Bioregulators and Rubber Synthesis in the Guayule Plant - American

20 Bioregulators and Rubber Synthesisinthe Guayule Plant

Downloaded by CORNELL UNIV on September 23, 2016 | http://pubs.acs.org Publication Date: July 6, 1984 | doi: 10.1021/bk-1984-0257.ch020

H. YOKOYAMA, W. J. HSU, Ε. HAYMAN, and S. POLING Agricultural Research Service, Fruit and Vegetable Chemistry Laboratory, U.S. Department of Agriculture, Pasadena, C A 91106

Bioregulators caused a significant increaseinthe synthesis of rubber in the guayule plant. This was accomplished without altering the microstructure (cis1,4 isoprene) of the polyisoprene molecule. The carbon13 nuclear magnetic resonance spectra show a complete absence of signals attributable to structural isomers and confirm that guayule rubber from treated plants is a highly stereospecific polymer composed entirely of cis-1,4 isoprene units. The gel permeation chromatog raphy results suggest that these bioregulators induce new rubber molecules rather than chain extension of rubber molecules at the surface of existing rubber par ticles. N a t u r a l p o l y i s o p r e n e r u b b e r is d i s t r i b u t e d w i d e l y in t h e p l a n t k i n g dom. About 2000 s p e c i e s o f p l a n t s a r e known t o s y n t h e s i z e r u b b e r . However, o n l y a few e v e r produce r u b b e r in s u b s t a n t i a l amount f o r commercial u s e . Two o f t h e s e , t h e r u b b e r t r e e Hevea b r a z i l i e n s i s , and t h e g u a y u l e shrub P a r t h e n i u m argentatum, have been c o n t i n u i n g sources of n a t u r a l rubber. These two p l a n t s have c o n t r a s t i n g c l i m a t i c requirements. Hevea is n a t i v e t o e q u a t o r i a l l o w l a n d r a i n f o r e s t s in t h e Amazon b a s i n . On t h e o t h e r hand, t h e g u a y u l e p l a n t grows w i l d in t h e u p l a n d p l a t e a u a r e a s o f Mexico and Texas w i t h sub t r o p i c a l - t e m p e r a t e c l i m a t e s and meager r a i n f a l l . Despite these d i f f e r e n c e s , t h e two p l a n t s produce a s i m i l a r r u b b e r . The p h y s i c a l , c h e m i c a l , and m e c h a n i c a l p r o p e r t i e s o f Hevea r u b b e r and g u a y u l e rubber a r e s i m i l a r ( 1 ) . Guayule

Plant

The g u a y u l e p l a n t is a member o f t h e s u n f l o w e r f a m i l y Compositae and b e l o n g s t o t h e genus P a r t h e n i u m . The g u a y u l e p l a n t is P a r t h e n i u m argentatum, so d e s i g n a t e d because o f a s i l v e r y sheen on its g r e y g r e e n f o l i a g e , and is one o f 16 s p e c i e s o f P a r t h e n i u m . I t is t h e o n l y Parthenium s p e c i e s known t o produce r u b b e r in any a p p r e c i a b l e q u a n t i t y ( 2 ) . U n l i k e t h e r u b b e r in Hevea and o t h e r l a t e x - p r o d u c i n g

This chapter not subject to U.S. copyright Published 1984, American Chemical Society

Ory and Rittig; Bioregulators ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

BIOREGULATORS: CHEMISTRY AND USES

246

plants in the family Euphorbiaceae, rubber in the guayule plant is not contained in ducts but in single thin-walled parenchyma c e l l s of the cortex, p i t h , and vascular rays of the stems and roots, and to a much lesser extent in the leaves (3). The rubber appears as membrane bound p a r t i c l e s (4), A bushy perennial shrub, guayule, could be c u l t i v a t e d in its native habitat and in the warmer areas of C a l i f o r n i a , Arizona, and New Mexico. It could also be c u l t i v a t e d in the subtropical-temperate regions of Asia, A u s t r a l i a , and A f r i c a .


