Whatman No. 1 filter paper and the filtrate analysed for proline and hyp. Proline and hyp de termination Assays were carried-out according to Bates et al. [211 and Woessner [22.23]. respectively. L-Proline, 4-hydroxy-L-proline, Chloramine-T,
29 ninhydrin and L-(dimethylamino)benzaldehyde were obtained from Sigma chemical company. Methyl-cellosolve was purchased from Merck. Other laboratory chemicals used were analytically pure. Deionized glass distilled water was used in all analytical procedures.
Stem volume Stem length and radius at the base were measured and the volume was calculated from the geometrical formula 1/3 n'r2h, where r is the radius at the base of the stem and h is stem length. Results and Discussion
Proline and hyp in several species The concentrations of proline and hyp in cotton, peanut, and sunflower are shown in Table I. Proline concentration in the leaf of cotton (110.3 ± 1.4 n m o l ' m g -~ dry wt.), peanut (113.9 ± 19.9 nmol" mg -~ dry wt.) and sunflower (136.7 ± 6.3 nmol. mg -~ dry wt.) were approximately 3-fold higher than in the root or stem. Hydroxyproline concentration in the root of these species was 38.1, 57.8 and 30.1 nmol- mg -t dry wt., respectively. It was approximately 6-fold higher than in the stem and leaf. Hyp is a protein amino acid, a product of
posttranslational modification of protein proline [9]. It has been reported in several plant glycoproteins, but its highest concentration was found in cell wall extensins [8]. The results obtained indicated either a higher content of extensin in root cell walls or higher extent of hydroxylation of the same amount of extensin in the root as in the other organs of the plants. There was a general trend of increase in the concentration of proline in the salt treated plants, which was more prominent in sunflower (Table I). There was an increase in proline concentration in the root from 59.2 ± 5.0 to 74.2 ± 10.8 nmol'mg -~ dry wt., in the stem from 33.8 ± 4.6 to 48.4 ± 0.6 n m o l ' m g -~ dry wt. and from 136.7 ± 10.3 to 178 ± 10.8 n m o l ' m g -~ dry wt. in the leaf (Table I). This may reflect accumulation of proline in the free amino acids pool, a well reported phenomenon, under osmotic stress conditions [24]. However, because the free amino acid pool is usually small, large changes of proline concentration in this pool must occur in order to be reflected in proline concentration of plant dry matter hydrolysates. In the other species (cotton and peanut) the increase in proline concentration was slight or non-detectable. It may be suggested also that proline accumulation in sunflower reflected inhibition of proline
Table I.
Effect of salt s t r e s s on proline and hyp concentrations in acid hydrolysates of dry m a t t e r of cotton, peanut and sunflower. Each mean represents three determinations. Means without ± S.D. are of duplicate determinations. Plant part
Treatment species
Proline (nmol • m g : dry wt.)
Hyp (nmo] • m g ~ dry wt.)
Control
Control
Saline
'-Saline
Root
Cotton Peanut Sunflower
44.3 ± 75.7 ± 59.2 ±
0.4 2.6 5.0
49.3 ± 3.6 71.3 ± 0.2 74.2 ± 10.8
38.1 57.8 30.1 ± 3.3
37.4 ± 0.1 62.2 ,~3.6 ± 3.7
Stem
Cotton Peanut Sunflower
41.1 ± 65.9 ± 33.8 ±
3.5 2.6 4.6
38.9 ± 72.8 ± 48.4 ±
8.3 0.9 6.6
6.5 ± 0.3 13.1 ± 0.9 6.4 ± 0.2
7.9 ± 0.1 14.8 ± 0.7 6.9 -+. 0.3
Leaf
Cotton Peanut Sunflower
96.6 ± 3.5 127.3 ± 0.I 178.0 ± 10.3
6.4 ± 0.2 8.8 ± 0.2 7.8 ± 0.3
6.9 ± 0.3 10.2 ± 0.I 8.3 ± 0.6
110.3 ± 1.4 113.9 ± 19.9 136.7 ± 6.3
