Planta (Berl.) 122, 245--254 (1975) 9 by Springer-Verlag 1975

The Metabolism of Indoleacetic Acid by Barley Grains* A n n Minchin ** a n d M a t t h e w A. H a r m e y Department of Botany, University College, Belfield, Stillorgan Road, Dublin 4, Eire Received 15 August, accepted 30 August, 1974 Summary. I t has been shown that indoleacetic acid (IAA) does not occur in developing grains of Hordeum vulgate L. (barley), but that an unidentified indolic compound does. This compound, designated 'A', was also found to be a product of the metabolism of exogenous IAA by barley. The expression of the gibberellic acid effect was delayed for at least 8 h if grains were imbibed in a solution of IAA, and during this time, the IAA was metabolised. The enzyme system involved could be peroxidase, which was active in the grains at all stages of their development and at maturity, and partially purified extracts of peroxidase were found to have considerable IAA oxidase activity.

Introduction I t is possible t h a t indolic c o m p o u n d s could m o d e r a t e t h e effects of gibberellins in t h e m a t u r e b a r l e y grain, i n h i b i t i n g g e r m i n a t i o n u n t i l t h e indoles h a d been d e g r a d e d or metabolised. I t is n o t k n o w n to w h a t e x t e n t I A A or its conjugates exist in b a r l e y grains b u t Tafuri (1966) r e p o r t e d a rise a n d fail in t h e c o n c e n t r a t i o n of I A A in maize e m b r y o s as t h e y d e v e l o p e d from anthesis to m a t u r i t y , a n d a similar m e c h a n i s m m i g h t exist in barley. The m e t a b o l i s m of exogenous I A A b y a wide range of higher p l a n t s has been described b y Good et al. (1956), A n d r e a e a n d V a n Ysselstein (1956), Mino (1970), a n d Zenk (196Q. The p r o d u c t s of this m e t a b o l i s m h a v e been r e p o r t e d as i n d o l a c e t a m i d e , indolea c e t y l a s p a r t a t e , catechol, indole glucose a n d 3 m e t h y l o x i n d o l e , which h a v e been identified m a i n l y on t h e basis of t h e i r Rfs, developed colour a n d U V spectra. The o b j e c t of t h e p r e s e n t e x p e r i m e n t s is to assess t h e occurrence of I A A a n d its m e t a b o l i t e s in developing b a r l e y grains, in order to see if t h e n a t u r a l d o r m a n c y of freshly h a r v e s t e d b a r l e y could be e x p l a i n e d b y t h e level of I A A in t h e grains. The m e t a b o l i s m of exogenous I A A b y e n d o s p e r m halves of b a r l e y is also inv e s t i g a t e d a n d t h e m e t a b o l i t e s described. The role of peroxidases in this m e t a b olism is e x a m i n e d in order to assess t h e p o s s i b i l i t y t h a t these enzymes r e g u l a t e t h e c o n c e n t r a t i o n of I A A in developing a n d g e r m i n a t i n g grains. ) I a t e r i a l s and Methods Extraction o] Plant Tissues. Developing barley grains were extracted at weekly intervals from anthesis to harvesting and the extracts analysed for the presence of IAA or its conjugates. Following the procedure proposed by Veen (1966) 100 g of grains were frozen in * Abbreviations: DMABA = dimethyl aminobenzaldehyde, dMACA = Dimethyl amino cinnamylaldehyde, GA = gibberellic acid, IAA = indole acetic acid. ** Present address: Department of Agriculture, University of Reading, Earley Gate, Reading RG6 2AT, U.K.

