Planta (Berl.) 112, 243--252 (1973) 9 by Springer-Verlag 1973
Effects of Hydroxyproline on the Growth of Excised Root Segments of Pisum sativum under Aseptic Conditions D. Vaughan and Evelyn Cusens The Macaulay Institute for Soft Research, Craigiebuckler, Aberdeen, AB9 2 QJ, U.K. Received April 3, 1973 Summary. The cis and trans isomers of 4-hydroxy-T.-proline stimulated the extension growth of excised 2 4 mm pea root segments during culture. Increase in the uptake and subsequent incorporation of [ltCJleueine into proteins was inhibited by both n-isomers, and so also were changes in chloride uptake capacity and in protein metabolism measured in terms of invertase and peroxidase activities. Changes in [ltC]proline uptake and incorporation, and in respiration, were unaffected. Proline had no effect on changes in extension growth or protein metabolism but did prevent the effects of both hydroxyproline isomers. Azetidine-2-carboxylie acid inhibited extension growth and all the aspects of protein metabolism studied, the effects again being all prevented by proline. It is suggested that hydroxyproline enhances growth by interfering with protein synthesis in the cell walls.
Introduction Several amino analogues inhibit changes in the extension growth and protein metabolism of isolated pea root segments incubated under aseptic condir (Vaughan and Cusens, 1970, 1973), whereas most amino acids have no effect. I n contrast to this, hydroxyproline, which is present as a natural constituent in some plan~ proteins (Lamport, 1965), stimulates extension growth, and when applied externally often appears to act as an amino acid analogue. Thus hydroxyproline inhibits auxin-induced growth of Avena coleoptfles (Cleland, 1963; Norris, 1967) and the growth of a variety of callus tissues (Holleman, 1967; Lamport, 1965), both effects being prevented b y proline. This suggests t h a t hydroxyproline m a y act on some aspect of protein synthesis, as postulated for amino acid analogues (Fowden, 1963), The effects of 4-hydroxy-L-proline on the growth and protein metabolism of isolated pea root segments have therefore been studied. Two isomers were used; the naturally occurring trans form, which is formed in plant tissues b y ~he hydroxylation of proCein.bound proline (see
244
D. Vaughan and E. Cusens:
L a m p o r t , 1965), a n d the cis isomer, which is i n c o r p o r a t e d directly into proteins where it replaces proline (Cleland a n d Olson, 1968). Azetidine2-carboxylic acid, which replaces proline i n n e w l y synthesised protein of some p l a n t tissues (Fowden, 1963) was also used i n some experiments. Materials and Methods Unless otherwise stated, seeds of Pisum sativum (car Meteor) were germinated for 2 days, and cut under the aseptic conditions described elsewhere (Vaughan and Cusens, 1973). Segments 2 mm long, cut from the region 2-4 mm behind the root tip, were used to study extension growth since there is no cell division in these segments under the incubation conditions used (Vaughan and Cnsens, 1973). Between 10 and 20 excised segments were placed in 10 ml of previously autoclaved medium in 150 ml conical flaks and shaken on a reciprocating shaker at 100-110 rpm at 25~ C. All the media contained 2 per cent (w/v) sucrose and were supplemented with proline, hydroxyproline and azetidine-2-carboxylic acid as required. Concentrations of 3 miV[ L-hydroxyproline isomers and azetidine-2-carboxylie acid were sufficient to produce the maximal growth effects without resulting in the death of the tissue. At the end of the incubation period, solutions were tested for infection as described by Vaughan and Cusens (1973). Segment lengths were measured as described by Heyes and Vaughan (1967). Changes in chloride uptake capacity and in invertase activity were determined as described by Vaughan and Cusens (1973). Peroxidase activity in the cytoplasmic fraction was determined at 20~ by a modification of the method Ponting and Joslya (1948) using a Pye-Unicam Automatie Enzyme System. Groups of 10 segments were homogenized in 10 ml of 0.05 M citrate-phosphate buffer, pH 5.0, centrifuged at 1000 g for 5 rain, and the final volume of the supernatant made up to 20 ml. Using the AC60 chemical processing unit, 0.5 ml of enzyme solution was added to 0.8 ml of 0.5 ~ citratephosphate buffer, pH 5.0, followed 15 rain later by 3.0 ml of 0.25 per cent (w/v) guaiaeol in 0.1 M citrate-phosphate butter, pH 5.0. Finally 0.4 ml of 0.1 M hydrogen peroxide was added and the solutions transferred automatically to a Pye-Unicam Spl800 speetrophotometer, where the reaction mixture was retained in the cuvette (1 em light path) for 2 min using an A062 enzyme programmer. The rate of colour development was measured at 470 nm. Peroxidase activity is expressed as the rate of eolour development (during the first 100 seconds of the reaction when the rate was approximately linear) per segment at 20~ C. Changes in the respiration rate of excised 2-4 mm segments were measured using conventional Warburg manometry. Groups of 20 segments were incubated continuously for 24 h at 25~ C in 3 ml of autoclaved medium in Warburg flasks. After 9 h the air in the flasks was replaced by flushing with compressed air sterilized by passing through a cotton wool filter. Uptake and incorporation of uniformly labelled [140]L-leueine into proteins was determined by incubating groups of 10 segments in a medium containing 10 ml of 2 per cent sucrose and 10 ~M labelled amino acid (activity 0.2 ~Ci/ml) for 1 h at 25~ O as described elsewhere (Vaughan and Cnsens, 1973). For measurements of the incorporation of [14CJL-leueineand [140]L-prolineinto the proteins of various sub-cellular fractions, groups of 10 segments, labelled as above were homogenized at 2~ C in 9 ml of a medium comprising 0.5 IV[ sucrose, 4 mM KH2POa, 2.5 ml Tris, 10 ~1~ MgSO4 and 0.1 mY unlabelled amino acid (pH 7.0). Five sub-cellular fractions were obtained by suecessive eentrifugations at i000 g f0r 10 mii~ (FI); 3[0000g for 15 rain (F2), 30000 g for 15 min (F3), and
