Gibberellins in Higher Plants: The Biosynthetic Pathway Leading to

Chapter 3

Gibberellins in Higher Plants: The Biosynthetic Pathway Leading to GA 1

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch003

C. R. Spray and B. O. Phinney Department of Biology, University of California, Los Angeles, CA 90024

The gibberellins (GAs) are a class of plant hormones that were first isolated as metabolites of the fungus, Gibberella fujikuroi. Thereafter they were found to be widespread among higher plants. The GAs have been shown to control a number of biological functions, including shoot elongation, fruit set and the de novo synthesis of α-amylase. Recent advances in technology have provided methods leading to the identification of 72 GAs. Also biosynthetic studies have integrated these GAs into a limited number of pathways, all of which originate from the common diterpene precursor, GA -aldehyde. In maize shoots, only a single pathway exists, which leads to the biologically active gibberellin, GA . The availability of GA-deficient mutants (dwarf mutants) of maize has provided a unique opportunity to analyze the biosynthetic pathway at both the chemical and genetic level. The results have led to the conclusion that only one gibberellin (GA) is active per se in the control of shoot elongation in maize. This unitary control is probably widespread among higher plants. 12

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The sequence of events that l e d to the discovery of the g i b b e r e l l i n s (GAs) has recently been reviewed by Phinney (1). While the GAs were f i r s t discovered as secondary metabolites of the fungus, Gibberella f u j i k u r o i , they have now been shown to be present i n most higher plants (both gymnosperms and angiosperms). Their recent i d e n t i f i c a t i o n from ferns and psilophytes (2,3) suggests that the GAs may be ubiquitous to the plant kingdom. G i b b e r e l l i n s have yet to be i d e n t i f i e d from l i v e r w o r t s , mosses, algae and bacteria. Of the 72 g i b b e r e l l i n s i d e n t i f i e d by gas chromatography-mass spectrometry (GC-MS), eleven are found only i n the fungus, G. f u j i k u r o i , f o r t y seven i n higher plants while fourteen a d d i t i o n a l GAs are common to both groups. The g i b b e r e l l i n s are t e t r a c y c l i c diterpene acids, having i n common the carbon skeleton, 0097-6156/87/0325-0025$06.00/0 © 1987 American Chemical Society

Fuller and Nes; Ecology and Metabolism of Plant Lipids ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

ent-gibberellane. The g i b b e r e l l i n s are then divided into two major groups, the ent-gibbere1lanes (the C O Q ' C A S ) and the ent-20-norgibberellanes (the C^g-GAs) (see Figure 1). Within the two groups, the GAs d i f f e r from each other i n a l i m i t e d number of ways, e.g. the number and p o s i t i o n of hydroxyl groups, the degree of unsaturation, and the oxidation state of carbon-19 and carbon-20. In order to s i m p l i f y the g i b b e r e l l i n nomenclature, the t r i v i a l names G A i _ are now used (4), with each newly characterized member being assigned the next a v a i l a b l e number. In general, an i n d i v i d u a l plant species contains a l i m i t e d number of g i b b e r e l l i n s (usually less than f i f t e e n ) ; i n many higher plants apparently only one of these, GA^, i s a c t i v e per se i n the c o n t r o l of shoot elongation; the other GAs of the shoot are a c t i v e as biosynthetic precursors to GA^, or b i o l o g i c a l l y i n a c t i v e as branch metabolites from the main pathway. The generalization, "one active GA for shoot elongation", originated from studies with maize (Zea mays) where dwarfing genes were shown to block s p e c i f i c steps i n the early-13-hydroxylation pathway leading to GA^ (5-8). This generalization may be widespread among higher plants since dwarf mutants have been found to c o n t r o l steps of t h i s same pathway i n pea (9), r i c e (10) and tomato (11). The absolute i d e n t i f i c a t i o n of the s p e c i f i c g i b b e r e l l i n s native to a plant i s c e n t r a l to the analysis of GA biosynthesis because the enzymes c o n t r o l l i n g s p e c i f i c steps i n the pathway have been shown to be non-specific, i . e . non-native GAs are metabolized to non-native products when added to either the fungus or to the plant (12). Most of the d e f i n i t i v e information on the i d e n t i f i c a t i o n of GAs from plants comes from studies using developing embryos and endosperm, which are r e l a t i v e l y r i c h sources of GAs, with l e v e l s as high as 10 to 100 milligrams GA per kilogram fresh weight. In contrast, vegetative green shoots may contain less than 1 microgram GA per kilogram fresh weight (1^3). Such low l e v e l s have long frustrated plant p h y s i o l o g i s t s because i t i s the shoot that shows dramatic elongation responses to applied g i b b e r e l l i n s . Much of the e a r l y work on the presence and l e v e l s of g i b b e r e l l i n s i n higher plants was based on bioassay data. While such bioassays are generally s p e c i f i c f o r the c l a s s of diterpenes, the g i b b e r e l l i n s , they are e s s e n t i a l l y useless i n determining kinds and l e v e l s of s p e c i f i c g i b b e r e l l i n s . In recent years p h y s i o l o g i c a l and biochemical research on plant hormones has been r e v o l u t i o n i z e d by the use of high performance l i q u i d chromatography (HPLC) as a key p u r i f i c a t i o n step, and c a p i l l a r y GC-MS, f o r the i d e n t i f i c a t i o n of s p e c i f i c GAs. Thus picogram l e v e l s of a p a r t i c u l a r GA can now be detected i n a sample background of 1 milligram (14). As a r e s u l t , endogenous GAs are being i d e n t i f i e d and q u a n t i f i e d from the shoots of an increasing number of higher plants (e.g. 2,15-18). (For d e t a i l e d discussions of the c r i t e r i a f o r the unambiguous i d e n t i f i c a t i o n of g i b b e r e l l i n s see references 14,19,20). A flow sheet summarizing the p u r i f i c a t i o n steps leading to the i d e n t i f i c a t i o n of endogenous GAs i n maize i s shown i n Figure 2. GC-MS i s also c r i t i c a l to metabolic studies. For example, a plant can be fed a double labeled substrate (e.g. [ ^^C,%]-GAjj). The radioisotope ( [ % ] ) can be used to track the substrate and metabolites during p u r i f i c a t i o n while the stable isotope ( [ ^ C ] )


