Biosynthesis of Phytohormones in Algae - Springer Link

ISSN 10214437, Russian Journal of Plant Physiology, 2012, Vol. 59, No. 5, pp. 595–610. © Pleiades Publishing, Ltd., 2012. Original Russian Text © A.A. Kiseleva, E.R. Tarachovskaya, M.F. Shishova, 2012, published in Fiziologiya Rastenii, 2012, Vol. 59, No. 5, pp. 643–659.

REVIEWS

Biosynthesis of Phytohormones in Algae A. A. Kiseleva, E. R. Tarachovskaya, and M. F. Shishova St. Petersburg State University, Univesitetskaya nab. 7/9, St. Petersburg, 199034 Russia; email: [email protected] Received November 27, 2011

Abstract—Like in the case of higher plants, algal growth and development are controlled by the hormonal regulatory system. Essentially all known phytohormones were identified in various algal taxa, and the range of their physiological activities was confirmed. At the same time, our knowledge of enzymes involved in the phytohormone synthesis in algae is rather limited. Data concerning genes encoding these enzymes are still more fragmentary. Current data about proteomes of some algae allow the revealing of amino acid sequences with homology to those of the higher plant enzymes and their conserved domains. Keywords: algae, phytohormones, IAA, cytokinins, ABA, gibberellins DOI: 10.1134/S1021443712050081

INTRODUCTION

AUXINS

The study of plant hormone metabolism attracts the attention of many researchers [1]. At present, the pathways of phytohormone biosynthesis in higher plants are sufficiently well studied; the data concern ing the control of these processes at the gene level are actively accumulated. At the same time, specific fea tures of hormone metabolism in different groups of algae remain largely unknown. Information about the control of phytohormone biosynthesis in them is so far fragmentary and not systemized [2]. The reasons for this is obviously a great diversity of these photoau totrophs and also a numerous methodological difficul ties arising in the work with these organisms. Cur rently, essentially all known phytohormones are found in the members of various algal groups [3–5]. Although the functioning of a comprehensive hor monal system in these organisms is not still finally proven, the role of phytohormones in the regulation of key metabolic processes in algae is no longer in doubt [6–11].

Auxin is one of the most important regulators of plant growth and development. It controls such pro cesses as expansion growth, vascular tissue formation, root development, and many others [12]. Auxin was detected in higher plants, algae, microorganisms, fungi, and even animals [13, 14]. The concentration of this hor mone in algal tissues is somewhat lower than in higher plants [12]. Thus, IAA content in the zygotes of brown algae, Fucus vesiculosus and F. disticus, is 2–9 ng/g fr wt [8, 15]; in other representatives of brown algae, IAA concentration is on the average 1–4 ng/g fr wt; in red algae – 5–10 ng/g fr wt, and in green algae – 11– 12 ng/g fr wt [16]. However, in some species (for example, in the red alga Polysiphonia urceolata), the content of this hormone attains 110 ng/g fr wt, which is markedly higher than values characteristic of angiosperms (25–30 ng/g fr wt) [17]. Effects of auxin on algal growth and development correspond as a whole to the spectrum of its physiolog ical action in higher plants [5]. In some algal species, auxin stimulates rhizoid formation, like this occurs in mosses [8]. In the cells of Chara globularis (Charo phyta), auxin treatment resulted in similar changes of the cytoskeleton as in angiosperms [10]. In the apical and intercalary zones of the thallus of the red alga Grateloupia dichotoma, the phytohormone stimulated cell division and elongation and/or suppressed branching, which also resembles processes character istic of angiosperms [18]. Auxin determined zygote polarization of fucoid algae, which is confirmed by a disturbance of zygote normal development in the pres ence of inhibitors of IAA polar transport [8, 15]. In spite of the fact that the structure of IAA, the most widespread natural phytohormone, was estab

The objective of this work was a systematization of knowledge of phytohormone biosynthesis in algae and also a comparison of amino acid sequences with homology to those of enzymes involved in the hor mone synthesis in higher plants. To this end, NCBI and Genbank data were used. Abbreviations: CPS—copalyl pyrophosphate synthase; CYP79B2/CYP79B3—cytochrome P450 79B2/B3; GGPP— geranyl geranyl pyrophosphate; NCED—9сisepoxycarotenoid dioxygenase; NIT1—nitrilase 1; PDS—phytoene desaturase; PSY—phytoene synthase; SDR—shortchain dehydroge nase/reductase; TDC—tryptophan decarboxylase; ZEP—zea xanthin epoxidase.

