Chemical and Biological Aspects of Abscisic Acid - American

5 Chemical and Biological Aspects of Abscisic Acid

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 10, 2015 | http://pubs.acs.org Publication Date: September 27, 1979 | doi: 10.1021/bk-1979-0111.ch005

JAN A. D. ZEEVAART MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824

Historical Background Among the plant hormones discussed in this Symposium, the growth inhibitor abscisic acid is the most recently discovered one. Work in the 1950s and early 1960s by three groups, work ing on apparently unrelated problems, ultimately resulted in the isolation and identification of abscisic acid in each case (see reviews 1, 2, 3, 4): (a) The search for an abscission -promoting hormone by Addicott and associates at the University of California at Davis led to the isolation in crystalline form of an active compound, called abscisin II, from young cotton fruits (5, 6) for which in 1965 the correct structural formula was proposed (7). Abscisic acid (ABA) was later proposed as the t r i v i a l chemical name for abscisin II (8). (b) Wareing and coworkers at the University of Aberystwyth in Wales were at tempting to isolate a dormancy-inducing substance from trees (9); the active chemical, isolated from leaves of Acer pseudoplatanus, turned out to be identical to ABA (10). (c) Rothwell and Wain (11) at Wye College in Kent, U. Κ., following earlier work by van Steveninck in New Zealand, had as objective the identification of a substance which stimulated flower and fruit drop in yellow lupin. This lupin-abscission factor was identi fied as ABA in 1966 in three different laboratories (12, 13, 14). Other evidence suggesting the existence of growth i n h i b i tors in plants came from analyses of acidic plant extracts by bioassay. This work indicated the presence of a zone in chromatograms with growth-inhibitory activity which was designated as i n h i b i t o r 3. The most a c t i v e component of i n h i b i t o r 3 was l a t e r shown to be ABA (15). Although ABA was o r i g i n a l l y discovered as an a b s c i s s i o n a c c e l e r a t i n g and dormancy-inducing substance, i t soon became c l e a r that i t has many other p h y s i o l o g i c a l e f f e c t s i n p l a n t s . When s y n t h e t i c (+)-ABA became widely a v a i l a b l e , i t was e s t a b l i s h e d that ABA i s a potent i n h i b i t o r in various bioassays and 0-8412-0518-3/79/47-lll-099$05.00/0 © 1979 American Chemical Society

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PLANT GROWTH

SUBSTANCES

counteracts the e f f e c t s of growth-promoting hormones ( a u x i n , g i b b e r e l l i n , c y t o k i n i n ) to which a p a r t i c u l a r organ or t i s s u e responds (16). However, to what extent endogenous ABA funct i o n s as an i n h i b i t o r i n i n t a c t plants i s s t i l l not c l e a r . More recent work i n d i c a t e s that ABA plays an important r o l e i n p l a n t s as a s t r e s s hormone (17, 18).


P r o p e r t i e s of A b s c i s i c A c i d ABA i s a c a r b o x y l i c a c i d which at pH 3.0 p a r t i t i o n s r e a d i l y i n t o organic solvents such as d i e t h y l ether and e t h y l acetate. The molecule has one asymmetric carbon atom at C - l and e x h i b i t s therefore o p t i c a l a c t i v i t y . The n a t u r a l l y o c c u r r i n g enantiomer i s d e x t r o r o t a t o r y and has a s i n i s t e r (S) c o n f i g u r a t i o n (Figure 1). ABA absorbs i n the u l t r a v i o l e t , the maximum varying somewhat with the pH o f the s o l u t i o n . The n e u t r a l methyl ester has a maximum a b s o r p t i o n at 265 nm (E = 20,900) (2). ABA shows e x c e p t i o n a l l y high o p t i c a l a c t i v i t y with extrema at 289 and 246 nm ([a]289 nm = + 2 4 , 0 0 0 ° ; [ ]246 nm = - 6 9 , 0 0 0 ° ) . T h i s property has been used to q u a n t i t a t i v e l y measure the amounts of ABA present i n p u r i f i e d plant e x t r a c t s (15; see also Table 1). In the side chain around C-2 the c o n f i g u r a t i o n can be e i t h e r c i s or t r a n s . By convention 2-cis-ABA i s simply c a l l e d ABA (81). The isomer with the trans c o n f i g u r a t i o n i s c a l l e d 2-trans-ABA (t-ABA) (Figure 1). In s o l u t i o n l i g h t catalyzes the i s o m e r i z a t i o n of the 2 - c i s double bond to e s t a b l i s h a 1:1 r a t i o of ABA and £-ABA. Consequently, samples should be kept i n darkness as much as p o s s i b l e during e x t r a c t i o n and p u r i f i c a t i o n to avoid anomalous r e s u l t s . In s e v e r a l growth i n h i b i t i o n assays the unnatural ( - ) - e n antiomer was as a c t i v e as (+)-ABA. However, (-)-ABA was much l e s s a c t i v e than (+)-ABA i n c l o s i n g stomata of detached b a r l e y leaves (see 3) . When assayed i n darkness (to avoid photoisom e r i z a t i o n ) t_-ABA was completely i n a c t i v e (2). f