245
Effects of Hydroxyproline on Root Segments
140000 g for 90 rain, yielding debris (F4) and supernatant (F5) fractions (Heyes and Vaughan, 1967). All fractions were treated with 10 ml of 10 per cent (w/v) trichloracetie acid overnight at 4~ and centrifuged at 2500 g for 10 rain. The pellets were resuspended and heated at 70~ C for 10 rain in 10 ml of 5 per cent (w/v) trichloracetic acid to remove nucleic acids, then brought to an ether-dried pellet by successive centrifugations at 1000g in 2 • of ethanol, 10ml of ethanol/ diethyl ether (1:1 v/v) and 10 ml of diethyl ether. Each pellet was dissolved in 1.5 ml of 80 per cent (v/v) formic acid, brought to dryness on aluminium planchets, and the radioactivity determined with a gasflow mieromil window detector (Nuclear Chicago). All radioactive substances were supplied by the Radioaehemieal Centre, Amersham. Unless otherwise stated in the tables the results are the mean values of at least 3 separate experiments. Results
E//ects o/Proline, Hydroxyproline and Azetidine-2-Carboxylic Acid on Extension Growth. T h e cis a n d trans isomers of 4-hydroxy-L-proline s t i m u l a t e d ex t e n s io n g r o w t h of 2 - 4 m m excised p ea r o o t s e g m e n t s d u r i n g i n c u b a t i o n w h e n c o m p a r e d w i t h segments i n c u b a t e d in m e d i a c o n t a i n i n g only sucrose (Table 1). I n c o n t r a s t a z e t i d i n e - 2 - e a r b o x y l i e acid i n h i b i t e d Table 1. Effect of proline, 4-hydroxy-L-protine and azetidine-2-earboxylie acid on the percentage increase in the length of 2 4 mm excised pea root segments incubated in 2 per cent sucrose Supplement to culture medium
Time from excision (h) 3
6
None--sucrose only
15.0
38.0
9
18
24
Percentage increase 3~
65.0
110.0
120.0
Proline (PRO)
16.0
40.0
70.0
105.0
115.0
3 m ~ Trans-hydroxyproline ( Trans-HYP)
22.0
50.0
85.0
135.0
150.0
3 mM Cis-hydroxy-proline (Cis-HYP)
23.0
55.0
94.0
145.0
165.0
3 mM Azetidine-2-COOtt (AZE)
13.0
26.0
32.0
53.0
56.0
3 mM PRO ~- 3 mM Trans-HYP
18.0
43.0
67.0
99.0
119.0
3 mM PRO ~- 3 mM Cis-HYP
16.0
45.0
66.0
103.0
121.0
3 mM PRO -~ 3 mM AZE
14.0
37.0
72.0
101.0
109.0
3 mM AZE ~- 3 m_M Trans-tIYP
12.0
29.0
36.0
59.0
62.0
3 mM Allo-])-hydroxyproline
17.0
43.0
66.0
109.0
122.0
21.0
52.0
87.0
131.0
154.0
(Allo-HYP)
3 mM Trans-HYP -t- 3 mM AIIo-HYP
246
D. Vaughan and E. Cusens:
Table 2. Effect or proline, hydroxyproline and azetidine-2-carboxylic acid on changes in invertase and peroxidase activities of 2-4 mm excised pea root segments during incubation in 2 per cent sucrose Supplement to culture medium
Time from excision (h) 0
3
6
9
18
2.8
1.4
L~vertase activity (mg sucrose hydrolysed/10 segments/h) None--sucrose only
1.9
1.7
2.4
3 mM Proline (PRO)
1.9
1.8
2.3
3.0
1.5
3 my[ Allo-l)-hydroxyproline
1.9
1.7
2.5
2.6
1.5
3 my[ Trans-hydroxyproline (Trans-HYP) 3 my[ Cis.hydroxyproline
1.9
1.7
1.5
1.3
0.9
1.9
1.6
1.4
1.1
0.7
3 my[ Azetidine-2-COOH (AZE)
1.9
1.2
0.9
0.6
0.4
3 m ~ PRO + 3 my[ Trans-HYP
1.9
1.6
2.1
2.4
1.2
3 my[ PRO + 3 mM AZE
1.9
1.7
1.9
2.4
1.3
Peroxidase activity a (Increase in Ea~0 nm/segment/100s) None---sucrose only
0.9
1.0
1.2
1.5
2.7
3 m ~ Proline
0.9
0.9
1.3
1.7
2.5
3 m ~ Trans-hydroxyproline
0.9
0.9
1.0
1.1
1.8
3 my[ Azetidine-2-COOIt
0.9
--
1.1
--
1.0
3 my[ PRO + 3 mY[ Trans-HYP
0.9
1.2
1.3
1.8
2.4
3 mM PRO + 3 my[ AZE
0.9
--
1.4
--
2.3
a Mean values of 2 experiments.
e x t e n s i o n g r o w t h e v e n in th e presence of h y d r o x y p r o l i n e . A l t h o u g h proline h a d no effect on e x t e n s i o n g r o w t h it did p r e v e n t t h e effects of b o t h h y d r o x y p r o l i n e isomers a n d of a z c t i d i n e - 2 - c a r b o x y l i c acid. Allo4-hydroxy-])-proline w h i c h was also w i t h o u t effect on e x t e n s i o n g r o w t h did n o t p r e v e n t t h e s t i m u l a t i o n p r o d u c e d b y t h e L - h y d r o x y p r o l i n e isomers. Metabolic Changes during Extension Growth. S e g m e n t s i n c u b a t e d in sucrose w i t h or w i t h o u t proline showed a decrease in i n v e r t a s e a c t i v i t y during t h e first 3 h, followed b y a n increase to a m a x i m u m a t 9-12 h, af t er Which t h e r e was again a decrease (Table 2). B o t h L - h y d r o x y p r o l i n e
Effects of ttydroxyproline on Root Segments
247
Table 3. Effect of proline and hydroxyproline on chloride uptake and respiration of 2 4 mm pea root segments during incubation in 2 per cent sucrose Supplement to culture medium
Time from excision (h) 0
6
12
18
24
Chloride uptake (nm~esa6CI/10 segments/h) None--sucrose only
53
82
97
64
43
3 mY[ Proline (PP~O)
53
87
94
60
45
3 mM Trans-hydroxyproline
53
56
39
28
21
(Trans-}IYP) 3 mM Cis hydroxyproline 3 mM PRO + 3 mM Trans-HYP
53
47
41
24
20
53
79
92
65
47
Respiration ~ (VI 02/i0 segments/h) None--sucrose only
18
21
25
23
17
3 ml~I Proline
18
20
25
24
19
3 mM Trans-hydroxyproline
18
19
22
22
15
3 mM PRO + 3 m_~ Trans-HYP
18
22
23
24
17
a Mean values of 2 experiments.