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lished still in 1930, the metabolism of this hormone is discussed until now [19]. Some details of IAA biosyn thesis is not elucidated conclusively even for higher plants. Their identification is complicated by the func tioning of several pathways of auxin biosynthesis in plant tissues; the preferred usage of each of them may reflect different developmental stages of the organism or be the response to the impact of environmental fac tors [20, 21]. Usually, two main pathways of IAA bio synthesis are distinguished: tryptophandependent (Fig. 1) and tryptophanindependent (Fig. 2) [19]. Four compounds produced from tryptophan are known, which conversions may result in the IAA for mation [22]. A key reaction of the indole3pyruvate pathway is a conversion of tryptophan into indole3 pyruvate catalyzed by tryptophan aminotransferase. Further, indole3pyruvate is subjected to decarboxy lation with the formation of indole3acetaldehyde, which oxidation results in the IAA formation. These reactions are catalyzed, respectively, by indole3 pyruvate carboxylase and indole3acetaldehyde oxi dase [23]. Another pathway includes the formation of indole3acetaldoxime under the influence of P450 monooxigenase (CYP79B2/CYP79B3). Indole3ace taldoxime may be a precursor of several compounds, which are finally transformed into IAA. Among them are indole3acetaldehyde, which is conversed into auxin in the unknown enzymatic reaction [24], and idole3acetonitrile transformed into IAA by nitrilases (NIT). Finally, indole3alkylthiohydroximate could be produced from imdole3acetaldoxime. The last reaction is a key one in the “glucosinolate loop” and is catalyzed by the enzyme CYP83B1 (SUR2) [25]. A variation of this pathway is the formation of indole3 acetaldoxime from Nhydroxytryptamine, which is synthesized from tryptamine by monoxigenase YUCCA (in rice and arabidopsis) [26] or FLOOZY (in petunia) [27]. Tryptamine is produced in the process of tryptophan decarboxylation by tryptophan decar boxylase. The compounds initiating the tryptophaninde pendent pathway of auxin biosynthesis may be indole or indole3glycerophosphate, intermediates in the pathway of tryptophan synthesis [28]. They produce indole3acetonitrile or indole3acetaldehyde. Enzymes catalyzing these conversions are not still studied [25, 29]. At present, genes encoding enzymes functioning at various steps of auxin biosynthesis are intensively stud ied [30]. The main enzymes of auxin biosynthesis are flavindependent monooxygenases, tryptophan ami notransferase, various cytochromes P450, tryptophan decarboxylase, CS lyases (SUR1), and NITs [23, 24]; mutations in genes encoding these enzymes induce substantial disturbances in plant organism develop ment. Further, we will analyze just these enzymes (Table 1). YUCCA. It was shown that in Arabidopsis thaliana several enzymes fulfill this function. When algal amino

acid sequences were alternatively compared using the BlastP program with arabidopsis sequences presented in Table 1, amino acid sequences differing only in the Evalue parameter (i.e., the degree of matching signif icance) were revealed. Like the enzyme YUCCA, they belong to the family of flavincontaining monooxyge nases (Table 1). Note that Evalue is low, which proves a homology between these sequences and indicates a possibility of the involvement of these proteins in the fulfilling similar function as arabidopsis proteins. In addition, the characteristics of these amino acid sequences presented in the NCBI database also indi cate a similarity in the functions of these proteins. FLOOZY. Since this protein is absent from arabi dopsis but fulfills the same function as YUCCA in Petunia × hybrida, the amino acid sequence of petunia FLOOZY was used as a matrix for the search in BlastP. Since both these enzymes, YUCCA and FLOOZY, belong to the same family of flavincontaining monooxygenases and their amino acid sequences have substantial homology (Evalue = 4e170), it is not sur prising that the spectrum of algal enzymes found by the search in BlastP against each of these sequences is essentially completely overlapped. Tryptophan aminotransferase. The comparative analysis performed with the BlastP program did not reveal algal sequences with a significant degree of sim ilarity. Subsequent acquaintance with the characteris tic of sequences with the lowest Evalues did not gave positive results as well, i.e., it was not shown that these pro teins could fulfill the function of tryptophan aminotrans ferase. Therefore, it may be suggested that, in tested algae with sequenced proteomes, the indole3pyruvate path way of auxin biosynthesis does not operate. CYP79B2 and CYP79B3. Further comparison of sequences of CYP79B2 and CYP79B3 enzymes revealed proteins belonging to the same family of cyto chrome P450, but the degree of their similarity to enzymes of A. thaliana was insignificant. In addition, tryptophan hydroxylation was not among their func tions. Note also that the results of the BlastP search for CYP79B2 and CYP79B3 enzymes do not differ, which is related to the fact that in A. thaliana they manifest not only functional but also structural homology. Tryptophan decarboxylase. In addition to the enzyme of Chlamydomonas reinhardtii presented in the table, we found also other proteins close to A. thaliana tryptophan decarboxylase; however, it was not found that they can decarboxylate tryptophan to tryptamine, most of them are decarboxylases of aro matic amino acids, such as tyrosine. CSlyase. Similarly as in A. thaliana, these algal enzymes revealed using BlastP belong to a vast family of aspartate aminotransferases, but their involvement in auxin biosynthesis was not demonstrated. Cytochrome P450 CYP83B1 (SUR2). We could not find any amino acid sequence significantly homol ogous to that of A. thaliana CYP83B1. Since this