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3. SPRAY AND PHINNEY

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Gibberellins in Higher Plants

Figure 1. The carbon skeleton and numbering system f o r the g i b b e r e l l i n s . A l l the known GAs are either C o-GAs ( a ) , or C^g-GAs ( b ) , i n which carbon-20 has been l o s t with the formation of a 19,10y lactone. (For the structures of the 72 GAs see references 14,57,39). 2




Plant material I Freeze Homogenise i n 80% MeOH Extract 24h., 3). However, i t was not u n t i l 1983 that the pathway i t s e l f was shown to be present (27). The pathway (Figure 4) i s i n i t i a t e d by the formation of GA^ which i s then oxidized s e q u e n t i a l l y at carbon-20 to the alcohol (GA15 [opened lactone]) and to the aldehyde (GA24). Loss of carbon-20 combined with formation of a 19,10 lactone gives the bioactive g i b b e r e l l i n , GAg. In addition, the aldehyde of GA24 may be further oxidized to the acid (GA25) which i s apparently not further metabolized; also GAg may be hydroxy lated at the 2(Sp o s i t i o n to give the b i o l o g i c a l l y inactive GA51.




to

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The Early-3-hydroxylation Pathway (Figure 5) This pathway i s present i n the fungus (G. f u j i k u r o i ) . A l l of the members shown i n figure 5 are endogenous to the fungus; also at l e a s t eighteen of the actual or p o t e n t i a l steps i n t h i s pathway have been analyzed by substrate/product feeding studies using [^^C]-labeled substrates. In t h i s pathway GA^2-aldehyde i s 33-hydroxylated to GA^-aldehyde which, i n turn, i s oxidized at carbon-7, g i v i n g GA^^. G i b b e r e l l i n A^^ i s then metabolized v i a an undefined s e r i e s of intermediates to give the C^g g i b b e r e l l i n , GA4. G i b b e r e l l i n A4 may be further metabolized, e i t h e r by 13-hydroxylation to GA^, or by 1,2-dehydrogenation to GA7. G i b b e r e l l i n A7 i s 13-hydroxylated to GA3. (This fungus i s used i n the commercial production of the a g r i c u l t u r a l l y important C^g g i b b e r e l l i n s , GA3, GA4, GA7 and GAg). There are several branch steps from the main early-3-hydroxylation pathway; e.g. GA^4 may be 16-hydroxylated ( G A ^ ) ) oxidized at carbon-20 (GA36 and GA13); i n a d d i t i o n , GA4 may be a-hydroxylated at carbon-1 (GA^g) or carbon-2 (GA47). G i b b e r e l l i n and GA36 are shown as branches from the main pathway since they are apparently not metabolized when fed to the fungus (31.) • This pathway has been presented and discussed i n greater d e t a i l i n several reviews (34-36). The f i r s t two members of the early-3-hydroxylation pathway are the C20 g i b b e r e l l i n s , GAj^-aldehyde and GA^. Since these two g i b b e r e l l i n s have yet to be i d e n t i f i e d from higher p l a n t s , there i s no d i r e c t evidence f o r the presence of the pathway i n higher plants; the n a t u r a l occurrence of GAs that would be found l a t e i n the pathway ( i . e . C^g-GAs) i s not c r i t i c a l evidence f o r the presence of the pathway, since C^g-GAs can have more than one biosynthetic o r i g i n . Likewise, presumptive pathways based on feeding studies with GA^4-aldehyde or GA^^ as substrates do not provide d i r e c t evidence f o r the presence of the pathway, because of substrate n o n - s p e c i f i c i t y . (As mentioned e a r l i e r , the fungus w i l l convert non-native g i b b e r e l l i n s through a series of non-native intermediates to non-native g i b b e r e l l i n products [12]). Feeds and refeeds of radiolabeled GA^4-aldehyde and i t s metabolites, to c e l l - f r e e systems from pumpkin endosperm, c l e a r l y document metabolism through an early-3-hydroxylation pathway i n t h i s system (37). However, the b i o l o g i c a l s i g n i f i c a n c e of t h i s pathway i n the i n t a c t pumpkin plant has yet to be established. o r

Other Pathways Six 12a-hydroxylated g i b b e r e l l i n s have been i d e n t i f i e d as native to pumpkin (38). Their structures show them to be 12a-hydroxy derivatives of the C20 g i b b e r e l l i n s , GA^2> GA^, GA37, GA^3 (named GA3g), and the C^g g i b b e r e l l i n s , GA4 (named GA5g), and GA34 (named GA^g). Thus they are p o t e n t i a l members of an early-12a-hydroxylation pathway analogous i n oxidation and hydroxylation pattern to the early-3-hydroxylation pathway; a l t e r n a t i v e l y , they may be a s e r i e s of s i n g l e step metabolites that branch from an early-3-hydroxylation pathway. Recent and extensive radiolabeled feeds to c e l l - f r e e systems from pumpkin suggest both p o s s i b i l i t i e s (37,38). In t h i s system the conversions are h i g h l y




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pH dependent. The r e l a t i o n s h i p of the 12a-hydroxylated g i b b e r e l l i n s to a s p e c i f i c pathway i s , as yet, unresolved. There are a number of a d d i t i o n a l g i b b e r e l l i n s native to higher plants with unique hydroxylation patterns ( f o r example, hydroxylated at positions 1, 11, 123, or 15) that f i t into no known pathways (2,3,39,40). These GAs cannot be assigned to a biosynthetic pathway u n t i l radiolabeled feeding studies have been made using the plants from which the g i b b e r e l l i n s were i s o l a t e d .


