How do cytokinins affect the cell?

ISSN 1021-4437, Russian Journal of Plant Physiology, 2009, Vol. 56, No. 2, pp. 268–290. © Pleiades Publishing, Ltd., 2009.Original Russian Text © G.A. Romanov, 2009, published in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 295–319.

268

1

HISTORY OF CYTOKININ DISCOVERY

Cytokinins (initially termed kinins

2

) were discov-ered in the laboratory of F. Skoog (United States) asearly as half-century ago [1]. However, only now, in the21th century, the basics of their molecular action startto be disclosed. The first artificially obtained cytokinin(kinetin) initially displayed its biological activity in theaxenic plant tissue culture as a compound promotingcell division and callus growth. Considering the factthat G. Haberlandt (Germany) supposed the occurrenceof such regulators in plants as early as in the beginningof the 20th century (see [2]), cytokinins were hastilyadded to the list of the basic plant hormones. It seemsnow evident that this was made somewhat prematurelybecause, at that time, the presence of cytokinin-likecompounds in plants and their intracellular biosynthe-sis were not strictly proven.

In fact, the first natural cytokinin zeatin was isolatedfrom maize (

Zea mays

) embryos by D. Letham et al.(Australia) [4, 5] almost ten years after the first syn-thetic cytokinin kinetin was obtained by Skoog et al.,whereas cytokinin biosynthesis in plants was proven byJapanese researchers only in 2001 [6, 7], i.e., already inthe 21th century.

1

After the materials of Chailakhyan Lecture delivered in 2006.

2

It would be more correctly to designate them as phytokinins, thusindicating both their functions and their belonging to the plantkingdom, as was suggested by Köhler and Conrad in 1966 [3]).

CYTOKININ STRUCTURE

Figure 1 presents the structures of the basic cytoki-nins. Naturally occurring cytokinins are relatively sim-ple adenine derivatives modified on the nitrogen atomat the position 6 of the six-member heterocycle. In mostcytokinins, a short aliphatic chain of the isopentenylresidue is attached to adenine at this position. Hor-mones, in which this chain is not modified, belong tothe group of isopentenyl-type (iP-type) cytokinins.However, there are enzymes in plants hydroxylating thealiphatic chain on its terminal carbon [8]. Cytokininswith hydroxylated aliphatic chain were termed zeatins(Z-type cytokinins). Two zeatin stereoisomers are pos-sible:

trans

-zeatin and

cis

-zeatin. In

trans

-zeatin, thehydroxyl group of the isopentenyl side chain is orientedaway from the adenine heterocycle.

trans

-zeatin isbelieved to be the most widespread and active plantcytokinin. In

cis

-zeatin, in contrast, the end hydroxylgroup is oriented toward the adenine ring, so that thehydrogen bond can be formed between the hydrogen ofthe OH-group and the nitrogen atom at position 1 of theadenine heterocycle (Fig. 1) [9]. Among other naturalcytokinins, there are such compounds as dihydrozeatin(with a reduced double bond in the side chain), 6-ben-zyladenine (BA), and topolin (an aromatic cytokininfirstly found in poplar plants with the 3-hydroxybenzylresidue instead of aliphatic chain [10]).

Cytokinins and their analogs can be produced byrelatively simple chemical synthesis. Therefore,numerous synthetic cytokinins differing in their activityhave been produced. In practice, most easily synthe-sized and stable cytokinins, such as kinetin, BA, and/orisopentenyladenine, are usually applied.

Along with derivatives of adenine, cytokinin activ-ity was detected in synthetic derivatives of phenylurea

How Do Cytokinins Affect the Cell?

1

G. A. Romanov

Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia;fax: 7 (495) 977-8018; e-mail: [email protected]

Received June 30, 2008

Abstract

—The lecture presents modern knowledge of the mechanisms of cytokinin perception and signaltransduction to the genes of primary and secondary responses. It also demonstrates the relations between therapid cytokinin-induced processes and cytokinin-induced physiological effects. The characteristics of the cyto-kinin regulatory system and its role in the control of plant growth and development are discussed.

Key words: cytokinin - receptor - signal transduction - primary response genes - control of gene expression -plant cell - long-distance communication

DOI:

10.1134/S1021443709020174

LECTURES

Abbreviations

: ARR—Arabidopsis response regulator; BA—ben-zyladenine; DZ—dihydrozeatin; GUS—

β

-glucuronidase; iP—isopentenyladenine; PLD—phospholipase D; Z—zeatin.

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

Vol. 56

No. 2

2009

HOW DO CYTOKININS AFFECT THE CELL? 269

[11]. Some phenylurea derivatives, such as N-phenyl-N'-(1,2,3-thidiazol-5-yl)urea (thidiazuron) or N-phe-nyl-N'-(2-chloro-4-pyridyl)urea (4PU-30), displayhigh physiological activity, similar to that of the mostactive natural cytokinins [12, 13].

CYTOKININ OCCURRENCE

Cytokinins are ubiquitous in the biosphere. They aredetected in essentially all organisms, including human,and also in water and soil. Cytokinin biosynthesisoccurs in higher plants, mosses, algae, some bacteria,fungi, and even some insects, plant parasites. Fromthese organisms, cytokinins penetrate into soil and var-ious water reservoirs, where they can also be found.Small amounts of cytokinins have been detected in ani-mals as well, although it is unlikely that they fulfill thehormonal function in them. Namely, some tRNA iso-forms contain isopentenylated at position 6 adeninebases near the anticodon [14]. These modified bases areusually

cis

-zeatin or isopentenyladenine as well as theirmethylthio derivatives. At tRNA breakdown, smallamounts of free cytokinins are released and might inprinciple affect the metabolism of the competent cells.However, plant tRNA evidently is not a major andessential cytokinin source [15]. The enzymes cata-lyzing cytokinin biosynthesis were also detected incytokinin-producing bacteria. As distinct from the freeiP-cytokinins, the hydroxylation of which in plantsresults in the formation of even more active

trans

-zeatin, the hydroxylation of iP within the tRNA pro-duces

cis

-zeatin displaying a low activity [14].The content of cytokinins in the plant internal liq-

uids (saps) is usually low, at the levels of nanomoles ortens of nanomoles. The xylem sap contains more cyto-

kinins of Z-type (usually their ribosides), whereas thephloem sap contains more iP cytokinins [16].

CYTOKININ PHYSIOLOGICAL ACTION

Cytokinins influence numerous growth and devel-opmental processes in plants [17–19]; the character oftheir action depends on their concentrations. One of themost important cytokinin effects is stimulation of celldivision, due to which cytokinins were firstly identified.Many cytokinin effects manifesting at the organismlevel, for example, the formation of vascular tissues inthe roots, are determined just by cytokinin capability ofstimulation of definite precursor cell proliferation.

Plant growth along the longitudinal axis is deter-mined by functioning of the shoot and root apical mer-istems, which are under the control of cytokinins andauxins [2, 20, 21]. At physiological concentrations,cytokinins activate shoot apical meristem and shootgrowth but suppress root meristem and root growth[22, 23]. Cytokinins promote not only cell division butalso cell expansion, especially in the leaves and cotyle-dons. Along with auxins, cytokinins determine theplant phenotype to a great degree: they stimulategrowth of lateral shoots but suppress lateral root initia-tion. Together with auxins and gibberellins, cytokininsare implicated in the regulation of source–sink rela-tions, enhancing sink capacity of particular organs ortheir parts. In the culture of plant calli or tissues, cyto-kinins induce shoot formation. Cytokinins facilitatephotosynthesis via activation of chloroplast differentia-tion frequently occurring even in darkness. In manyplants, cytokinins retard senescence of leaves, bothdetached and on intact plants. In plants capable of sym-biosis with nitrogen-fixing bacteria, cytokinins and the

iP tZ cZ

BA

N

NH

N

HN O

N

N

N NH

N

HN

N

N NH

N

HNOH

N

N NH

N

HN

OH

N

N NH

N

HN

NH

NH N

NS

O

Kin

TD

12 3 4

56

78

9

Fig. 1.

Structures of typical cytokinins.BA—6-benzyladenine; Kin—kinetin; iP—isopentenyladenine; cZ—

cis

-zeatin; tZ—

trans

-zeatin; TD—thidiazuron.

270


Vol. 56

No. 2

2009

ROMANOV

systems of their perception are required for triggeringsymbiotic nodule formation [24, 25]. In many plants,cytokinins stimulate seed germination and pigment for-mation. In mosses, such as

Funaria

, cytokinin treat-ment of protonema induces bud development, produc-ing a leafy sporophyte [26]. In contrast, at high concen-trations, cytokinins inhibit growth and can even haveapoptotic effects [27, 28]: they can suppress rootgrowth, cell culture growth, and even induce leafabscission.

The established cytokinin effects served as a basisfor the development of bioassays for these phytohor-mones [29].

Cytokinin physiological effects at the cellular levelare summarized below:

(a) Stimulation of cell division;(b) Activation of metabolite attraction (sink effect);(c) Differentiation of plastids;(d) Increasing the cell volume;(e) Biosynthesis of pigments;(f) Retardation of senescence (at low concentra-

tions);(g) Induction of apoptosis (at high concentrations);(h) Shoot differentiation in calli.

CYTOKININ PRODUCTION AND APPLICATION

Many renowned companies, including well-knowninternational corporations, such as Sigma, Dushefa,Fluka, and Calbiochem, are engaged in cytokinin pro-duction. The firm OlChemim (Czech Republic) is spe-cialized to produce various forms of super pure cytoki-nins. Cytokinins are important constituents of the planttissue culturing media. They are widely applied in stud-ies with plant tissue cultures, micropropagation of valu-able plants, and production of transgenic plants. Cyto-kinins and their analogs are used for the formation ofsampling crowns, for the shift from male to female sexin some vegetables (cucumber), for senescence retarda-tion of cut flowers and vegetables, and for seed dor-mancy release [30]. Apoptotic effect of synthetic cyto-kinins (thidiazuron) is applied for cotton defoliation[31], which facilitates cotton harvesting. A possibilityof cytokinin usage for improving cereal yield, rice forexample, was proven experimentally. Some positiveeffects of cytokinins on the yield of some fruit trees,apple in particular, were reported. The involvement ofcytokinins in improving plant tolerance to abioticstresses, like drought, was demonstrated, that makesvery promising the usage of these phytohormones inthis field.

In recent years, cytokinins are used as constituentsof cosmetic creams. According to the data obtained byfirms-producers, the long-term application of cytoki-nins improves the skin structure and reduces the signsof aging. However, it is still before time to considerthese results as reliable. Along with these studies,

investigations in the last decade clearly demonstratedantiproliferative effects of some cytokinins and theiranalogs on animal tumor cells; therefore, these com-pounds were suggested for usage as anticancer prepara-tions [32–34]. Some cytokinin-like compounds arealready used in medicinal practice.

