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Plant Molecular 26: 1459-1481, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 1459

Plant conjugation

Gtlnther Sembdner*, Rainer Atzorn and Gernot Schneider Institut far Pflanzenbiochemie, Weinberg 3, D-06018 Halle, Germany (* author for correspondence)

Received and accepted 11 October 1994

Key words: , conjugation, , , , , , brassinolide

Introduction (including structural elucidation, synthesis etc.) and, more recently, their (including Plant are an unusual group of second- for conjugate formation or hydrolysis), ary plant constituents playing a regulatory role in and their genetical background. However, the plant growth and development. The regulating most important biological question concerning the properties appear in course of the biosynthetic physiological relevance of plant hormone conju- pathways and are followed by deactivation via gation can so far be answered in only a few cases catabolic processes. All these metabolic steps are (see Conjugation of ). There is evidence in principle irreversible, except for some processes that conjugates might act as reversible deactivated such as the formation of ester, glucoside and storage forms, important in hormone 'homeosta- amide conjugates, where the free parent com- sis' (i.e. regulation of physiologically active hor- pound can be liberated by enzymatic hydrolysis. mone levels). In other cases, conjugation might For each class of the plant hormones so-called accompany or introduce irreversible deactivation. 'bound' hormones have been found. In the early The difficulty in investigating these topics is, in literature this term was applied to hormones part, a consequence of inadequate analytical bound to other low-molecular-weight substances methodology. However, the advent of analytical or associated with macromolecules or struc- techniques such as HPLC-MS or capillary tures irrespective of whether structural elucida- electrophoresis-MS may help to resolve matters. tion had been achieved. After the characteriza- tion of the first gibberellin (GA) glucoside - GA8- 2-O-fl-D-glucoside from maturing of Conjugation of auxins Phaseolus coccineus [175, 176] - the term GA conjugate was used for a GA covalently bound to It is the main intention in this section to review another low-molecular-weight compound [184]. the conjugation of naturally occurring auxins; the Subsequently, the term was extended to all other numerous data on conjugates of synthetic auxins groups of plant hormones [ 178], including their will not be discussed. In addition to , precursors and metabolites as well as to second- catabolism is another way to control the levels of ary plant constituents in general. free -3-acetic acid (IAA), and conjugation Plant hormone conjugates have been studied represents one important aspect of IAA catabo- intensively during the past decades and good lism. However, at least some IAA conjugates are progress was made concerning their not merely irreversibly deactivated end products

[223] 1460 of but instead act as temporary stor- lic acid [3, 151]. It has been proposed that a spe- age forms, from which IAA can be released via cial 'IAA-oxidase' is responsible for these hydrolysis. Convincing data about IAA metabo- conversions [9, 50, 70]. However, there is a dis- lism in Zea mays suggest that in seedlings conju- crepancy between results of in vitro oxidation gate hydrolysis in the represents the under different conditions and the relatively low dominating source of free IAA in the occurrence of these catabolites in plant tissues [5, 6, 7]. It is not known whether this mechanism [1431. is valid for higher in general. The main products of the non-decarboxylation After the first comprehensive review about pathway, which appears to operate in many plant 'bound auxins' in 1982 [27], the number of iden- species, are oxindole-3-acetic acid and dioxin- tified IAA catabolites increased, as documented dole-3-acetic acid [66, 141, 142]. In Zea mays, by several subsequent reviews [4, 8, 88, 143, 152]. 7-hydroxylation and subsequent glucosylation of Major catabolic routes (Fig. 1) are (1)oxidative oxindole-3-acetic acid have also been observed decarboxylation of IAA, (2) non-decarboxylative [126]. The concentrations of both substances ex- oxidative catabolism and (3) ester and ceeded the levels of free IAA about ten-fold, im- conjugation. The latter can be divided into for- plying that this is a major route for the inactiva- mation of conjugates from which IAA hydrolysis tion of IAA. In contrast, there are many data is still possible and into compounds where IAA indicating that the formation of both ester and was inactivated via oxidation after conjugate for- amino acid IAA conjugates is associated with a mation. transport function rather than modes of auxin The formation and physiological significance of inactivation [5, 12, 90]. IAA conjugation is of primary importance in this article, but from a regulatory point of view it is necessary to discuss briefly alternative routes of Ester conjugates IAA degradation. The oxidative decarboxylation pathway is catalysed by peroxidases, leading in Most of the available information on synthesis several plant species to products such as indole- and hydrolysis of IAA esters (see Fig. 2) comes 3-methanol [16, 149, 194] and indole-3-carboxy- from experiments with Zea mays [4, 5, 7, 8, 27],

Oxidative [ decarboxylation

14 /

I Am~oadd conjugafiom I Oy~n:

/ ~ Dioxindoles

Fig. 1. Main routes of IAA metabolism in higher plants.