a

Methods for Detection and Measurement of A b s c i s i c A c i d A v a r i e t y of methods has been employed to detect and quant i f y ABA. During the e a r l y i s o l a t i o n procedures i n v e s t i g a t o r s were guided by measuring a b s c i s s i o n - a c c e l e r a t i n g e f f e c t s i n the c o t t o n expiant a b s c i s s i o n assay or growth i n h i b i t i o n i n the wheat c o l e o p t i l e or r i c e seedling assay (Table I ) . More s e n s i t i v e bioassays such as i n h i b i t i o n of frond m u l t i p l i c a t i o n i n Lemna grown under a s e p t i c c o n d i t i o n s (24), or stomatal closure i n Commelina epidermal s t r i p s (25) have been developed more recently. The drawback of a l l bioassays i s that they are l a b o r i o u s and time-consuming. Moreover, i d e n t i f i c a t i o n of ABA i n bioassays i s only t e n t a t i v e . Many other chemicals, besides


5. ZEEVAART Table I .

Various Methods Employed for Detection and Measurement of A b s c i s i c Acid

Detection

Methods Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 10, 2015 | http://pubs.acs.org Publication Date: September 27, 1979 | doi: 10.1021/bk-1979-0111.ch005

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limits

References

Bioassays: 5-10 ng per a b s c i s s i o n zone

(5,

10-20 ng/ml

(20,

Rice s e e d l i n g : growth of second l e a f sheath

80 ng/ml

(23)

Lemna growth

0,02-0.03 n g / f l a s k

(24)

0.02-0.1 ng/ml

(25)

Spectropolarimetry

200-500 ng/ml

(15,

Gas chromatography* with flame i o n i z a t i o n detector

5-50 ng per tion

(27, 28., 30)

Cotton expiant

abscission

Wheat c o l e o p t i l e

growth

Commelina stomatal

aperture

*

injec-

6,

19)

2±>

26)

0.005-0.05 ng per injection

(27, 33)

Combined gas chromatography*mass spectrometry

10-30 ng per tion

(20, 29, 34)

High-performance chromatography

1-2 ng per tion

Gas chromatography with e l e c t r o n capture detector

liquid

injec-

0.1-0.3 ng per sample

Rad i o immunoa s say

ABA methylated

injec-

or

(35,

!!>

3(6,

(38, 29)