MECHANISM OF CYTOKININ ACTION

Cytokinin-Sensitive Genes

In the course of many decades after cytokinin dis-covery, it was still unclear what an intracellular targetfor their action is, whether they influence gene activitydirectly, and when they do, what the mechanism ofcytokinin signal transduction is. Certainly, these topicswere vividly discussed in scientific circles; however,the persuading data about the occurrence of genes ofprimary response to cytokinins and those concerningcytokinin receptors appeared relatively recently.In 1998, in the laboratories of J. Kieber (United States)and T. Sugiyama (Japan), the genes directly activatedby cytokinin were discovered in Arabidopsis and maize[35, 36]. One of the firstly identified cytokinin-sensitivegenes was the gene encoding so-called response regu-lator, which was designated as

ARR5

(from

ArabidopsisResponse Regulator 5

). The transcription of this genewas rapidly activated by cytokinin, and this activationwas insensitive to protein synthesis inhibitor cyclohex-imide, i.e., it did not require the synthesis of any pro-tein. Thus, the

ARR5

gene is a primary target for cyto-kinin, which implies that the promoter of this gene issensitive to cytokinin. In fact, the isolated promoter ofthe

ARR5

gene fused with the reporter

GUS

gene ren-dered cytokinin dependency on the expression of thelatter gene in plants [37].

For quantification of particular gene expression, weadapted the model system on the basis of Arabidopsisseedlings in water culture [38]. We used transgenicArabidopsis plants with the promoter of the cytokinin-sensitive

ARR5

gene fused with the reporter

GUS

gene[37]. This construct permits monitoring promoterexpression and determining its localization in plant tis-sues and organs. In the absence of exogenous cytoki-nins, expression of the

ARR5

promoter was observedmainly at the sites of active cytokinin synthesis (Fig. 2),e.g. in the root tip (the main site) and in the shoot apicalmeristem. Substantial staining was also observed in tis-sues along the root vascular system, which evidentlyreflects the pathway of cytokinin xylem transport fromthe root into the stem. However, most seedling tissuesremained unstained.

Plant treatment with cytokinin resulted in the activa-tion of the reporter gene in all plant parts, which waswell detectable at the histochemical level by GUS-staining (Fig. 2). Quantitative data about temporal anddose dependences of GUS activation by cytokinins andabout specificity of this activation are presented in Fig. 3.These data show that the strongest activation of the


Vol. 56

No. 2

2009


ARR5

promoter occurred after treatment with 5

µ

M BA(Fig. 3a); activation was induced only by cytokinins butnot by adenine or other phytohormones and growth reg-ulators (Fig. 3b). GUS activation could not be detecteduntil after a certain lag-period (Fig. 3c). During thisperiod, some changes in the transcription rate couldoccur. Indeed, direct measurements of

ARR5

mRNA bynorthern-blot showed its rapid accumulation duringlag-period. In our experiments, the highest mRNAamount was attained within 45 min of cytokinin treat-ment; however, as soon as in 15 min, the level of induc-tion reached 80% of the highest one (Fig. 4a). Theresults of our experiments are in good agreement withthe results of similar experiments performed with Ara-bidopsis seedlings grown and treated with cytokininunder different conditions [35, 37]. All these resultspermitted a supposition that other primary responsegenes would respond to cytokinin as rapidly in thismodel system. Indeed, the analysis of changes in thetranscription rate revealed other Arabidopsis genes dis-playing similar expression profiles following the cyto-kinin treatment (Fig. 4b, [37]). A comparison of thetranscript levels of the

ARR5

gene per se and the

GUS

gene under the control of the

ARR5

promoter showedthat cytokinin induced their accumulation similarly(Fig. 4c, [40]). Also, the effects of other active com-pounds on the accumulation of these transcripts wereessentially similar. Since transcripts of the foreign

GUS

gene are hardly subjected to any specific action in theplant cell, we can assume that the amount of the

ARR5

mRNA is controlled by cytokinins only at the level oftranscription (more precisely, transcription activation).Otherwise, some cytokinin-dependent posttranscrip-tional modifications of the

ARR5

gene transcriptswould change the pattern of this gene induction as com-pared to the foreign

GUS

gene.

On the basis of kinetic data on the

ARR5

gene acti-vation, we attempted to reveal the entire spectrum ofthe genes of primary response to cytokinin [41]. To thisend, we used ATH1 microchips (Affymetrix, UnitedStates) with fixed oligonucleotides of nearly 24000Arabidopsis genes, i.e., almost entire genome of thisplant [42]. To identify so-called “early” genes, i.e., pri-mary response genes, Arabidopsis seedlings weretreated with cytokinin at its optimum concentration for15 min. To reveal the “late” genes responding to cytokinin,the seedling were incubated with hormone for 2 h. There-after, mRNA was isolated from cytokinin-treated andcontrol seedlings; this RNA served as a template for thesynthesis of fluorescent-labeled cDNAs, which contentwas analyzed using microchips. In such a way, onecould evaluate the level of particular transcripts.

Approximately 11500 genes, i.e., nearly half of allthe genes represented on the microchip, manifestedtheir expression. For the first 15 min of cytokinin treatment,approximately 80 genes displayed more than 1.8-foldchanges in the expression level. This is less than 1% oftotal number of active genes. Among these 80 genes,70 were activated by cytokinin, whereas 11 genes wererepressed. Such a low percentage of primary responsegenes is characteristic not only of Arabidopsis. Up tothat time, we have already studied rapid cytokinineffects on tobacco genome activity using methods ofDifferential Display and Representational DifferenceAnalysis. In this case, the proportion of cytokinin-sen-sitive early genes was also less than 1% of the totalnumber of genes [43, 44].

What can we say about Arabidopsis genes directlyactivated by cytokinin? Among them, there are sevenresponse regulator genes closely related to the

ARR5

gene (Table 1). These genes influence the functioningof the two-component system of signal transduction wewill discuss later. A great part of the genes directly con-trolled by cytokinins are the genes for transcription fac-tors and other regulatory proteins (Table 1). Transcrip-tion factors encoded by these genes compriseAPETALA2 (AP2), the HAT22 factor includinghomeodomain and leucine zipper, the bHLH factorwith a helix–loop–helix motif, the Myb transcriptionfactor, the factor with a typical NAC domain, and alsothe protein with zinc fingers. These protein factors dif-fer in their structure and could recognize differentnucleotide sequences, i.e., interact with promoters ofdiverse genes [45, 46]. A rapid cytokinin induction ofvarious transcription factors creates conditions for mas-sive and coordinated changes in the expression of thefollowing set of genes (so-called late genes), directly orindirectly controlled by these factors.

Along with the genes for transcription factors, cyto-kinin-dependent genes for regulatory proteins display-ing protein kinase or phosphatase activity (serine/thre-onine protein kinase, CBL-interacting protein kinase 3,putative tyrosine phosphatase) were found among earlyresponse genes (Table 1). These proteins could be

Control Treatment withcytokinin

Fig. 2.

GUS activity in control and 5

µ

M BA-treated three-day-old etiolated seedlings of transgenic

ARR5::GUS

Arabidopsis.Histochemical GUS staining was performed after [39].Arrows indicate zones of staining in control seedlings.

272


Vol. 56

No. 2

2009

ROMANOV

GU

S sp

ecifi

c ac

tivity

, uni

ts

×

10

–3

0 2 4 60

2

4 (c)

Control

10

–12

10

–10

0

8

12 (‡)

Control

4

10

–8

10

–6

Cyt

okin

ins

200 400 600 800

Compound

0

Gro

wth

Gro

wth

regu

lato

rsre

gula

tors

+ B

A

H

2

OBAKin

t

ZiPTDiPiPRAdoAde

H

2

OBANAA2,4-DABAGA

3

BLJASASpm

H

2

OBANAA2,4-DABAGA

3

BLJASASpm

(b)

BA concentration, M

Time, h GUS specific activity, % of control

Fig. 3.

Quantitative characteristics of

ARR5

promoter induction in

ARR5::GUS

Arabidopsis seedlings treated with 5

µ

M BA.GUS activity was determined fluorometrically [39].(a) Dose dependence; (b) hormonal specificity; (c) kinetics of activation.ABA—abscisic acid; Ade—adenine; Ado—adenosine; BA—6-benzyladenine; BL—epibrassinolide; 2,4-D—2,4-dichlorophe-noxyacetic acid; GA

3

—gibberellic acid; iP—isopentenyladenine; iPR—isopentenyladenosine; JA—jasmonic acid; Kin—kinetin;NAA—naphtylacetic acid; SA—salicylic acid; Spm—spermine; TD—thidiazuron;

tZ

—

trans

-zeatin. All compounds were at con-centration of 5

µ

M, except SA and Spm (50

µ

M).

involved in the modulation of transcription factor activ-ity and/or activity of signal transduction proteins, thusaffecting the transcription rate of a great number ofgenes. Another group of primary response genes com-prises genes of directed protein degradation. This sys-tem attracts now a considerable attention in connectionwith the mechanisms of hormonal regulation in plants.It has been found recently that the molecular mecha-nisms of auxin and gibberellin action are tightly con-nected with degradation of proteins, the repressors ofhormone-dependent gene transcription [47–49].Involvement of the 26S proteasome subunit in cytoki-nin signaling has been reported as well [50]. In this con-nection, the observed changes in the expression of fourgenes involved in the directed protein degradation(E2, ubiquitin-conjugating enzyme 16, lectin-like pro-tein of F-box, ATP-dependent subunit of protease, andSKP1 interacting with F-box protein) could result in theshift in the content of transcription factors in thenucleus and, as a consequence, in changes in the rate ofthe gene transcription controlled by these factors.

Thus, the functional analysis has revealed that aconsiderable portion of genes of the primary responseto cytokinin is involved in the control of gene expres-sion. We have also analyzed the pattern of Arabidopsisgene expression after a longer period of hormone treat-

ment. This pattern differed substantially from that ofthe early response. Direct measurements of the levels ofgene expression following 2 h of cytokinin treatmentrevealed a great number of genes, the transcription ofwhich has changed by this time under the influence ofhormone. It appeared that there were more than 1500 ofsuch late genes responding to cytokinin, which isapproximately by 20 times more than the primaryresponse genes. Interestingly, most late genes wererepressed by cytokinins, whereas among early genes,those activated by cytokinins prevailed. This meansthat, at the level of structural gene transcription, 2-htreatment with cytokinin induced not so much genomeactivation but rather its repression. Therefore, althoughcytokinins are believed to be hormones-stimulators,cytokinin-induced activation of distinct, although veryimportant, metabolic pathways is not directly corre-lated with the degree of entire genome activation: thecausal relationships here are much more complex. Itseems likely that the activation of some dominatingmetabolic processes in the cell requires inevitably sup-pression of many other processes, i.e., a definite reduc-tion of metabolic diversity.

It has been supposed earlier that phytohormones,cytokinins in particular, change genome activitythrough transcriptional cascades [51], when expression


Vol. 56

No. 2

2009


100

0H

2

O

ARR5

NMMA

100

0 2

% o

f m

axim

um

Time of BA treatment, h

1 3 4 5

806040

ARR5

20

(a) (b)

Time of BA treatment

0 15

′

45

′

2 h 4 h 8 h 24 h

ARR5

Actin

2

0 15 45 120

Time of BA treatment, min

At3g03850 (

ARR5

)

At1g03850 dlutaredoxin

At2g26980 (

CIPK3

)

At3g16770 (AP2-fam.)