[2241 1461

indole-3-acetyl-myo-inositol-galactoside myo-inositol was converted, which contrasts with the first glucosylation steps where there was al- (-arabinoside) most always complete conversion of the substrate [4, 29, 30]. The conjugating enzymes are soluble and can be separated from each other by Sepha- dex G 150 [4]. However, further characterization of IAA ester-forming enzymes is still lacking. Interesting information on IAA metabolism as indole-3-acetyl-myo-inositol ox an important regulative element has come from experiments with genetically manipulated plants I where the auxin biosynthesis genes from the of were expressed to ob- tain auxin overproducing plants [e.g. 68, 186, 187, x 189]. Quantitative determinations of bound and mdole-3-acetylglue~e free IAA showed an increase of both forms, but often conjugates accumulated to a higher extent. i In most cases the identity of the IAA conjugates was not determined, but there is some evidence [188] that they consist at least partly of ester H compounds, although IAA amino acid conjugates were the main products. Experiments of this kind indole-3-acetic acid are a powerful way to show how plant cells can Fig. 2. Formation of IAA ester conjugates. regulate the levels of active auxins and how they deal with excess production of IAA. It also opens possibilities for a better access to the metabo- although IAA esters have been found in many lizing enzymes. other plant species [8, 21,143]. The first evidence The release of IAA from ester conjugates has for an IAA-glucoside in plants was presented by been studied extensively in the maize coleoptile Zenk in 1961 [233]. In Zea mays kernels, 1-O- [7, 8]. A combination of quantification and turn- (indole-3-acetyl)-/3-D-glucose [43 ] and 2-O-(in- over studies revealed that most of the free IAA in dole-3-acetyl)-myo-inositol have been detected as the copeoptile tips of growing did not well as 5-0-/3-1-arabinopyranosyl-2-O-(indole-3- originate from de novo synthesis but from ester acetyl)-myo-inositol and 5-galactopyranosyl-2-O- hydrolysis in the endosperm. Similar studies with (indole-3-acetyl)-myo-inositol [25, 203, 204]. In other species are not known, so whether this addition, a high-molecular-weight IAA ester of a source of IAA is widespread in young seedlings cellulosic glucan has been detected in extracts remains to be determined. from maize [ 139]. Numerous feeding experiments in combination The enzymology of IAA ester formation was with biological activity determinations and sub- studied by Michalczuk and Bandurski [ 104, 105], sequent analysis of metabolites [8, 28, 46, 125] using crude cell-free preparations from immature indicate that the high physiological activity of IAA kernels of sweet maize which converted (1)2- esters is indirect, resulting from release of free 14C-IAA and UDPG to IAA-/3-D-glucopyrano- IAA. The state of knowledge about enzymes side and IAA-myo-inositol, and (2)UDP-galac- which can hydrolyse IAA from esters is once tose and IAA-myo-inositol to IAA-myo-inositol- again confined to maize, and apart from studies galactose and IAA-myo-inositol-arabinose [4, 6, using more or less crude preparations, 30]. Typically not more than 20~/o of the IAA- not much information is available.

[2251 1462

Amide conjugates Cohen [ 11 ] detected an IAA with a mo- lecular weight of about 5 kDa in Phaseolus, and There are two types of amide conjugates formed the presence of an IAA glycoprotein conjugate with IAA in which either the indole ring of the has also been reported [137]. IAA remains unchanged or oxindole or dioxin- The function of amide conjugates is not fully dole derivatives are synthesized after formation understood. The peptide bond-forming enzymes of the peptide bond (Fig. 3). IAA-aspartate from are not, as yet, well-characterized, and it has not of soybean was the first amino acid conju- been possible to produce these conjugates in vitro. gate to be identified conclusively [26]. This form There is evidence from many experiments mostly of IAA conjugation occurs in legume seeds [4], confined to plant cultures [e.g. 90] or seeds but has been observed in other species too, for [e.g. 12] that hydrolysis of IAA-aspartate takes example in shoots ofPinus silvestris [ 1] and place to a high extent. This might explain its high of [21 ]. IAA-glutamate conjugates are less biological activity, which also applies to other common [45], and to date no other IAA amino IAA amino acid conjugates [89]. Not much is acid conjugates have been detected in higher known about the hydrolysing enzymes. There are plants. However, there are several reports about only few data about isolation and characteriza- larger amide conjugates. For instance, Bialek and tion of a crude extract from Phaseolus [ 10, 143].

~ COOt.I H IAA 1 --~CO--N.-~--c.,--coo.

H Indole-3-acetyl-asparticacid / OH COOH COOH ~ ~~o co--"- Ic"-c",--coo" H H ~dole-~-~l-L~l)~rUe acid Dio]dndole-3-acetyl-aspartic acid 1 ~OI~ COOI'I

H Fig. 3. Formation of IAA amino acids conjugates.