trimethylsilylated



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PLANT GROWTH

SUBSTANCES

ABA, when present i n s u f f i c i e n t l y high c o n c e n t r a t i o n s , can also cause ( n o n - s p e c i f i c ) i n h i b i t i o n of growth. Nowadays ABA i s t h e r e f o r e p r e f e r a b l y detected and measured by one of s e v e r a l methods which make use of c e r t a i n unique p r o p e r t i e s of the ABA molecule. Spectropolarimetry i s based on the l a r g e s p e c i f i c r o t a t i o n i n the u l t r a v i o l e t (see above). This method is s p e c i f i c for ABA, but not very s e n s i t i v e (Table I ) . For gas chromatography v o l a t i l e d e r i v a t i v e s such as the methyl ester of ABA (27, 29, 30) or t r i i r e t h y l s i l y l a t e d ABA (20, 28) must be prepared. ABA i s a molecule with a high e l e c t r o n a f f i n i t y so that the methyl ester can be measured with a gas chromatograph equipped with an e l e c t r o n capture d e t e c t o r . Inj e c t i o n s of as l i t t l e as 5 pg of Me-ABA cause a detector r e sponse (27). Metabolites of ABA such as phaseic and d i h y d r o phaseic a c i d can a l s o be measured by t h i s method (33). The most conclusive method to i d e n t i f y ABA i s by combined gas chromatography-mass spectrometry (20, 29, 33). L i t t l e et a l . (34) monitored the current of a s i n g l e i o n , v i z . that of the base peak, m/e 190, i n the mass spectrum of Me-ABA, for q u a n t i t a t i v e determinations of ABA i n the cambium of Picea sitchensis. However, few l a b o r a t o r i e s are able to c a r r y out analyses by t h i s method on a routine b a s i s . The use of h i g h performance l i q u i d chromatography f o r measuring ABA has been reported by s e v e r a l workers (Table I ) . However, i n view of the many substances i n plant e x t r a c t s that absorb i n the u l t r a v i o l e t region of the spectrum, t h i s method cannot be considered as c o n c l u s i v e . A radioimmunoassay which i s h i g h l y s p e c i f i c for ABA, has been developed r e c e n t l y (38, 39). This method appears very a t t r a c t i v e when l a r g e numbers of samples have to be analyzed r o u t i n e l y f o r ABA content. It i s obvious that unequivocal i d e n t i f i c a t i o n of small q u a n t i t i e s of ABA can only be accomplished by combined gas chromatography-mass spectrometry. However, once t h i s has been accomplished i n a p a r t i c u l a r system, r o u t i n e measurements w i l l i n the future probably mostly r e l y on gas chromatography with e l e c t r o n capture detector and on radioimmunoassay. Occurrence ABA i s ubiquitous i n higher p l a n t s . It has also been identified i n gymnosperms, f e r n s , h o r s e t a i l s , lycopods and mosses, but not i n l i v e r w o r t s (_3> 39). In the l a t t e r group l u n u l a r i c a c i d appears to take the place of ABA as a growth i n h i b i t o r (3). In a recent report ABA was i d e n t i f i e d as a metabolite of the fungus Cercospora r o s i c o l a (40). This observation has been confirmed i n our l a b o r a t o r y (Zeevaart, unpublished r e s u l t s ) . The a v a i l a b i l i t y of a microorganism that produces ABA, o f f e r s unique o p p o r t u n i t i e s for b i o s y n t h e t i c and genetic studies of



5. ZEEVAART

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Abscisic Acid

ABA formation that cannot be r e a d i l y conducted with higher plants. S i m i l a r studies with the fungus G i b b e r e l l a f u j i k u r o i , which produces g i b b e r e l l i n s , have g r e a t l y advanced our knowledge of t h i s group of hormones as reported by Phinney in t h i s Symposium. ABA has been detected i n a l l organs of higher p l a n t s , as w e l l as i n phloem and xylem sap (3). However, the concentrat i o n s of ABA can g r e a t l y vary from organ to organ. Fruits, young seeds and buds u s u a l l y have a high ABA content. In leaves the ABA content per unit weight i s highest i n the youngest l e a v e s ; i t d e c l i n e s as the leaves expand (Figure 2 ) . Biosynthesis

of A b s c i s i c A c i d

The i n c o r p o r a t i o n of l a b e l from mevalonate into ABA, a s e s q u i t e r p e n o i d , has been demonstrated i n d i f f e r e n t parts of p l a n t s (e._g. 41). This i n d i c a t e s that ABA can be synthesized throughout the p l a n t . In a d d i t i o n to the d i r e c t i n c o r p o r a t i o n of three isoprene u n i t s , derived from mevalonate, into ABA, an i n d i r e c t b i o s y n t h e t i c pathway v i a carotenoids has been proposed. This idea stems from the f i n d i n g that x a n t h o p h y l l s , i n p a r t i c u l a r v i o l a x a n t h i n , can e i t h e r photochemically or enzymat i c a l l y be converted to the n e u t r a l i n h i b i t o r xanthoxin (42) (Figure 3 ) . When labeled xanthoxin was fed i n the t r a n s p i r a t i o n stream to bean or tomato shoots, c a . 10% was converted to ABA over an 8-hr period (43). However, the importance of the b i o s y n t h e t i c route to ABA v i a xanthophylls and xanthoxin i n normal metabolism remains to be e s t a b l i s h e d , and most of the evidence favors the d i r e c t synthesis route v i a a precursor (see 2). So f a r , only one i n v i t r o system for ABA b i o s y n t h e s i s has been described (44). In t h i s study c h l o r o p l a s t s i s o l a t e d from r i p e n i n g avocado f r u i t s incorporated mevalonate into ABA. Upon l y s i n g the c e l l - f r e e system i n d i l u t e b u f f e r , i n c o r p o r a t i o n of l a b e l i n t o ABA increased c o n s i d e r a b l y , i n d i c a t i n g that the c h l o r o p l a s t membrane was a major b a r r i e r to the penetration of mevalonate. Although these p r e l i m i n a r y r e s u l t s demonstrate that ABA can be synthesized w i t h i n the c h l o r o p l a s t , the p o s s i b i l i t y that synthesis can also take place outside the c h l o r o p l a s t s , can by no means be r u l e d out. Metabolism of A b s c i s i c A c i d f