Plant Bioregulation The single major obstacle to commercial production of guayule rubber is the low y i e l d . Thus, this has stimulated research towards increasing y i e l d from this plant. T r a d i t i o n a l breeding programs and a v a r i e t y of more exotic hybridization techniques may not be the only ways to increase the y i e l d of rubber in the guayule plant. A promising approach to improving the y i e l d c h a r a c t e r i s t i c s is through bioregulation of the synthesis of polyisoprenes to cause an accumul a t i o n of increased amounts of rubber. This approach is based on the discovery of bioregulators that stimulate the production of additional quantities of tetraterpenoids in plant tissues (5). Bioregulators A series of bioregulators were developed for possible use in i n f l u encing rubber formation in guayule. Thus f a r , one of the most e f f e c t i v e bioregulators is DCPTA (2-diethylaminoethyl-3,4-dichlorophenylether) as shown in Table I (6). Table I.

Strain

212 228 230 234 239 241 242

Bioinduction of Rubber in Stem and Branch Tissues of Guayule (18 months old) by 2-Diethylaminoethyl3,4-Dichlorophenylether. The Plants were Treated with 2000 ppm of Bioregulator, 1000 ppm Isopropanol, and 500 ppm 0rtho-X77. A l l Plants were Harvested 40 Days after Treatment. Each Result Represents the Mean of 6 Plants.

Rubber Content (mg/g

dry

wt.)

Control

Treated

58 61 42 52 57 48 51

146 100 77 92 122 122 108


20.

YOKOYAMA ET AL.

247

Rubber Synthesis in the Guayule Plant

A number of benzylalkylamines and benzylfurfurylamines were synthesized (7). £-Bromobenzylfurfurylamine and N-methylbenzylhexylamine caused marked increases in the content of rubber in guayule (Tables I I and I I I ) .


Table I I .

Strain

228 239 234 89

Table I I I .

Strain

Bioinduction of Rubber in Stem and Branch Tissues of Guayule (18 months old) by £-bromobenzylfurfurylamine. The Plants were Treated with 2000 ppm of Bioregulator, 1000 ppm of Isopropanol and 500 ppm of 0rtho-X77. A l l Plants were Harvested 40 Days after Treatment. Each Result Represents the Mean of 6 Plants. Rubber Content (mg/g

dry wt.)

Control

Treated

58 52 54 72

146 121 162 207

Bioinduction of Rubber in Stem and Branch Tissues of Guayule (10 months old) by N-methylbenzylhexylamine. The Plants were Treated with 2000 ppm of Bioregulator, 1000 ppm of Isopropanol and 500 ppm of OrthoX77. A l l Plants were Harvested 40 Days after Treatment. Each Result Represents the Mean of 5 Plants. Rubber Content (mg/g

dry wt.)

Control

Treated

39

131

593

At lower l e v e l s of concentration, the bioregulators appear to have an e f f e c t on the t o t a l rubber content as shown in Table IV (8).

American Chemical Society Library 1155 16th St. N. W. Ory and Rittig; Bioregulators Washington, D. C.Society: 20031 ACS Symposium Series; American Chemical Washington, DC, 1984.


248 Table IV.

Effects of Several Bioregulators on Rubber Content of Guayule


Treatment

Control 250 ppm of 2-(3,4-dichlorophenoxy)triethylamine 125 ppm of 2- ( 3,4-dichlorophenoxy)triethylamine 500 ppm of 2- (2,4-dichlorophenoxy)triethylamine 1000 ppm of N-methylbenzylhexylamine 500 ppm of 2- (3,4-dimethylphenoxytriethylamine

Rubber Content a

15.5 ± 3.9 23.6 ± 6.5

5.3 ± O.7 5.0 ± 1.4

23.5 ± 6.2

6.7 ± O.5

24.3 ± 4.2

6.0 ± 1.5

26.4 ± 7.4

6.6 ± O.2

29.6 ± 8.4

6.6 ± O.7

Grams of rubber per plant ± the standard deviation for four to s i x plants. Percent of rubber ± the standard deviation for four to six plants. D