At3g18780 (

‡ctin 2)

(c)

BA BA+ BA+1-but

50

H2O

GUS

NMMABA BA+ BA+

1-but

Tra

nscr

ipt

con

tent

, %Gene

Fig. 4. Effect of 5 µM BA on the level of transcripts of cytokinin-sensitive genes in Arabidopsis seedlings.(a) Kinetics of ARR5 transcript accumulation (top—Northern-blot analysis; bottom—quantitative results); (b) kinetics of transcriptaccumulation of several cytokinin-sensitive genes; (c) the content of the ARR5 (left) and ARR5 promoter-driven GUS (right) genetranscripts in 35 min of BA treatment; some of the treatments were performed in the presence of various inhibitors. NMMA(5 mM)—an inhibitor of NO synthase; 1-but—butan-1-ol (1%), an inhibitor of phosphatidic acid production by PLD.

of the small number of rapidly activated genes encod-ing transcription factors and/or their modulators resultsfurther in the changes of expression of a great set ofgenes involved in the realization of global physiologi-cal programs. The results of our and several analogousinvestigations are in good agreement with this supposi-tion. In particular, Rashotte et al. [52] used microchipscontaining DNA replica of a great number (althoughnot a complete set) of Arabidopsis genes (~8300 genes)and found only several tens of genes, the transcriptionof which was consistently changed (increased ordecreased) in response to cytokinin treatment. A greatportion of rapidly activated genes continued to respondto cytokinin even in the presence of cycloheximide(blocking protein biosynthesis). This permitted theauthors to refer to these early genes as to the genes ofprimary response to cytokinin. Among them, manygenes encoding proteins-regulators of transcriptionwere detected, including those belonging to the AP2transcription factors. Further studies in this laboratory[53] have ascertained that cytokinin not only activatedthe genes encoding AP2 transcription factors but alsostimulated a rapid translocation of these factors into thenucleus.

Cytokinin Receptors; Receptor Mutants

How does the cytokinin signal reach its primaryintracellular target, i.e., primary response genes?In 1996, Japanese researcher T. Kakimoto [54] sup-posed for the first time that sensor histidine kinasemight be a cytokinin receptor. In 2001, this researcherand independently T. Mizuno and his coworkers provedthat sensor histidine kinase CRE1/AHK4 is a cytokininreceptor in Arabidopsis [55, 56]. At present it is com-monly accepted that in Arabidopsis there are three sen-sor histidine kinases closely related in their structure,which function as cytokinin receptors: CRE1/AHK4,AHK3, and AHK2. Structurally similar cytokininreceptors were identified in plant species taxonomicallydistinct from Arabidopsis, such as maize and rice [57–59]. In Arabidopsis with all three cytokinin receptorsinactivated, plant sensitivity to this hormone was lost,and primary response genes ceased to respond to cyto-kinin [60–62]. Such triple mutants are tiny plantletswith a low viability and mainly sterile. Interestingly, indouble and especially in triple receptor mutants, a sharpincrease in the Z-type cytokinins has been detected.However, no signs of excessive cytokinin action wereobserved [62], thus indicating that Arabidopsis plants

274

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 56 No. 2 2009

ROMANOV

Table 1. Arabidopsis genes of primary response to cytokinin related to transcription regulation

AGI id * Function

Response regulators

At3g48100 response regulator 5 (ARR5), A type







Transcription factors

At1g76410 RING protein with “zinc fingers” (putative)

At5g13330 AP2 domain containing protein (putative)

At4g37790 homeobox protein HAT 22

At2g18300 bHLH transcription factor (bHLH064 putative)

At3g16770 AP2 domain containing transcription factor RAP2.3

At1g08810 transcription factor from myb family

At4g26150 GATA protein with “zinc fingers”

Regulatory enzymes

At4g23290 putative serine/threonine protein kinase

At2g26980 CBL-interacting protein kinase 3 (CIPK3)

At5g16480 putative tyrosine phosphatase

At1g75440 E2, ubiquitin-conjugating enzyme 16 (UBC16)

At3g61060 protein of F-box (lectin-like)

clpP ATP-dependent subunit of protease

At2g02310 protein of F-box (SKP1-interacting partner)

* Direction of changes: increase; decrease.



do not contain any other cytokinin receptors apart fromthose three sensor histidine kinases.

Figure 5 shows the structure of the CRE1/AHK4cytokinin receptor. This sensor histidine kinase consistsof several domains. At its N-terminal region, twohydrophobic segments that determine receptor mem-brane localization are positioned. It was initiallybelieved that the cytokinin receptors are localized in theplasmalemma. In that case, the region of the polypep-tide chain positioned between hydrophobic segmentsshould be localized outside the plasmalemma, i.e., out-side the cell. This extracellular part is homologous tothe ligand-binding domains found in many other recep-tors of prokaryotes, single-celled eukaryotes, andplants; it is so-called CHASE (Cyclase/Histidinekinase-Associated Sensing Extracellular) domain [63,64]. As will be shown below, it is just the CHASEdomain that is responsible for recognition of and bind-ing the cytokinins. The central part of the protein,located inside the cell, comprises several domains,one of which displays histidine kinase activity. At theedge of this domain, a so-called conserved histidineresidue (i.e., histidine within the conserved sequence-ATVSHEIRTP-) is located; it can be phosphorylatedby using ATP as the phospho-donor. In the C-terminalregion, a typical receiver domain with the conservedaspartate residue is positioned; it can receive phosphatefrom phosphohistidine. According to the currentknowledge, when a cytokinin interacts with its receptor,the latter produces a dimer, and its histidine kinaseactivity is induced. As a result, the conserved histidineresidue within histidine kinase (transmitter) domain isphosphorylated, and this high-energy phosphoryl groupis then transferred to the conserved aspartate residue ofthe receiver domain on the C-terminus of the protein(Fig. 5). Thus, during hormone perception, the eventsoccurring within the protein start at its N-terminus andprogress toward its C-terminus: hormone bindingoccurs at the N-terminus, phosphorylation occurs in thecentral part of the molecule, and this phosphoryl groupis then transferred to the region near the C-terminus.Arabidopsis cytokinin receptors AHK2 and AHK3 areclose in their structures to the CRE1/AHK4 receptor(identical in amino acid sequence by 52–54%), but they

evidently differ in the number of transmembrane seg-ments adjacent to the CHASE domain (see below).

In order to study the properties of cytokinin recep-tors in more detail, we used a model system based ontransgenic bacteria expressing particular cytokininreceptors [56, 65, 66]. In these bacteria (Escherichiacoli), one of their own histidine kinases (RcsC) close inits structure to Arabidopsis histidine kinases wasabsent. In the intact bacterial cell, this bacterial histi-dine kinase RcsC transfers a signal to the promoter ofthe cps-operon. It turned out that ArabidopsisCRE1/AHK4 sensor histidine kinase, when expressedin bacteria, could replace the absent bacterial histidinekinase RcsC and transfer its signal to the cps-operon,using the bacterial pathway of signal transduction.However, it is of importance that this signal transduc-tion from CRE1/AHK4 operated only in the presenceof active cytokinins [56, 65, 66]. This shows that a plantreceptor in bacteria is in the functionally active stateand is capable of cytokinin recognition. To better mon-itor the cytokinin receptor activity, the reporter lacZ(galactosidase) gene under the control of the same cps-promoter was inserted into the bacterial genome [56](Fig. 6). Thus, a simple testing of galactosidase activitypermitted us to evaluate functional activity of the cyto-kinin receptor in bacteria. On the basis of these mea-surements, we compared the efficiency of a great num-ber of cytokinins and structurally close ligands upontreatment bacteria expressing two different cytokininreceptors, AHK3 or CRE1/AHK4 [66]. Although vari-ous cytokinins display generally similar relative activi-ties in both test-systems, it turned out that the observedresponses somewhat differed depending on the cytoki-nin applied. In particular, it was true for compounds,such as dihydrozeatin, cis-zeatin, and ribosides. Thus,we could suppose definite differences in the ligandspecificity between closely related cytokinin receptorsfrom one and the same plant species [66].

The step of specific hormone–receptor interaction isthe key one for hormonal signal perception and itstransduction inside the cell. Therefore, it is of impor-tance to know characteristics of this interaction [67].For more precise analysis of ligand specificity of cyto-kinin receptors and their principal properties, we ana-

Fig. 5. Domain structure of the CRE1/AHK4 cytokinin receptor of Arabidopsis.Protein domains: TM—transmembrane; LB—ligand-binding (CHASE); HK—histidine kinase; Ac—acceptor. Ck—cytokinins;H—conserved histidine residue; D—conserved aspartate residue; N and C—N- and C-termini of the protein. Arrows in the middleand on the right indicate the site of phosphorylation and transfer of high-energy phosphate (�P).

N ë

ATP

CHASE H

TMLBíå HK

~P

D

Ck

Äc

276


ROMANOV

RcsC-receptor

YojN

Äçä-receptor

Äçä

plasmidPINIII ∆EH

Nucleoid

RcsB

cps

LacZ

E. coli

~P

~P

Fig. 6. Schematic representation of transgenic bacterial cell (E. coli), expressing cytokinin receptors and sensitive to cytokinins.YoiN—transporter of active phosphate (~P) to the transcription factor RscB, the regulator of the cps promoter; RcsC—hybrid his-tidine kinase of the bacterium, analogous to the AHK receptor.

lyzed receptor capability of specific binding of the cor-responding ligands. We performed these studies usingthe same bacterial strains expressing individual cytoki-nin receptors of Arabidopsis. The receptors expressedin transgenic bacteria were functionally active and werepresent at the concentrations sufficient for measuringtheir hormone-binding parameters. Since E. coli has nosystems for specific binding and metabolizing of cyto-kinins of its own, the side effects do not seem veryprobable.

The results obtained with highly radioactive 3H-trans-zeatin showed substantial and significant differencebetween total and nonspecific binding of the labeledzeatin, indicative of its specific binding by the livingtransgenic bacteria. This specific binding depended onthe receptor type and the bacterial clone used; it wasabsent from the control clones expressing empty vectorand present only in the clones expressing plant cytoki-nin receptors (Fig. 7a) [68, 69]. Cytokinin binding washighly cytokinin-specific: many other phytohormoneswere tested along with cytokinins, but cytokinins werethe only ones that interacted with these receptors (Fig. 7b).It is of interest that physiological concentrations of bivalentcations (Ca2+, Mg2+, and Mn2+) did not essentially affectcytokinin binding by either receptor.

Binding of cytokinins to their receptors was usuallyeasily reversible. Once the complex between labeledzeatin and the receptor was formed, the addition of coldzeatin caused rapid label replacement. In the case ofCRE1/AHK4, the replacement of labeled hormone by

unlabeled one occurred to the greater extent comparedto the AHK3 receptor. The receptor affinity for zeatinwas determined by addition of various concentrationsof unlabeled hormone to the constant amount of itsradioactive counterpart (Fig. 8) [67–69]. Linear depen-dences were obtained on Scatchard plots, indicating thepresence of the single binding sites without any signs ofcooperativity of this binding. The calculated dissocia-tion constants (KD) for zeatin were 1–2 nM for AHK3and 2–4 nM for CRE1/AHK4, which is characteristicof high-affinity hormone–receptor interactions. More-over, these KD values correspond to already determinedcytokinin concentrations in planta. Endogenous con-centrations of trans-zeatin and isopentenyladenine inwild type Arabidopsis plants, recently determined indifferent laboratories [62, 70], varied from 0.7 to2.5 nM, which is close to the measured KD values.