The Early-13-hydroxylation Pathway (Figure 6) The g i b b e r e l l i n s that comprise t h i s pathway are both unique to higher plants ( i . e . not present i n the fungus) and widespread among higher plants. The pathway i t s e l f has been shown to be present i n maize (Zea mays) (7,T7,41), and pea (Pisum sativum) (27,42). This report w i l l consider only the early-13-hydroxylation pathway f o r maize. The f i r s t clue f o r the presence of the pathway i n maize came from the i d e n t i f i c a t i o n of eight 13-hydroxylated GAs from very young t a s s e l s and vegetative shoots (T7,41^,43). No other GAs were found i n these extracts. A comparison of the structures of these eight g i b b e r e l l i n s c l e a r l y placed them i n a s i n g l e hypothetical pathway o r i g i n a t i n g from the common GA-precursor, GA^-aldehyde ( i . e . GA53, GA44, 19» 1 7 [branch?], GA , GA g [branch?], GA^, and GAg [branch?]) (5). The order of four s p e c i f i c steps has now been established from radiolabeled feeds t o maize seedlings, where the fate of the l a b e l was followed a f t e r predetermined periods of incubation. The s p e c i f i c steps between GAi -aldehyde and GA53 are s t i l l unresolved; i n a d d i t i o n , the steps GA44 to GA^g; GA^g to GA17; and GA^g to G A Q have yet to be demonstrated. This c l a s s i c a l approach (feed of labeled substrate followed by i d e n t i f i c a t i o n of labeled immediate product, etc.) was greatly f a c i l i t a t e d by the use of a series of GA-mutants that lack e i t h e r a l l or s p e c i f i c combinations of endogenous GAs. These f i v e mutants, dwarf-1, dwarf-2, dwarf-3, dwarf-5 and anther ear-1 (Figure 7), are simple recessives, n o n - a l l e l i c to each other and expressed as a dwarf phenotype from the e a r l y seedling stage to maturity; i n the dark as w e l l as i n the l i g h t (44). They are GA mutants i n that each dwarf mutant resumes normal growth i n response to exogenous GA; a constant supply of g i b b e r e l l i n i s necessary f o r t h i s normal growth (5,45). No other plant or animal hormone e l i c i t s such a response. The pattern of response to each member of the pathway varies depending on the mutant being tested (5) (see Figure 8). Such d i f f e r e n t i a l growth responses are expected only when the terminal g i b b e r e l l i n i n the main pathway i s bioactive per se. Other members ( g i b b e r e l l i n s and t h e i r precursors) are bioactive by t h e i r metabolism to the terminal b i o a c t i v e g i b b e r e l l i n . Thus a l l members of the pathway subsequent to a genetic block would be b i o a c t i v e , and those before the block i n a c t i v e . I t i s the pattern of response f o r each mutant that suggests the p o s i t i o n i n the pathway blocked by each mutant. I n i t i a l l y , growth response data were used to suggest the s p e c i f i c p o s i t i o n blocked by each of the f i v e dwarf mutants. Subsequently, radiolabeled feeds, using e i t h e r [ ^C]- or [ C , H ] precursors, have provided d e f i n i t i v e evidence f o r the positions GA

G A