17 Plant~(Berl.),Vol. 122

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A. Minchin and M. A. ttarmey

liquid .Y, ground and refluxed in acetonitrile for 2 h. The solution was filtered, the volume evaporated to dryness under reduced pressure at 45 ~ and the residue taken up in 1 ml of acetonitrile for chromatography. This method of extraction was also used in the labelling experiments. When investigating the metabolism of IAA in mature grains the extraction procedure of Ueda and Bandurski (1969) was followed as up to 1 kg of grain was used. After treatment the grain was blotted dry, ground to a fine powder in a Sorvall macerator and the powder extracted by agitation for I h in 50% acetone (5 1 for 1 kg grain). The solution was filtered twice to remove plant material. 6N NaOH was added to make the filtrate IN with respect to NaOtI and the alkali hydrolysis terminated after 30 rain by the addition, over ice, of 50% H2SO 4 to bring the solution to p H 2. The solution was then extracted three times with peroxide free ether and the combined ether solutions evaporated to dryness. The residue was taken up in ethanol or methanol prior to chromatography and scanning UV spectra. Application o/ Labelled IAA. [1-i4C] IAA and [2-14C] IAA were obtained in a benzene solution from the Radiochemical Centre, Amersham (England) with a specific activity of 190 [xCi mg -i. The benzene was evaporated under a stream of nitrogen and the residue taken up in phosphate buffer (0.05 M pH 7.2) and added to cold IAA to give a final solution of known molarity. 250 grains were incubated in 10 ml of this solution; a) immediately after dehusking, or b) after imbibition, for various periods of time at 30 ~ Chromatography. Dried extracts from treated grains were taken up in methyl cyanide, ethanol or methanol and chromatographed on silica gel coated glass plates (Merck). Chromatograms were developed in four solvent systems: 1. Isopropanoh ammonia: t t 2 0 - - 8 : 1 : 1 ; 2. Chloroform: methanol: glacial acetic acid--95:20:5; 3. Chloroform: glacial acetic acid-95:5; 4. Ethyl acetate: methylethylketone: formic acid: H 2 0 - - 5 : 3 : 3 : 1 . Various standards were included to assist identification and the plates were developed in saturated tanks at room temperature. After separation the position of the spots and standards was determined by spraying with Van Urk's reagent (1 g 5-DMABA in 50 ml concentrated HC1 then added to 50 ml ethanol---Stahl, 1965) or with the DMACA reagent (1 g DMACA in 100 ml concentrated HC1 Meudt and Powell-Gaines, 1967), both of which were made up freshly before use. The Van Urk solution reacts with IAA and indole compounds to produce blue, pink or purple spots, but the DMACA solution reacts only with IAA oxidation products. After chromatography of labelled extracts on silica gel, the plates were dried to remove all traces of ammonia and autoradiograms made to locate the labelled spots. Agfapan 1000 professional film strips (Agfa-Gevaert) were stuck onto glass sheets of the same size as the chromatogram and the film placed in contact with the chromatogram, wrapped in black polythene and exposed for 2 or 3 days. The above procedure was carried out in total darkness as the film is sensitive even to a green or yellow safety light. The film was developed in Agfa Rodinol developer at 18 ~ for 14 min. The exposed areas of the film were then compared with the chromatogram and the spots eluted from the silica gel for further analysis.

Results I n n o n e of t h e e x t r a c t s f r o m d e v e l o p i n g b a r l e y g r a i n s was I A A d e t e c t e d a f t e r c h r o m a t o g r a p h y . H o w e v e r a n i n d o l i c c o m p o u n d , S p o t A, w h i c h r a n a h e a d of t h e I A A s t a n d a r d i n all s o l v e n t s a n d h a d a n RIA A of 1.8 was f o u n d in i m m a t u r e grains, a n d p e r s i s t e d as t h e g r a i n s m a t u r e d . I t a p p e a r e d o n l y in s m a l l q u a n t i t i e s , h o w e v e r , a n d d i d n o t a t a n y s t a g e y i e l d e n o u g h for c h e m i c a l c h a r a c t e r i s a t i o n . T h e m e t a b o l i s m of e x o g e n o u s I A A b y e n d o s p e r m h a l v e s of b a r l e y was inv e s t i g a t e d u s i n g l a b e l l e d I A A . T h i s w a s c o n s i d e r e d n e c e s s a r y b e c a u s e t h e endosperm halves and grains secreted a substance which interfered with the tradit i o n a l c o l o u r t e s t s for I A A d e t e c t i o n , t h e r e f o r e a false p i c t u r e w o u l d e m e r g e if t h e s e w e r e solely r e l i e d on. T h e s e c r e t e d s u b s t a n c e w a s c o n c e n t r a t e d b y freezed r y i n g t h e s o l u t i o n in w h i c h g r a i n s h a d b e e n b a t h e d a n d a l t h o u g h it d i d n o t m o v e f r o m t h e s t a r t o n a t h i n - l a y e r c h r o m a t o g r a p h i c p l a t e in 3 d i f f e r e n t s o l v e n t s ,