30 hydroxylation under salt stress rather than accumulation of free proline. However, there were no differences in proline concentration between salt-treated (35.1 ± 1.6 nmoi. mg -~ dry wt.} and control (33.1 ± 1.3 nmoi'mg -~ dry wt.) in the purified cell wall fraction of root (Table II), which represented the protein bound amino acids. Therefore, it appears that the accumulation of proline in sunflower was in the free amino acid pool and not due to inhibition of peptidyl-proline hydroxylation. The effect of salt treatment on hyp concentration in any of the organs of the species tested was not significant (Table I). Assuming hyp as a marker for extensin, the results suggested that there was no effect of the salt on the hydroxylation of proline in extensin already present in the cell wall. This is consistent with the current view that the posttranslational hydroxylation of proline in the extensin precursor occurs in the endoplasmic reticulum before being transported to the cell wall [25]. Furthermore, from the fact that there were no major changes in proline and hyp concentrations during growth under stress conditions (Fig. 1} we conclude that the hydroxylation of proline was not affected in the newly made extensin. The effect of salt stress on proline and hyp concentrations in the purified cell wall fraction of sunflower roots showed no significant effect on both proline and hyp concentrations, although a slight increase in hyp occured (Table II}. The lack of difference in proline concentration between control and salt treatment in the purified cell wall fraction of sunflower (Table II) in contrast to the significant difference found in dry matter hydrolysates (Table I), may reflect the removal T a b l e II. Effect of salt s t r e s s on proline and concentration in s u n f l o w e r root purified cell wall. Treatment (NaCI mmol" 1"9
0 I00
A m i n o acid
(nmol
•
hyp
25 C 0L,.
x
0
15
L--
R T
~
5 w u.
,
I
~
l
,
,
I
,
.
,
I
.
.
A
c 250
-6 &.
Q.
50 20
25
30
Time {doys} Fig. I. Effect of salt s t r e s s on proline (Al and hyp (B* concentration in the dry m a t t e r of s u n f l o w e r s e e d l i n g s d u r i n g growth, a, proline; b, hyp. Leaf. • - • ; S t e m , @ - @; Root, &-&. (Filled s y m b o l s a r e for NaCI t r e a t m e n t (I00 retool" l q and open s y m b o l s a r e for control (0 mmol • I q.
rag" dry wt.)
Proline
Hyp
33.1 ± 1.3 35.1 ± 1.6
54.8 ± 7.2 63.6 ± 5.8
of the free amino acid pool in the purification steps {see Materials and Methods}. This showed more specifically than the dry matter hydrolysates (Table I) that hydroxylation of cell wall peptidyi-proline was not affected by salt stress.
31 Table III. Effect of salt stress on sunflower stem volume, dry matter content and number of leaves.
3
Treatment NaCI (retool • II)
Stem
No. of leaves
4
Volume (ml)
Dry matter (rag)
0 I00
21.3 ± 5.2 3.8 ± 1.6
719 ± 186 88 ± 33
17.7 ± 1.0 12.7 ± 1.4
5
The effect of salt stress (100 mmol" I-I NaCl) for 16 days on sunflower development is shown in Table III. Stem volume decreased from 21.3 ± 5.2 ml to 3.8 ± 1.6 ml mainly because of shortening of the stem. The number of cells in a cross-section was virtually unaffected by the salt treatment (data not shown}, therefore it appears that the salt stress inhibited cell elongation, dry matter content of the stem declined from 719 ± 186 mg to 88 ± 33 mg and number of leaves per plant was 18 in control and 13 in the salt stressed plants (Table III). A similar trend of reduction in dry matter content of root and leaves was obtained (data not shown}. The salt treatment had a major developmental effect on sunflowers in reducing the number of leaves and the size of the plants. There were no major changes in proline and hyp concentration in hydrolysates of sunflower seedlings during growth between day 18 and 32 (Figs. la and lb). It may be suggested that during maturation under salt stress cell wall protein synthesis and hydroxylation were inhibited to the same extent. We propose that changes in the physicochemical properties of plant cell wall accompanying the osmotic adjustment should be sought in other posttranslational modifications of extensin(s), either glycosylation or inter- and/or intramolecular cross-linking in the cell wall.