The 2,4-dichloro and 3,4-dimethyl analogs of DCPTA appeared to increase the t o t a l rubber content of the plants. Mode of Action of Bioregulators The e f f e c t of bioregulators on the increases in t o t a l y i e l d of rubber per plant is limited to a great extent by the a v a i l a b i l i t y of storage areas for the newly synthesized rubber molecules. Studies on native guayule and some hybrids of guayule and other Parthenium species have shown greater v a r i a b i l i t y of amounts of parenchyma tissues (which act as storage areas for rubber) present in the plants (9). Studies also showed that newly induced rubber molecules formed after the young plant was treated with the bioregulator £-bromobenzylfurfurylamine are stored in the parenchymatous c e l l s which did not have a notable quantity of rubber before the treatment (10). In these studies, the bioinduction of rubber was observed over a period of time (55 days) by taking tissue s l i c e s of the stems at i n t e r v a l s of 0, 13, 24, 34, and 55 days after treatment. Micrographs taken at 24 and 34 days showed s i g n i f i c a n t increases in rubber content; the dark areas stained with Sudan black in Figures 1, 2, and 3. No increases in rubber content were noted between 0 and 13 days and between 34 and 55 days. Bioinduction appeared to cease after a l l of the c e l l s which are capable of



249


20. YOKOYAMA ET AL.

F i g u r e 1. Cross s e c t i o n o f a young guayule stem 13 days a f t e r treatment w i t h £-bromobenzyIfurfurylamine. No i n c r e a s e in rubber c o n t e n t (dark s p o t s ) is observed when compared t o c r o s s s e c t i o n a t 0 days. Cross s e c t i o n a t 0 day is i d e n t i c a l t o one at 13 days.




250

F i g u r e 2. Rubber c o n t e n t (dark s p o t s ) 24 days a f t e r t r e a t m e n t . C r o s s s e c t i o n o f young stem show rubber c o n t e n t i n c r e a s i n g in response t o a p p l i c a t i o n o f b i o r e g u l a t o r £-bromobenzylfurfurylamine.




YOKOYAMA ET AL.

F i g u r e 3. Rubber c o n t e n t (dark s p o t s ) 34 days a f t e r treatment w i t h p - b r o m o b e n z y l f u r f u r y l a m i n e . Cross s e c t i o n o f young stem at 55 days a f t e r treatment shows no i n c r e a s e in rubber c o n t e n t .


252


p r o d u c i n g and s t o r i n g r u b b e r have been f i l l e d w i t h r u b b e r . Approxi m a t e l y a t h r e e - f o l d i n c r e a s e in the number o f r u b b e r p a r t i c l e s was seen. F i n d i n g s thus f a r i n d i c a t e t h a t the g u a y u l e p l a n t has a c e r t a i n c a p a c i t y to form and s t o r e r u b b e r , and t h i s is dependent on the particular strain. B i o i n d u c t i o n will n o t be f e a s i b l e beyond the b i o l o g i c a l p o t e n t i a l u n l e s s t h i s p o t e n t i a l is i n c r e a s e d by cell d i f f e r e n t i a t i o n o r enlargement. The l e v e l s of s e v e r a l enzymes i n v o l v e d in the s y n t h e s i s o f c i s - p o l y i s o p r e n e and t r a n s - f a r n e y l p y r o p h o s p h a t e (FPP) from m e v a l o n i c a c i d (MVA) in the stems of DCPTA-treated and c o n t r o l p l a n t s a r e shown in T a b l e V (11).


T a b l e V.

The enzymic

E f f e c t o f DCPTA Treatments on the A c t i v i t y of Enzymes I n v o l v e d in the S y n t h e s i s o f c i s P o l y i s o p r e n e and FPP from MVA in Guayule stems. a c t i v i t i e s a r e a v e r a g e s of two r e p l i c a t e s

each.