We studied ligand specificity of interaction on thebasis of the ability of cytokinins and their analogs tocompete with labeled zeatin for binding to the receptor[68, 69] by using classical radioligand method (Fig. 9)[67]. It turned out that both receptors displayed highaffinity for trans-zeatin (Fig. 9a). All modifications oftrans-zeatin, even the minor ones, substantiallyreduced its capability of receptor binding. The removalof zeatin aliphatic side chain, leading to the formationof adenine, resulted in an almost complete loss of itscapacity to interact with the receptor. Glucosylation ofthis side chain exerted similar effect. Isomerization ofthe side chain, i.e., relocation of OH-group from trans-



into cis-position sharply reduced molecule bindingwith the receptor, although this binding was still signif-icant. Surprisingly, BA, adenine with the aromatic sideresidue, which was traditionally considered as one ofthe most potent cytokinins, demonstrated only a mod-erate binding activity.

Most importantly, we found clear differencesbetween AHK3 and CRE1/AHK4 receptors withrespect to their ligand specificity [69]. Cytokinins ofthe iP-type, isopentenyladenine and its riboside, boundto the CRE1/AHK4 receptor with a rather high affinitybut relatively weakly to the AHK3. On the other hand,DZ, a product of hydrogenation of a double bond in theside chain, interacted much stronger with the AHK3than with CRE1/AHK4. Testing these compounds in awide range of concentrations clearly demonstrated dif-ferences between receptors in specificity of ligandbinding (Fig. 9b). The label replacement curves for iPand DZ mirrored each other in the case of AHK3 andCRE1/AHK4 receptors. On the basis of the competitioncurves obtained for each receptor, we constructed therows of affinity of ligand–protein interactions (Fig. 9c).It is worth mentioning that these affinity rows wereclose to the rows of hormonal activity determined in thefunctional test of promoter activation by the same cyto-kinins. The affinity rows obtained by the radioligandmethod are also well correlated with physiological

activities of these phytohormones established earlier innumerous bioassays. Our recent results indicated thatligand specificity of binding is a highly conservativefeature, even in receptors with mutations in the CHASEdomain [71]. All these results show that the data onreceptor ligand specificity obtained by radioligandmethod in the bacterial system reflect correctly theirfunctional properties in the plant cell.

Replacement curves obtained enabled us to calcu-late the affinity constants of various cytokinins for thereceptors (Table 2). It is evident that these constantshave a wide range of values and could differ more than1000-fold for various compounds. The affinity for somemolecules, e.g. iP and DZ, substantially differedbetween receptors, the differences attaining even anorder of magnitude.

Naturally, these differences depend on the structureof the hormone-binding center in the molecule of thereceptor protein. So far we could not describe the cyto-kinin-binding sites of the receptors, although the factthat just the CHASE domain forms the cytokinin-bind-ing site is established reliably. Several receptor muta-tions that reduce or suppress cytokinin binding areknown up to now, all of them being positioned withinthe CHASE domain (Fig. 10) [72]. If the entire cyto-plasmic part of the cytokinin receptor (CRE1/AHK4 inthis case) is deleted, the remaining N-terminal fragment

Bou

nd 3 H

-hor

mon

e, c

pm

PINIII-clones0

200

400

600

800

1000(a) 1

23

Äçä4-clones Äçä3-clones(control)

0

200

400

600

800

(b)

AHK3

tZ IAA 2,4-D ABA GA3 cAMPSpm Control

0

500

1000

1500

2000

CRE1/AHK4

Fig. 7. Characteristics of highly-radioactive 3H-trans-zeatin binding by intact transgenic bacteria (E. coli) expressing cytokininreceptors of Arabidopsis, AHK3 and CRE1/AHK4.(a) (1) Total, (2) nonspecific, and (3) specific binding in AHK-expressing and control clones; (b) hormonal specificity of receptors.ABA—abscisic acid; cAMP—cyclic AMP; 2,4-D—2,4-dichlorophenoxyacetic acid; GA3—gibberellic acid; IAA—indole-3-aceticacid; Spm—spermine; tZ—trans-zeatin. Each compound administered at the concentration of 8.6 µM, except Spm (17.3 µM).

278


ROMANOV

of the protein containing the CHASE domain was stillable to bind cytokinin at the level close to that of theintact receptor, when expressed in E. coli. In contrast,the isolated C-terminal fragment was essentially devoidof hormone binding ability. All these data indicateunambiguously that the CHASE domain is a hormone-binding region of the protein. The precise structure ofthe binding site would be evidently deciphered uponcrystallization of the receptor protein or its CHASE-containing fragment.

Now we can only propose more or less crediblespeculations about changes in the receptor structureupon binding of the cytokinin. As evident from above,the fact of high affinity binding of the hormone by thereceptor is established quite reliably. Thus, the simplesthypothesis of cytokinin action on the receptor is that,upon the formation of the complex between the hor-mone and its binding site, as in each high-affinity inter-action, free energy of the complex increases, and it isused for changes in the structure of the receptor protein,possibly, the removal of some steric barriers for dimerformation. A further step is likely to be a dimerizationof the hormone–receptor complexes; proteins, the con-stituents of this dimer could use another one (more pre-cisely, conserved histidine of another protein) as a sub-

strate for the kinase activity of their own histidinekinase domain. Thereafter, the phosphorylated histi-dine transfers its activated phosphate to the conservedaspartate residue at the C-terminal region of the protein.This activated phosphate serves as a specific signallabel in the cytokinin signal transduction inside the cell.However, how does this “hot” phosphate reach thenucleus and activate there the primary response genes?

Transduction and Attenuation of the Cytokinin Signal

Cytokinin receptors, being sensor histidine kinasesin their structure, belong to the proteins of so-called two-component system of signal transduction (see Fig. 5).Such systems are widely spread and well studied inbacteria [73]. The classical two-component systemcomprises sensor histidine kinase (receptor) andresponse regulator (transcription factor). Under theinfluence of specific signal, receptor produces a dimer;it is phosphorylated and then transfers its “hot” phos-phate to the aspartate residue of the response regulator.The latter comprises a DNA-binding domain; its acti-vated form binds to a definite DNA sequence in the pro-moter of the corresponding gene or operon, thus acti-vating it or, in contrast, repressing it. Transcription fac-tors close in their structure to bacterial responseregulators were found in Arabidopsis and other plantsas well (Fig. 11) [58, 59, 74–76].

However, these factors, like other chromatin-associ-ated proteins, are localized in the nucleus, whereas sen-sor histidine kinases, according to recent data, areincorporated in the cell membranes, both plasmalemmaand internal membranes [71, 77]. Therefore, in orderthe activated phosphate could reach its final target, e.g.transcription factors of the two-component system (inArabidopsis, these factors are called ARR-B), it shouldbe transferred from either of these membranes to thenucleus by some transporter. In Arabidopsis, five suchtransporters were identified; they were called phospho-transmitters (Fig. 11a). Such transmitters get an acti-vated phosphate from the receptor aspartate and attachit to the conserved histidine in their molecule; thereaf-ter, they are translocated to the nucleus, where this acti-vated phosphate is further transferred to the responseregulator of type B. ARR-B comprise a so-calledGARP domain capable of site-specific binding to DNA.Upon phosphorylation, ARR-B acquires an ability tobind to DNA and interact with the specific consensusregions within the gene promoter (Fig. 11b), which aresupposed to include short AT-rich sequences [52, 74,78, 79]. Recently, it has been established that a consen-sus sequence of the transcription factor ARR1 is a pal-indrome or pseudopalindrome of the AAGATC/TTTtype [80]. Such a structure of the recognition siteimplies DNA interaction with the ARR-B dimers.

As a result of phosphorylation of ARR-B transcrip-tion factors, several tens of genes, the promoters ofwhich contain nucleotide sequences recognizable by

5 10 15 20Bound 3H-trans-zeatin, nM

0

0.5

1.0

1.5

2.0

2.5

25

0.08

0.05

0.04

0.03

0.02

0.01

0.12 0.16 0.200.040

CRE1/AHK4

KD = 3.1 nåSpec

ifica

lly b

ound

3 ç-z

eatin

, cpm

× 1

0–2

Bou

nd/u

nbou

nd

0

2

4

6

8

0.0250.020

0.015

0.010

0.005

0.04 0.060.020

AHK3

KD = 1.8 nå

Bound 3H-zeatin, nM0.07

Bound 3H-zeatin, nM

Bou

nd/u

nbou

nd

Fig. 8. Dose-dependence of 3H-trans-zeatin interactionwith cytokinin receptors AHK3 and CRE1/AHK4 and itsanalysis in Scatchard plot.



CRE1/AHK4

10 100 1000

Äçä3

20

10 100 1000

40

60

80

100

Bou

nd 3 H

-hor

mon

e, %

(a)

0.4

0.1 1 10

0.8

1.2

1.6

Bou

nd 3 H

-hor

mon

e,

(b)

cpm

× 1

0–3

1 2

3

100 10000.01 0.1 1 10

1 3

2

100 10000.01

tZOGAdeAcZiPRBAcZiPDZtZRTDtZ

Concentration of unlabeled ligand, nM

(c)

10000

Fig. 9. Ligand specificity of the cytokinin receptors AHK3 and CRE1/AHK4.(a, b) Competition curves for 3H-trans-zeatin replacement with unlabeled ligands; (c) rows of cytokinin affinity for receptors. Ade—adenine; AcZ—acetyl-O-zeatin; BA—6-benzyladenine; cZ—cis-zeatin; DZ—dihydrozeatin; iP—isopentenyladenine; iPR—iso-pentenyladenosine; TD—thidiazuron; tZ—trans-zeatin; tZOG—trans-zeatin-O-glucoside; tZR—trans-zeatin riboside.(1) tZ; (2) DZ; (3) iP.

CRE1/AHK4 N

TM LB íå HK Ac

Ckç DCHASE

198

W244A

K297AT301IF304A

R305A

T317A 411

Fig. 10. Mutations in the CRE1/AHK4 cytokinin receptor defective in cytokinin binding.Bold rings designate the sites of amino acid replacement on definite positions within the CHASE domain. Other designations as on Fig. 5.

280


ROMANOV

these factors, are activated or repressed as soon as inseveral minutes after the cytokinin perception. It is justthese early genes, or primary response genes, that werementioned in preceding section.

Such is the schema of the cytokinin signal transduc-tion, which has now become canonical (Fig. 11). Cyto-kinins were shown not to affect substantially transcrip-tion of the genes related to the two-component systemper se, which are involved in the transduction of thecytokinin signal [41, 52]. Some positive relationshipwas observed only for the CRE1/AHK4 histidinekinase. It seems likely that the basic two-componentsystem is ever present and ready to function indepen-dently of the cytokinin concentration in the cell and tis-sue. However, there are response regulators of anothertype, related to the two-component system, which arestrongly influenced by cytokinins. These are responseregulators of A type (ARR-A); they are closely relatedto the response regulators of B type, from which theydiffer by the absence of the DNA-binding domain[58, 59, 74–76]. It seems evident that A-type responseregulators are not transcription factors. However, hav-ing a functional receiver domain, these regulators arecapable of receiving the activated phosphate from thephosphotransmitters like B-type response regulators.This assigns the role of A-type response regulators asattenuators of cytokinin action: the active synthesis ofthese regulators is supposed to result in scavenging agreat amount of activated phosphates directed to thenucleus, thus lowering the level of cytokinin-inducedtranscription. This can account for a peculiar characterof the kinetics of the typical primary response geneARR5 transcription induction: a sharp rise followed bya rapid decrease in the amount of its transcripts (Fig. 4)[41], and this has been demonstrated also by otherresearchers [35, 37].