6
7
8 9
10 11
12
13
14
15
16
17
References 18 1
2
P.M. Hasegawa, R.A. Bressan and A.K. Handa, Cellu lar mechanisms of salinity tolerance. HortScience. 21 {1986} 1317- 1324. E. Van Volkenburgh and J.S. Boyer, Inhibitory effects of water deficit on maize leaf elongation. Plant Physiol., 77 (1985) 190 - 194.
19
J.M. Cutler, D.W. Rains and R.S. Loomis, The impor tance of cell size in the water relations of plants. Phy. siol. Plant., 40 (1977) 255 - 260. N. Sakural, S. Tanaka and S. Kura/shi, Changes in wall polysaccharides of squash (Ctwarbita maxima Duch.) hypocotyls under water stress condition. I. Wall sugar composition and growth as affected by water stress. Plant Cell Physiol., 28 (1987) 1051 - 1058. N. Sakural, S. Tanaka and S. Kuraishi, Changes in wall polysaccharides of squash (Cucurbita maxima Duch.) hypocotyls under water stress condition. 2. Composi. tion of pectic and hemicellulosic polysaccharides. Plant Cell Physiol., 28 (1987) 1059- 1070. C.S. Bozarth, J.E. Mullet and J.S. Boyer, Cell wall proteins at low water potentials. Plant Physiol.. 85 (1987) 261 - 267. N.M. Iraki, N. Singh. R.A. Bressan and N.C. Carpita, Alteration of the physical and chemical structure of the primary cell wall of growth.limited plant cells adapted to osmotic stress. Plant Physiol.. 91 (1989) 39 -47. G.I. Cassab and J.E. Varner, Cell wall proteins. Annu. Rev. Plant Physiol. Plant Mol. Biol., 39 (1988) 321 - 353. D.T.A. Lamport, Hydroxyproline-O-glycosidic linkage of the plant cell wall glycoprotein extensin. Nature, 216 (1967) 1322- 1324. R.E Cleland. Cell wail extension. Annu. Rev. Plant Physiol.. 22 (1971) 197 - 222. D. Savada. F. Walker and M.J. Chrispeels. Hydroxy. proline-rich cell wall protein (extensin): biosynthesis and accumulation in growing pea epicotyls. Dev. Biol.. 30 (1973) 42 - 48. T. Hnson and S. Wada, Role of hydroxyproline-rich cell wall protein in growth regulation of rice coleoptiles grown on or under water. Plant Cell Physiol.. 21 (1980} 511 - 524. T. Fujii, M. Shimokoiyama and H. Ichimura, Stimulation of A ~,ena coleoptiie growth by reduction of oxygen supply. Planta, 115 (1974) 345 - 354. N.M. Barnett, Dipyridylinduced cell elongation and inhibition of cell wall hydroxyproline biosynthesis. Plant Physiol., 45 (1970) 188 - 191. D. Vaughan and E. Cusens, Protein bound hydroxypro. line and root extension growth. Biechem. Soc. Trans., 2 (1974) 124- 126. S. Rose.John and H. Kende, Effect of submergence on the cell wall composition of deep.water rice internodes. Plant Physiol., 76 {1984) 106 - 11 I. A. Darvill, M. McNeil, P. Albersheim and D.P. Delmer, in: N.E. Tolbert (Ed.). The Biochemistry of Plants. Vol. I, Academic Press, New York, London, 1980, p. 92. E.J. Hewitt, Sand and water culture methods used in the study of plant nutrition. Technical communication no. 22 (revised). commonwealth bureau of horticultural and plantation crops. East Malling. Commonwealth Agricultural Bureaux. Farnham Royal, England, 1966. H.S. [.ips, Fertilization and saline water irrigation. Scientific Report to AID.CI)R, 1988.