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OH O NH2

N H

tryptophan

tryptophan aminotransferases

tryptophan decarboxylase (TDC)

OH O N H tryptamine

O

N H indole3 pyruvic acid

cytochromes P450 (CYP79B2/CYP79B3)

NH2

monooxygenase (YUCCA/FLOOZY)

O OH NH2

NH Nhydroxytryptamine

indole3pyruvate decarboxylase

CSlyase (SUR1) cytochrome P450 (CYP83B1/SUR2)

N N OH H indole3acetaldoxime

glucosinolates mirosinase

?

?

H

N H

O

N H indole3acetaldehyde

N

indole3acetonitril

indole3acetaldehyde oxidase

nitrilase (NIT)

O OH N H indole3acetic acid Fig. 1. Tryptophandependent pathway of IAA biosynthesis.

enzyme is a key one for reactions of the glucosinolate loop, it seems likely that this pathway of auxin biosyn thesis does not operate in algae. Nitrilase (NIT1). As evident from Table 1, BlastP analysis revealed in Thalassiosira pseudonana an enzyme characterized by nitrilase activity leading to the IAA formation. For other algal enzymes presented in Table 1, it was shown that they are nitrilases and even close in their structure to NIT1, but so far to speak with confidence about their role in the auxin biosynthesis is too early. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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Analyzing the data presented in Table 1, it can be concluded that the most likely pathway of auxin bio synthesis include tryptamine and further indole3 acetonitrile because the enzymes of just this pathway were detected in some algae belonging to different tax onomic groups. GIBBERELLINS The regulatory action of gibberellins (GAs) is well studied for higher plants; however, only scarce data No. 5

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KISELEVA et al. O

OH CH2 O

O

OH

OH

chorismate anthranylate synthase

O OH NH2

anthranylate phosphoribosyl transferase

O OH O NH

HO P O O

OH

H

H

OH

H OH

H

N(5'phosphoribosyl)anthranylate isomerase

O

OH NH OH OH O

O

P HO

OH

1(ocarboxyphenylamino)1 deoxyribuloso5phosphate indole3 glyceriphosphate synthase

CO2

OH

OH OH HO

P

N H

O

OH

N H

O

glyceraldehyde3 phosphate

tryptophane synthase α

O N H indole3acetaldehyde

O

indole3 pyruvic acid

indole3glyceriphosphate

H

decarboxylase

?

aldehyde oxidase

O

N H

indole Ser H2O

tryptophane synthase β

N N H indole3acetonitrile

nitrilase

N H

OH

indole3acetic acid

O OH NH2

NH tryptophan Fig. 2. Tryptophanindependent pathway of IAA biosynthesis. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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Table 1. Key enzymes of tryptophandependent IAA biosynthesis Enzyme

Organism

Protein

E value*

Arabidopsis thaliana

NP_194980.1 NP_193062.1 NP_171955.1 NP_196693.1 NP_199202.1 NP_197944.2 NP_180881.1 NP_194601.1 NP_171914.1 NP_175321.1 NP_173564.1

Ostreococcus lucimarinus

XP_001422130.1

2e24

Ectocarpus siliculosus

CBJ33551.1

4e23

Ostreococcus tauri

XP_003084158.1

7e23

Chlorella variabilis

EFN57954.1

3e22

Petunia × hybrida

AAK74069.1


XP_001422130.1

1e21

Ostreococcus tauri

XP_003084158.1

8e21


EFN57954.1

2e18

Tryptophan aminotransferase (WEI8/SAV3/TAR)


NP_177213.1

Cytochrome P450 (CYP79B2/CYP79B3)


NP_195705.1 NP_179820.2

Tryptophan decarboxylase (TDC)


CAB81456.1

Chlamydomonas reinhardtii

XP_001690025.1


NP_179650.1 NP_973489.1


XP_001415929.1 XP_001420314.1

4e79 8e25

Micromonas sp.