ABA i s metabolized v i a the unstable intermediate 6 - h y droxymethyl-ABA, more r e c e n t l y c a l l e d hydroxyabscisic acid (HOABA) by H i r a i ejt a l . (45), to phaseic a c i d (PA). In c e r t a i n p l a n t s the l a t t e r compound i s further converted to 4 - d i h y d r o phaseic a c i d (DPA) which accumulates as the end product. The ABA-> PA DPA pathway (Figure 4) operates i n beans (46), i n pea seedlings (47), i n ash seeds (48), i n castor bean (49), and f



PLANT GROWTH

( + )-Abscisic acid Figure 1.

SUBSTANCES

2-trans-obscisic acid

Structures of (+)-abscisic acid (ABA) and of 2-tra.ns-abscisic acid (t-ABA)

/

L e a f Size Plant Physiology

Figure 2A. Change in abscisic acid content of Xanthium leaves with age. Fresh and dry weight and abscisic acid content of 10 leaves of different ages (51).


5.

ZEEVAART

105

Abscisic Acid

ι

ι

r


Β

Leaf

Size Plant Physiology

Figure 2B. Change in abscisic acid content of Xanthium leaves with age. Abscisi acid content of leaves of different ages expressed per unit fresh and dry weight

Figure 3. Structures of cis,tYans-xanthoxin and trans,trans-xanthoxin



106

PLANT GROWTH

Figure 4. Metabolic pathway of abscisic acid (ABA) via the unstable intermediate 6 -hydroxymethyl-ABA to FA and DPA f

SUBSTANCES

DPA



5. ZEEVAART

Abscisic Acid

i n the endosperm of immature E c h i n o c y s t i s f r u i t s (50). From t h i s l a t t e r m a t e r i a l a c e l l - f r e e system has been prepared which converted ABA to PA and DPA. The enzyme p r e p a r a t i o n was separated by c e n t r i f u g a t i o n i n t o a p a r t i c u l a t e f r a c t i o n with ABA-hydroxylating a c t i v i t y and a soluble Ρ Α - r e d u c i n g a c t i v i t y (30). I n s i g n i f i c a n t amounts of DPA were detected i n leaves of Xanthium strumarium (51)* This r a i s e s the p o s s i b i l i t y that i n some plants conversion of PA to DPA i s not the p r i n c i p a l meta b o l i c route for PA d e g r a d a t i o n . When racemic ABA was fed to p l a n t s , only the (+)-enantiomer was metabolized to PA and DPA (2, 33). In a d d i t i o n , a con jugate with g l u c o s e , a b s c i s y l - 3 - D - g l u c o p y r a n o s i d e was formed, i d e n t i c a l to the glucose ester of ABA i s o l a t e d from the f r u i t s of yellow l u p i n (52). However, the n a t u r a l glucose ester y i e l d e d e x c l u s i v e l y (+)-ABA on h y d r o l y s i s , whereas the glucose e s t e r produced a f t e r feeding of (+)-ABA gave predominantly (-)-ABA (2, 33). Thus, (+)-ABA can be both metabolized to PA and conjugated with glucose, whereas (-)-ABA i s only conjuga ted. L i k e w i s e , _t-ABA i s only conjugated into the glucose ester (2, 53). Another conjugate of ABA, v i z . 3 -hydroxy-3~methy1g l u t a r y l h y d r o x y a b s c i s i c a c i d (HMG-HOABA) has r e c e n t l y been i s o l a t e d from seeds of Robinia pseudoacacia (45). P h y s i o l o g i c a l Roles of A b s c i s i c A c i d As discussed above, ABA was discovered as a substance that promotes a b s c i s s i o n and as a substance associated with the on set of dormancy i n woody p e r e n n i a l s . Since then i t has become c l e a r that ABA i s widely d i s t r i b u t e d i n higher p l a n t s , but l i t t l e evidence has accumulated that ABA, as an endogenous hormone, i s involved i n the r e g u l a t i o n of e i t h e r a b s c i s s i o n or dormancy (see 16, 54, 55). ABA i s u s u a l l y c l a s s i f i e d as an i n h i b i t o r that counteracts the e f f e c t s of growth-promoting sub stances ( 16). While t h i s appears to be true for growth sub stances a p p l i e d to excised p a r t s , i t i s not evident that t h i s a l s o p e r t a i n s to the endogenous hormones i n whole p l a n t s . For example, d e s p i t e a very high ABA content (Figure 2), young leaves expand r a p i d l y which argues against a r o l e for ABA as an endogenous i n h i b i t o r . It i s p o s s i b l e , of course, that i n the absence of any ABA young leaves would expand more r a p i d l y than they normally do, but without s p e c i f i c i n h i b i t o r s for ABA syn t h e s i s , or A B A - d e f i c i e n t mutants, t h i s idea cannot be tested at present. In a recent review ( 16) of i t s p h y s i o l o g i c a l f u n c t i o n s , evidence was presented that ABA plays a r o l e i n the geotropic response of roots and i n tuber formation. It may have a r o l e i n other growth and developmental processes as w e l l , but so far the evidence i s i n c o n c l u s i v e . A most i n t e r e s t i n g development i n work on ABA has been the