32 20
21
22
23
K.W. Talmadge, K. Keegstra, W.D. Bauer and P. Albersheim. The structure of plant cell walls. Plant Physiol., 51 (1973) 158 - 173. L.S. Bates. R.P. Waldren and I.D. Teare, Rapid deter. ruination of free proline for water stress studies. Plant and Soil, 39 (1973} 205 - 207. J.F. Woessner, The determination of hydroxyproline in tissue and protein samples containing small propor. tions of this imino acid. Arch. Biochem. Biophys.. 93 (1901) 4 4 0 - 447. H. Stegemann. Mikrobestimung yon hydroxyprolin
24
25
mit Chloramin-T und p-dimethylaminobenzaldehyd. Hoppe-Seylers Z. Physiol Chem., 311 (1958} 41 - 45. C.R. Stewart. Proline accumulation: Biochemical aspects, in: L.G. I)aleg and D. Aspinall (Eds.I, The Phy siology and Biochemistry of Drought Resistence in Plants, Academic Press, Sydney, 1981. p. 242. D.G. Robinson, M. Andreae and A. Sauer, Hydroxypro line-rich glycoprotein biosynthesis: A comparison with that of collagen, in: C.T. Brett and J.R. Hillman (Eds.). Biochemistry of Plant Cell Walls. Cambridge Univer. sity Press, London, New York, 1985. p. 155.
27
Elsevier Scientific Publishers Ireland Ltd.
H Y D R O X Y P R O L I N E A N D P R O L I N E C O N T E N T O F CELL W A L L S OF S U N F L O W E R , P E A N U T A N D COTTON G R O W N U N D E R SALT S T R E S S
A. GOLAN-GOLDHIRSH', B. HANKAMER and S.H. LIPS
Plant Adaptation Researck Unit. Tke Jacob BlauJtein Institute for Desert Researc~ Ben.Gu)~on Unit)ersity o/tke Negev. Sede Boqer C.mnp~84993(Is~eU (Received December 8th, 1989) (Revision received February 2nd. I990) (Accepted February 2nd. 1990)
Proline and hydroxyproline content of the cell walls of peanut (A ~acku bypoga~a L. cv. Shulamit), cotton (Gossypium kirsuturn L. cv. sj) and sunflower (Heliantku.s annum) L. ¢v. Saffola S0222) were determined. Proline concentration in the leaves of all plants species tested was approximately 110 nmol •mg ~ dry matter and higher than in the root and stem. Hydroxyproline concentration was highest in the root of all species tested and was between 30 and 60 nmol" mg "~ dry matter. This was 5 - 10 times higher than the concentration in the stem and leaf. There was no significant effect of salt stress on proline and hydroxy. proline concentration in "purified cell wall fraction" of sunflower. Salt stress (100 mmol • 1~ NaCI} reduced substantially plant growth of 53-day~)ld sunflowers, stem volume decreased from 21~3 ± 5.? ml to 3.8 ± 1.6 ml, dry matter content of stem decreased from 719 ± 186 mg to 88 ± 33 mg and the number of leaves per plant decreased from 18 to 13. These results suggest that inhibition of plant growth, by salt stress, was accompanied by inhibition of cell wall proteins (extensins) synthesis. Therefore, changes in the physicochemical properties of cell wall accompanying the osmotic adjustment should be sought in other posttranslatiortal modifications of extensin(s}, either glycosylation or inter, and/or intramolecular cros.q-linking in the cell wall.
Key worda: Heliantkus annuu~ L.; Aracb/m hF/>ogoea L.; GossFpium kirsutum I,.; extensin(s); hydroxyproline; proline.
Introduction A reduction in cell size has often been noted in field grown plants and in plant cell cultures, which developed under conditions of water stress [1~ Restricted cell expansion associated with osmotic adjustment has been suggested as an adaptive mechanism to counteract reduced rates of solute uptake [2]. A significantly thicker cell wall was observed in stressed plants compared to controls [3]. If osmotic stress suppresses plant growth through an effect on cell elongation, the cell wall structure may change under such conditions. Changes in the polysaccharides fraction of cell wall under water stress were indeed reported recently
• Author to whom all correspondence should be addressed.