[226] 1463

Interestingly, no common peptidases or - to numerous plant tissues almost exceeds the ases are able to cleave the amide bond of such number of known endogenous cytokinin metabo- IAA conjugates, indicating that the enzyme in- lites. Therefore it is much more difficult to clas- volved is very specific. On the other hand, kinetic sify the many products of cytokinin metabolism. studies in soybean seedlings show that the IAA- The scheme illustrated in Fig. 4 is an extension to aspartate pool increases during proposals by Horgan [62]. [4, 12], and its level found in the shoots of 7-day One type of metabolism involves the cleavage old seedlings is about twice the level found in the of the N 6 side chain which results in a complete dry . The site of compartmentation of IAA loss of biological activity. The enzyme involved in amino acid conjugates is not known in general. this reaction is called 'cytokinin oxidase' and has In recent years evidence arose about subse- been characterized in various plant species [e.g. quent metabolization of IAA amino acid conju- 19, 20, 22, 119, 133]. Since this type of metabo- gates. Studies by Tsurumi and Wada [ 198, 199, lism is not a form of conjugation, it will not be 200, 201] have shown that oxidation of IAA- discussed further. This applies also to other kinds aspartate represents an important pathway of ir- of side-chain modification that do not involve reversible IAA inactivation in . The first step conjugation. is conjugation with aspartate, followed by oxida- The second type of metabolism comprises the tion of the indole ring at two positions (Fig. 3), interconversions of cytokinin bases, nucleosides and subsequent glucosylation, but this last step and nucleotides. The 9-ribosides and their 5'- does not seem to be obligatory. Metabolites of mono, di- and triphosphates are amongst the most similar structures have been found in Dalbergia [ abundant naturally occurring and 116, 128 ] and tomato [ 21 ]. After feeding tritium- metabolites, and they exist in the in labelled IAA and IAA-aspartate to protonemata apparent equilibrium [62, 98]. Obviously several of the moss Funaria hygrometrica more than 80~o enzymes involved in adenylate metabolism will of the radioactivity was found in compounds co- utilize the cytokinins as substrates and, so far, all chromatographing with dioxindole aspartate [ 15 ]. of the detected enzymes exhibit lower affinities for Apart from dioxindole derivatives, a similar ox- the cytokinins than for adenine or adenosine. For indole conjugate has been found in tomato [ 144]. instance, Chen and co-workers [24] in their stud- The steps of synthesis are similar, starting with ies on preparations from germ cells dis- the peptide bond formation. The exact structure covered an adenosine phosphorylase which of the final product is not clear yet, but it seems converts 2-isopentenyladenine (2iP) to 2-isopen- to be a small peptide of still unknown amino acid tenlyadenosine (2iPA), an adenosine kinase [23] sequence. In contrast to simpler amino acid con- which converts directly 2iPA to 2iPMP, and an jugates, it is possible to synthesize the compound adenosine phosphoribosyltransferase which con- in vitro with a crude enzyme extract. verts directly 2iP to 2iPMP. The significance of such conversions is not completely understood, but Laloue and Pehte [75] have shown that to- Conjugation of cytokinins bacco cells are impermeable to cytokinin nucle- otides but not to bases and ribosides. During the past decade, more progress was made Apart from the different types of cytokinin glu- in the field of cytokinin metabolism than in the cosylation, which are the most prominent cyto- field of cytokinin biosynthesis which is the subject kinin conjugates and will be discussed below in of controversal discussion, as reflected in several more detail, N-alanyl conjugation and O-acetyl- reviews [62, 81, 96, 98]. Unlike to the situation ation [98] are also reported (see Fig. 4). After for other plant hormone classes, the number of feeding of zeatin (Z) and BA, their alanyl conju- metabolites obtained after feeding of synthetic cy- gates have been found in Lupinus [133] and in tokinins such as benzyl adenine (BA) or immature apple seeds [44]. The alanyl conjugate

[227] 1464

C~OH HNSH:OH

9-Ala-Z ~c°°H ml,

o, CH:O~ OH HN._/~ ~°H .el~OH

"q.s? c,o, , ~.J>-.¢, 3 ~~-~OH Z-O-G Z OH OH

N-glueosides: Z-7-G Z-9-G

Fig. 4. Survey on conjugation of cytokinins, shown for zeatin.

of Z is also an endogenous compound in Lupinus Cytokinin glucosides [ 192]. An enzyme, fl-(6-allylaminopurine-9-yl)- adenine synthase, has been partly purified from Cytokinin glucosides are of widespread distribu- developing Lupinus seeds [44]. These conjugates tion in many plant species [98], and glucosylation are extremely stable [49, 130, 133], suggesting is possible at four positions (Fig. 4). Not all struc- that their production represents a form of irre- tures identified are reflected in this article so that versible conjugation. the below discussion represents only a small facet The cytokinin ribosides should not be regarded of all known substances. as real conjugates since it is still an open question N-glucosides are known in 3-, 7-, and 9-posi- whether they are active per se or via release of the tion of the purine ring. Z-7-G was the only de- free bases. A novel form of is Z-O- tectable cytokinin in radish seedlings [193], xyloside from Phaseolus [41]. The corresponding Z-9-G was the major compound in Vinca rosea enzyme, O-xylosyl-transferase, is well-character- crown gall tissue [ 177] and a minor compound in ized and has been purified to homogenity; also a maize kernels [ 193]. After feeding of iP and iPA monoclonal antibody was raised against the en- to cytokinin-dependent cells, iP-7-G was zyme [113, 114], and prospects of obtaining a the major metabolite [77], as was BA-7-G after cDNA clone would appear to be good. BA feedings [49, 76]. In de-rooted radish seed- lings, three N-glucosides (BA-3-G, BA-7-G, BA-