107

108

PLANT GROWTH

SUBSTANCES


discovery that ABA plays an important r o l e i n stomatal closure and thus i n reducing the l o s s of water from plants under stress. Consequently, ABA has been assigned a r o l e as a s t r e s s hormone (17). The Role of A b s c i s i c A c i d as a S t r e s s Hormone. The r o l e of ABA i n water s t r e s s has been studied most e x t e n s i v e l y , a l though there i s also evidence for involvement of ABA i n other s t r e s s e s such as s a l i n i t y , mineral d e f i c i e n c y , high osmotic c o n c e n t r a t i o n , and w a t e r - l o g g i n g . A review on the r o l e of ABA i n s t r e s s phenomena has appeared r e c e n t l y (18). There are two basic observations with respect to the r o l e of ABA i n water s t r e s s : (a) Exogenously a p p l i e d (+)-ABA causes c l o s u r e of the s t o mata i n many p l a n t s , thus reducing t r a n s p i r a t i o n . The response of detached leaves to (+)-ABA supplied v i a the t r a n s p i r a t i o n stream was very r a p i d , the stomata of c e r t a i n species s t a r t i n g to close 3 to 10 minutes a f t e r ABA was introduced in the i r r i g a t i o n water (56, 57, 58). Stomatal closure in bean, corn and rose leaves s t a r t e d when the ABA l e v e l reached approximately twice the normal endogenous concentration (57), but i n Xanthium an increase i n the ABA of the whole l e a f by only 1-2% was s u f f i c i e n t to cause a stomatal response (58). Upon withdrawal of the (+)-ABA s o l u t i o n , the stomata s t a r t e d to open w i t h i n 5 minutes (56). Thus, the response of stomata to (+)-ABA i s both r a p i d and r e v e r s i b l e . An e f f e c t on stomatal aperture can also be observed i n i s o l a t e d epidermal s t r i p s that are f l o a t i n g on a buffer s o l u t i o n ; t h i s phenomenon has been used as a very s e n s i t i v e bioassay for ABA (Table I ) . When ABA was added to the s o l u t i o n , the stomatal aperture s t a r t e d to d e c l i n e w i t h i n a few minutes, due to the l o s s of K ions from the guard c e l l s , r e s u l t i n g i n a decreased turgor i n these c e l l s (59). (b) When plants s t a r t to w i l t , there i s a l a r g e accumula t i o n of ABA i n the leaves and also i n other organs such as stems, a p i c e s , f l o w e r s , f r u i t s , seeds, and roots ( β . ] | . 18, 41, 60). An example of ABA accumulation i n detached and w i l t e d Xanthium leaves i s given i n Figure 5. Following w i l t i n g the ABA content increased over a 6-hr period from 200 to 1350 ng ABA per g f r e s h weight, and then l e v e l e d o f f . A 50% increase was d i s c e r n a b l e a f t e r 30 minutes. In bean seedlings a 1 . 5 - f o l d increase i n ABA has been observed 10 minutes a f t e r the onset of s t r e s s (61) which was c o r r e l a t e d with increased l e a f r e s i s t a n c e ( 1 . β _ . decreased stomatal a p e r t u r e ) . However, such c o r r e l a t i o n s have not always been found, and Walton et_ al. (62) have sugges ted that i t i s not the t o t a l ABA content that counts i n d e t e r mining stomatal a p e r t u r e , but rather the rate of ABA s y n t h e s i s . Drought-induced ABA accumulation i s common i n mesophytes; i t i s l e s s pronounced i n hygrophytes, p a r t i c u l a r l y i n submerged leaves (18, 41). The r a p i d increase i n ABA f o l l o w i n g s t r e s s occurs presumably by de novo s y n t h e s i s , and not through release +