However, the question arises whether the two-com-ponent system of bacterial type is the only one involvedin the transduction of the cytokinin signal? We per-formed the pharmacological analysis of the effects ofvarious inhibitors and activators of particular signalingpathways of the eukaryotic type in the model systemsAmaranthus [81] and ARR5::GUS Arabidopsis seed-lings [38]. Among a great number of various compounds,the effects of inhibitors of protein phosphatases, proteinkinases, and phospholipases D and C, donors of nitrogenoxide and inhibitors of its synthase, antagonists of calciumand calmodulin, activators of G-proteins and many othercompounds were analyzed. The list of inhibitors affect-ing or not transgenic Arabidopsis plants is presented inTable 3. It turned out that the primary alcohols werecapable of suppressing the cytokinin action, whereasstructurally similar secondary alcohols were inactiveunder the same conditions. The ability of phospholi-pases D (PLD) to interact with primary but not second-ary alcohols is a well known fact [82–84]. As a result ofits interaction with primary alcohols, PLD ceases toproduce phosphatidic acids, its basic product, produc-ing instead phophatidyl-alcohols (Fig. 12). Phospha-

tidic acids act as the second messengers in transductionof many signals, hormonal in particular [85, 86]. There-fore, specific suppression of some process by primarybut not secondary alcohols is indicative of PLDinvolvement in the process.

We performed a special study to elucidate a possi-bility of PLD involvement in the cytokinin signal trans-duction. Primary alcohols (ethanol, propan-1-ol, butan-1-ol) manifested a capability to suppress the cytokininaction when applied at low concentrations (startingfrom 0.1–0.2%). Butan-1-ol was more efficient thanalcohols of lower molecular weights (Fig. 13a), whichis in agreement with the properties of the typical PLD[87]. Using a model system of Amaranthus seedlingsand kinetics-inhibitor analysis, we demonstrated thatprimary alcohols (butan-1-ol) exerted their effectsrather early, before transcription was activated [81]. Inthe model system of Arabidopsis seedlings, we showedthat butan-1-ol suppressed the accumulation of cytoki-nin-dependent transcripts (Figs. 13b, 13c) [38, 87].These data are in agreement with the supposition thatPLD operates at the stage of the cytokinin signal trans-duction preceding the onset of the transcription rise. Itwas also established that not only primary alcohols butalso other PLD inhibitors can reduce a degree of theARR5::GUS transgene induction up to the control level[89]. In other experiments on Amaranthus seedlingsperformed in cooperation with the laboratory ofV.S. Kravets in the framework of the INTAS programwe obtained a direct evidence about PLD activationduring the early period of the cytokinin action: thisresponse has manifested as soon as in 10 min after the

Table 2. Apparent affinity constants (KD) of cytokinins forAHK3 and CRE1/AHK4 Arabidopsis receptors

Cytokinin Abbre-viation

~KD, nM

AHK3 (“leaf”)

CRE1/AHK4 (“root”)

trans-Zeatin tZ 1.3 3.9Thidiazuron TD 13 40trans-Zeatin riboside tZR 15 50Dihydrozeatin DZ 50 400N6-(∆2-isopente-nyl)adenine

iP 150 17

cis-Zeatin cZ 375 830N6-benzyladenine BA 1050 300N6-(∆2-isopente-nyl)adenosine

iPR ≥2300 130

trans-Zeatin-O-acetyl tZOAc ≥2300 ≥2750trans-Zeatin-O-gluco-side

tZOG nd nd

Adenine Ade nd nd

Note: nd—not determined because too low affining.



AD P/Q-rich domainD D K GARP

(a)

(Plasma) membrane

Cytokininreceptors:sensorhistidinekinases (3)

Cytoplasm–Nucleus

Nucleus

His-phosphotransmitters (5)

Type-Bresponseregulators (11)

Type-A response regulators (11)

íå LB íå HKCHASE

Ac

DH N G1 G2

T955 A1009T278 G470

AHK21173aa

AHK31176aa

CRE1/AHK41057aa*

F

CHASE

CHASE

RD

RD

B-type ARR,382–690 aa

NLS NLS OD NLS

AD GARP P/Q-rich domain

XHQXKGSSXS

(b)

Cytokinins

Plasmalemma

Cytoplasm

Nucleus

ARR-B

GenomeTranscription

ARR-A

D K

H N G1 G2F

H N G1 G2F

R

D D K

D D K

T

H

D D K

D D KA-type ARR,122–259 aa

AHPs127–157 aa

AD

PAHK

AHP

Fig. 11. Two-component system of cytokinin signal transduction in Arabidopsis (modified from [72]).(a) Structure of proteins belonging to this system. Protein size (number of amino acid residues, aa) is designated on the left and thenumber of this type of proteins, on the right. NLS—signal for nuclear localization; —conserved histidine residue; —con-served aspartate residue. AD, F, G1, G2, GARP, N, P/Q-rich—conserved amino acid sequences. All other abbreviations as definedin the Fig. 5b. The scheme of intracellular transduction of the cytokinin signal. AHKs—receptors; AHPs—phosphotransmitters;ARRs-B—response regulators (transcription factors); open circles—activated phosphate (~P).

H D

phytohormone was added to the seedlings [90, 91]. It issupposed that the activity of cytokinin-sensitive PLDdepends on phosphatidylinositol-4,5-diphosphate

(PIP2). At the same time, physiologically active con-centrations of butan-1-ol per se did not exert any spe-cific effect on hormone–receptor interaction [69]. All

282


ROMANOV

these and some other results argue for PLD involve-ment just in the process of intracellular transduction ofthe cytokinin signal.

So far, we could not identify the precise site and roleof PLD in the cytokinin signal transduction; furtherinvestigations are needed for this purpose. Phosphatidicacid, a PLD product, could directly interact with vari-ous proteins and affect their biological activity [92]. Onthe other hand, phosphatidic acids are capable to redis-tribute some of the proteins among various cell com-partments, particularly, by anchoring proteins in themembranes [93]. Such redistribution could facilitate or,in contrast, hamper protein–protein interactionsinvolved in signal transduction. In such a way, phospha-tidic acids could, in particular, regulate the activity ofprotein phosphorylation cascades [94]. In addition,PLD activity could be required for structural rearrange-ments of receptors within the membrane after theirinteraction with the hormone.

As regard to other efficient inhibitors, such as cal-cium chelators (EDTA, BAPTA), polyamines (sper-mine), or inhibitors of NO-synthase (L-NMMA), addi-tional studies did not confirm their involvement in cyto-kinin signal transduction [38, 40, 81, 95, 96], indicatingtheir relatively nonspecific inhibitory action on the laterstages of gene response. Mastoparan, a peptide activa-tor of heterotrimeric G-proteins, in a wide range of con-centrations did not reproduce cytokinin effects, whichargues against the involvement of G-protein-dependenttransmembrane receptors in cytokinin signaling. On the

whole, the data obtained permit us to consider the cyto-kinin signal transduction to be a rather complex pro-cess, that includes not only the elements of the bacterialtwo-component system but also the components of theeukaryotic-type systems, particularly those related toPLD activity.

SOME CONSEQUENCES OF MOLECULAR DISCOVERIES

The Pathway from the Signal to the Effect

The recent advances have improved our understand-ing of the molecular mechanisms that underlie cytoki-nin physiological action in the plant. Using a modelArabidopsis plant, it was established that the main cyto-kinin signaling pathway in plant cell is the one thatinvolves membrane histidine kinases as receptors andthe two-component system as a signal transducer to thelimited number of primary response genes. Responsesof these genes to cytokinin are determined by their pro-moters and occur at the level of transcription initiation.This is a very rapid process occurring within few min-utes, and the magnitude of the gene response is limitedby the feedback mechanisms. Cytokinin receptors areevidently able to interact not only with the extracellularhormones in the apoplast but also with the intracellularhormones in the symplast or endoplast, because a con-siderable proportion of the receptors were detectedinside the cell. Many genes of primary response to cyto-kinin encode regulatory proteins, which induce global

Table 3. Pharmacological (inhibitor) analysis of ARR5::GUS activation by cytokinin (BA) in Arabidopsis seedlings

Inhibitor Concentration Induction, % Inhibitor target

BA (without inhibitor) 5 µM 100

Butan-1-ol ~1% 22 ± 2 phospholipase D

Propan-1-ol ~1% 29 ± 3 phospholipase D

Butan-2-ol ~1% 80–100 isomer of butan-1-ol

Propan-2-ol ~1% 80–100 isomer of propan-1-ol

EDTA 10 mM 0 divalent cations

BAPTA 10 mM 2 ± 2 calcium

W7 500 µM 0 calmodulin

Calyculin A 2 µM 9 ± 1 protein phosphatase (1 = 2A)

Okadaic acid 2 µM 67 ± 6 protein phosphatase (2A > 1)

Tautomycin 0.5–2 µM 100 protein phosphatase (1 > 2A)

K252a 4 µM 40 ± 19 protein kinases

Genistein 50–500 µM 100 tyrosine kinases

Roskovitine 10–100 µM 100 cyclin-dependent kinases

L-NMMA 10 mM 2.5 ± 2 NO synthase

Spermine 50 mM 3.5 ± 1 unknown

U73122 10 µM 70 ± 3 phospholipase C

Wortmannin 1–4 µM 100 phosphatidyl inositol 3-kinase

Propanolol 250 µM 17 ± 3 phosphatidic acid phosphatase



secondary changes in gene expression by the cascademechanism. In such a way, regulation of gene expres-sion at various levels is possible. For example, in somecases cytokinins regulate gene expression not only atthe level of transcription but also at the level of mRNAstability [97]. Direct cytokinin effects on protein syn-thesis by ribosomes [14, 98, 99], and even on posttrans-lational protein stability [100, 101] were also reported.In Arabidopsis, cytokinin activated the expression ofAtSAHH1 and AtADK1 genes controlling the level ofglobal genome methylation [102], which in turnaffected gene expression [103]. Other levels of cytoki-nin action are also possible, but are poorly studied yet.

The outcome of the cytokinin signal transduction issuch changes in gene expression that result in some typ-ical physiological effect. So far, underlying molecularprocesses for only few rapid cytokinin effects havebeen elucidated. For example, activation of only smallnumber of genes is sufficient for cytokinin stimulationof pigment synthesis. Particularly, in Arabidopsis, cyto-kinins enhanced coordinated expression of four genesof anthocyanin biosynthesis; two of them were acti-vated at the transcription level whereas two others,posttranscriptionally [104].

Induction of cell division requires far more molecu-lar participants. In Arabidopsis, no less than 50 variousproteins are involved in the G1/S and G2/M switches ofthe cell cycle; among them 30 cyclins, 11 cyclin-depen-dent protein kinases, and several inhibitors of thesekinases were identified [105, 106]. Cytokinins act dif-ferently in different cells: in the cell culture, they, as a

rule, stimulate division, whereas during initiation of lat-eral roots, they, in contrast, block pericycle cell division[107]. Some data indicate that cytokinins could stimu-late biosynthesis of particular cyclins and affect thephosphorylation level of the cell cycle proteins; how-ever, these processes are not studied comprehensivelyso far. At super-high concentrations, cytokinins inducecell apoptosis not only in plants [27, 28] but also in ani-mals [33, 34]. Further investigations of transcriptomesand proteomes of particular organs, tissues, and cellswill help us to track the changes in activities of the keygenes and proteins, which ultimately result in the real-ization of physiological programs by a given phytohor-mone.