isomers p r o d u c e d a continuous decrease in t h e e n z y m e a c t i v i t y during i n c u b a t i o n , b a t t h e decrease was g r e a t e s t in m e d i a containing azetidine2 - c a r b o x y l i c acid. Allo-D-hydroxyproline h a d no effect on t h e developm e n t of i n v e r t a s e a c t i v i t y . The soluble p e r o x i d a s e a c t i v i t y of 2 - 4 m m excised segments increased during i n c u b a t i o n in sucrose (Table 2). This increase was smaller in t h e presence of trans-hydroxyproline a n d was p r e v e n t e d b y azetidine2-earboxylic acid. A l t h o u g h proline h a d no effect on t h e d e v e l o p m e n t of i n v e r t a s e a n d soluble p e r o x i d a s e activities i t d i d p r e v e n t t h e i n h i b i t o r y effects of L - h y d r o x y p r o l i n e a n d azetidine-2-carboxylic acid. S i m i l a r l y the d e v e l o p m e n t of t h e c a p a c i t y of segments t o t a k e u p chloride (Table 3) was i n h i b i t e d b y b o t h L - h y d r o x y p r o l i n e isomers, their effect again being p r e v e n t e d in t h e presence of proline. I n c o n t r a s t h y d r o x y p r o l i n e h a d v i r t u a l l y no effect on changes in r e s p i r a t i o n r a t e (Table 3). Changes in Protein Synthesis during Extension Growth. I n v e s t i g a t i o n s using u n i f o r m l y labelled [14C]leueine showed t h a t when segments were i n c u b a t e d in sucrose media, w i t h or w i t h o u t proline, t h e r e was a n increase in t h e i r c a p a c i t y t o t a k e u p leucine d u r i n g t h e first 9 h followed
248
D. Vaughan and E. Cusens:
Table 4. Effect of proline and hydroxyproline on [A] the uptake and [B] the incorporation into proteins of [14C]leucine by 2-4 mm excised pea root segments incubated in 2 per cent sucrose Groups of 10 segments were labelled in 10 ml of 2 per cent sucrose containing 10 y2r [14C]leucine (0.2 ~Ci/ml) for 1 h at 25~ C. Supplement to culture medium
Time from excision (h) 0
3
6
9
18
[A] Uptake into segments miz moles leueine/10 segments/h None--sucrose only 3 mM Proline
3 mM Trans-hydroxyproline
950 950 950
1060 1000 1100
1520 1490 1260
1910 1820 1520
480 510 450
670 640 590
210 250 180
[B] Incorporation into proteins None--sucrose only 3 mlVIProline 3 mlVI Trans-hych'oxyproline
300 300 300
380 430 360
520 510 440
Mean values of 2 experiments.
b y a decrease (Table 4). Although trans-hydroxyproline was s o m e w h a t inhibitory it did not prevent an increase in leucine u p t a k e capacity during the first 9 h. The p a t t e r n of incorporation of [14C]leucine into segment proteins during incubation was similar to those for the u p t a k e values but a b o u t 70 per cent lower (Table 4). Comparison of the segments which h a d been incubated in sucrose for 6 h with the unineubated 0 hour controls (Table 5) showed increased incorporation of [14C]leueine in all the sub-cellular fractions. Proline h a d no effect on this increase, b u t with trans-hydroxyproline it was smaller in all fractions. I n segments incubated for 6 h with azetidine-2-cartJoxylic acid the leucine incorporation into all fractions was less t h a n in the unineubated 0 hours controls. Proline prevented the effects of trans-hydroxyproline a n d azetidine-2-earboxylie acid. I n c u b a t i o n in sucrose also increased the ability of the segments to incorporate [14C]proline (Table 6). The main difference, compared with [14C]leucine, was the larger a m o u n t of [14C]proline which was incorporated into the 1000 (F1) f r a c g o n s containing the cell walls. Cis and trans-hydroxyproline isomers were w i t h o u t effect on this increase, but azetidine-2-carboxylic acid again resulted in a decrease in the incorporation of [14C]proline into all fractions compared with the freshly cut segments.
249
Effects of Hydroxyproline on Root Segments
Table 5. Effect of proline, hydroxyproline and azetidine-2-carboxylic acid on the incorporation of [14CJleucine into the proteins of various sub-cellular fractions of 2-4 mm pea root segments incubated in 2 per cent sucrose Groups of 10 segments were incubated for 6 h in the appropriate medium, then labelled in 10 ml of 2 per cent sucrose containing 10 y_M [14C]leucine (0.2 ~Ci/ml) for 1 h at 25~ C and sub-cellular fractions obtained by differential centrifugation. Supplemented to culture medium
cpm/10 segments 1000g (F1)
10000ff 30000g 140000g Supernatant Total for (F2) (F3) (F4) (F5) fractions
None--sucrose only 3 m~r Proline (PRO) 3 mM Trans.hydroxyproline 3 mM[ Azetidine-2-COOH (AZE) 3 mM PRO + 3 mM: Trans-HYP 3 m~i PI~O ~ 3 mM AZE (Time 0 h controls)
4830 4510 4020 1870 4300 4100 3390
1690 1880 1320 780 1610 1350 930
3160 3290 2440 1470 3220 2950 2300
3490 3370 2890 1620 3230 3100 2550
12070 13050 10270 4830 12810 12170 7100
25240 26100 20940 10570 25170 23670 16270
Mean values of 2 experiments.
Table 6. Effect of hydroxyproline and azetidine-2-carboxylic acid on the incorporation of [l~C]proline into the proteins of various sub-cellular fractions of 2-4 mm pea root segments incubated in 2 per cent sucrose Labelling conditions as described for leucine in Table 5. Supplement to culture medium
1000 g (F1)
10000 g 30000 g 140000 g Supernatant Total for (F2) (F3) (F4) (F5) fractions
None--sucrose only 3m~r Trans-hydroxyproline 3 mM Cis-hydroxyproline 3 ~ Azetidine-2-COOH (Time 0 h controls)
12750 11590 11920 5850 7910
1340 1170 1440 650 860
2 030 1870 1750 1200 1560
1950 1730 1850 950 1440
4870 4300 4760 2470 3350
22940 20660 21720 11120 15120
Discussion Previous work (Vaughan a n d Cusens, 1970 a n d 1973) showed t h a t a m i n o acid analogues i n h i b i t the extension growth of excised pea root segments b y interfering with p r o t e i n synthesis. The i n h i b i t o r y effects of azetidine-2-carboxylic acid were p r e v e n t e d b y proline suggesting t h a t this analogue interferes with some aspect of p r o t e i n synthesis i n v o l v i n g proline. I n this c o n n e c t i o n F o w d e n (1963) has shown t h a t azetidine2-earboxylic acid i n h i b i t e d the growth of m u n g b e a n seedlings a n d replaced u p to 95 per cent of proline i n n e w l y synthesized proteins.