5. ZEEVAART

Abscisic Acid

109

from a conjugated form of ABA (26, 41). A number of workers (63, 64, 65) have suggested that there i s a c r i t i c a l » t h r e s h o l d l e a f water p o t e n t i a l i n the -10 to -12 bar range at which the ABA l e v e l s of leaves s t a r t to i n c r e a s e . However, more recent evidence i n d i c a t e s that zero turgor i s the c r i t i c a l parameter at which the ABA content s t a r t s to increase (66, 67_). Following recovery from water s t r e s s the ABA l e v e l s of leaves d e c l i n e r a p i d l y ( £ . £ . 61, 63, 68). This i s i l l u s t r a t e d i n Figure 6 f o r Xanthium l e a v e s . After detached leaves had been i n the w i l t e d state for 5 h o u r s , the ABA content had i n creased to 3000 ng per g f r e s h weight. Upon submerging such w i l t e d leaves i n d i s t i l l e d water for 5 minutes to regain t u r gor, a s i g n i f i c a n t drop i n the ABA l e v e l was observed a f t e r 2 hours and a f t e r 4 hours i t had e s s e n t i a l l y returned to the p r e stress value. This i n d i c a t e s that r a p i d ABA synthesis ceases as soon as turgor i s regained while rapid ABA degradation con tinues u n t i l the excess ABA has been removed. This decrease i n ABA i s accompanied by a t r a n s i e n t increase in PA as demonstra ted i n leaves of grapevine (69) and Xanthium (Zeevaart, unpub lished results). Thus, following a period of drought rewatering w i l l r e s u l t i n a r a p i d disappearance of the excess ABA. However, the stomata do not open for s e v e r a l more days and photosynthesis remains reduced (70). This s o - c a l l e d after e f f e c t of moisture s t r e s s was thought to be due to i n h i b i t i o n of photosynthesis by the accumulated PA (69), but t h i s hypothe s i s has been disproven r e c e n t l y (see next s e c t i o n ) . Research on ABA has p r a c t i c a l i m p l i c a t i o n s for A g r i c u l t u r e since water i s a l i m i t i n g f a c t o r for crop production i n many areas of the world. On an experimental scale ABA and c e r t a i n d e r i v a t i v e s have been a p p l i e d to crop plants as " a n t i t r a n s p i r ants" (71, 72). In short-term experiments t r a n s p i r a t i o n was c o n s i d e r a b l y reduced without much e f f e c t on the rate of photo synthesis. Thus, a p p l i e d ABA increased the water-use e f f i c i e n cy of p l a n t s . Another development which may be u s e f u l i s the f i n d i n g that a d r o u g h t - t o l e r a n t corn v a r i e t y produced more ABA upon w i l t i n g than d i d two s e n s i t i v e c u l t i v a r s (73). The enhanced a b i l i t y to accumulate ABA might be used as a marker for breed ing d r o u g h t - t o l e r a n t p l a n t s . Roles of M e t a b o l i t e s of A b s c i s i c A c i d . Nothing i s known about the p h y s i o l o g i c a l r o l e of PA and DPA i n p l a n t s , although these two metabolites of ABA have been tested in several b i o a s says r e c e n t l y . In the cotton expiant a b s c i s s i o n assay PA had one-tenth of the a c t i v i t y of ABA (19). PA and DPA were e q u a l l y e f f e c t i v e i n i n h i b i t i n g α - a m y l a s e s e c r e t i o n by b a r l e y aleurone l a y e r s treated with g i b b e r e l l i n A3; DPA had approximately one-tenth of the a c t i v i t y of ABA i n t h i s system (74). The e f f e c t of PA on growth of bean embryos was n e g l i g i b l e (75).