Metabolism of Indoleaeetic Acid by Barley Grains

247

Table 1. Radioactivity in the medium containing labelled IAA in which endosperm halves had been incubated for different periods of time. 20 ~1 samples of the medium were counted and 10 lzl of the grain extract. (0.68 tzCi was recovered from grains fed 1.0 fzCi) Time

CPM medium

0 hours 4 hours 18 hours 48 hours

18780 T zl@: 14760T- 106 9 610 T- 240 2310~- 30

CPM grain extract

105170 ~ 280

and had therefore a different Rf value to any published for indole compounds, t gave a positive colour reaction to the test for oxidation products of IAA. A UV spectrum of the bathing solution of grains showed a peak at 277 rim. The secretion of this compound, which remains unidentified, varied with different temperatures of incubation with a Q10 of 1.3 and did not take place at all from grains which had been killed by heat treatment. When IAA labelled at the 1-C or 2-C positions was fed to endosperm halves or whole grains, it was taken up rapidly and subsequently metabolised. Some decarboxylation took place in the medium, showing that the enzymes necessary for this must be readily activated and near the surface of the grain, but the greater part of the applied IAA went into the grain (Table 1). 4, 18 and 48 h after incubation the grains were extracted in acetonitrile to determine the fate of the IAA. An autoradiogram of the chromatogram after separation of the extract shows two principal products: Spot 'A' (RIAA 1.8) and Spot 'B' (RIAA 0.18 in isopropanol ammonia I-I~O) (Fig. 1). The figure shows that product A is the first to be formed from the applied IAA and that as time goes on the product A diminishes and product B increases in concentration. The same products were formed whether IAA labelled at the 1-C or 2-C positions was used, but there were no products formed when labelled IAA was supplied to grains that had been killed by heat treatment, showing that it was an active metabolism of IAA that was taking place in the live grains and not a mere chemical decomposition. Charaeterisation of the products A and B showed after purification and spectroscopic analysis that ' B ' was indole acetylaspartate (Mollan et al., 1972). Product 'A' has not been identified, as sufficient quantities could not be obtained for chemical characterisation and its Rf was not similar to any of the indolic standards available. I t was labelled when either [1-1~C]IAA or [2A4C] IAA was applied and would therefore appear to be an addition product of IAA. I t was indolic in character and corresponded in Rf and colour development to product 'A' isolated from developing seeds. I t was formed during the first hours of incubation following application of exogenous IAA and later appeared to be converted to product B. A UV spectrum of 'A' eluted in methanol from chromatographic paper shows it has a peak at 290 nm and a broad peak between 278 and 283 nm. This was not similar to the spectra of any standard available to us although indole had certain similarities. 17"

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A. Minehin and M. A. Harmey

Fig. 1. Conversion of [1-1~C]IAA by endosperm halves to the products A and indole acetylaspartate. Samples of 30 halves were incubated aerobically at 30~ C, extracted at intervals and the extracts separated by thin layer chromatography. The autoradiogram shows (from the left) IAA and its products A and IAA-asp. from extracts after 4, 18 and 48 h incubation

I n view of the fact t h a t no I A A was detectable i n the grain at a n y stage of d e v e l o p m e n t , it can h a r d l y be considered to have a role in the n a t u r a l regul a t i o n of the GA response or to be one of the ' i n h i b i t o r s ' which give rise to dorm a n c y . However the onset of t h e GA response of endosperm halves, as m e a s u r e d b y peroxidase secretion ( H a r m e y a n d Murray, 1968; M u r r a y a n d H a r m e y , 1970), which n o r m a l l y is detectable after 8 h, was delayed to 16 h w h e n the endosperm halves h a d been i m b i b e d in 100 [xg ml -~ I A A (Fig. 2). This lag period was most likely used to metabolise the I A A to a less toxic form. E x p e r i m e n t s