DCPTA Enzyme Control

nmol MVA k i n a s e IPP i s o m e r a s e Rubber t r a n s f e r a s e FPP s y n t h e t a s e

mg-l

43.8 4.1 4.5 6.4

Treated

Control

Protein h ~ l

nmol g ~ l

66.7 7.5 9.1 20.2

233.9 19.8 21.9 31.5

Treated

F r e s h Wt

h"

1

439.0 39.8 48.3 107.3

A f t e r 120 days of growth f o l l o w i n g DCPTA a p p l i c a t i o n , the stems of t r e a t e d p l a n t s c o n t a i n e d about a 2 - f o l d i n c r e a s e in rubber ( T a b l e VI) and a 1 . 5 - f o l d g r e a t e r MVA k i n a s e a c t i v i t y than d i d the control plants. The a c t i v i t i e s of i s o p e n t e n y l p y r o p h o s p h a t e (IPP) isomerase and rubber t r a n s f e r a s e were doubled in the stems of the DCPTA-treated p l a n t s and t h e r e was about a 3 - f o l d s t i m u l a t i o n of FPP s y n t h e t a s e in the b i o r e g u l a t o r - t r e a t e d guayule p l a n t s .

Rubber Q u a l i t y L i k e Hevea r u b b e r , g u a y u l e r u b b e r is a polymer o f the s i m p l e 5c a r b o n i s o p r e n e m o l e c u l e and has the c i s - 1 , 4 shape ( F i g u r e 4). The i s o p r e n e u n i t s a r e j o i n e d t o g e t h e r end-to-end to form a g i a n t molec u l e c o n t a i n i n g t e n s of thousands of c a r b o n atoms in a l i n e a r c h a i n i d e n t i c a l t o t h a t o f Hevea r u b b e r and w i t h s i m i l a r m o l e c u l a r weight

(12). Any improvement of y i e l d of r u b b e r in g u a y u l e must be accomp l i s h e d w i t h o u t d i m i n i s h i n g the p h y s i c o - c h e m i c a l c h a r a c t e r i s t i c s o f rubber. The p r e c i s e s t e r e o c h e m i s t r y must be m a i n t a i n e d u n a l t e r e d . These a r e e s s e n t i a l r e q u i r e m e n t s f o r c o m m e r c i a l i z a t i o n in the b i o i n d u c t i o n of r u b b e r f o r m a t i o n . Rubber samples i s o l a t e d from g u a y u l e



20.

YOKOYAMA ET AL.


253

p l a n t s t r e a t e d w i t h DCPTA were compared t o t h o s e from u n t r e a t e d p l a n t s a s w e l l as Hevea r u b b e r . Carbon-13 n u c l e a r m a g n e t i c r e s o nance (NMR) s p e c t r a ( F i g u r e 5) a r e i d e n t i c a l and c o n f i r m t h e s t r u c t u r a l and g e o m e t r i c a l p u r i t y o f g u a y u l e r u b b e r i s o l a t e d from t r e a t e d plants. The NMR s p e c t r a show a complete absence o f s i g n a l s a t t r i b u t a b l e t o s t e r e o o r s t r u c t u r a l i s o m e r s , namely t r a n s - 1 , 4 i s o p r e n e u n i t s , d e m o n s t r a t i n g t h a t g u a y u l e r u b b e r from t r e a t e d p l a n t s is a h i g h l y s t e r e o s p e c i f i c polymer composed e n t i r e l y o f c i s - 1 , 4 i s o p r e n e units. The NMR s p e c t r a c o n f i r m t h a t improvement o f r u b b e r y i e l d is accomplished without a l t e r i n g the m i c r o s t r u c t u r e of the rubber. The m o l e c u l a r weight d i s t r i b u t i o n o f g u a y u l e r u b b e r and Hevea r u b b e r was examined by g e l p e r m e a t i o n chromatography (GPC) as shown in F i g u r e 6. The d i s t r i b u t i o n o f m o l e c u l a r w e i g h t s o f t h e p o l y i s o prene c h a i n in r u b b e r from u n t r e a t e d p l a n t s is i d e n t i c a l t o t h a t from t r e a t e d p l a n t s and b o t h a r e unimodal ( F i g u r e 7 ) . The GPC r e s u l t s suggest t h a t t h e s e b i o r e g u l a t o r s i n d u c e new r u b b e r m o l e c u l e s r a t h e r than a c h a i n e x t e n s i o n o f rubber molecules a t the s u r f a c e o f e x i s t i n g rubber p a r t i c l e s .

Table VI.

E f f e c t o f DCPTA on t h e A c c u m u l a t i o n of Rubber in Guayule P l a n t s .