250
D. Vaughan and E. Cusens:
Proline also prevents the enhancement of extension growth caused by L-hydroxyproline suggesting that T.-hydroxyproline is interfering with proline involvement in protein synthesis. This implies that externally supplied hydroxyproline behaves in the same way as an amino acid analogue in this system. There was, however, no evidence that hydroxyproline interfered with the incorporation of [14C]proline into pea segments, an observation also reported for sycamore cell suspensions by Holleman (1967). Although the increase in the development of [laC]leucine incorporation in segments incubated in hydroxyproline was reduced by only 20-25 per cent, the presence of azetidine-2-carboxylic acid resulted in less incorporation than in the freshly cut tissue (Table 5). Indeed Cleland (1967) has shown that hydroxyproline is not a general inhibitor of respiration or protein synthesis in the Avena coleoptile. The effects of hydroxyproline on pea root segments present a dilemma, since extension growth is enhanced while certain changes in protein metabolism are inhibited. Like azetidine-2-earboxylie acid, cis-4-hydroxy-L-proline can replace proline directly in plant proteins (Cleland and Olson, 1968), whereas trans-4-hydroxy- L-proline is first converted into proline (Cleland and Olson, 1968), incorporated into proteins as proline and subsequently hydroxylated to trans-hydroxyproline, probably in specific proteins (Lamport, 1965). However, some direct incorporation of trans-hydroxyproline into proteins does occur (Holleman, 1967; Cleland and Olson, t968). It is, however, unlikely that hydroxyproline enhances extension growth by interfering with all protein synthesis involving proline. This view is strengthened by the observation that azetidine-2-earboxylic acid inhibits extension growth even in the presence of hydroxyproline. It is likely that stimulation of growth by hydroxyproline results from a more specific effect of the imino acid on protein synthesis since transhydroxyproline is located almost entirely in cell-wall proteins (Lamport, 1965). Lamport (1965) has proposed a structural role for wall-bound hydroxyproline and has shown that cellulose micro-fibrils are cross-linked by a glyeoprotein consisting of galaeto-araban units and hydroxyprolinerich peptides. The extent of this cross-linking is probably a factor controlling extensibflity of the cell wall. In pea roots some 85 per cent of wall-bound hydroxyproline is linked to arabinose (Lamport and Miller, 1971). Cleland and Karlsnes (1967) proposed that in etiolated peas an increase in cell-wall bound hydroxyproline with age may be a factor controlling the termination of growth, although definite evidence to support this suggestion is lacking. Pertinent to this, l~idge and Osborne (1970) have suggested that the reduction of elongation in etiolated pea
Effects of ttydroxyproline on Root Segments
251
epicotyls as a result of t r e a t m e n t w i t h e t h y l e n e could be r e l a t e d t o t h e " p r e m a t u r e " increase in wall h y d r o x y p r o l i n e . A l t h o u g h h y d r o x y p r o l i n e does n o t affect t h e t o t a l a m o u n t of proline i n c o r p o r a t e d into t h e p r o t e i n s of p e a r o o t segments i t is p r o b a b l e t h a t e x t e r n a l l y - s u p p h e d h y d r o x y p r o l i n e inhibits t h e f o r m a t i o n of h y d r o x y proline from p r o t e i n - b o u n d proline, as shown for A v e n a coleoptiles b y Cleland (1967). Such a m e c h a n i s m w o u l d allow for a n e n h a n c e d cell elongation a n d a s o m e w a h t i n h i b i t e d d e v e l o p m e n t of c y t o p l a s m i c protein m e t a b o l i s m where h y d r o x y p r o l i n e could replace proline d i r e c t l y in these p r o t e i n s (Holleman, 1967; Cleland a n d Olson, 1968). H y d r o x y proline, therefore, provides a m e a n s of s t u d y i n g which facets of p r o t e i n synthesis are concerned in cell e x p a n s i o n a n d this a s p e c t of t h e w o r k will be t h e s u b j e c t of a f u r t h e r c o m m u n i c a t i o n . We should like to thank Dr. I. R. MacDonald for the determination of chloride uptake capacity and Mrs. L. M. Smith for technical assistance.
References Cleland R. : ttydroxyproline as an inhibitor of auxin-induced cell elongation. Nature (Lond.) 200, 908-909 (1963). Cleland R. : Inhibition of cell elongation in Arena coleoptile by hydroxyproline. Plant Physiol. 42, 271-274 (1967). Cleland R.: Distribution and metabolism of protein-bound hydroxyproline in an elongating tissue, the Avena coleoptile. Plant Physiol. 43, 865-870 (1968). Cleland R., Karlsnes, A. M. : A possible role of hydroxyproline-containing protein in the cessation of cell elongation. Plant Physiol. 42, 669-671 (1967). Cleland R., Olson, A. C. : Direct incorporation of hydroxyproline into Arena coleoptile proteins. Biochemistry 7, 1745-1751 (1968). Fowden L. : Amino acid analogues and the growth of seedlings. J. exp. Bot. 14, 387-398 (1963). tteyes, J. K., Vaughan, D. : The effects of 2-thiouracil on growth and metabolism in the root. 11. The metabolism of isolated root segments. Proc. roy. Soc. B 169. 89-105 (1957). Holleman, J.: Direct incorporation of hydroxyproline into protein of sycamore cells incubated at growth-inhibitery levels of hydroxyproline. Proc. nat. Acad. Sei. (Wash.) 57 50-54 (1967). Lamport, D. T. A.: The protein component of primary cell walls. Adv. bot. t~es. 2, 151-218 (1965). Lamport, D. T. A., Miller, D. It. : Hydroxyproline arabinosides in the plant kingdom. Plant Physiol. 42, 481-486 (1971). Nooden, L., Thimann, K. V. : Action of inhibitors of RNA and protein synthesis on cell enlargement. Plant Physiol. 41, 157-164 (1966). Norris, W. E. : Reversal of hydroxyproline-induced inhibition of elongation of Avena coleoptiles. Plant Physiol. 42, 481-486 (1967). Olson, A. C. : Proteins and plant cell walls. Proline to hydroxyproline in tobacco suspension cultures. Plant Physiol. 39, 543-550 (1964).
252 D. Vaughan and E. Cusens: Effects of Hydroxyproline on Root Segments Ponting, J.D., Joslyn, M.A.: Ascorbic acid oxidation and browning in apple tissue extracts. Arch. Biochem. Biophys. 19, 47-63 (1948). Ridge, I., Osborne, D. J. : Hydroxyproline and peroxidases in cell walls of Pisurn sativum: Regulation by ethylene. J. exp. Bot. 21, 843-856 (1970). Vaughan, D., Cusens, E. : An effect of amino acid analogues on extension growth in pea root segments under aseptic conditions. Biochem. J. 120, 18-19P (1970). Vaughan, D., Cusens, E. : Effects of amino acid analogues on growth and protein metabolism in Pea roots. Ann. Bot. (in press).