[4,5~ The effect of salt stress on the protein fraction of cell wall has been reported scantily {6,7]. Extensin is a major plant cell wall glycopr~ tein in dicots. In many plant species examined so far, it was found to be an hydroxyproline(hyp)-rich (46 s o l % ) glycoprotein [8~ Of the total cell hyp, 95% was found in the cell wall fraction [9]. Therefore, hyp was often used as a marker for extensin. Several reports indicated that increased hyp content was correlated with cessation of growth [10]. Growth inhibiting agents increased hyp content in excised pea epicotyls, while o,a'. dipyridyl, an iron chelator, blocked hyp formation and overcame the effect of growth inhibitors [11]. The levels of cell-wall bound hyp inversely correlated with growth rates [12]. Inhibition of proline hydroxylation in the pres-
0168-9452/90/$03.50 (c~"19~) Elsevier Scientific Publishers Ireland IAd. Printed and Published in Ireland
28 ence of low POz or a,a'-dipyridyl was correlated with increased growth [13,14]. Hydroxyproline content of cell walls of Pisum satitmm increased in successive segments cut basipetally from the root tip [15] also indicating that decreased growth rate is associated with high levels of hyp. In contrast to these findings, which suggested that high hyp is associated with cessation of growth it was reported that cell walls of rapidly growing, submerged internodes of rice plants contained more hyp and had a higher hydration capacity than control internodes [16}. The significance of hyp may not be the same in monocotyledons as in dicotyledons since monocotyledons contain much less {10-20 times) hyp [17}. The aim of this work was to evaluate the changes in cell wall proteins induced by salt stress, by determination of the changes in hyp and proline concentrations. Materials and Methods
Plant materials Peanut (Arachis hypogaea L. cv. Shulamit), and cotton (Gossypium hirsutum L. cv. sj) were grown in aerated Long Ashton (LA) [18] nutrient solutions containing 4 m m o l ' l -~ nitrate, as Ca(NOs)~ in the greenhouse. The medium of salt-treated peanut and cotton contained 100 mmol" 1-1 and 200 retool. I-~ sodium chloride. Nutrient solutions were changed weekly and water loss was replenished daily [19]. Plants were harvested after 16 days, separated into root, stem and leaves and dried at 70°C for 48 h. The dry material was ground to fine powder and saved desicated at room temperature until analysed. Sunflowers (Helianthus annuus L. cv. Saffola S0222) were grown and treated as described above, except that the medium contained 15 mmol. 1-~ nitrate, the salt treatment contained 100 mmol.l -~ NaCI. Sample plants were removed during development for analysis. Cell wall extraction Cell walls of sunflower root were extracted according to Talmadge et al. [20], with the fol-
lowing modifictions: roots were washed in ice cold water, frozen in liquid nitrogen and freeze dried. The dry tissue was ground to fine powder and stored desicated at room temperature until used. Cell wall extraction was carried out on 1-g samples of dry powder. It was washed twice in 500 ml of water containing 10 retool" 1~ NaHSO 3. The residue was resuspended in 40 ml cold washing solution (10 mmol" 1-1 NaHSO s and 0.5% Nonidet). The suspension obtained was passed through a Carver Laboratory French Press Model M at 10 0 0 0 - 3 0 000 psi and the extruded emulsion passed directly into 460 ml cold washing solution. The cell wall fraction was collected on a miracloth filter, resuspended in 200 ml of washing solution and sonicated in a Labsonic 1510 for 1 rain at 140 W. The washed cell wall fraction was centifruged at 8000 x g at 4°C for 5 rain. The resulting pellet was sonicated and centrifuged as before and washed three times in water, followed by 5 washes in 40 ml methanol/chlorform (1:1) mixture, then washed 3 times in acetone and dried at 75°C for 2 h. Completion of cell breakdown was verified microscopically. The resulting pellet referred to as the "purified cell wall fraction" was used for hydrolysis.
Acid hydrolysis of dry matter and purified cell waU fraction A sample of plant dry material or purified cell wall fraction (25-50 rag) was suspended in HCI 6 N (2 ml), in screw-capped pyrex tubes and heated at ll0°C for 20 h. In preliminary experiments it was found that the release of proline and hyp was complete after 8 h and the amino acids were stable for at least 30 h under the conditions of hydrolysis. Hydrolysates were cooled to room temperature, their pH adjusted to 6 - 7 and their volume made-up to 10 ml with water. This solution was filtered through Whatman No. 1 filter paper and the filtrate analysed for proline and hyp. Proline and hyp de termination Assays were carried-out according to Bates et al. [211 and Woessner [22.23]. respectively. L-Proline, 4-hydroxy-L-proline, Chloramine-T,
29 ninhydrin and L-(dimethylamino)benzaldehyde were obtained from Sigma chemical company. Methyl-cellosolve was purchased from Merck. Other laboratory chemicals used were analytically pure. Deionized glass distilled water was used in all analytical procedures.