[228] 1465

9-G) were found [83]. BA-7-G was also found in ber of different endogenous cytokinins which can corn tissue cultures [49, 132]. In common, vary greatly from one plant species to another. N-glucosides are extremely stable in plant tissues But because of the substantial progress in recent [62, 98], and their biological activity is consider- years in isolating conjugating enzymes, as well as ably lower than the activity of their free bases cytokinin oxidase, there are good prospects to [82], perhaps of possible side chain cleavage by elucidate the quantitative relationships between cytokinin oxidases. N-glucosylation might repre- different metabolic routes of cytokinin conjuga- sent an irreversible form of cytokinin conjugation. tion. Also promising, are the increasing reports Side-chain glucosylation leads to the other form about cytokinin levels in transgenic plants where of cytokinin glucosides, the O-glucosides. The the isopentenyltransferase gene from Agrobacte- Z-O-glucosides are abundant in Lupinus rium tumefaciens is overexpressed [e.g. 99, 123]. [191, 192], Phaseolus [113, 114, 130], and Vinca In general, such transformed plants show elevated rosea [ 177]. The O-glucosides are less stable than total endogenous cytokinin levels and abnormal N-glucosides; for example they can be hydrolyzed [94]. However, not much is known by almond fl-glucosidase (Emulsin) [98]. Z-O-G yet about the rates of metabolism in such systems. hydrolysis was observed after feeding in detached of Lupinus luteus [ 133], primary leaves of [ 130], and in Vinca rosea crown Conjugation of gall [63]. Interestingly, they cannot be inactivated by cytokinin oxidases [97, 177]. Since the structural identification of the first gib- There are somewhat contrasting results about berellin (GA) glucoside, GA8-2-O-fl-D-glucoside the biological activity of cytokinin-O-glucosides: (GAs-2-O-G), from maturing fruits of Phaseolus Letham [82] found them as equally active as the coccineus [ 175, 176, 184], a series ofGA glucosyl free bases, whereas Kleczkowski et al. [67] re- conjugates have been isolated and structurally ported higher activity for the bases. On the other elucidated; in addition, acyl and alkyl GA deriva- hand, Mok and co-workers [ 112, 114] detected tives have been found. Today, the conjugation much higher biological activity for the glucosides, process is considered to be an important aspect suggesting that they might be active per se and not of GA metabolism in plants. The field of GA via release. Most of the authors regard them as conjugation has been reviewed previously in a genuine cytokinin storage forms from which the general way [ 163, 168] as well as in the context bases are liberated and so regulate the levels of of special biochemical and physiological pro- active cytokinins [e.g. 62]. In this context, the cesses [39, 78, 148, 155, 163, 182, 183]. finding by Brzobohaty etal. [18] about a The most common GA conjugates isolated fl-glucosidase from maize which is from plants are those in which the GAs are con- able to release active cytokinins from conjugates nected to glucose. These conjugates can be di- is of interest. vided into two groups: glucosyl ethers (or Much progress has been made in isolating and O-glucosides), where a hydroxy group of the GA characterizing O-glucosyltransferase in Phaseolus skeleton is linked to the glucose, and glucosyl [ 114]. The enzyme was purified to homogenity esters, in which the glucose is attached via the [41, 93], and it has high substrate specificity, uti- GA-C-7-carboxyl group. So far, the conjugating lizing trans-zeatin but neither dihydro-zeatin, cis- moiety has had a fl-D-glucopyranose struc- zeatin nor zeatin riboside [92, 115]. The molecu- ture. A summary of the isolated and structurally lar mass was about 50 kDa. As already mentioned elucidated GA glucosyl conjugates and some ad- for O-xylosyltransferase, a monoclonal antibody ditional conjugates is given in Table 1. In the case was raised against this enzyme. of the GA-O-glucosides the glucose moiety is In summary, the metabolic picture of cytoki- linked either to 2-O-, 3-O-, 11-O-, 13-O- or 17- nins remains complex because of the large num- O-position of the parent GA (see Fig. 5). From [229] 1466

OH o.O O. 20H

CH20H HO --~..~ 0 GA3s-11 -O-glucoeide

- i"i.. .-J" ......

HO o. -_.. /' ...... :...... i...... / 0 "° ~o. GAll-2-O-glu¢oside ~ ..o.--" ....." 0 H -" J=CH=

...... ==,,,-.r GA=o - 13 - 0 - gl u c osi d • 17 ...... GA2=-13-O-gluco=ide CHzOH C H 2 HO 0 0 ..:" ~: .... H " ...-3

GA:s-3-O'glucosideGA1-3"O'gluc°side...... /.''"...... / /: ',

GA1 glucosyl ester I i 16'17"H2"10'17-dihydr°xy'GA4 GA, glucosyl ester I i 17-O-giucoside GAs glucosyl ester I i GA= glucosyl ester J i GA3? glucosyl ester li GA3s glucoeyl ester [::' GA44 glucosyl e=ter J

Fig. 5. Schematic structures of endogenously occurring gibberellin glucosyl conjugates. the occurrence of GA conjugates in various spe- sides (GA-O-G), GC-MS of permethylated de- cies of higher plants it can be assumed that GA rivatives has provided reliable data [ 145, 156, 160, conjugates are distributed ubiquitously [ 121]. In 164, 166, 171], while with GA glucosyl esters addition to the GA conjugates shown in Table 1, (GA-GE), LC-MS is now the favoured approach there are many other reports of the occurrence of [ 117, 118, 122]. Partial synthesis of numerous conjugates in which identification has been based GA-O-glycosyl derivatives has provided both solely on chromatographic parameters or the standards and labelled compounds for use both identification of the parent GA after hydrolysis. as internal standards and as substrates in meta- The same also applies to the numerous putative bolic studies [58, 162, 163, 170, 172, 173]. GA conjugates detected in metabolic studies Knowledge of the enzymology of GA conjuga- [168]. Further characterization of these com- tion is still limited except for some data on the pounds is largely dependent upon access to ap- biosynthesis and metabolism of GA glucosyl con- propriate standards and progress in analytical jugates.In maturing fruits of Phaseolus coccineus a methodology. For the analysis of GA-O-gluco- GA glucosylating activity was found [69, 120,

[230] 1467

Table 1. Naturally occurring GA conjugates.