Metabolism of Indoleacetic Acid by Barley Grains

249

Table 2. Inhibition of GA response lcy IAA. Endosperm halves were imbibed in sterile solutions of IAA, at different concentratiors, for 20 hours at 5 ~ C. 20 halves were then incubated, after rinsing, in 5 ml of 1 ~g/ml -i GA and peroxidase secreted into the medium measured after 24 hours. The experiment was carried out in triplicate IAA concentration (imbibing soln.) Peroxidase AA~60 rain -1 0.1 m1-1 IAA concentration (imbibing soln.)

lmgm1-1

0 . 5 m g m l -i

1 0 0 ~ g m l -i

5 0 ~ g m l 1 10~zgml 1 5~zg m1-1

0

0

0.33

0.45

0.88

0.87

1 v-g ml 1

0.5 ~g ml -i

0.1 gg m1-1

Imb. H20 Inc. H20

Imb. H20 Inc. GA

0.84

0.67

0.78

0.04

0.77

Peroxidase AA46 o min -i

0.1 ml -i

Table 3. Physiological activity of products A and B The products of IAA metabolism, A and B, were eluted from thin layer plates, evaporated to dryness and the residue redissolved in water. Endosperm halves were imbibed in these solutions (three replicates of each) and then incubated in 0.01 y.g m1-1 GA. The peroxidase secreted by 20 halves was measured after 24 hours at 30 ~ C. Imbibing solution

H20

Spot A

Spot B

Blank silica gel

Imbibed and incubated It20

Peroxidase AA~6o m i ~ 1 0.1 ml 1

0.145 0.145 0.147

0.135 0.141 0.137

0.0 ~, 0.01 0.03

0.11 0.15 0.14

0.01 0.005 0.01

u s i n g a r a n g e of I A A c o n c e n t r a t i o n s (Table 2) s h o w e d t h a t c o n c e n t r a t i o n s b e l o w 10 [zg m l 1 h a d no i n h i b i t o r y effect. T h e effects of p r o d u c t s ' A ' a n d ' B ' on t h e G A r e s p o n s e w e r e m e a s u r e d w h e n e n d o s p e r m h a l v e s h a d b e e n i m b i b e d in a q u e o u s s o l u t i o n s of ' A ' a n d ' B ' a f t e r e l u t i o n f r o m t h i n l a y e r plates. T h e r e s u l t s ( T a b l e 3) s h o w t h a t t h e i n d o l e a c e t y l a s p a r t a t e is a n e f f e c t i v e i n h i b i t o r of t h e r e s p o n s e b u t t h a t p r o d u c t A, w h i c h was a t a m u c h l o w e r c o n c e n t r a t i o n , h a d no effect. A t t e m p t s to f i n d a n e n z y m e s y s t e m , a c t i v e in t h e g r a i n s d u r i n g t h e e a r l y s t a g e s of i m b i b i t i o n w h i c h t o o k p a r t in t h e m e t a b o l i s m of t h e e x o g e n o u s I A A

and the formation of its conjugates, led to the isolation of peroxidase. This enzyme was extracted from grains at all stages of imbibition and after ammonium sulphate precipitation the extracts were separated by disc eleetrophoresis on polyacrylamide gels (Davis, 1964). Dry grains were found to have one isoenzyme and during imbibition this increased to 9 bands all detected by the action of the isoenzymes in reducing guaiacol in the presence of 1-1202. These extracts were also tested for IAA oxidase activity using the method of Krupsagar

250

A. Minehin and M. A. Harmey 1.5

T~

o

0~

.S

/

i

~,,

/

I ,.-4"

I 20

10 hrs

of

I 30 in GA

inc

I 40

Fig. 2. Secretion of peroxidase by endosperm halves. The endosperm halves were imbibed in H~O (...), or 100 ~gm1-1 IAA (,~,), incubated in 10-eg m1-1 GA and the peroxidase secreted was measured at intervals. The open symbols represent peroxidase secreted by endosperm halves imbibed as above then incubated in H20. The points are the means of six samples. Vertical bars ~: S.D. OD = A

C

A

D Ft.