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P L A N T G R O W T H SUBSTANCES

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Figure 5. Accumulation of abscisic acid in detached mature leaves of Xanthium strumarium after the fresh weight had been reduced by 10%

Figure 6. Decrease in abscisic acid con tent of wilted leaves of Xanthium after stress was relieved. Detached leaves were wilted by reducing the fresh weight by 10%. Stress was relieved after 5 hr by submerging leaves into distilled water for 5 min.

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DPA d i d not cause stomatal closure i n any of the species t e s t e d , while the e f f e c t of PA ranged from a response as rapid as that caused by ABA i n Commelina, to a l e s s r a p i d closure than a f t e r ABA treatment i n Amaranthus, Hordeum, Xanthium, and Zea, to no response at a l l i n V i c i a (76). Kriedemann et_ a l . (69) have proposed that endogenous PA f u n c t i o n s as an i n h i b i t o r of photosynthesis following r e l i e f of water s t r e s s when t h i s metabolite accumulates. This proposal was based on the observation that plant e x t r a c t s containing PA s t r o n g l y i n h i b i t e d photosynthesis in detached leaves of several species (69). However, subsequent work with c r y s t a l l i z e d PA has shown that i t was not PA that i n h i b i t e d photosynthesis, but r a t h e r i m p u r i t i e s that were present i n the solvents used (76). Thus, the p h y s i o l o g i c a l r o l e of PA, i f any, remains to be determined. Concluding Remarks Although much has been learned about the chemistry and physiology of ABA since i t s discovery i n 1965, many unsolved problems remain. Degradation of ABA has been f a i r l y w e l l worked out, but b i o s y n t h e s i s i s s t i l l poorly understood. Of p a r t i c u l a r importance would be to discover the sensing mechanism for s t r e s s - i n d u c e d ABA accumulation. If turgor i s the c r u c i a l f a c t o r , the plasma membrane might be i n v o l v e d , since at zero turgor t h i s o r g a n e l l e i s no longer pressed against the c e l l wall. On the other hand a c e r t a i n amount of ABA i s always produced i n f u l l y t u r g i d c e l l s , thus suggesting that there may be two d i f f e r e n t mechanisms for ABA s y n t h e s i s : one that operates i n t u r g i d c e l l s , and another one which becomes a c t i v a t e d only i n c e l l s under s t r e s s c o n d i t i o n s . One f u r t h e r problem i s the l a r g e overshoot i n ABA product i o n in wilted leaves. With a p p l i e d ABA a doubling of the ABA content of the l e a f i s u s u a l l y adequate for stomatal c l o s u r e , while increases up to 4 0 - f o l d have been reported i n w i l t e d leaves. However, e x t r a c t i o n s of whole leaves do not take into account the l o c a t i o n of ABA w i t h i n the l e a f . Perhaps much of the hormone i s sequestered i n a compartment that has no access to the guard c e l l s . Thus, i t would be of much importance to determine the d i s t r i b u t i o n of ABA at the t i s s u e l e v e l as w e l l as i t s i n t r a c e l l u l a r l o c a t i o n . Since ABA i s a small waters o l u b l e molecule, conventional f r a c t i o n a t i o n techniques may not be s u i t a b l e to determine i t s d i s t r i b u t i o n i n various organelles. A h i g h l y s p e c i f i c immunological method for d e t e c t i o n of ABA has r e c e n t l y been developed (38, 39). It i s conceivable that t h i s technique could be further developed for determining the c e l l u l a r l o c a l i z a t i o n of ABA as has already been done for the photoreceptor phytochrome (77, 78). Acknowledgement - My research reported i n t h i s paper was supported by the U. S. Department of Energy under Contract EY-76-C-02-1338 and by grant no. PCM-7807653 from the National Science Foundation.


111

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Chemical and Biological Aspects of Abscisic Acid - American

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