Conjugate Plant source/Reference

GA-O-glucosides Gal-3-O-glucoside Dolichos lablab [220], Hordeum vulgare [ 169], Phaseolus coccineus [156, 158, 182], Zea mays [ 169] GAl-13-O-glucoside Phaseolus coccineus [ 156] 3-epiGAi-3-O-glucoside Phaseolus coccineus [ 156] GA3-3-O-glucoside Pharbitis nil [221,223, 226], Quamoclit pennata [212] 16,17-H 2, 16,17-dihydroxy-GA4-17-O-glucoside Oryza sativa [211 ] GA 5-13-O-glucoside Phaeolus coccineus [ 156] GAs-2-O-glucoside Althea rosea [52], Hordeum vulgare [169], Pharbitis nil [225, 226], Phaseolus coccineus [ 156], Phaeolus vulgaris [55, 57], Zea mays [169] GA20-13-O-glucoside Hordeum vulgate [ 169], Pisum sativum [ 171], Triticum aestivum [80], Zea mays [ 167, 169] GA26-2-O-glucoside Pharbitis nil [221,225, 226] GA27-2-O-glucoside Pharbitis nil [221,225, 226] GA29-2-O-glucoside Hordeum vulgate [169], Pharbitis nil [221], Phaseolus coccineus [ 156], Pisum sativum [ 171 ], Zea mays [169] GA29-13-O-glucoside Hordeum vulgare [169], Pisum sativum [171], Zea mays [169] GA35-11-O-glucoside Cytisus scoparius [216, 217]

GA B-D-glucopyranosyl esters GA 1 glucosyl ester Phaseolus vulgaris [55, 56, 57] GA 4 glucosyl ester Phaseolus vulgans [55, 56, 57] GA 5 glucosyl ester Pharbitis purpurea [210] GA 9 glucosyl ester Picea sitchensis [85, 117], Pseudotsuga menziesii [42, 103], Pinus concorta [42] GA37 glucosyl ester Phaseolus vulgaris [55, 56, 57] GA38 glucosyl ester Phaseolus bulgaris [55, 56, 57] GA44 glucosyl ester Pharbitis purpurea [210]

GA alkyl ester GA 1 n-propyl ester Cucumis sativus [ 53 ] GA 3 n-propyl ester Cucumis sativus [ 53 ] GA~ methyl ester L ygodium japonicum [215] GA73 methyl ester Lygodiumjaponicum [214, 215] GA88 methyl ester L ygodium japonicum [213]

GA acyl derivatives GA3-3-O-acetate fujikuroi [ 174] GA39-3-O-isopentanoate maxima [ 13]

GA-related conjugate Gibberethione Pharbitis nil [227]

180], which has been shown to be a glucosyl- albeit less efficiently, GA 7 and GA30, forming ex- transferase located exclusively in the pericarp. clusively the 3-O-fl-D-glucopyranosides [69]. This enzyme preferentially utilizes UDP-glucose This substrate specificity, however, contradicts as a glucose donor and accepts GA3 as well as, the fact that GA3 glucose conjugates have to date [231] 1468 not been identified as endogenous constituents in occurrence of GA glucosyl conjugates and their this plant material. As a consequence, the physi- facile metabolic formation provoke the assign- ological significance of the GA 3 glucosylation re- ment of some distinct physiological functions, mains unclear. Enzyme preparations from coty- which, due to a lack of convincing evidence, are ledons of 24h imbibed seeds of Phaseolus still contradictory and speculative.The loss of coccineus transform labelled GA 4 to GA 1, GA34 , biological activity in the course of the conjugation GA 4 glucosyl ester and a GA34 glucoside process and the increased polarity of GA glucosyl [35,202]. Cell-free systems from germinating conjugates are considered to favour GA conju- peas (Pisum sativum) metabolize labelled GA12- gates for being deposited into the . From aldehyde into a GA12-aldehyde glucosyl ester the occurrence of GA conjugates in bleeding conjugate [64]. Cytosolic enzyme fractions from of , a possible function in the long-distance cells of Lycopersicon peruvianum grown in suspen- transport has been suggested [37, 38]. It also has sion cultures have been shown to specifically been suggested that the glucosyl moiety of GA transform GA 7 and GA 9 to the corresponding conjugates may cause a distorted orientation of glucosyl esters in the presence of UDP-glucose the GA within the membrane, which [180]. prohibits the appropriate binding to an assumed U sing radioactively labelled GA-O-G and GA- receptor [ 190]. Because of their preferential for- GE, the hydrolytic cleavage within several bioas- mation and accumulation during seed maturation say systems was found to parallel the biological it has been proposed that GA glucose conjugates activities of the conjugates [58, 84]. These find- may function as storage products [79, 80, 169]. ings have led to the suggestion that GA glucose This, however, applies only to conjugates of bio- conjugates per se are biologically inactive. Any logically active GAs, where hydrolysis, for ex- response obtained in the assay then reflects the ample during early stages of seed germination, degree of hydrolysis and the activity of the re- gives raise to free GAs prior to the onset of de- leased parent GA [179]. In such circumstances, novo GA biosynthesis. the occurrence of a series of specific In the case of 2fl-hydroxylated GAs, which are fl-glucosidases might be anticipated. In keeping themselves biologically inactivated metabolites, with this possibility GAs-2-O-G is as active as conjugation may be a step within the process of GAs after application to dwarf rice seedlings further catabolism. The easy formation and hy- [34], whereas GA3-3-O-G is much less active drolysis of GA glucosyl conjugates, which means than its aglycon [222]. It is therefore of relevance reversible deactivation/activation, is also dis- that a fl-glucosidase fraction from dwarf rice cussed in connection with the regulation of free leaves hydrolyses GAs-2-O-G 200 times faster GA pools. The rapid exchange of pools of GA than GA3-3-O-G [153]. Furthermore, in extracts glucosyl ester, GA glucoside and free GAs has from maturing pods of Phaseolus coccineus, a been shown in maize seedlings [164]. There are soluble fl-glucosidase has been detected which also indications that, in the case of Brassica mu- exhibits a high hydrolysing activity toward the tants, different conditions may influence GA endogenous GAs-2-O-G. This GAs-2-O-G hy- metabolism including the formation of GA con- drolysing activity decreases during pod matura- jugates [ 147]. The tentative physiological roles of tion [ 154], which appears to be functionally re- GA glucose conjugates will only be clarified if lated to the increase in GA glucosylating activity appropriate methods for identification and quan- in the same tissue [69]. Fungal fi-glucosidases, tification of pool sizes become available and are such as cellulase efficiently hydrolyse GA-13-O- used to investigate physiologically relevant pro- glucosides [157]. In contrast, enzyme prepara- cesses. Special attention should be paid to the tions from plants exhibit only low activity problem of compartmentalization, which invari- [153, 182] with this naturally occurring group of ably makes it always difficult to measure specific GA conjugates [156, 169, 171]. The ubiquitous pools. [232] 1469