XP_002507098.1 XP_002502760.1

5e77 8e25

Micromonas pusilla

XP_003056607.1 XP_003060871.1

9e74 2e25

Ostreococcus tauri

XP_003074502.1 XP_003083519.1

2e72 2e22

Phaeodactylum tricornutum

XP_002186145.1 XP_002176258.1

8e56 2e25

Thalassiosira pseudonana

XP_002289601.1 XP_002295148.1

5e46 8e26

Volvox carteri

XP_002946497.1

4e27


EFN59424.1

1e24


CBJ48382.1 CBN75546.1

8e24 1e23

Flavincontaining monooxidase (YUCCA)

Flavincontaining monooxidase (FLOOZY)

CSlyase (SUR1)


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Table 1. (Contd.) Enzyme

Organism

Protein

Cytochrome P450 CYP83B1 (SUR2)


NP_194878.1

Nitrilase (NIT1)


NP_001078234.1

E value*

NP_566868.3 Thalassiosira pseudonana

XP_002290043.1

7e19


XP_002183613.1

2e20


CBJ25483.1

5e20

Micromonas sp.

XP_002503164.1

7e17


EFN54567.1

2e16

Volvox carteri

XP_002948137.1

2e14

XP_002956542.1

8e14

XP_003064056.1

7e14

XP_003061179.1

6e13

Micromonas pusilla

* Evalue is a parameter that describes a degree of the homology between aligned amino acid sequences of algae and A. thaliana. This means that the lower the Evalue the higher is the “significance” of the match. We used NCBI and GenBank databases. When the sequence was found in both bases, the NCBI sequence identification number was put into the table. When the sequence was found only in GenBank database, its identification number was put into the table.

occur about their possible influence on algal growth. Gibberellinlike activity was demonstrated for the fol lowing algal species: Fucus vesiculosus, F. spiralis (Phaeophyceae), Tetraselmis spp., Caulerpa paspal oides (Chlorophyta), Hypnea musciormis (Rhodo phyta) [6, 30, 31]. The GA content in tissues of some algae, such as Enteromorpha is on average 100 mg/kg fr wt [32]. Since treatments of brown and red algae with GAs enhanced their growth, it may be suggested that, in these algae, GAs control growth of axial structures, like this occurs in higher plants [5]. From the chemical point of view, GAs are diterpe nes, and the early stages of GA formation occur through one of two pathways of isoprenoid synthesis: through mevalonic acid or methylerythrito phosphate [33, 34]. The analysis of published data shows that, in both higher plants and algae, GAs are predominantly synthesized through the methylerythrito phosphate pathway operating in plastids [35–37]. A key reaction of GA biosynthesis is cyclization of geranylgeranyl pyrophosphate (GGPP) resulting in the formation of entkaurene. This twostage process is catalyzed by two enzymes: copalyl pyrophosphate synthase (CPS) and entkaurene synthase. GGPP cyclization occurs in proplastids of growing tissues. Data concerning

localization of enzymes catalyzing further reactions of GA biosynthesis indicate that these processes occur on the plastid outer membrane and in the endoplasmic reticulum [38, 39]. The mechanism of entkaurene transport from plastids is so far not very clear [40]. ent Kaurene is converted into physiologically active GAs through the series of oxidative reactions catalyzed by enzymes of two types. First reactions are catalyzed by cytochrome P450dependent monooxygenases, which results in the formation of GA12 and its 13hydroxy lated analog GA53. Further GA conversions, in partic ular into their inactive forms, are catalyzed by soluble cytosolic dioxygenases using 2oxoglutaric acid as a cosubstrate. Among these enzymes are GA20 oxi dase, 3βhydroxylase, 2oxoglutoratedependent dioxygenase [41–43]. The scheme of GA biosynthesis is presented in Fig. 3. Table 2 presents data about iden tified amino acid sequences of enzymes presumably involved in GA biosynthesis. Copalyl pyrophosphate synthase. entKaurene syn thase, and oxidase of entkaurenoic acid. Regretfully, we can not found in algae sequences homologous to these arabidopsis enzymes catalyzing early stages of GA biosynthesis. Nevertheless, it can be suggested that this phenomenon argues for the shortage of available