Fr.

9

Front

Fr

m

. $ spacer gel

Fig. 3. Drawings of gels showing the peroxidase isoenzymes separated when samples from the peaks A, B, C and D eluted from a C ~ cellulose column were applied. The intensities of the bands are represented as follows: black bands, heavy staining; striped bands, medium staining; stippled bands, light staining

a n d Sequiera (1969), b a s e d on t h e changing s p e c t r u m of t h e a s s a y m e d i u m on a d d i t i o n of enzyme. Some o x i d a t i v e a c t i v i t y was f o u n d in all t h e b a r l e y extracts. I n o r d e r to d e t e r m i n e if t h i s a c t i v i t y was due to a n y p a r t i c u l a r isoenzyme or group of isoenzymes, a n d to e l i m i n a t e interference f r o m oxidase i n h i b i t o r s in t h e crude e x t r a c t , it was s e p a r a t e d on c a r b o x y m e t h y l cellulose (CIV[) columns ( S h a n n o n et al., 1966). F o u r p e r o x i d a s e p e a k s were e l u t e d f r o m t h e column a n d w h e n t h e e n z y m e in t h e s e was c o n c e n t r a t e d a n d s e p a r a t e d on a c r y l a m i d e gels each one was f o u n d t o be c o m p o s e d of m o r e t h a n one i s o e n z y m e - - F i g . 3. The e n z y m e f r o m these p e a k s was also t e s t e d for I A A oxidase a c t i v i t y a n d i t was

Metabolism of Indoleacetic Acid by Barley Grains

251

Fig. 4. Autoradiograms of ~ thin-layer plate showing the reaction products of IAA produced in vitro. Enzyme extracted from barley was incubated with labelled IAA plus co-factors and after 6 h samples were chromatographed using solvent system 1. The autoradiograms show (1) [I-14C]IAA; (2) [1-1aC]IAA plus enzyme, and (3) [1-14C]IAA plus boiled enzyme. For comparison (4) a sample of extract of grains to which [1-14C]IAA had been supplied before incubation, is included

found that those from peak C contained the highest ratio of IAA oxidase activity (Table 4) to peroxidase activity. A number of papers, (Endo, 1968; 1V[ino, 1970, and Meudt and Gaines, 1967) show that peroxidase might act as an IAA oxidase. In the present experiments the maximum IAA oxidase activity was only obtained after the addition of H20 ~ to the assay solution. I t is possible that there is competition between the peroxidase and IAA oxidase aspects of the enzyme for available H~Ot At low concentrations this is used preferentially by the peroxidase and the IAA oxidase activity is only detected when more is added. The results obtained from tests on the enzyme eluted from the OM column strongly suggest that some peroxidase isoenzymes can act as IAA oxidases, whereas others cannot. In vitro metabolism of labelled IAA was carried out using enzyme concentrated from a crude extract of barley grains and both 1-1aC and 2-14C labelled IAA. The enzyme, labelled IAA, co-factors and tt~O 2 were incubated for 3 h, then an aliquot of the reaction mixture was chromatographed on thin layer plates. On development one plate was exposed to photographic film and the other

252

A. Minchin and M. A. Harmey

Table 4. Peroxidase and IAA oxidase activity of a partially purified extract of barley grains and of peaks of activity eluted from a CM cellulose column to which the extract had been applied

Extract B C D

Perox mg protein -1 (zlA46o mi~ 1)

IAA ox mg protein -1 (zJA24~ rain-1 • 102)

Ratio IAA: perox • 102

16.85 110.5 26.5 36.7

87.3 711.0 291.0 57.4

5.18 6.44 11.0 1.56

one sprayed with Van Urk's reagent. The products were not sufficiently concentrated to develop colour with this reagent, but the film, on development, showed that I A A had been metabolised to a certain extent, product ' A ' having been formed when either [1-14C]IAA or [2-14C] IAA was used. In neither ease did product B appear (Fig. 4). Discussion