Conjugation of abscisic acid PA and DPA can be conjugated to esters of the fl-D-glucopyranoside type [60, 111], whereas Whereas the main route of abscisic acid (ABA) glucose esters of these metabolites have not been biosynthesis in higher plants was until recently a found. matter of discussion [32, 229, 231], a lot of in- The earliest feeding experiments showed that formation was available on ABA metabolites and considerable amounts of ABA were subjected to conjugates [see 108, 109, 206] shortly after ABA conjugation [ 108]. The glucose ester (ABAGE) was first discovered [31,127]. Two methods of was the first identified conjugate of ABA [72], ABA degradation are known (Fig. 6). One pos- and later investigations showed that it was sibility is the conversion to phaseic acid (PA) and synthesized in fruits of several plant spe- dihydrophaseic acid (DPA), with subsequent cies [109, 123, 146]. ABA-fl-glucopyranoside conjugation, the other route is the direct forma- (ABAG) is another quantitatively important con- tion of ABA conjugates. Interestingly, most of the jugate. After being originally isolated from apple presently known ABA metabolites had been seeds [86], it now seems to be ubiquitously dis- characterized before 1984 (see [87]), and recent tributed in germinating seeds. In germinating bar- developments have been confined to their pos- ley grain both esters account for up to 20 ~o of the sible functions rather than the discovery of new total metabolites [36, 61 ]. structures. The knowledge of the enzymes involved in Metabolism of ABA to phaseic acid and re- ABA conjugation is still very poor [87]. A glu- lated compounds seems to be the main inactiva- cosyltransferase has been described, but in no tion pathway [138, 232]. It leads over 6-hy- case a substantial release of ABA from conju- droxymethyl-ABA to PA, DPA and some polar gates has been detected. This indicates that ABA conjugates, with a side-branch from 6-hydro- conjugation is probably an irreversible process xymethyl-ABA to fl-hydroxy-fl-methyl-glutaryl- which contrasts with the properties of similar hydroxy-ABA [ 59]. PA is usually present in plant conjugates of other plant hormones. This is in tissues in small amounts [87, 196], whereas ac- keeping with data on the biological activity of cumulation of DPA and also its conjugates has ABA conjugates. Whereas PA seems to have a been observed in many plants, especially in as- similar activity to ABA in stomata closure [207] sociation with [144, 229] and at some and inhibition of a-amylase synthesis [61 ], ABA stages of germination [36, 61 ]. To a lesser extent, conjugates are inactive.

ABAGE ABAG Conjugation of \ J ((-)-JA) and its stereo isomer ( + )-7-iso-JA (synonymous with ( + )-2-epi-JA) are the major representatives of a group of native plant bioregulators called jasmonates. They are widespread in the plant kingdom and exert vari- ABA ous physiological activities when applied exog- /l enously to plants [ 181 ]. Their functional role as 6-hydroxymethyI-AnA native regulators is being studied intensively and PA ~ conjugates evidence is given for their involvement in pro- cesses such as plant [134, 140, 205] and the formation of vegetative storage organs epi-DPA • DPA DPA-glucoside [71, 94]. Even more striking is the potential role Fig. 6. Overview over the metabolism of abscisic acid. of jasmonates in the signalling of external stress

[233] 1470 impulses, such as [48] and tion of the hydroxylated metabolites gives either [51 ] attack, mechanical forces (touch [208]) and 11-O- or 12-O-glucosides, or 6-O-glucosides of osmotic stress [136], to give internal stress re- cucurbic acid-related structures. sponses usually measured as activation or expres- Metabolic conjugation with amino acids of ei- sion of specific genes and formation of charac- ther non-metabolised jasmonates or of their side- teristic [ 181]. chain hydroxylated derivatives is widespread in plants. In the case of barley shoots, valine, iso- Metabolical formation of jasmonate conjugates leucine, and leucine conjugates have been identi- fied [ 100, 101,102]. In cell suspension cultures of Investigations on the metabolic transformation of tomato and potato [65], instead of amino acid exogenously applied jasmonates using excised conjugate formation, conjugation at C1 with sug- shoots of barley seedlings, tomato and potato ars took place, yielding JA glucosyl and gentio- plants [100, 101, 102], as well as cell suspension biosyl ester as the major metabolites. In a cell cultures of tomato, potato [65 ], and Eschscholtzia culture suspension of Eschscholtzia, JA was [209], showed that conjugation is common in metabolized to the 11-O-fl-D-glucoside of 11-(R)- plant tissues either without or after other meta- OH-JA [209]. bolic transformations. According to these results, summarized in Fig. 7, major metabolic steps (oth- ers than conjugation) are hydroxylation at C11 Natural occurrence of jasmonate conjugates and (usually) or C12 yielding the ll-OH or 12-OH their possible physiological role derivatives (tuberonic acid-related) and reduction of the C6 keto group resulting in cucurbic acid- With the exception of the sugar conjugates formed related metabolites. Conjugation by O-glucosyla- in cell suspensions, conjugates detected as

Esterification (methyt-, glucosyl, geatobiosyl)

C-I

C-6 O ! II O (O-glucogdatiea) Reduction +

1 ~.~ Hydroxylation C - 11 (-)-Jasmonic acid (+)-7-iso-Jasmonic acid C - 12

I C-1

Amino acid conjugation (Val, Lee, he)

Fig. 7. Survey on metabolic routes ofjasmonic acid.