The d a t a a r e t h e average o f t h r e e r e p l i c a t e s . days a r e averages o f two r e p l i c a t e s each. Induction Period

Treat ment

from 120

Percent Rubber

mg/plant

g

%

15.9 20.5

O.30 O.31

11.0 11.5

76.0 125.0

O.69 1.04

90 90

13.4 14.4

168.0 379.0

1.25 2.63

120 120

13.3 14.3

377.0 748.0

2.83 5.23

Control DCPTA

30 30

Control DCPTA

60 60

Control DCPTA Control DCPTA

5.21 6.61

....-H C 2

H C 3

F i g u r e 4.

Rubber Content

Dry Wt

d

Data

Simple

5-carbon

C H

2

—

Η

isoprene u n i t of rubber.



254


If

-I

F i g u r e 5. Comparison o fC.-13n u c l e a r magnetic resonance s p e c t r a of guayule rubber from u n t r e a t e d p l a n t s and p l a n t s t r e a t e d w i t h DCPTA ( 2 - d i e t h y l a m i n o e t h y l - 3 , 4 - d i c h l o r o p h e n y l e t h e r ) .

Columns:

19

20

23

25

27 29 31 e l u t i o n volume (ml)

33

yStyragel

35

F i g u r e 6. G e l permeation chromatography (GPC) o f guayule rubber and Hevea rubber. The GPC a n a l y s i s was conducted u s i n g a Waters model 6000 chromatography w i t h d i f f e r e n t i a l r e f r a c t i v e index d e t e c t o r , s o l v e n t system of t e t r a h y d r o f u r a n ( a t 28°C.) s t a b i l i z e d w i t h 200 ppm o f 2 , 6 - d i - t e r t - b u t y l - 4 - e t h y l p h e n o l and a s e t of 5 u - S t y r a g e l columns each w i t h a normal p o r o s i t y o f 1 0 , 1 0 , 1 0 , 1 0 , and 500&. 5

3


20.

YOKOYAMA ET AL.


255


Columns: μ5ϋ3π,β1 Solvent : Tetrahydrofuran Flow rate: 1 ml/mln.

elution

volume (ml)

F i g u r e 7. GPC comparison of guayule rubber from u n t r e a t e d p l a n t s and p l a n t s t r e a t e d w i t h DCPTA.

R e f e r e n c e to a p r o d u c t name o r company does n o t i m p l y endorsement o f t h a t p r o d u c t o r company by t h e U S . Department o f A g r i c u l t u r e t o t h e e x c l u s i o n o f o t h e r s t h a t may be a v a i l a b l e . Q

Literature 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12.

Cited

McIntyre, D. In "Guayule"; Guzman, W., Ed., Consejo Nacional de Ciencia y Technologia: Mexico, 1978; Chap. 2 Hammond, B. L., Polhamus, L. G. U.S. Department of Agriculture Technical B u l l e t i n No. 1329, 1965; pp. 5-7. C u r t i s , O. P. Plant Physiology 1947, 22, 33. G i l l i l a n d , M. G.; Van Staden, J. Z. Pflanzenphysiol. Bd. 1983 110, 285. Yokoyama, Η., Hsu, W. J., Poling, S., Hayman, E. Proc. Int. Soc. C i t r i c u l t u r e 1977, p. 717. Yokoyama, H.; Hayman, E., Hsu, W. J., Poling, S.; Bauman, A. Science 1977, 197, 1076. Poling, S.; Hsu, W. J., Yokoyama, H. Phytochemistry 1980, 19, 1677. Hayman, E., Yokoyama, H., Gold, S. J. Agric. Food Chem. 1983, 31, 1120. Mehta, I. Amer. J. Botany 1982, 69, 502. Bauer, T., Glaeser, R.; Yokoyama, H. Unpublished data. Benedict, C. R., Reibach, P. H., Madhavan, S., Stipanovic, R. V., Keihly, J. Η., Yokoyama, H. Plant Physiol. 1983, 72, 897. Campos-Lopez, E., Palacios, J. J. Polymer Sci. 1976, 14, 15.

RECEIVED April 20, 1984 Ory and Rittig; Bioregulators ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

Bioregulators and Rubber Synthesis in the Guayule Plant - American

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