Stem volume Stem length and radius at the base were measured and the volume was calculated from the geometrical formula 1/3 n'r2h, where r is the radius at the base of the stem and h is stem length. Results and Discussion
Proline and hyp in several species The concentrations of proline and hyp in cotton, peanut, and sunflower are shown in Table I. Proline concentration in the leaf of cotton (110.3 ± 1.4 n m o l ' m g -~ dry wt.), peanut (113.9 ± 19.9 nmol" mg -~ dry wt.) and sunflower (136.7 ± 6.3 nmol. mg -~ dry wt.) were approximately 3-fold higher than in the root or stem. Hydroxyproline concentration in the root of these species was 38.1, 57.8 and 30.1 nmol- mg -t dry wt., respectively. It was approximately 6-fold higher than in the stem and leaf. Hyp is a protein amino acid, a product of
posttranslational modification of protein proline [9]. It has been reported in several plant glycoproteins, but its highest concentration was found in cell wall extensins [8]. The results obtained indicated either a higher content of extensin in root cell walls or higher extent of hydroxylation of the same amount of extensin in the root as in the other organs of the plants. There was a general trend of increase in the concentration of proline in the salt treated plants, which was more prominent in sunflower (Table I). There was an increase in proline concentration in the root from 59.2 ± 5.0 to 74.2 ± 10.8 nmol'mg -~ dry wt., in the stem from 33.8 ± 4.6 to 48.4 ± 0.6 n m o l ' m g -~ dry wt. and from 136.7 ± 10.3 to 178 ± 10.8 n m o l ' m g -~ dry wt. in the leaf (Table I). This may reflect accumulation of proline in the free amino acids pool, a well reported phenomenon, under osmotic stress conditions [24]. However, because the free amino acid pool is usually small, large changes of proline concentration in this pool must occur in order to be reflected in proline concentration of plant dry matter hydrolysates. In the other species (cotton and peanut) the increase in proline concentration was slight or non-detectable. It may be suggested also that proline accumulation in sunflower reflected inhibition of proline
Table I.
Effect of salt s t r e s s on proline and hyp concentrations in acid hydrolysates of dry m a t t e r of cotton, peanut and sunflower. Each mean represents three determinations. Means without ± S.D. are of duplicate determinations. Plant part
Treatment species
Proline (nmol • m g : dry wt.)
Hyp (nmo] • m g ~ dry wt.)
Control
Control
Saline
'-Saline
Root
Cotton Peanut Sunflower
44.3 ± 75.7 ± 59.2 ±
0.4 2.6 5.0
49.3 ± 3.6 71.3 ± 0.2 74.2 ± 10.8
38.1 57.8 30.1 ± 3.3
37.4 ± 0.1 62.2 ,~3.6 ± 3.7
Stem
Cotton Peanut Sunflower
41.1 ± 65.9 ± 33.8 ±
3.5 2.6 4.6
38.9 ± 72.8 ± 48.4 ±
8.3 0.9 6.6
6.5 ± 0.3 13.1 ± 0.9 6.4 ± 0.2
7.9 ± 0.1 14.8 ± 0.7 6.9 -+. 0.3
Leaf
Cotton Peanut Sunflower
96.6 ± 3.5 127.3 ± 0.I 178.0 ± 10.3
6.4 ± 0.2 8.8 ± 0.2 7.8 ± 0.3
6.9 ± 0.3 10.2 ± 0.I 8.3 ± 0.6
110.3 ± 1.4 113.9 ± 19.9 136.7 ± 6.3
30 hydroxylation under salt stress rather than accumulation of free proline. However, there were no differences in proline concentration between salt-treated (35.1 ± 1.6 nmoi. mg -~ dry wt.