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BIOSYNTHESIS OF PHYTOHORMONES IN ALGAE CH3

CH3

O HO

O P CH3

O

HO

CH2

CH3

copalyl pyrophosphate synthase

H3C

601

entkaurene synthase

CH3

CH3

CH3

O P CH3

H3C

OH

geranylgeranyl pyrophosphate

CH3

H3C

copalyl pyrophosphate

entkaurene entkaurene oxidase

CH3

CH2

H3C

O

HO

CH2

CH3

CH2

OH

O

H3C

H

H3C

O

HO

GA12 aldehyde

OH

CH3

O

HO

entkaurenoic acid

ent7αhydroxykaurenoic acid

CH2

CH2

GA13 hydroxylase H3C

O

O

HO

OH

O OH

GA12

GA53 GA20 oxidase

GA20 oxidase

O

OH

O CH2

CH2

O

O O

CH3

O

CH3

OH

OH

GA9

GA20 GA3βhydroxylase

GA3βhydroxylase

OH

O

O

CH2

CH2 O

O HO

HO O

CH3

O

H3C

OH

OH

GA4

GA1

GA2 oxidase

GA2 oxidase

O

OH

O HO CH2

CH2

O

O

HO

HO O

CH3

O

CH3

OH

OH

GA34

GA8

Fig. 3. Gibberellin biosynthesis. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

O

HO

CH3

H3C

CH3

oxidase of entkaurenoic acid

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Table 2. Enzymes of gibberellin biosynthesis Enzyme

Organism

Protein

E value

Copalyl pyrophosphate synthase


NP_192187.1

entKaurene synthase


NP_178064.1

entKaurene oxidase


NP_197962.1

Ostreococcus tauri

XP_003082726.1

8e30


XP_001422903.1 XP_001420992.1

7e33 2e32

Micromonas pusilla

XP_003058421.1

8e24

Oxidase of entkaurenoic acid


NP_180803.1 NP_172008.1

GA20 oxidase


NP_194272.1 NP_199994.1 NP_176294.1 NP_175075.1 NP_196337.1


XP_001695234.1


XP_002286453.1

2e28**


XP_002176333.1

2e27**

Micromonas pusilla

XP_003058775.1

2e26**


NP_173008.1 NP_178150.1 NP_193900.1 NP_178149.1


XP_002288613.1

4e34


CBN78512.1

3e33


XP_002186205.1 XP_002176333.1 XP_002286453.1

1e30 1e26 5e26

Volvox carteri f. nagariensis

XP_002949877.1

2e25


NP_177965.1 NP_174296.1 NP_181002.1 NP_175233.1 NP_171742.1 NP_175509.1 NP_001154262.1


CBJ26610.1 CBJ26611.1

6e32 7e33


EFN50719.1 EFN51620.1

2e32 4e29


XP_002186205.1

8e28

Micromonas pusilla

XP_003058775.1

2e27


XP_002288613.1

4e27

GA3βhydroxylase

GA2 oxidase

** Explanations in the text. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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Table 3. Enzymes of cytokinin biosynthesis Enzyme Isopentenyltransferase

Organism

Protein


NP_177013.1

Micromonas sp.

XP_002499449.1 XP_002500269.1

2e25 2e23

Micromonas pusilla

XP_003061927.1 XP_003055760.1

3e24 2e20


EFN51365.1

2e23

Ostreococcus tauri

XP_003078218.1 XP_003080095.1

2e20 1e04


CBJ48350.1

1e17

Volvox carteri f. nagariensis

XP_002949722.1

2e16


XP_001418572.1

2e11


XP_002289575.1

8e09


XP_002177482.1

3e07

proteomic data but not for the absence of correspond ing enzymes in algae. GA20 oxidase. This enzyme has been earlier char acterized in a green alga C. reinhardtii; its functioning as GA20 oxidase was proven. Nevertheless, it mani fested only weak similarity with A. thaliana enzyme with similar function. In this connection, we decided to use BlastP search instead of early used algorhythm; the C. reinhardtii but not A. thaliana amino acid sequence was used as a matrix. As a result, we identi fied some sequences, which manifested a high homol ogy to C. reinhardtii sequence (low Evalues). GA3βhydroxylase, GA2 oxidase. In A. thaliana this function is fulfilled by several enzymes. The search performed with alternative usage of all sequences pre sented in the table as a matrix revealed some identical sequences. Evalues presented in Table 2 refer to sequences NP_173008.1 and NP_1777965.1. Note that the analysis of GA20 oxidase, GA3β hydroxylase, and GA2 oxidase revealed one and the same algal proteins. This may be explained by the fact that all analyzed enzymes and detected algal proteins belong to a single family of oxygenases. Perhaps in the future the functions of these proteins will be estab lished more precisely and their direct involvement in GA biosynthesis will be proven. In general, protein sequences identified in algae and homologous enzymes of the late stages of GA bio synthesis in A. thaliana are overlapped completely. Thus, it may be suggested that the pathway of GA bio synthesis in algae does not differ from that in higher plants. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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E value