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Figure 6. The early-13-hydroxylation pathway f o r maize (Zea mays). The dwarf-1 block has been defined chemically. The dwarf-2 and dwarf-3 blocks are based on bioassay data only. The i n i t i a l steps subsequent to GA^2-aldehyde Y known, hence the alternate routes v i a GA^ or GA53-aldehyde. G i b b e r e l l i n A2g-catabolite has been detected as a metabolite from feeds of [ 17-^ C,%2]GA2o to normal and dwarf-1 seedlings ( 7 ) . a r e

n o t

e t

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Figure 7. The f i v e mature dwarf mutants and normal maize (Zea mays). The height of the mutants varies from 1/4 to 1/5 that of the normals.


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ECOLOGY

A N D METABOLISM

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LIPIDS

Figure 8. The d i f f e r e n t i a l growth response of the dwarf-5 and dwarf-1 mutants to G A ^ Q . The dwarf-5 mutant ( l e f t set: dwarf-5; dwarf-5, - K ^ Q J Normal, - K ^ Q ; Normal) responds by normal growth to treatment with G A , the dwarf-1 mutant (right set: dwarf-1; dwarf-1, +GA2Q; Normal, + G A ; Normal) does not respond when treated with the same l e v e l of G A ^ .


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blocked by the dwarf-5 and dwarf-1 mutants. This report w i l l analyze the dwarf-5 and dwarf-1 systems only. The dwarf-5 mutant. The dwarf-5 mutant controls the c y c l i z a t i o n step, CPP to ent-kaurene ( 2 2 ) . This conclusion i s drawn from three l i n e s of evidence: F i r s t , seedlings respond by normal growth to members of the main pathway, including ent-kaurene ( 5 , 4 6 ) . These data suggest that the mutant blocks an early step i n the pathway, before ent-kaurene. Second, c e l l - f r e e systems from normal seedlings synthesize ent-kaurene from radiolabeled GGPP, CPP and MVA, whereas c e l l - f r e e systems from dwarf-5 seedlings synthesize ent-kaurene from these precursors at l e v e l s o n e - f i f t h that of the normal ( 2 2 ) . I t i s i n t e r e s t i n g that the mutant system accumulates the isomer, ent-isokaurene, an isomer present i n only trace amounts i n the normal system; (ent-isokaurene i s b i o l o g i c a l l y i n a c t i v e [ 4 7 ] ) . Presumably, the formation of the b i o l o g i c a l l y i n a c t i v e ent-isokaurene i s favored i n the mutant as a consequence of the g e n e t i c a l l y a l t e r e d ent-kaurene synthetase B (Figure 9 ) ( 2 2 ) . The t h i r d l i n e of evidence comes from the analysis of the endogenous GAs present i n normal and dwarf-5 seedlings ( 4 3 ) . G i b b e r e l l i n Aj_, G A Q , GA^g, GAjj and GA44 have been i d e n t i f i e d from normal seedlings, whereas dwarf-5 seedlings give no evidence f o r the presence of four of these GAs (trace amounts of G A Q were found i n the dwarf-5 e x t r a c t ) . These r e s u l t s would be expected i f the dwarf-5 mutant controls the c y c l i z a t i o n of CPP to ent-kaurene. The dwarf-5 mutant has been used extensively to define the s i n g l e steps, CPP to ent-kaurene ( 2 2 ) ; ent-kaurenol t o ent-kaurenal, ent-kaurenal t o ent-kaurenoic acid ( 4 8 ) ; GA53 to GA44 and G A Q to GA ( 1 7 ) ; G A to GA ( 7 ) ; and GA to GAg ( 4 3 , 4 9 , 5 0 ) . For each step a radiolabeled substrate was fed to the dwarf-5 mutant and the l a b e l recovered i n the immediate product. 2