Effects of Hydroxyproline on the Growth of Excised Root Segments of Pisum sativum under Aseptic Conditions D. Vaughan and Evelyn Cusens The Macaulay Institute for Soft Research, Craigiebuckler, Aberdeen, AB9 2 QJ, U.K. Received April 3, 1973 Summary. The cis and trans isomers of 4-hydroxy-T.-proline stimulated the extension growth of excised 2 4 mm pea root segments during culture. Increase in the uptake and subsequent incorporation of [ltCJleueine into proteins was inhibited by both n-isomers, and so also were changes in chloride uptake capacity and in protein metabolism measured in terms of invertase and peroxidase activities. Changes in [ltC]proline uptake and incorporation, and in respiration, were unaffected. Proline had no effect on changes in extension growth or protein metabolism but did prevent the effects of both hydroxyproline isomers. Azetidine-2-carboxylie acid inhibited extension growth and all the aspects of protein metabolism studied, the effects again being all prevented by proline. It is suggested that hydroxyproline enhances growth by interfering with protein synthesis in the cell walls.
Introduction Several amino analogues inhibit changes in the extension growth and protein metabolism of isolated pea root segments incubated under aseptic condir (Vaughan and Cusens, 1970, 1973), whereas most amino acids have no effect. I n contrast to this, hydroxyproline, which is present as a natural constituent in some plan~ proteins (Lamport, 1965), stimulates extension growth, and when applied externally often appears to act as an amino acid analogue. Thus hydroxyproline inhibits auxin-induced growth of Avena coleoptfles (Cleland, 1963; Norris, 1967) and the growth of a variety of callus tissues (Holleman, 1967; Lamport, 1965), both effects being prevented b y proline. This suggests t h a t hydroxyproline m a y act on some aspect of protein synthesis, as postulated for amino acid analogues (Fowden, 1963), The effects of 4-hydroxy-L-proline on the growth and protein metabolism of isolated pea root segments have therefore been studied. Two isomers were used; the naturally occurring trans form, which is formed in plant tissues b y ~he hydroxylation of proCein.bound proline (see
244
D. Vaughan and E. Cusens:
L a m p o r t , 1965), a n d the cis isomer, which is i n c o r p o r a t e d directly into proteins where it replaces proline (Cleland a n d Olson, 1968). Azetidine2-carboxylic acid, which replaces proline i n n e w l y synthesised protein of some p l a n t tissues (Fowden, 1963) was also used i n some experiments. Materials and Methods Unless otherwise stated, seeds of Pisum sativum (car Meteor) were germinated for 2 days, and cut under the aseptic conditions described elsewhere (Vaughan and Cusens, 1973). Segments 2 mm long, cut from the region 2-4 mm behind the root tip, were used to study extension growth since there is no cell division in these segments under the incubation conditions used (Vaughan and Cnsens, 1973). Between 10 and 20 excised segments were placed in 10 ml of previously autoclaved medium in 150 ml conical flaks and shaken on a reciprocating shaker at 100-110 rpm at 25~ C. All the media contained 2 per cent (w/v) sucrose and were supplemented with proline, hydroxyproline and azetidine-2-carboxylic acid as required. Concentrations of 3 miV[ L-hydroxyproline isomers and azetidine-2-carboxylie acid were sufficient to produce the maximal growth effects without resulting in the death of the tissue. At the end of the incubation period, solutions were tested for infection as described by Vaughan and Cusens (1973). Segment lengths were measured as described by Heyes and Vaughan (1967). Changes in chloride uptake capacity and in invertase activity were determined as described by Vaughan and Cusens (1973). Peroxidase activity in the cytoplasmic fraction was determined at 20~ by a modification of the method Ponting and Joslya (1948) using a Pye-Unicam Automatie Enzyme System. Groups of 10 segments were homogenized in 10 ml of 0.05 M citrate-phosphate buffer, pH 5.0, centrifuged at 1000 g for 5 rain, and the final volume of the supernatant made up to 20 ml. Using the AC60 chemical processing unit, 0.5 ml of enzyme solution was added to 0.8 ml of 0.5 ~ citratephosphate buffer, pH 5.0, followed 15 rain later by 3.0 ml of 0.25 per cent (w/v) guaiaeol in 0.1 M citrate-phosphate butter, pH 5.0. Finally 0.4 ml of 0.1 M hydrogen peroxide was added and the solutions transferred automatically to a Pye-Unicam Spl800 speetrophotometer, where the reaction mixture was retained in the cuvette (1 em light path) for 2 min using an A062 enzyme programmer. The rate of colour development was measured at 470 nm. Peroxidase activity is expressed as the rate of eolour development (during the first 100 seconds of the reaction when the rate was approximately linear) per segment at 20~ C. Changes in the respiration rate of excised 2-4 mm segments were measured using conventional Warburg manometry. Groups of 20 segments were incubated continuously for 24 h at 25~ C in 3 ml of autoclaved medium in Warburg flasks. After 9 h the air in the flasks was replaced by flushing with compressed air sterilized by passing through a cotton wool filter. Uptake and incorporation of uniformly labelled [140]L-leueine into proteins was determined by incubating groups of 10 segments in a medium containing 10 ml of 2 per cent sucrose and 10 ~M labelled amino acid (activity 0.2 ~Ci/ml) for 1 h at 25~ O as described elsewhere (Vaughan and Cnsens, 1973). For measurements of the incorporation of [14CJL-leueineand [140]L-prolineinto the proteins of various sub-cellular fractions, groups of 10 segments, labelled as above were homogenized at 2~ C in 9 ml of a medium comprising 0.5 IV[ sucrose, 4 mM KH2POa, 2.5 ml Tris, 10 ~1~ MgSO4 and 0.1 mY unlabelled amino acid (pH 7.0). Five sub-cellular fractions were obtained by suecessive eentrifugations at i000 g f0r 10 mii~ (FI); 3[0000g for 15 rain (F2), 30000 g for 15 min (F3), and