[234] 1471 metabolites after exogenous application of jas- and 9,10-dihydro-JA were isolated from the fun- monates are known to occur as native constitu- gus Gibberellafujikuroi [33, 106]. The isoleucine ents in plants (Fig. 8). Thus, the 6-O-glucoside of conjugate of ( - )-JA was found also in of cucurbic acid was found in pumpkin seeds [73] Pinus mugo. JA-Ile inhibits the pollen germina- and the 12-O-fl-D-glucopyranoside of 12-OH-JA, tion, whereas free JA is neither inhibiting nor the aglucon of which is designated tuberonic acid, stimulating (Kn6fel, unpublished results). The re- occurs as a native compound in potato [228]) suits indicate speculation about the possible role and Jerusalem artichoke [95] that induces tuber of JA-Ile in regulation of this process; however, formation [71]. Several (S)-amino acid conju- besides pollen germination also senescence gates of JA and other jasmonates have been [ 140] has to be considered as a process affected. found: (Tyr), tryptophane (Trp), and Good evidence for a physiological role of jas- phenylalanine (Phe) conjugates in of the monate conjugates in stress signalling [ 135, 136, broad bean [17, 159], isoleucine (Ile) conjugates 181]) comes from the following results. in fruits as well as Ile, leucine (Leu) and valine 1. Like free jasmonates, in barley leaf segments (Val) conjugates in young leaves of this plant jasmonate conjugates, such as the naturally oc- [ 159]. Whether this distribution pattern of amino curring ( - )-JA-(S)-IIe, were found to be active in acid conjugates in different broad bean organs is inducing so-called jasmonate-inducible proteins related to any physiological role, is a matter for (JIPs), whereas the (-)-JA-Trp is of very low study. A phenylalanine conjugate of 12-acetoxy- activity [ 54]. JA is known to occur in Praxelis [14], and the 2. In barley leaves osmotic stress by sorbitol, isoleucine conjugates of ( - )JA, ( + )-7-iso-JA, mannitol, sucrose, fructose etc. leads to JIPs

oi GI¢ /Gio o

Cucurbic acid-O-.glucoside 12-.B-D-glucopyranosyl.-JA (-)-9,10-Dihydro-JA-He

o

(+)-7-iso-JA-Ele (-)-~.-Aceto~A-Phe methyl ester (-)-.IA-S-amlno acid conjugates

e~ Ib

3,7-Didehydro-JA-ne N-[3-oxo-2(penten-2-yl)-cydopeat- 1-yl-propionyl] -isoleucine Fig. 8. Structures of endogenously occurring jasmonates. [235] 1472 together with the accumulation of both free acid Conjugation of and conjugated jasmonates like JA-Ile, JA-Leu, JA-Val [74]. Only a few papers have so far dealt with conju- According to the basic definition for conjugates gation of the brassinosteroids (Fig. 9). This field (see Introduction), the methyl esters of jas- is in its very infancy [91] and the seemingly low monates have also to be considered as conju- concentration of conjugates may gates, although they differ markedly from all other well represent a major difficulty in their detection conjugate types. The JA methyl ester has been and analysis. shown to possess physiological potencies of the same order, or even higher, than the free acid, *%~, 23 probably depending on differences in the uptake OH and on the plant species used. The occurrence of OH JA methyl ester is well established in essential oils o , I I I -..~c~ ~ ~ CH20H [40], but its physiologically relevance in plant tis- R2 sues [197] must still be confirmed (use of metha- HO nol extraction could cause artefact formation from other endogenous ester conjugates). Neverthe- 1 R 1 ~ OH; R2 ~ H less, volatile either applied 2 R~ = H; R 2 = OH through the or released from Artemi- sia leaves was able to induce proteinase inhibitor (PI) [47, 48, 150]. Thus, methyl jasmonate might be a volatile signal in interplant communication, ...... OH released in response to wounding or other stress situations. HO..,,,,~ In summary, jasmonates are, like classic phy- H0"" tohormones, transformed metabolically to conju- 0 gates, the types of which resemble those of aux- ins and gibberellins. Concerning jasmonate conjugation, apparently amino acid conjugates nH . OH dominate. Some of them are of high activity when exogenously applied. They are widely distributed, and their endogenous levels increase rapidly in HO.,~ 6 response to external stress. How they are involved H0"" in the transduction chain between external stress 0 impulse and internal stress response has to be studied further. Even more speculative is the physiological role of JA amino acid conjugates in pollen germination and senescence processes. O-glucosides represent another important group ofjasmonate conjugates they might be of physi- ological relevance in regulation of tuber forma- tion. Whether they are special transport remains an open question. Of further interest is H the existence of jasmonate methyl ester conju- 5 R = Louryl gates. These volatile compounds exhibit high 8 R = Myristyl physiological potency and, thus, qualify as poten- Fig. 9. Structures of endogenously occurring brassinosteroid tial air-borne signals. conjugates.