Cytokinin Involvement in Long-Distance Communication

Functioning of cytokinins as hormones implies theirbiosynthesis in one part of the plant with subsequenttransport to and signaling in another part. However, thesituation gets complicated by the occurrence of multi-ple sites of cytokinin synthesis and by the cytokininpresence in both plant transport channels, i.e., xylemand phloem [16]. In the meantime, cytokinins werelong and on reasonable basis considered as root hor-mones, which transfer the information about the extentof root well-being and its nutritional status (nitrogen, inparticular) to the shoots (leaves) [2, 15, 20]. How doshoot (leaf) cells distinguish cytokinins coming fromthe root and those synthesized in the shoot and trans-ported along the phloem?

Let us remember now about peculiarity of the wholeorganization of cytokinin signaling in plants. As dis-tinct from many other hormones (auxin, ethylene,ABA), cytokinins are represented by a number of iso-forms, with a possibility of their particular interconver-sions. The spectra of these isoforms in the xylem andthe phloem differ substantially [16, 108]. Xylem cyto-kinins are represented mainly by Z-type cytokininswith a prevalence of trans-zeatin riboside. Zeatin cyto-kinins are synthesized mainly in the root tip, whereCYP735A enzymes are expressed; these enzymeshydroxylate terminal, trans-isomerized carbon of theside chain of isopentenyl cytokinins [7]. Phloem cyto-kinins are hydroxylated to a lesser extent and are repre-sented mostly by iP-cytokinins (mainly isopentenylad-enine riboside). The content of trans-zeatin in thephloem is low. Also, large amount of cis-zeatin ispresent in the xylem and especially in the phloem exu-date; however, keeping in mind its low biological activ-ity, the contribution of cis-zeatin to the total Arabidop-sis cytokinin activity is unlikely to be significant.

On the other hand, perception of various cytokininisoforms by the cell depends on the set of the receptorspresent. As was discussed above, the basic cytokininreceptors of Arabidopsis, AHK3 and CRE1/AHK4, dis-play similar high affinity for trans-zeatin and its ribo-side but differ strongly in their affinity for iP-cytokinins

Phospholipids

Aqueous medium Presenceof primary alcohols

Phospholipase D

Phosphatidicacids Phosphatidyl-alcohols

Regulatory processes

Fig. 12. Scheme of phosphatidic acids (signal messengers)formation by phospholipase D and of blocking this processby primary alcohols.

284


ROMANOV

[69]. The CRE1/AHK4 receptor expressed mainly inthe root and determining cytokinin effect on the under-ground organs (Table 4) manifests high affinity for isopen-tenyladenine, close to that for trans-zeatin (Table 2). Thus,this receptor is sensitive to both root-derived Z-type andleaf-borne iP cytokinins, coming from the shoot alongthe phloem. The AHK3 receptor, predominantlyexpressed in the shoot (leaf mesophyll) and determiningcytokinin effects on the aboveground organs (Table 4),displays low affinity for the phloem iP cytokinins(Table 2); hence, it is tuned to respond to root-derivedzeatin cytokinins transported to the shoot along thexylem (see the scheme in Fig. 14). Therefore, in thiscase, cytokinin exerts long-distance action. In general,functional heterogeneity of cytokinin receptors couldbe related to their role in the interorgan communication

and could coordinate the functional integrity of theplant organism.

Signal Pathway Interaction

The cytokinin regulatory system functions in theplant in an incessant (or permanent) contact with other,primarily hormonal, regulatory systems. The tightinteractions between cytokinins and auxins, both ago-nistic and antagonistic, are well known; the mode ofinteraction depends on the type of the process con-trolled by these phytohormones. Cytokinins and auxinscreate a countercurrent regulatory conduit that largelydetermines the rate of bipolar growth and total architec-ture of the shoot and root systems [2, 20, 21, 108, 109].Molecular basics of interaction between cytokinin and

GU

S ac

tivity

, %

20

0.2 0.4 0.6 0.8 1.00

40

60

80

100

0

Alcohol concentration, %

(a)

1 23

4

GU

S ac

tivity

20

40

60

80

100 (b)

Con

tent

of

20

40

60

80100 (c)

AR

R5

mR

NA

H2O BA BA+1-but

BA+2-but

%

Fig. 13. Suppression of cytokinin (BA, 5 µM) effect by primary alcohols on the model of ARR5::GUS Arabidopsis.(a) Concentration dependences for some alcohols; (b, c) effects of butan-1-ol (1-but) and butan-2-ol (2-but) (1%) on (b) GUS activ-ity and (c) the content of ARR5 gene transcripts.(1) 1-but (2) methanol; (3) ethanol; (4) 2-but.

Table 4. The role of particular AHK receptors in the control of cytokinin-dependent processes in Arabidopsis*

Plant material Controlled process Receptor involvement

Shoot leaf and shoot growth AHK3 > AHK2 � AHK4hypocotyl growth AHK3 � AHK2, AHK4chloroplast development AHK3 > AHK2 � AHK4shoot deetiolation AHK3 > AHK2, AHK4tolerance to far red illumination AHK3 > AHK2, AHK4leaf senescence AHK3 � AHK2, AHK4chlorophyll protection AHK3 > AHK2 � AHK4

Root root growth (inhibition) AHK4 > AHK3, AHK2adventitious and lateral root formation AHK4 > AHK3 � AHK2

Cell culture cell division AHK4 > AHK3 � AHK2plastid development AHK4 > AHK3 � AHK2

* On the basis of studies with Arabidopsis double ahk mutants [57–59].



auxin signal transduction pathways, especially duringregulation of cell proliferative activity, are now beingactively studied [105, 111].

Also long known is the antagonism between thecytokinins and ABA, manifesting in such processes asstomata functioning, seed germination, and chloroplastdifferentiation [112]. In the course of plant develop-ment, cytokinin frequently opposes to ethylene, whichactivates the genetic program of plant senescence [113,114]; still, under definite conditions, cytokinin canaccelerate ethylene production [100]. Much less isknown about the interactions of the cytokinins withother phytohormones, gibberellins in particular,although their opposite effects on some biochemicaland physiological processes have been reported [81,115–117]. Our results regarding the cytokinin influenceon Arabidopsis transcriptome [41] partially improvedour understanding of the processes of the cytokinininteraction with other hormones at the gene level. Cyto-kinin was shown to enhance the content of transcriptsfor the ethylene receptors, ETR2 and ERS2, and alsoEIN3, a key transcription factor for ethylene-dependentgenes. This indicates a possibility of a cytokinin-induced increase in the cell sensitivity to ethylene. Onthe contrary, cytokinins were shown to act as negativeregulators with respect to gibberellins. Cytokinins sup-

pressed the expression of the genes for biosynthesis ofactive gibberellins, encoding GA20 oxidase and 3β-hydro-lase (GA4). In addition, cytokinins accelerated expres-sion of the GAI and RGA genes, whose products sup-press GA signaling [41].

Cytokinins often simulate light effects [17–19],though antagonistic influences of cytokinins and lightsensor phytochrome B during potato tuberization werealso reported [118, 119].

The investigations of ligand-binding properties ofcytokinin receptors [68, 69] revealed novel possibilityof signaling pathway interactions at the level of hor-monal signal perception. For example, auxin-inducedapoplast acidification could change the sensitivity tocytokinins of some AHK-receptor located in the plas-malemma and thus affect the efficiency of cytokininsignaling.

CONCLUSION

Cytokinins, classical phytohormones, represent animportant class of hormones-stimulators favoring plantactive metabolism and growth. Their specific feature isthe occurrence in multiple isoforms, hence the com-plexity of their biosyntheses and further metabolism.Essential absence of natural mutants in the cytokininregulatory system and a great number of genes (morethan a hundred) involved in its functioning prove itsimportance for plant growth and development. A greatadvance of recent years was an identification of genesand proteins of this system, including the primaryresponse genes and receptors. It was just these investi-gations at the gene level, as well as obtaining the corre-sponding mutants, that permitted a reliable determina-tion of the biological function of proteins related to thisregulatory system.

As was indicated above, the cytokinin system con-trols physiological processes in the plant in cooperationwith other hormonal and nonhormonal regulatory sys-tems. General principles of its arrangement and func-tioning evidently have many common features withorganization of other systems of hormonal regulation inplants.

Firstly, this is the cytokinin polyfunctionality: thesehormones affect a great number of physiological pro-cesses and exert diverse effects (pleiotropic action).Theoretically, multifaceted cytokinin action mightresult from multiplicity of their isoforms, so that eachcytokinin structural form would be responsible for adefinite metabolic process. However, so far there is nofirm basics to believe that cytokinin isoforms differqualitatively; in contrast, a huge experimental materialindicates that all cytokinins are functionally active,although to a different degree, in the realization of samephysiological programs. Therefore, a diversity of cellresponses to cytokinin should depend on both the spec-ificity of the cell system of signal perception, and thecell competence to respond to cytokinin by changes in

Äçä3

ZR, Z

Shoot

Root

iP iPR

CRE1/AHK4

ZR

, Z x

ylem

Phlo

em i

P, iP

R

Phlo

em i

P, iP

R

Fig. 14. Hypothetical scheme of long-distance cytokininaction in the plant, as exemplified by Arabidopsis.Z, ZR—(trans-)Z-type cytokinins transported from the rootto the shoot along the xylem; iP, iPR—iP-type cytokininssynthesized in the shoot and transported along the phloem.Dominating receptor in the shoot (leaf)—AHK3; in theroot, CRE1/AHK4. Thick bilateral arrows correspond tostrong hormone–receptor interaction, thin arrows—to rela-tively weak interaction.

286


ROMANOV

the expression of the primary and secondary responsegenes. As was exemplified by Arabidopsis, the plant hasseveral receptors for a single phytohormone, whoseratio depends on the type of organ and tissue. Qualita-tive differences in the phytohormone perceptionmachinery in different tissues and organs and the cas-cade principle of cell transcriptome response areimportant factors of hormone polyfunctionalityin planta, which explains a well known fact that muchlower diversity of hormones is sufficient for plants ascompared to animals [51, 120].

Another important feature of the cytokinin regula-tory system is its high reliability, which manifests atvarious levels of plant organization; its novel elementsare revealed constantly with widening the methodolog-ical possibilities of research. The reliability at the celllevel is related to the limits of both upper and lower ofprimary response gene expression, whose exceedingcould be lethal for the plant. Overexpression of the pri-mary response genes is blocked by transcription ofARR-A type pseudoregulators, whereas the absence ofthe cytokinin signaling might be partially compensatedby the background signaling from other sensor histi-dine kinases, such as CKI1 [54] or ethylene receptorETR1 [121]. Local excess of the cytokinins is compen-sated by the activation of enzymes of their inactivationor catabolism, such as cytokinin oxidase/dehydroge-nase and others. Disturbance in the cytokinin homeo-stasis induces plant physiological responses leading toits restoring. For example, ectopic expression of cyto-kinin oxidase/dehydrogenase, reducing the concentra-tion of endogenous cytokinins, induces enhancedgrowth of the root system [22, 23]. An increase in thevolume of the root meristems, in turn, could lead toadditional synthesis of cytokinins transported furtherover the entire plant. On the other hand, at cytokininshortage, the volume of the shoot meristem reduces,and this could decrease the level of auxin synthesis,thus facilitating a rapid restoration of the auxin–cytoki-nin balance. Another example is found in mutatedplants where the genes for cytokinin receptors wereswitched off: they respond to a decrease in the receptorlevel by an increase in the concentration of endogenouscytokinins [62], which should compensate to somedegree the weakening of their perception machinery.This is evidently the reason why Arabidopsis knock-outmutants deficient in two of three receptors manifestonly small phenotypic changes as compared with thewild-type plants [60–62].