245
Effects of Hydroxyproline on Root Segments
140000 g for 90 rain, yielding debris (F4) and supernatant (F5) fractions (Heyes and Vaughan, 1967). All fractions were treated with 10 ml of 10 per cent (w/v) trichloracetie acid overnight at 4~ and centrifuged at 2500 g for 10 rain. The pellets were resuspended and heated at 70~ C for 10 rain in 10 ml of 5 per cent (w/v) trichloracetic acid to remove nucleic acids, then brought to an ether-dried pellet by successive centrifugations at 1000g in 2 • of ethanol, 10ml of ethanol/ diethyl ether (1:1 v/v) and 10 ml of diethyl ether. Each pellet was dissolved in 1.5 ml of 80 per cent (v/v) formic acid, brought to dryness on aluminium planchets, and the radioactivity determined with a gasflow mieromil window detector (Nuclear Chicago). All radioactive substances were supplied by the Radioaehemieal Centre, Amersham. Unless otherwise stated in the tables the results are the mean values of at least 3 separate experiments. Results
E//ects o/Proline, Hydroxyproline and Azetidine-2-Carboxylic Acid on Extension Growth. T h e cis a n d trans isomers of 4-hydroxy-L-proline s t i m u l a t e d ex t e n s io n g r o w t h of 2 - 4 m m excised p ea r o o t s e g m e n t s d u r i n g i n c u b a t i o n w h e n c o m p a r e d w i t h segments i n c u b a t e d in m e d i a c o n t a i n i n g only sucrose (Table 1). I n c o n t r a s t a z e t i d i n e - 2 - e a r b o x y l i e acid i n h i b i t e d Table 1. Effect of proline, 4-hydroxy-L-protine and azetidine-2-earboxylie acid on the percentage increase in the length of 2 4 mm excised pea root segments incubated in 2 per cent sucrose Supplement to culture medium
Time from excision (h) 3
6
None--sucrose only
15.0
38.0
9
18
24
Percentage increase 3~
65.0
110.0
120.0
Proline (PRO)
16.0
40.0
70.0
105.0
115.0
3 m ~ Trans-hydroxyproline ( Trans-HYP)
22.0
50.0
85.0
135.0
150.0
3 mM Cis-hydroxy-proline (Cis-HYP)
23.0
55.0
94.0
145.0
165.0
3 mM Azetidine-2-COOtt (AZE)
13.0
26.0
32.0
53.0
56.0
3 mM PRO ~- 3 mM Trans-HYP
18.0
43.0
67.0
99.0
119.0
3 mM PRO ~- 3 mM Cis-HYP
16.0
45.0
66.0
103.0
121.0
3 mM PRO -~ 3 mM AZE
14.0
37.0
72.0
101.0
109.0
3 mM AZE ~- 3 m_M Trans-tIYP
12.0
29.0
36.0
59.0
62.0
3 mM Allo-])-hydroxyproline
17.0
43.0
66.0
109.0
122.0
21.0
52.0
87.0
131.0
154.0
(Allo-HYP)
3 mM Trans-HYP -t- 3 mM AIIo-HYP
246
D. Vaughan and E. Cusens:
Table 2. Effect or proline, hydroxyproline and azetidine-2-carboxylic acid on changes in invertase and peroxidase activities of 2-4 mm excised pea root segments during incubation in 2 per cent sucrose Supplement to culture medium
Time from excision (h) 0
3
6
9
18
2.8
1.4
L~vertase activity (mg sucrose hydrolysed/10 segments/h) None--sucrose only
1.9
1.7
2.4
3 mM Proline (PRO)
1.9
1.8
2.3
3.0
1.5
3 my[ Allo-l)-hydroxyproline
1.9
1.7
2.5
2.6
1.5
3 my[ Trans-hydroxyproline (Trans-HYP) 3 my[ Cis.hydroxyproline
1.9
1.7
1.5
1.3
0.9
1.9
1.6
1.4
1.1
0.7
3 my[ Azetidine-2-COOH (AZE)
1.9
1.2
0.9
0.6
0.4
3 m ~ PRO + 3 my[ Trans-HYP
1.9
1.6
2.1
2.4
1.2
3 my[ PRO + 3 mM AZE
1.9
1.7
1.9
2.4
1.3
Peroxidase activity a (Increase in Ea~0 nm/segment/100s) None---sucrose only
0.9
1.0
1.2
1.5
2.7
3 m ~ Proline
0.9
0.9
1.3
1.7
2.5
3 m ~ Trans-hydroxyproline
0.9
0.9
1.0
1.1
1.8
3 my[ Azetidine-2-COOIt
0.9
--
1.1
--
1.0
3 my[ PRO + 3 mY[ Trans-HYP
0.9
1.2
1.3
1.8
2.4
3 mM PRO + 3 my[ AZE
0.9
--
1.4
--
2.3
a Mean values of 2 experiments.
e x t e n s i o n g r o w t h e v e n in th e presence of h y d r o x y p r o l i n e . A l t h o u g h proline h a d no effect on e x t e n s i o n g r o w t h it did p r e v e n t t h e effects of b o t h h y d r o x y p r o l i n e isomers a n d of a z c t i d i n e - 2 - c a r b o x y l i c acid. Allo4-hydroxy-])-proline w h i c h was also w i t h o u t effect on e x t e n s i o n g r o w t h did n o t p r e v e n t t h e s t i m u l a t i o n p r o d u c e d b y t h e L - h y d r o x y p r o l i n e isomers. Metabolic Changes during Extension Growth. S e g m e n t s i n c u b a t e d in sucrose w i t h or w i t h o u t proline showed a decrease in i n v e r t a s e a c t i v i t y during t h e first 3 h, followed b y a n increase to a m a x i m u m a t 9-12 h, af t er Which t h e r e was again a decrease (Table 2). B o t h L - h y d r o x y p r o l i n e
Effects of ttydroxyproline on Root Segments
247
Table 3. Effect of proline and hydroxyproline on chloride uptake and respiration of 2 4 mm pea root segments during incubation in 2 per cent sucrose Supplement to culture medium
Time from excision (h) 0
6
12
18
24
Chloride uptake (nm~esa6CI/10 segments/h) None--sucrose only
53
82
97
64
43
3 mY[ Proline (PP~O)
53
87
94
60
45
3 mM Trans-hydroxyproline
53
56
39
28
21
(Trans-}IYP) 3 mM Cis hydroxyproline 3 mM PRO + 3 mM Trans-HYP
53
47
41
24
20
53
79
92
65
47
Respiration ~ (VI 02/i0 segments/h) None--sucrose only
18
21
25
23
17
3 ml~I Proline
18
20
25
24
19
3 mM Trans-hydroxyproline
18
19
22
22
15
3 mM PRO + 3 m_~ Trans-HYP
18
22
23
24
17
a Mean values of 2 experiments.