CYTOKININS At present, both isoprenoid and aromatic cytoki nins and also some their conjugates were detected in algae, which indicates the presence in them of the complex system of interconversions of these com pounds and regulation of their activities [5]. Different cytokinins were identified in tissues of the members of genera Cladophora, Ulva, Chlorella (Chlorophyta), Fucus, Ecklonia, Laminaria (Phaeophyta), Porphyra, Corallina, Gigartina (Rhodophyta), Euglena (Eugle nophyta), and some others [9, 43, 44]. In different algal species, the concentration of cytokinins varies from 13 to 453 pmol/g dry wt [18]. It is worth men tioning that algae evidently are not characterized by a great diversity of cytokinin conjugates [9]. Apparently, the function of inactive glucosides regulating the level of hormones in the cells is partially fulfilled by cis zeatin prevailing in algae; it is much less active than transzeatin. The effects of cytokinins on algal growth and devel opment (stimulation of cell division, growth activation, intensification of some photosynthetic processes) cor respond completely to the spectrum of cytokinin bio logical action in higher plant tissues [46–48]. The synthesis of isoprenoid cytokinins in higher plants can occur through two different pathways. A direct route or de novo biosynthesis includes the for mation of N6isopentenyladenosine monophosphate (i6AMP) from AMP and pyrophosphate. The key reaction of biosynthesis is catalyzed by isopentenyl transferase [49]. The second pathway of cytokinin for mation includes changes in the structure of tRNA comprising ciszeatin. In spite of evident progress in the deciphering the routes of hormone formation, the No. 5

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Table 4. Enzymes of ABA biosynthesis Enzyme

Organism

Phytoene synthase (PSY) Arabidopsis thaliana Muriella zofingiensis Haematococcus pluvialis

Dunaliella bardawil

Dunaliella sp. Dunaliella salina Auxenochlorella protothecoides Chlamydomonas reinhardtii Volvox carteri f. nagariens Chlorella variabilis Ostreococcus tauri

Micromonas sp. Micromonas pusilla

Phytoene desaturase (PDS)

Pavlova lutheri Ectocarpus siliculosus Phaeodactylum tricornutum Bigelowiella natans Arabidopsis thaliana Muriella zofingiensis Haematococcus pluvialis Chlorella variabilis Auxenochlorella protothecoides Volvox carteri f. nagariensis Chlamydomonas reinhardtii Dunaliella salina

Dunaliella bardawi Ectocarpus siliculosus Porphyra yezoensis Micromonas pusilla Thalassiosira pseudonana Phaeodactylum tricornutum

Protein

E value

NP_001154715.1 CBW37867.1 AAW28851.1 AAK15621.1 AAY53806.1 ABY50091.1 ABY50090.1 AAB51287.1 ACN62390.1 ACN62391.1 ABE97388.1 AAT46069.1 AAT28184.1 ADC32152.1 AAT38473.1 XP_001701192.1 XP_002956783.1 EFN51796.1 XP_003079460.1 XP_003082791.1 XP_003080885.1 XP_002508518.1 XP_003057282.1 XP_003063807.1 ABA55571.1 CBJ31337.1 XP_002178776.1 AAP79176.1 NP_193157.1 ABR20877.1 AAV37090.1 CAA60479.1 EFN58267.1 ADC32153.1 XP_002948155.1 XP_001690859.1 ADD52599.1 ABB51091.1 AAY26317.1 ABH09129.1 CAA75094.1 CBN77338.1 ACI45964.1 XP_003055146.1 XP_003063922.1 XP_002291632.1 XP_002184512.1 XP_002180171.1 XP_002180171.1


1e148 1e145 1e145 5e145 8e142 4e141 3e140 2e32 5e31 3e141 6e141 1e135 9e140 3e138 2e137 2e137 1e136 9e136 2e44 1e07 6e134 2e133 2e48 3e95 2e90 3e87 1e44 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6e158 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Vol. 59

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Table 4. (Contd.) Enzyme

Organism

Protein

Micromonas sp.

Zeaxanthin epoxidase (ZEP)

Ostreococcus lucimarinus Ostreococcus tauri Mantoniella squamata Bigelowiella natans Arabidopsis thaliana Chlorella variabilis Chlamydomonas sp. Chlamydomonas reinhardtii Volvox carteri f. nagariens Micromonas pusilla Micromonas sp. Ostreococcus lucimarinus Ostreococcus tauri Phaeodactylum tricornutum

Thalassiosira pseudonana Ectocarpus siliculosus Guillardia theta 9cisepoxicarotenoid dioxigenase (NCED)

Arabidopsis thaliana Chlorella variabilis Ectocarpus siliculosus

Volvox carteri f. nagariensis Chlamydomonas reinhardtii Micromonas pusilla Ostreococcus lucimarinus Micromonas sp. Phaeodactylum tricornutum Xanthoxi dehydrogenase Arabidopsis thaliana like SDR (dehydrogena Chlorella variabilis se/reductase) Ectocarpus siliculosus