[236] 1473

Although brassinosteroid molecules contain Schulze A: Genetics, chemistry, and biochemical physi- a series of functional groups, only hydroxyl ology in the study of hormonal homeostasis. In: Kars- sen CM, van Loon LC, Vreugdenhil D (eds) Progress in groups at C-23 and C-25 have been found to Plant Growth Regulation, pp. 1-2, Kluwer Academic be linked to glucosyl moieties. Extracts from Publishers, Dordrecht (1992). seeds of Phaseolus vulgaris have been shown to 6. Bandurski RS, Schulze A: Concentrations of indole-3- contain 23-0-fl-D-glucosyl-25-methyl dolichos- acetic acid and its esters in Avena and Zea. Plant Physiol terol (Fig. 9-1) and its 2fl isomer (Fig. 9-2) [218, 54:257-262 (1974). 7. Bandurski RS, SchulzeA, Desrosiers M, Jensen P, 219, 224]. Epel B: Relationship between stimuli, IAA and growth. Conjugates of brassinosteroids have also been In: Pharis RP, Rood SB (eds) Plant Growth Substances detected in metabolic studies. After feeding 1988, pp. 341-352, Springer-Verlag, Berlin/Heidelberg/ brassinolide to mung beans, the corresponding New York (1990). 23-O-fl-D-glucosyl conjugate (Fig. 9-3) was iden- 8. Bandurski RS, Schulze A, Domagalski W, Komoszyn- ski M, Lewer P, Nonhebel H: Synthesis and metabo- tiffed [195], while cell cultures of Lycopersicon lism of conjugates of indole-3-acetic acid. In: esculentum transform 24-epi-brassinolide to the Schreiber K, Schtltte HR, Sembdner G (eds) Conju- 25-O-fl-D-glucosyloxy derivative (Fig. 9-4) [ 165 ]. gated Plant Hormones: Structure, Metabolism and Although 23-O-glucosyl brassinolide is as active Function, pp. 11-20. VEB Deutscher Verlag der Wis- as the free brassinolide in the rice lamina inclina- senschaften, Berlin (1987). 9. Beffa R, Martin HV, Pilet P-E: In vitro oxidation of in- tion test, there has been discussion suggesting doleacetic acid by soluble auxin-oxidases and peroxi- that 23-O-glucosylation ofbrassinosteroids repre- dases from maize . Plant Physiol 94:485-491 sents a regulatory deactivation step [195]. Re- (1990). cently, two acyl conjugates of brassinosteroids 10. Bialek K, Cohen JD: Hydrolysis of an indole-3-acetic have been identified carrying the conjugation acid amino acid conjugate by an enzyme preparation from Phaseolus vulgaris. Plant Physiol 75 (suppl): 108 moieties at the 3-hydroxy group. These 3fl-O- (1984). lauryl and 3fl-O-myristyl derivatives ofteasterone 11. Bialek K, Cohen JD: Isolation and partial characteriza- (Fig. 9-5 and 9-6) were isolated from pollen of tion of the major amide-linked conjugate of indole-3- Lilium longifolium [2]. acetic acid from Phaseolus vulgaris. Plant Physiol 88: 99-104 (1986). 12. Bialek K, Cohen JD: Free and conjugated indole-3- acetic acid in developing bean seeds. Plant Physiol 91: References 775-779 (1989). 13. Blechschmidt S, Castel U, Gaskin P, Hedden P, Grae- 1. Andersson B, Sandberg G: Identification of endogenous be JE, MacMillan J: GC/MS analysis of the plant hor- N-(3-indoleacetyl) aspartic acid in Scots pine (Pinus sil- mones in seeds of Cucurbita maxima. 23:553-558 (1984). vestris L.) by combined -mass spec- 14. Bohlmann F, Wegner P, Jakupovic J, King RM: Struk- trometry, using high-performance liquid chromato- tur und Synthese von N-(Acetoxy)-jasmonoylphenyla- graphy for quantification. J Chromatogr 238:151-156 (1982). laninmethylester aus Praxelis clematidea. Tetrahedron 2. Asakawa S, Abe H, Natsume M: New acyl conjugated 40:2537-2540 (1984). brassinosteroids from Lily pollen. XVth International 15. Bopp M, Atzorn R: Hormonelle Regulation der Congress (Yokohama 1993), Abstract 4163 Moosentwicklung. Naturwissenschaften 79:337-346 (1993). (1992). 3. Badenoch-Jones J, Summons RE, Rolfe BG, Le- 16. Brown BH, Crozier A, SandbergG: Catabolism of thamDS: Phytohormones, Rhizobium mutants, and indole-3-acetic acid in fractions from light- nodulation in legumes. IV. Auxin metabolites in pea root grown Pisum sativum L. seedlings. Plant Cell Environm nodules. J Plant Growth Regul 3:23-29 (1984). 9:527-534 (1986). 4. Bandurski RS: Metabolism of indole-3-acetic acid. In: 17. Brtlckner C, Kramell R, Schneider G, Schmidt J, Pre- Crozier A, HillmanJR (eds) The Biosynthesis and iss A, Sembdner G, Schreiber K: N-[( - )jasmonoyl]-S- Metabolism of Plant Hormones, SEB-Series 23, and a related tryptophane conjugate from pp. 183-200, Cambridge University Press, Cambridge Viciafaba. Phytochemistry 27:275-276 (1988). (1984). 18. Brzobohaty B, Moore I, Kristoffersen P, Bako L, Cam- 5. Bandurski RS, Desrosiers MF, Jensen P, Pawlak M, pos N, Schell J, Palme K: Release of active cytokinin by

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