isomers p r o d u c e d a continuous decrease in t h e e n z y m e a c t i v i t y during i n c u b a t i o n , b a t t h e decrease was g r e a t e s t in m e d i a containing azetidine2 - c a r b o x y l i c acid. Allo-D-hydroxyproline h a d no effect on t h e developm e n t of i n v e r t a s e a c t i v i t y . The soluble p e r o x i d a s e a c t i v i t y of 2 - 4 m m excised segments increased during i n c u b a t i o n in sucrose (Table 2). This increase was smaller in t h e presence of trans-hydroxyproline a n d was p r e v e n t e d b y azetidine2-earboxylic acid. A l t h o u g h proline h a d no effect on t h e d e v e l o p m e n t of i n v e r t a s e a n d soluble p e r o x i d a s e activities i t d i d p r e v e n t t h e i n h i b i t o r y effects of L - h y d r o x y p r o l i n e a n d azetidine-2-carboxylic acid. S i m i l a r l y the d e v e l o p m e n t of t h e c a p a c i t y of segments t o t a k e u p chloride (Table 3) was i n h i b i t e d b y b o t h L - h y d r o x y p r o l i n e isomers, their effect again being p r e v e n t e d in t h e presence of proline. I n c o n t r a s t h y d r o x y p r o l i n e h a d v i r t u a l l y no effect on changes in r e s p i r a t i o n r a t e (Table 3). Changes in Protein Synthesis during Extension Growth. I n v e s t i g a t i o n s using u n i f o r m l y labelled [14C]leueine showed t h a t when segments were i n c u b a t e d in sucrose media, w i t h or w i t h o u t proline, t h e r e was a n increase in t h e i r c a p a c i t y t o t a k e u p leucine d u r i n g t h e first 9 h followed
248
D. Vaughan and E. Cusens:
Table 4. Effect of proline and hydroxyproline on [A] the uptake and [B] the incorporation into proteins of [14C]leucine by 2-4 mm excised pea root segments incubated in 2 per cent sucrose Groups of 10 segments were labelled in 10 ml of 2 per cent sucrose containing 10 y2r [14C]leucine (0.2 ~Ci/ml) for 1 h at 25~ C. Supplement to culture medium
Time from excision (h) 0
3
6
9
18
[A] Uptake into segments miz moles leueine/10 segments/h None--sucrose only 3 mM Proline
3 mM Trans-hydroxyproline
950 950 950
1060 1000 1100
1520 1490 1260
1910 1820 1520
480 510 450
670 640 590
210 250 180
[B] Incorporation into proteins None--sucrose only 3 mlVIProline 3 mlVI Trans-hych'oxyproline
300 300 300
380 430 360
520 510 440
Mean values of 2 experiments.
b y a decrease (Table 4). Although trans-hydroxyproline was s o m e w h a t inhibitory it did not prevent an increase in leucine u p t a k e capacity during the first 9 h. The p a t t e r n of incorporation of [14C]leucine into segment proteins during incubation was similar to those for the u p t a k e values but a b o u t 70 per cent lower (Table 4). Comparison of the segments which h a d been incubated in sucrose for 6 h with the unineubated 0 hour controls (Table 5) showed increased incorporation of [14C]leueine in all the sub-cellular fractions. Proline h a d no effect on this increase, b u t with trans-hydroxyproline it was smaller in all fractions. I n segments incubated for 6 h with azetidine-2-cartJoxylic acid the leucine incorporation into all fractions was less t h a n in the unineubated 0 hours controls. Proline prevented the effects of trans-hydroxyproline a n d azetidine-2-earboxylie acid. I n c u b a t i o n in sucrose also increased the ability of the segments to incorporate [14C]proline (Table 6). The main difference, compared with [14C]leucine, was the larger a m o u n t of [14C]proline which was incorporated into the 1000 (F1) f r a c g o n s containing the cell walls. Cis and trans-hydroxyproline isomers were w i t h o u t effect on this increase, but azetidine-2-carboxylic acid again resulted in a decrease in the incorporation of [14C]proline into all fractions compared with the freshly cut segments.
249
Effects of Hydroxyproline on Root Segments
Table 5. Effect of proline, hydroxyproline and azetidine-2-carboxylic acid on the incorporation of [14CJleucine into the proteins of various sub-cellular fractions of 2-4 mm pea root segments incubated in 2 per cent sucrose Groups of 10 segments were incubated for 6 h in the appropriate medium, then labelled in 10 ml of 2 per cent sucrose containing 10 y_M [14C]leucine (0.2 ~Ci/ml) for 1 h at 25~ C and sub-cellular fractions obtained by differential centrifugation. Supplemented to culture medium
cpm/10 segments 1000g (F1)
10000ff 30000g 140000g Supernatant Total for (F2) (F3) (F4) (F5) fractions
None--sucrose only 3 m~r Proline (PRO) 3 mM Trans.hydroxyproline 3 mM[ Azetidine-2-COOH (AZE) 3 mM PRO + 3 mM: Trans-HYP 3 m~i PI~O ~ 3 mM AZE (Time 0 h controls)
4830 4510 4020 1870 4300 4100 3390
1690 1880 1320 780 1610 1350 930
3160 3290 2440 1470 3220 2950 2300
3490 3370 2890 1620 3230 3100 2550
12070 13050 10270 4830 12810 12170 7100
25240 26100 20940 10570 25170 23670 16270
Mean values of 2 experiments.
Table 6. Effect of hydroxyproline and azetidine-2-carboxylic acid on the incorporation of [l~C]proline into the proteins of various sub-cellular fractions of 2-4 mm pea root segments incubated in 2 per cent sucrose Labelling conditions as described for leucine in Table 5. Supplement to culture medium
1000 g (F1)
10000 g 30000 g 140000 g Supernatant Total for (F2) (F3) (F4) (F5) fractions
None--sucrose only 3m~r Trans-hydroxyproline 3 mM Cis-hydroxyproline 3 ~ Azetidine-2-COOH (Time 0 h controls)
12750 11590 11920 5850 7910
1340 1170 1440 650 860
2 030 1870 1750 1200 1560
1950 1730 1850 950 1440
4870 4300 4760 2470 3350
22940 20660 21720 11120 15120
Discussion Previous work (Vaughan a n d Cusens, 1970 a n d 1973) showed t h a t a m i n o acid analogues i n h i b i t the extension growth of excised pea root segments b y interfering with p r o t e i n synthesis. The i n h i b i t o r y effects of azetidine-2-carboxylic acid were p r e v e n t e d b y proline suggesting t h a t this analogue interferes with some aspect of p r o t e i n synthesis i n v o l v i n g proline. I n this c o n n e c t i o n F o w d e n (1963) has shown t h a t azetidine2-earboxylic acid i n h i b i t e d the growth of m u n g b e a n seedlings a n d replaced u p to 95 per cent of proline i n n e w l y synthesized proteins.