Thirdly, like other phytohormones, cytokinins act asboth local and long-distance signals. Arabidopsis is atypical plant synthesizing most cytokinins in the roottip. These root-derived cytokinins are transported acro-petally along the xylem over the entire plant. In such away, information about the status of growing parts ofthe root system is transferred to the shoot. Thus, a def-inite gradient of cytokinin (and auxin) concentrations iscreated along the longitudinal plant axis; these gradi-ents serve as “a signal field” for patterning plant tissues

and cells. However, xylem transport is strongly depen-dent on external cues (temperature, humidity, or wind)and is therefore unstable. It seems likely that, in orderto provide more stable cytokinin concentrations in thezones of strong cytokinin requirement, e.g. in the zonesof dividing cells, plants developed local regions of thecytokinin synthesis in the shoot. These are primarily theapical meristem, including floral one, leaf phloem, lat-eral buds, zones of fruit abscission, and some others[16]. This means that cytokinins, being true phytohor-mones, combine endocrine action characteristic of hor-mones (i.e., long-distance effects) and paracrine action(i.e., local effects in the sites of their biosynthesis). Theoccurrence of local sites of cytokinin biosynthesisimproves the reliability of the entire system of cytoki-nin regulation.

In spite of enormous advances attained duringrecent decades in the investigation of molecular basicsof cytokinin signaling, both intracellular and in thewhole plant, many questions still wait for their answers.Nevertheless, new regularities revealed and, what ismost important, genes involved in the perception of thecytokinin signal and response to it open wide perspec-tives for further detailed studies and application of theobtained knowledge in plant industry.

ACKNOWLEDGMENTS

The author would like to express his gratitude toT. Schmülling (Free University of Berlin) for construc-tive discussion of this review topic and to the RussianFoundation for Basic Research for financial support(project nos. 04-04-49120, 07-04-00331, 07-04-91211-YaF, and 08-04-90429-Ukr).

REFERENCES

1. Miller, C.O., Skoog, F., von Saltza, N.M., andStrong, F.M., Kinetin, a Cell Division Factor fromDeoxyribonucleic Acid, J. Am. Chem. Soc., 1955,vol. 77, pp. 1329–1334.

2. Polevoy, V.V., Fitogormony (Phytohormones), Lenin-grad: Leningr. Gos. Univ., 1982.

3. Köhler, K.H. and Conrad, K., Ein quantitativer Phyto-kinintest, Biol. Rundschau., 1966, vol. 5, pp. 36–37.

4. Letham, D.S., Zeatin, a Factor Inducing Cell Division Iso-lated from Zea mays, Life Sci., 1963, vol. 2, pp. 569–573.

5. Letham, D.S., Shannon, J.S., and McDonald, I.R., TheStructure of Zeatin, a Factor Inducing Cell Division,Proc. Chem. Soc. London, 1964, pp. 230–231.

6. Kakimoto, T., Identification of Plant Cytokinin Biosyn-thetic Enzymes as Dimethylallyl Diphosphate:ATP/ADPIsopentenyltransferases, Plant Cell Physiol., 2001,vol. 42, pp. 677–685.

7. Takei, K., Sakakibara, H., and Sugiyama, T., Identifica-tion of Genes Encoding Adenylate Isopentenyltrans-ferase, a Cytokinin Biosynthesis Enzyme, in Arabidop-sis thaliana, J. Biol. Chem., 2001, vol. 276, pp. 26405–26410.



8. Takei, K., Yamaya, T., and Sakakibara, H., ArabidopsisCYP735A1 and CYP735 A2 Encode Cytokinin Hydroxy-lases That Catalyze the Biosynthesis of trans-Zeatin, J.Biol. Chem., 2004, vol. 279, pp. 41 866–41 872.

9. Korszun, Z.R., Knight, C., and Chen, C.M., A Stereo-chemical Model for Cytokinin Activity, FEBS Lett.,1989, vol. 243, pp. 53–56.

10. Strnad, M., The Aromatic Cytokinins, Physiol. Plant.,1997, vol. 101, pp. 674–688.

11. Bruce, M.I. and Zwar, J.A., Cytokinin Activity of SomeSubstituted Ureas and Thioureas, Proc. R. Soc. London,Ser. B, 1966, vol. 165, pp. 245–265.

12. Isogai, Y., Cytokinin Activities of N-Phenyl-N'-(4-Pyridyl)ureas, Metabolism and Molecular Activities ofCytokinins, Guern, J. and Peaud-Lenoel, C., Eds., Ber-lin: Springer-Verlag, 1981, pp. 115–128.

13. Shudo, K., Chemistry of Phenylurea Cytokinins, Cyto-kinins. Chemistry, Activity, and Function, Mok, D.W.S.and Mok, M.C., Eds., Boca Raton: CRC, 1994, pp. 35–42.

14. Romanov, G.A., Cytokinins and tRNA: A New Insightinto an Old Problem, Sov. Plant Physiol., 1990, vol. 37,pp. 922–935.

15. Sakakibara, H., Cytokinin Biosynthesis and Metabo-lism, Plant Hormones. Biosynthesis, Signal Transduc-tion, Action, Davies, P.J., Ed., Dordrecht: Kluwer, 2004,pp. 95–114.

16. Hirose, N., Takei, K., Kuroha, T., Kamada-Nobusada, T.,Hayashi H., and Sakakibara, H., Regulation of Cytoki-nin Biosynthesis, Compartmentalization and Transloca-tion, J. Exp. Bot., 2008, vol. 59, pp. 75–83.

17. Mok, M.C., Cytokinins and Plant Development—anOverview, Cytokinins. Chemistry, Activity, and Func-tion, Mok, D.W.S. and Mok, M.C., Eds., Boca Raton:CRC, 1994, pp. 155–166.

18. Taiz, L. and Zeiger, E., Plant Physiology, Sunderland(USA): Sinauer Associates, 2002.

19. Kuznetsov, Vl.V. and Dmitrieva, G.A., Fiziologiya ras-tenii (Plant Physiology), Moscow: Vysshaya Shkola,2006.

20. Romanov, G.A., A Model for Hormone-Organized Pro-liferative Growth: Analogy with Plant Growth, Ontoge-nez, 1992, vol. 23, pp. 228–236.

21. Romanov, G.A., A Model for Bipolar Plant-TypeGrowth: Role of Auxin–Cytokinin Countercurrent,Progress in Plant Growth Regulation, Karssen, C.M.,et al., Eds., Dordrecht: Kluwer, 1992, pp. 459–463.

22. Werner, T., Motyka, V., Strnad, M., and Schmülling, T.,Regulation of Plant Growth by Cytokinin, Proc. Natl.Acad. Sci. USA, 2001, vol. 98, pp. 10 487–10 492.

23. Werner, T., Motyka, V., Laucou, V., Smets, R., vanOnckelen, H., and Schmülling, T., Cytokinin-DeficientTransgenic Arabidopsis Plants Show Multiple Develop-ment Alterations Indicating Opposite Functions ofCytokinins in the Regulation of Shoot and Root Mer-istem Activity, Plant Cell, 2003, vol. 15, pp. 2532–2550.

24. Murray, J.D., Karas, B.J., Sato, S., Tabata, S.,Amyot, L., and Szczyglowski, K., A Cytokinin Percep-tion Mutant Colonized by Rhizobium in the Absence ofNodule Organogenesis, Science, 2007, vol. 315,pp. 101–104.

25. Tirichine, L., Sandal, N., Madsen, L.H., Radutoiu, S.,Albrektsen, A.S., Sato, S., Asamizu, E., Tabata, S., andStougaard, J., A Gain-of-Function Mutation in a Cyto-kinin Receptor Triggers Spontaneous Root NoduleOrganogenesis, Science, 2007, vol. 315, pp. 104–107.

26. Bopp, M. and Jacob, H.J., Cytokinin Effect on Branch-ing and Bud Formation in Funaria, Planta, 1986,vol. 169, pp. 462–464.

27. Mlejnek, P. and Prochazka, S., Activation of Caspase-Like Proteases and Induction of Apoptosis by Isopente-nyladenosine in Tobacco BY-2 Cells, Planta, 2002,vol. 215, pp. 158–166.

28. Carimi, F., Zottini, M., Formentin, E., Terzi, M., and LoSchiavo, F., Cytokinins: New Apoptotic Inducers inPlants, Planta, 2003, vol. 216, pp. 413–421.

29. Bioassays and Other Special Techniques for Plant Hor-mones and Plant Growth Regulators, Ch. 4, CytokininBioassays, Yopp, J.H., Aung, L.H., and Steffens, G.,Eds., Florida (USA): Plant Growth Regulator Society ofAmerica, 1986, pp. 63–84.

30. Sel’skokhozyaistvennaya biotekhnologiya (AgriculturalBiotechnology), Shevelukha, V.S., Ed., Moscow:Vysshaya Shkola, 2003.

31. Grossmann, K., Induction of Leaf Abscission in CottonIs a Common Effect of Urea- and Adenine-Type Cyto-kinins, Plant Physiol., 1991, vol. 95, pp. 234–237.

32. Vesel , J., Havlí ek, L., Strnad, M., Blow, J.J., Donella-Deana, A., Pinna, L., Letham, D.S., Kato, J., Deti-vaud, L., Leclerc, S., and Meijer, L., Inhibition ofCyclin-Dependent Kinases by Purine Analogues, Eur.J. Biochem., 1994, vol. 244, pp. 771–786.

33. Ishii, Y., Hori, Y., Sakai, S., and Honma, Y., Control ofDifferentiation and Apoptosis of Human Myeloid Leu-kemia Cells by Cytokinins and Cytokinin Nucleosides,Plant Redifferentiation-Inducing Hormones, CellGrowth Diff., 2002, vol. 13, pp. 19–26.

34. Spinola, M., Colombo, F., Falvella, S., and Dragani, T.A.,N6-Isopentenyladenosine: A Potent Therapeutical Agentfor a Variety of Epithelial Cancers, Int. J. Cancer., 2007,vol. 120, pp. 2744–2748.

35. Brandstatter, I. and Kieber, J.J., Two Genes with Simi-larity to Bacterial Response Regulators Are Rapidly andSpecifically Induced by Cytokinin in Arabidopsis, PlantCell, 1998, vol. 10, pp. 1009–1020.

36. Taniguchi, M., Kiba, T., Sakakibara, H., Ueguchi, C.,Mizuno, T., and Sugiyama, T., Expression of ArabidopsisResponse Regulator Homologs Is Induced by Cytokininsand Nitrate, FEBS Lett., 1998, vol. 429, pp. 259–262.

37. D’Agostino, I., Deruére, J., and Kieber, J.J., Character-ization of the Response of the Arabidopsis ARR GeneFamily to Cytokinin, Plant Physiol., 2000, vol. 124,pp. 1706–1717.

38. Romanov, G.A., Kieber, J.J., and Schmülling, T., ARapid Cytokinin Response Assay in Arabidopsis Indi-cates a Role for Phospholipase D in Cytokinin Signal-ling, FEBS Lett., 2002, vol. 515, pp. 39–43.