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The dwarf-1 mutant. The dwarf-1 gene controls the 3(3-hydroxylation of G A Q to GA^. The o r i g i n a l evidence comes from growth response data; i . e . GA^ i s a c t i v e , and G A O Q and i t s precursors i n a c t i v e ( i . e . less than 1% the a c t i v i t y of GA^). By contrast, these same GAs and t h e i r precursors are a l l a c t i v e i n stimulating elongation i n dwarf-5 seedlings (see Figure 8 ) . Conclusive evidence f o r the p o s i t i o n of the genetic block comes from feeds of double labeled G A Q to dwarf-1 seedlings. These seedlings do not metabolize G A Q to GA^, whereas dwarf-5 seedlings do metabolize G A Q to GA^ (and GA g) ( 7 ) . Analysis of the endogenous GAs i n dwarf-1 seedlings also supports the p o s i t i o n that the dwarf-1 mutant controls the 33-hydroxylation of G A Q to GA^. Extracts of dwarf-1 seedlings contain G A Q , GA^g, GA^y and GA44 - and no GAi, whereas a l l f i v e g i b b e r e l l i n s are present i n normal seedlings ( 4 3 ) . The absence of endogenous GA^ i n dwarf-1 plants would be expected i f the mutant blocks the conversion of G A Q to GA^. This mutant has been used to obtain data from radiolabeled feeds that document the steps G A Q to GA g ( 7 ) and GA^ to GAg ( 4 3 ) . 2

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ent-kaurene

£/7/-isokaurene

Figure 9. Proposed scheme f o r the conversion of CPP t o ent-kaurene and ent-isokaurene (22). In the dwarf-5 mutant the pathway to ent-isokaurene i s favored, ent-isokaurene i s b i o l o g i c a l l y inactive (47).




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General Comments Probably the most s i g n i f i c a n t conclusion that comes from our genetic and biochemical studies i s that GA^ must be the only g i b b e r e l l i n i n the early-13-hydroxylation pathway that i s active per se i n the c o n t r o l of shoot elongation i n maize. Other members of the pathway are active through t h e i r metabolism to GA^. This conclusion i s supported by data from three kinds of approaches:- b i o a c t i v i t y studies, radiolabeled feeds, and analysis of endogenous GAs i n mutant and normal seedlings. Unfortunately, the enzymological approach i s s t i l l complicated by low levels of a c t i v i t y and i n s t a b i l i t y of the enzymes (hydroxylases and oxidases) that catalyze the s p e c i f i c steps i n the pathway(s). This i s e s p e c i a l l y true f o r c e l l - f r e e systems o r i g i n a t i n g from young green shoots. Slow but steady progress i s now being made on p u r i f i c a t i o n steps by Graebe's group i n Gottingen (51), West s group at U.C.L.A. (52), Coolbaugh's group a t Iowa (53) and MacMillan s group at the University of B r i s t o l (54,55), which i s also approaching the problem through the use of monoclonal antibodies (56). Our group at U.C.L.A. i s using Robertson's mutator as a probe f o r cloning the dwarfing genes i n maize (6,8). f

1

Acknowledgment s Portions of t h i s work were supported by a grant to BOP from the National Science Foundation No. PCM 83-14338.

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