None of the methods used in attempts to extract IAA from mature and immature barley grains was successful. As other workers (Srivastava, 1963; Tafuri, 1966) have extracted IAA from as little as 10 g of maize, it is reasonable to suggest that IAA does not exist in the free state in barley grains. Thus, the hypothesis that immature grains do not respond to GA because of the inhibitory action of endogenous IAA, cannot be upheld. However, the absence of IAA in the grains could perhaps be explained by their ability to metabolize it at a very early stage. This ability was found in grains when they were first tested two weeks after anthesis. I t was also found that during maturation the grains were capable of a greater rate of IAA metabolism per unit protein than when they were fully mature. I t is therefore possible that in barley endogenous IAA is immediately converted to product A which, in fact, was found in grains to which no exogenous IAA had been added. Mature grains were also capable of metabolizing added IAA within two hours of incubation. The enzymes necessary for this must already be present and only require the addition of substrate and water for activation. I t is generally reported (MacLeod et al., 1964; Briggs, 1968; Varner et al., 1964; Pollard, 1969) that the development of enzyme activity and the secretion of metabolites from barley grains are dependent on GA, and that there is a lag phase of 8-10 h before the secretion takes place. However, peroxidase which is reported to be involved in IAA metabolism (Endo, 1968; Mino, 1970; Meudt and Gaines, 1967) is present in active form in dry grains and endosperm halves. The endosperm halves were found to be able to metabolize IAA whether GA had been added or not and the conversion of IAA to other forms took place during the lag period, showing that the system involved in IAA metabolism was independent of GA action. The significance of the products of this metabolism, ' A ' and indoleacetylaspartate, is difficult to define. Indoleacetylaspartate was found to inhibit the GA response in endosperm halves that had been imbibed in it. I t can therefore hardly be classed as a detoxification product. Product A may have greater sig-

Metabolism o5 Indoleae~tic Acid by Barley Grains

253

nificance as it was f o u n d to occur n a t u r a l l y in t h e grains, a n d f u r t h e r work w o u l d be f a c i l i t a t e d if it was identified. Other p r o d u c t s of I A A m e t a b o l i s m - indoleaeetomide, i n d o l e a e e t y l a s p a r t a t e , eatechol, pyrogallol, a n d 3 m e t h y l oxindole (Good et al., 1956; A n d r e a e a n d V a n Ysselstein, 1956; Mino, 1970) are n o t similar to p r o d u c t 'A' on t h e basis of t~fs, d e v e l o p e d colours a n d spectra. I t retains 1-14C a n d it therefore is more likely to be an a d d i t i o n p r o d u c t . The c o m b i n a t i o n of I A A w i t h myoinositol for storage in maize was r e p o r t e d b y B a n d u r s k i et al. (1969) b u t again t h e p a t h w a y in b a r l e y a p p e a r s to be different as p r o d u c t 'A' is n o t similar to t h e inositols of I A A . T h e role of p e r o x i d a s e in t h e m e t a b o l i s m of I A A in t h e grains could be inferred from t h e results obtained. I A A oxidase does n o t a p p e a r to be a stable e n z y m e a n d t h e m e a s u r e m e n t of its a c t i v i t y in v i t r o d e p e n d e d on t h e r e m o v a l of i n h i b i t o r s (Parish, 1968) a n d t h e a d d i t i o n of co-factors to f a v o u r its o x i d a t i v e r a t h e r t h a n p e r o x i d a t i v e a c t i v i t y . H o w e v e r it was shown t h a t some groups of p e r o x i d a s e isoenzymes h a d g r e a t e r I A A oxidase a c t i v i t y t h a n others, after t h e y h a d been s e p a r a t e d a n d purified on CM cellulose columns. E n d o (1968) r e p o r t e d similar findings on p e r o x i d a s e isolated from horse-radish. I n vitro e x p e r i m e n t s w i t h these enzymes showed t h a t t h e y could c o n v e r t labelled I A A to p r o d u c t A, which is also f o u n d to occur n a t u r a l l y in grains. As p e r o x i d a s e is p r e s e n t in developing a n d m a t u r e grains t h e r e is a strong p o s s i b i l i t y t h a t t h e y are indeed i n v o l v e d in r e g u l a t i n g t h e c o n c e n t r a t i o n of I A A or indolie c o m p o u n d s in developing a n d g e r m i n a t i n g grains.