} and control (33.1 ± 1.3 nmoi'mg -~ dry wt.) in the purified cell wall fraction of root (Table II), which represented the protein bound amino acids. Therefore, it appears that the accumulation of proline in sunflower was in the free amino acid pool and not due to inhibition of peptidyl-proline hydroxylation. The effect of salt treatment on hyp concentration in any of the organs of the species tested was not significant (Table I). Assuming hyp as a marker for extensin, the results suggested that there was no effect of the salt on the hydroxylation of proline in extensin already present in the cell wall. This is consistent with the current view that the posttranslational hydroxylation of proline in the extensin precursor occurs in the endoplasmic reticulum before being transported to the cell wall [25]. Furthermore, from the fact that there were no major changes in proline and hyp concentrations during growth under stress conditions (Fig. 1} we conclude that the hydroxylation of proline was not affected in the newly made extensin. The effect of salt stress on proline and hyp concentrations in the purified cell wall fraction of sunflower roots showed no significant effect on both proline and hyp concentrations, although a slight increase in hyp occured (Table II}. The lack of difference in proline concentration between control and salt treatment in the purified cell wall fraction of sunflower (Table II) in contrast to the significant difference found in dry matter hydrolysates (Table I), may reflect the removal T a b l e II. Effect of salt s t r e s s on proline and concentration in s u n f l o w e r root purified cell wall. Treatment (NaCI mmol" 1"9
0 I00
A m i n o acid
(nmol
•
hyp
25 C 0L,.
x
0
15
L--
R T
~
5 w u.
,
I
~
l
,
,
I
,
.
,
I
.
.
A
c 250
-6 &.
Q.
50 20
25
30
Time {doys} Fig. I. Effect of salt s t r e s s on proline (Al and hyp (B* concentration in the dry m a t t e r of s u n f l o w e r s e e d l i n g s d u r i n g growth, a, proline; b, hyp. Leaf. • - • ; S t e m , @ - @; Root, &-&. (Filled s y m b o l s a r e for NaCI t r e a t m e n t (I00 retool" l q and open s y m b o l s a r e for control (0 mmol • I q.
rag" dry wt.)
Proline
Hyp
33.1 ± 1.3 35.1 ± 1.6
54.8 ± 7.2 63.6 ± 5.8
of the free amino acid pool in the purification steps {see Materials and Methods}. This showed more specifically than the dry matter hydrolysates (Table I) that hydroxylation of cell wall peptidyi-proline was not affected by salt stress.
31 Table III. Effect of salt stress on sunflower stem volume, dry matter content and number of leaves.
3
Treatment NaCI (retool • II)
Stem
No. of leaves
4
Volume (ml)
Dry matter (rag)
0 I00
21.3 ± 5.2 3.8 ± 1.6
719 ± 186 88 ± 33
17.7 ± 1.0 12.7 ± 1.4
5
The effect of salt stress (100 mmol" I-I NaCl) for 16 days on sunflower development is shown in Table III. Stem volume decreased from 21.3 ± 5.2 ml to 3.8 ± 1.6 ml mainly because of shortening of the stem. The number of cells in a cross-section was virtually unaffected by the salt treatment (data not shown}, therefore it appears that the salt stress inhibited cell elongation, dry matter content of the stem declined from 719 ± 186 mg to 88 ± 33 mg and number of leaves per plant was 18 in control and 13 in the salt stressed plants (Table III). A similar trend of reduction in dry matter content of root and leaves was obtained (data not shown}. The salt treatment had a major developmental effect on sunflowers in reducing the number of leaves and the size of the plants. There were no major changes in proline and hyp concentration in hydrolysates of sunflower seedlings during growth between day 18 and 32 (Figs. la and lb). It may be suggested that during maturation under salt stress cell wall protein synthesis and hydroxylation were inhibited to the same extent. We propose that changes in the physicochemical properties of plant cell wall accompanying the osmotic adjustment should be sought in other posttranslational modifications of extensin(s), either glycosylation or inter- and/or intramolecular cross-linking in the cell wall.
6
7
8 9
10 11
12
13
14
15
16
17
References 18 1
2
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