250
D. Vaughan and E. Cusens:
Proline also prevents the enhancement of extension growth caused by L-hydroxyproline suggesting that T.-hydroxyproline is interfering with proline involvement in protein synthesis. This implies that externally supplied hydroxyproline behaves in the same way as an amino acid analogue in this system. There was, however, no evidence that hydroxyproline interfered with the incorporation of [14C]proline into pea segments, an observation also reported for sycamore cell suspensions by Holleman (1967). Although the increase in the development of [laC]leucine incorporation in segments incubated in hydroxyproline was reduced by only 20-25 per cent, the presence of azetidine-2-carboxylic acid resulted in less incorporation than in the freshly cut tissue (Table 5). Indeed Cleland (1967) has shown that hydroxyproline is not a general inhibitor of respiration or protein synthesis in the Avena coleoptile. The effects of hydroxyproline on pea root segments present a dilemma, since extension growth is enhanced while certain changes in protein metabolism are inhibited. Like azetidine-2-earboxylie acid, cis-4-hydroxy-L-proline can replace proline directly in plant proteins (Cleland and Olson, 1968), whereas trans-4-hydroxy- L-proline is first converted into proline (Cleland and Olson, 1968), incorporated into proteins as proline and subsequently hydroxylated to trans-hydroxyproline, probably in specific proteins (Lamport, 1965). However, some direct incorporation of trans-hydroxyproline into proteins does occur (Holleman, 1967; Cleland and Olson, t968). It is, however, unlikely that hydroxyproline enhances extension growth by interfering with all protein synthesis involving proline. This view is strengthened by the observation that azetidine-2-earboxylic acid inhibits extension growth even in the presence of hydroxyproline. It is likely that stimulation of growth by hydroxyproline results from a more specific effect of the imino acid on protein synthesis since transhydroxyproline is located almost entirely in cell-wall proteins (Lamport, 1965). Lamport (1965) has proposed a structural role for wall-bound hydroxyproline and has shown that cellulose micro-fibrils are cross-linked by a glyeoprotein consisting of galaeto-araban units and hydroxyprolinerich peptides. The extent of this cross-linking is probably a factor controlling extensibflity of the cell wall. In pea roots some 85 per cent of wall-bound hydroxyproline is linked to arabinose (Lamport and Miller, 1971). Cleland and Karlsnes (1967) proposed that in etiolated peas an increase in cell-wall bound hydroxyproline with age may be a factor controlling the termination of growth, although definite evidence to support this suggestion is lacking. Pertinent to this, l~idge and Osborne (1970) have suggested that the reduction of elongation in etiolated pea
Effects of ttydroxyproline on Root Segments
251
epicotyls as a result of t r e a t m e n t w i t h e t h y l e n e could be r e l a t e d t o t h e " p r e m a t u r e " increase in wall h y d r o x y p r o l i n e . A l t h o u g h h y d r o x y p r o l i n e does n o t affect t h e t o t a l a m o u n t of proline i n c o r p o r a t e d into t h e p r o t e i n s of p e a r o o t segments i t is p r o b a b l e t h a t e x t e r n a l l y - s u p p h e d h y d r o x y p r o l i n e inhibits t h e f o r m a t i o n of h y d r o x y proline from p r o t e i n - b o u n d proline, as shown for A v e n a coleoptiles b y Cleland (1967). Such a m e c h a n i s m w o u l d allow for a n e n h a n c e d cell elongation a n d a s o m e w a h t i n h i b i t e d d e v e l o p m e n t of c y t o p l a s m i c protein m e t a b o l i s m where h y d r o x y p r o l i n e could replace proline d i r e c t l y in these p r o t e i n s (Holleman, 1967; Cleland a n d Olson, 1968). H y d r o x y proline, therefore, provides a m e a n s of s t u d y i n g which facets of p r o t e i n synthesis are concerned in cell e x p a n s i o n a n d this a s p e c t of t h e w o r k will be t h e s u b j e c t of a f u r t h e r c o m m u n i c a t i o n . We should like to thank Dr. I. R. MacDonald for the determination of chloride uptake capacity and Mrs. L. M. Smith for technical assistance.
References Cleland R. : ttydroxyproline as an inhibitor of auxin-induced cell elongation. Nature (Lond.) 200, 908-909 (1963). Cleland R. : Inhibition of cell elongation in Arena coleoptile by hydroxyproline. Plant Physiol. 42, 271-274 (1967). Cleland R.: Distribution and metabolism of protein-bound hydroxyproline in an elongating tissue, the Avena coleoptile. Plant Physiol. 43, 865-870 (1968). Cleland R., Karlsnes, A. M. : A possible role of hydroxyproline-containing protein in the cessation of cell elongation. Plant Physiol. 42, 669-671 (1967). Cleland R., Olson, A. C. : Direct incorporation of hydroxyproline into Arena coleoptile proteins. Biochemistry 7, 1745-1751 (1968). Fowden L. : Amino acid analogues and the growth of seedlings. J. exp. Bot. 14, 387-398 (1963). tteyes, J. K., Vaughan, D. : The effects of 2-thiouracil on growth and metabolism in the root. 11. The metabolism of isolated root segments. Proc. roy. Soc. B 169. 89-105 (1957). Holleman, J.: Direct incorporation of hydroxyproline into protein of sycamore cells incubated at growth-inhibitery levels of hydroxyproline. Proc. nat. Acad. Sei. (Wash.) 57 50-54 (1967). Lamport, D. T. A.: The protein component of primary cell walls. Adv. bot. t~es. 2, 151-218 (1965). Lamport, D. T. A., Miller, D. It. : Hydroxyproline arabinosides in the plant kingdom. Plant Physiol. 42, 481-486 (1971). Nooden, L., Thimann, K. V. : Action of inhibitors of RNA and protein synthesis on cell enlargement. Plant Physiol. 41, 157-164 (1966). Norris, W. E. : Reversal of hydroxyproline-induced inhibition of elongation of Avena coleoptiles. Plant Physiol. 42, 481-486 (1967). Olson, A. C. : Proteins and plant cell walls. Proline to hydroxyproline in tobacco suspension cultures. Plant Physiol. 39, 543-550 (1964).
252 D. Vaughan and E. Cusens: Effects of Hydroxyproline on Root Segments Ponting, J.D., Joslyn, M.A.: Ascorbic acid oxidation and browning in apple tissue extracts. Arch. Biochem. Biophys. 19, 47-63 (1948). Ridge, I., Osborne, D. J. : Hydroxyproline and peroxidases in cell walls of Pisurn sativum: Regulation by ethylene. J. exp. Bot. 21, 843-856 (1970). Vaughan, D., Cusens, E. : An effect of amino acid analogues on extension growth in pea root segments under aseptic conditions. Biochem. J. 120, 18-19P (1970). Vaughan, D., Cusens, E. : Effects of amino acid analogues on growth and protein metabolism in Pea roots. Ann. Bot. (in press).