References Andreae, W. A., Van Ysselstein, M. W. H. : Studies on indole acetic acid metabolism. III. The uptake of IAA by pea epicotyls and its conversion to indoleacetylaspartate. Plant Physiol. 31, 235-249 (1956) Bandurski, R. S., Ueda, M., Nicholls, P. B. : Esters of indole acetic acid and myo-inositol. Ann. N. Y. Acad. Sci. 165, 655-667 (1969) Briggs, D. E. : e-amylase in germinating deeorticated barley. Phytochem. 7, 513-529 (1968) Davis, B. J. : Disc Electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404427 (1964) Endo, T. : Indole acetic acid oxidase activity in horseradish and other plant peroxidase isoenzymes. P1. and Cell Physiol. 9, 333-341 (1968) Good, N. E., Andreae, W. A., Van Ysselstein, M. W. H. : Studies on indole acetic acid metabolism. II. Some products of the metabolism of exogenous indole acetic acid in plant tissues. Plant Physiol. 31, 231-235 (1956) Harmey, M.A., Murray, A. : The effect of gibberellic acid on peroxidase levels in barley. Planta (Berl.) 83, 387-389 (1968) Krupasager, V., Sequeira, L.: Auxin destruction by Alarasmius perniciosus. Amer. J. Bot. 56, 390-397 (1969) MacLeod, A.M., Duffus, J . H . , Johnston, C.S.: Development of hydrolytic enzymes in germinating grain. J. Inst. Brew. 70, 521-528 (1964) Meudt, W. J., Powell-Gaines, T. : Studies on the oxidation of indole acetic acid by peroxidase enzymes. P1. Physiol. 42, 1395-1399 (1967) Mino, Y. : Studies on the destruction of indole acetic acid by a species of Arthrobactor. IV. Decomposition products. Plant and Cell Physiol. l l , 128-138 (1970) Mollan, R.C., Donnelly, D. M. X., Harmey, M.A.: Synthesis of indole-3-acetylaspartic acid. Phytochem. 11, 1485-1488 (1972) Murray, A., Harmey, M. A. : Peroxidase in barley endosperm as a bioassay for gibberellins. Sci. Proe. roy. Dublin Soc. 2, 275-281 (1970)

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Parish, H. W. : Studies on senescing tobacco leaf discs with special reference to peroxidase. Planta (Berl.) 82, 1-21 (1968) Pollard, C. J. : A survey of the sequence of some effects of gibberellic acid in the metabolism of cereal grains. Plant Physiol. 44, 1227-1232 (1969) Shannon, L.M., Kay, S., Lew, J . Y . : Peroxidase isoenzymes from horseradish roots. J. biol. Chem. 241, 2166-2172 (1966) Shrivastava, B. I. S. : Ether soluble and ether insoluble auxins from immature corn kernals. Plant Physiol. 88, 473478 (1963) Stahl, E. : Thin-layer chromatography, a laboratory handbook. Berlin-Heidelberg-New York: Springer 1965 Tafuri, F. : IAA determination in the kernals of four lines of corn and of their hybrids. Phytochem. 5, 999-1013 (1966) Ueda, M., Bandurski, R. S.: A quantitative estimation of alkali-labile IAA compounds in dormant and germinating maize kernals. Plant Physiol. 44, 1175-1181 (1969) Varner, J. E., Ram Chandra, G. : Hormonal control of enzyme synthesis in barley endosperm. Proc. nat. Aead. Sci. (Wash.) 52, 100-106 (1964) Veen, H. : Naphthylaeetie acid-l-14C in Coleus extracts. Aeta bot. neerl. 15, 419433 (1966) Zenk, )/[. H. : 1-(Indole-3-acetyl)-D-glucose, a new compound in the metabolism of indole acetic acid in plants. Nature (Lond.) 191, 165-177 (1961)

The metabolism of indoleacetic acid by barley grains.

It has been shown that indoleacetic acid (IAA) does not occur in developing grains of Hordeum vulgare L. (barley), but that an unidentified indolic co...
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