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E value 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5e112 1e61 7e109 1e100 4e96 2e47 3e91 5e78 1e70 1e82 3e76 6e80 2e79 4e22 1e96 1e96 2e93 1e90 2e85 6e76 1e71 3e52 7e36 3e43 1e39 3e24 7e36 5e28 2e25 2e24 1e23 1e23 8e24

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KISELEVA et al. N N O

O HO P O OH HO

AMP

N OH

NH2 N

N

H3C

O + O O P O P OH HO OH

NH

N

CH3

O isopentenyltransferase HO P O OH

O HO

CH3 N

N

H3C

OH O OH + HO P P O OH O

OH

N6isopentenyladenosine monophosphate

dimethylallyl pyrophosphate

Fig. 4. Cytokinin biosynthesis.

mechanisms of isoprenoid cytokinin biosynthesis are not elucidated reliable until now. First of all, this refers to the formation of aromatic hormones [49, 50]. It is suggested that the second indirect pathway including tRNA degradation is more characteristic for algae [9]. Table 3 presents the data to compare amino acid sequences of isopentenyltransferase in higher plants and some algae. For all algae presented in Table 3, enzymes operat ing as isopentenyltransferase were identified. Thus, it may be concluded that these autotrophic organisms synthesize cytokinins along the pathway analogous to that in higher plants, which was described above. ABSCISIC ACID Abscisic acid was found in many algae from differ ent taxonomic groups (about 100 species) [7, 51, 52]. Under normal conditions, the ABA concentration in algal cells is from 7 to 34 nmol/kg fr wt, which is much lower than on average in higher plants [11]. Similar content of ABA is characteristic of hydrophilic liver worts. Like in higher plants, the content of this hor mone in algal tissue increases markedly under stress conditions. Such an effect was observed in the mem bers of the genera Dunaliella (under the influence of salinity, medium alkalinization, and nitrogen starva tion), Chlorella (salinity, increased temperature, and high insolation), Stichococcus, Haematococcus (medium acidification, drought) [53, 54]. In the sporophytes of the brown alga Laminaria, the concen tration of endogenous ABA experienced seasonal changes positively correlated with the development of reproductive tissues [51]. It may be that in these algae one of the ABA functions is the regulation of sporo phyte transition from growth to the stage of propaga tion. ABA can be synthesized directly from isopentenyl pyrophosphate or it is produced due to the carotenoids degradation [55]. The first stage of the direct pathway of ABA biosynthesis is the synthesis of carotenoids. Like all isoprenoids, carotenoids are produced from isopentenyl pyrophosphate (IPP), which is synthe sized from pyruvate and glyceraldehyde3phosphate [55]. IPP is conversed into C20GGPP. The conver sion of GGPP into C40carotenoid phytoene is cata lyzed by phytoene synthase (PSY). Phytoene is con

versed into ζcarotene, lycopene, βcarotene, and fur ther until zeaxanthin. Phytoene desaturase (PDS) catalyzes conversion of phytoene into ζcarotene; it is an enzyme specific of the carotenoid synthesis. The first reaction assumed to be essential for ABA synthesis is a conversion of zeaxanthin into transviolaxanthin via twostep deepoxidation. However, this is a reac tion nonspecific for the synthesis of this phytohor mone because it is characteristic also for carotenoids formation. This process is catalyzed by zeaxanthin epoxidase (ZEP). Enzymes involved in the transvio laxanthin conversion into 9cisneoxanthin are not so far found. The following stage is the oxidative splitting of 9cisviolaxanthin and/or 9cisneoxanthin with the formation of xanthoxin, In this case, the key enzyme is 9cisepoxycarotenoid dioxygenase (NCED). There are three possible routes for ABA for mation from xanthoxin: via ABA aldehyde, xanthox inic acid, or abscisic alcohol. The last possibility is, as a rule, a minor one and operates usually only in the case of mutations preventing ABA synthesis along other pathways [56]. The conversion of xanthoxin into ABA aldehyde is catalyzed by the enzyme encoded in arabidopsis by the АВА2 gene; this enzyme is close to shortchain dehydrogenase/reductase (SDR). The enzyme conversing aldehyde into ABA manifesting sulfurase activity is encoded in arabidopsis by the ABA3 gene. Aldehyde oxidases (in arabidopsis these enzymes are encoded by AAO1–AAO4 genes) are also involved in the process of ABA synthesis. When these genes are inhibited, xanthoxin accumulates in plants, which permits for a suggestion that xanthoxin is a sub strate for aldehyde oxidase. Information about ABA synthesis in algae is rather fragmentary. Studying C. reinhardtii permits a sugges tion that neoxanthinmediated ABA synthesis oper ates in this unicellular green alga [57]. At the same time, studying the effects of salinity on the synthesis of this hormone in D. salina [58] indicated that ABA syn thesis occurred not via neoxanthin but via iononic derivatives using farnesyl diphosphate [11]. This path way of ABA synthesis may be characteristic of the members of Heterokontophyta and Rhodophyta where neoxanthin is absent [57]. Since the first stages of ABA biosynthesis coincide with those of carotenoids biosynthesis, it may be expected that similar enzymes catalyzing these reac


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geranylgeranyl pyrophosphate phytoene synthase (PSY)

phytoene phytoene desaturase(PDS)

ζcarotene lycopene βcarotene H3C CH3

CH3

CH3

CH3

HO

H3C

CH3

OH

CH3 H3C CH3

zeaxanthin zeaxanthinepoxidase (ZEP)

H3C CH3 O HO

CH3

CH3

OH

H3C

CH3

CH3

CH3 H3C CH3

antheroxanthin zeaxanthinepoxidase (ZEP)

H3C

H H

CH3

CH3

O HO

CH3

CH3

H3C

HO

CH3

H3C CH3

OH

O CH3

O

CH3

H H

transviolaxanthin

CH3 HH

OH

O

CH3

CH3 C

O

CH3 H3C CH3

HO

CH3

transneoxanthin

9cisepoxycarotenoid dioxygenase (NCED)

CH3

H3C CH3

C

O

O

xanthoxin H3C CH3

HO CH3

CH3

H3C CH3

9cisepoxycarotenoid dioxygenase (NCED)

CH3 H

9cisneoxanthin

CH3

H3C CH3

xanthoxin dehydrogenase (SDR)

O

O

xanthoxinic acid H3C CH3

O

CH3

OH

CH3HO

O

ABA aldehyde H3C CH3

aldehyde oxidase (AO)

CH3

OH CH3HO

CH3 H

CH3

OH O

O

abscisic acid

CH3

abscisic alcohol Fig. 5. ABA biosynthesis.


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HO CH3

CH3 H3C H3C

CH3

O HO

OH

CH3

CH3

O HO

CH3 H3C H3C

CH3

9cisviolaxanthin

HO CH3

OH

OH

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KISELEVA et al.

tions are present in a wide set of algae. As evident from Table 4, as the results of the analysis of A. thaliana amino acid sequences of enzymes PSY, PDS, and ZER, a substantial number of analogues were found in diverse algae; the low Evalues indicate their signifi cant similarity. At the same time, the homologues of enzymes specific for ABA biosynthesis, such as NCED and SDR, were also identified in various algae. In general, on the basis of information presented in Table 4, it may be suggested that, since algae comprise homologues of enzymes participating in ABA biosyn thesis, the pathway of this hormone biosynthesis in them is similar to that in higher plants. CONCLUSIONS Concluding the review of data concerning the enzymes of biosyntheses of auxin, gibberellins, cytoki nins, and ABA, it may be noted that available data are still too scarce for a comprehensive comparison between higher plants and algae and between different taxonomic groups of algae. Nevertheless, accumulated experimental data permit characterization of the main stages of the growth regulator syntheses. The most probable pathway of auxin biosynthesis is its formation through tryptamine and further through indole3ace tonitrile. Identified enzymes of GA biosynthesis oper ate during its final stages responsible for the formation of GA active forms. The participants of earlier stages require an additional identification. The homology of sequences involved in the key stage of cytokinin syn thesis argues for the similarity of this process in all plant organisms. And finally, essentially all enzymes of ABA synthesis were identified in algae belonging to different evolutionary groups. It might be that the wider spectrum of data concerning ABA metabolism is explained by intense studying of the mechanisms of algal adaptation to varying environmental conditions and/or deciphering the carotenoids synthesis. Thus, on the basis of the comparative analysis of homology between enzyme conserved domains, it may be con cluded that the homology between the pathways of the formation of abovelisted growth regulators arose dur ing the early stages of photoautotroph evolution. Dif ficulties in the identification of some enzyme (as in the case of gibberellins) may be explained by modification of biosynthetic routes during evolution. Certainly, the intensification of research in this field of plant biology is required for further deciphering of the stages of phy tohormone synthesis in different algal taxonomic groups. ACKNOWLEDGMENTS The authors are very grateful to Dr. V.V. Emel’yanov for valuable comments on the manuscript. This work was partially supported by the Russian Foundation for Basic Research (project no. 1004

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