39. Zvereva, S.D. and Romanov, G.A., Reporter Genes forPlant Genetic Engineering: Characteristics and Detec-tion, Russ. J. Plant Physiol., 2000, vol. 47, pp. 424–432.

40. Romanov, G.A., Lomin, S.N., Rakova, N.Yu., Heyl, A.,and Schmülling, T., Does NO Play a Role in Cytokinin

y c

288


ROMANOV

Signal Transduction? FEBS Lett., 2008, vol. 582,pp. 874–880.

41. Brenner, W., Romanov, G.A., Köllmer, I., Burkle, L.,and Schmülling, T., Immediate-Early and DelayedCytokinin Response Genes of Arabidopsis thalianaIdentified by a Genome-Wide Expression ProfilingReveal Novel Cytokinin-Sensitive Processes and Sug-gest Cytokinin Action through Transcriptional Cas-cades, Plant J., 2005, vol. 44, pp. 314–333.

42. Redman, J.C., Haas, B.J., Tanimoto, G., and Town, C.D.,Development and Evaluation of an Arabidopsis WholeGenome Affymetrix Probe Array, Plant J., 2004,vol. 38, pp. 545–561.

43. Romanov, G.A., Schafer, Z., Hevelt, A., Krolzik, S., andSchmülling, T., Cytokinin Action on mRNA Populationin Tobacco Cells, Tez. IV S"ezda Obshchestva fiziologovrastenii Rossii (Abst. IV Symp. Russ. Plant Physiol.Soc.), Moscow, 1999, p. 675.

44. Schäfer, S., Krolzik, S., Romanov, G.A., andSchmülling, T., Cytokinin-Regulated Transcripts inTobacco Cell Culture, Plant Growth Regul., 2000,vol. 32, pp. 307–313.

45. Patrushev, L.I., Iskusstvennye geneticheskie sistemy(Artificial Genetic Systems), Moscow: Nauka, 2004,vol. 1.

46. Lutova, L.A., Provorov, N.A., Tikhodeev, O.N.,Tikhonovich, I.A., Khodzhaiova, L.T., and Shish-kova, S.O., Genetika razvitiya rastenii (Genetics ofPlant Development), St. Petersburg: Nauka, 2000.

47. Dharmasiri, N., Dharmasiri, S., and Estelle, M., The F-Box Protein 1 Is an Auxin Receptor, Nature, 2005,vol. 435, pp. 441–445.

48. Kepinsky, S. and Leyser, O., The Arabidopsis F-BoxProtein 1 Is an Auxin Receptor, Nature, 2005, vol. 435,pp. 446–451.

49. Ueguchi-Tanaka, M., Ashikari, M., Nakajima, M.,Itoh, H., Katoh, E., Kobayashi, M., Ohow, T.Y.,Hsing, Y.I.C., Kitano, H., Yamaguchi, I., and Mat-suoka, M., GIBBERELLIN INSENSITIVE DWARF1Encodes a Soluble Receptor for Gibberellin, Nature,2005, vol. 437, pp. 693–698.

50. Smalle, J., Kurepa, J., Yang, J., Babiychuk, E., Kush-nir, S., Durski, A., and Viestra, R.D., Cytokinin GrowthResponses in Arabidopsis Involve the 26S ProteasomeSubunit RPN12, Plant Cell, 2002, vol. 14, pp. 17–32.

51. Romanov, G.A., The Phytohormone Receptors, Russ.J. Plant Physiol., 2002, vol. 49, pp. 552–560.

52. Rashotte, A.M., Carson, S.D.B., To, J.P.C., and Kie-ber, J.J., Expression Profiling of Cytokinin Action inArabidopsis, Plant Physiol., 2003, vol. 132, pp. 1998–2011.

53. Rashotte, A.M., Mason, M.G., Hutchison, C.E., Fer-reira, F.J., Schaller, G.E., and Kieber, J.J., A Subset ofArabidopsis AP2 Transcription Factors Mediates Cyto-kinin Responses in Concert with a Two-ComponentPathway, Proc. Natl. Acad. Sci. USA, 2006, vol. 103,pp. 11 081–11 085.

54. Kakimoto, T., CKI1, a Histidine Kinase HomologImplicated in Cytokinin Signal Transduction, Science,1996, vol. 274, pp. 982–985.

55. Inoue, T., Higuchi, M., Hashimoto, Y., Seki, M., Koba-yashi, M., Kato, T., Tabata, S., Shinozaki, K., and

Kakimoto, T., Identification of CRE1 as a CytokininReceptor from Arabidopsis, Nature, 2001, vol. 409,pp. 1060–1063.

56. Suzuki, T., Miwa, K., Ishikawa, K., Yamada, H.,Aiba, H., and Mizuno, T., The Arabidopsis Sensor His-Kinase, AHK4, Can Respond to Cytokinins, Plant CellPhysiol., 2001, vol. 42, pp. 107–113.

57. Yonekura-Sakakibara, K., Kojima, M., Yamaya, T., andSakakibara, H., Molecular Characterization of Cytoki-nin-Responsive Histidine Kinases in Maize. Differen-tial Ligand Preferences and Response to cis-Zeatin,Plant Physiol., 2004, vol. 134, pp. 1654–1661.

58. Pareek, A., Singh, A., Kumar, M., Kushwaha, H.R.,Lynn, A.M., and Singla-Pareek, S.L., Whole-GenomeAnalysis of Oryza sativa Reveals Similar Architectureof Two-Component Signaling Machinery with Arabi-dopsis, Plant Physiol., 2006, vol. 142, pp. 380–397.

59. Du, L., Jiao, F., Chu, J., Chen, M., and Wu, P., The Two-Component Signal System in Rice (Oryza sativa L.): AGenome-Wide Study of Cytokinin Signal Perception andTransduction, Genomics, 2007, vol. 89, pp. 697–707.

60. Higuchi, M., Pischke, M.S., Mahonen, A.P., Miyawaki, K.,Hashimoto, Y., Seki, M., Kobayashi, M., Shinozaki, K.,Kato, T., Tabata, S., Helariutta, Y., Sussman, M.R., andKakimoto, T., In Planta Functions of the ArabidopsisCytokinin Receptor Family, Proc. Natl. Acad. Sci. USA,2004, vol. 101, pp. 8821–8826.

61. Nishimura, C., Ohashi, Y., Sato, S., Kato, T., Tabata, S.,and Ueguchi, C., Histidine Kinase Homologs That Actas Cytokinin Receptors Possess Overlapping Functionsin the Regulation of Shoot and Root Growth in Arabi-dopsis, Plant Cell, 2004, vol. 16, pp. 1365–1377.

62. Riefler, M., Novak, O., Strnad, M., and Schmülling, T.,Arabidopsis Cytokinin Receptor Mutants Reveal Func-tions in Shoot Growth, Leaf Senescence, Seed Size,Germination, Root Development and Cytokinin Metab-olism, Plant Cell, 2006, vol. 18, pp. 40–54.

63. Anantharaman, V. and Aravind, L., The CHASEDomain: A Predicted Ligand-Binding Module in PlantCytokinin Receptors and Other Eukaryotic and Bacte-rial Receptors, Trends Biochem. Sci., 2001, vol. 26,pp. 579–582.

64. Mougel, C. and Zhulin, I.B., CHASE: An ExtracellularSensing Domain Common to Transmembrane Recep-tors from Prokaryotes, Lower Eukaryotes and Plants,Trends Biochem. Sci., 2001, vol. 26, pp. 582–584.

65. Yamada, H., Suzuki, T., Terada, K., Takei, K., Ish-ikawa, K., Miwa, K., Yamashino, T., and Mizuno, T.,The Arabidopsis AHK4 Histidine Kinase Is a Cytoki-nin-Binding Receptor That Transduces Cytokinin Sig-nals across the Membrane, Plant Cell Physiol., 2001,vol. 42, pp. 1017–1023.

66. Spíchal, L., Rakova, N.Y., Riefler, M., Mizuno, T.,Romanov, G.A., Strnad, M., and Schmülling, T., TwoCytokinin Receptors of Arabidopsis thaliana,CRE1/AHK4 and AHK3, Differ in Their Ligand Speci-ficity in a Bacterial Assay, Plant Cell Physiol., 2004,vol. 45, pp. 1299–1305.

67. Lomin, S.N. and Romanov, G.A., The Analysis of Hor-mone–Receptor Interaction. Theoretical and PracticalAspects, Russ. J. Plant Physiol., 2008, vol. 55, pp. 259–273.



68. Romanov, G.A., Spíchal, L., Lomin, S., Strnad, M., andSchmülling, T., A Live Cell Hormone-Binding Assay onTransgenic Bacteria Expressing a Eukaryotic Receptor Pro-tein, Anal. Biochem., 2005, vol. 347, pp. 129–134.

69. Romanov, G.A., Lomin, S.N., and Schmülling, T., Bio-chemical Characteristics and Ligand-Binding Proper-ties of Arabidopsis Cytokinin Receptor AHK3 Com-pared to CRE1/AHK4 as Revealed by a Direct BindingAssay, J. Exp. Bot., 2006, vol. 57, pp. 4051–4058.

70. Takei, K., Ueda, N., Aoki, K., Kuromori, T.,Hirayama, T., Shinozaki, K., Yamaya, T., and Sakak-ibara, H., AtIPT3 Is a Key Determinant of Nitrate-Dependent Cytokinin Biosynthesis in Arabidopsis,Plant Cell Physiol., 2004, vol. 45, pp. 1053–1062.

71. Lomin, S.N., Ligand-Binding Properties and Subcellu-lar Localization of Cytokinin Receptors, Cand. Sci.(Biol.) Dissertation, Moscow: Timiryazev Inst. PlantPhysiol. Russ. Acad. Sci., 2008.

72. Heyl, A., Wulfetange, K., Pils, B., Nielsen, N.,Romanov, G.A., and Schmülling, T., Evolutionary Pro-teomics Identifies Amino Acids Essential for Ligand-Binding of the Cytokinin Receptor CHASE Domain,BMC Evol. Biol., 2007, vol. 7, p. 62.

73. Mizuno, T., His–Asp Phosphotransfer Signal Transduc-tion, J. Biochem., 1998, vol. 123, pp. 555–563.

74. Heyl, A. and Schmülling, T., Cytokinin Signal Percep-tion and Transduction, Curr. Opin. Plant Biol., 2003,vol. 6, pp. 480–488.

75. Ferreira, F.J. and Kieber, J.J., Cytokinin Signaling,Curr. Opin. Plant Biol., 2005, vol. 8, pp. 518–525.

76. Müller, B. and Sheen, J., Arabidopsis Cytokinin Signal-ing Pathway, Science STKE, 2007, cm7.

77. Lomin, S.N., Sakakibara, H., and Romanov, G.A., TwoMaize Cytokinin Receptors, ZmHK1 and ZmHK2,Have Different Ligand-Binding Properties, Abst. II Int.Symp. “Plant Growth Substances: Intracellular Hor-monal Signaling and Applying in Agriculture”, Kiev:Polytechnika, 2007, p. 52.

78. Sakai, H., Aoyama, T., and Oka, A., Arabidopsis ARR1and ARR2 Response Regulators Operate as Transcrip-tional Activators, Plant J., 2000, vol. 24, pp. 703–711.

79. Hosoda, K., Imamura, A., Katoh, E., Hatta, T.,Tachiki, M., Yamada, H., Mizuno, T., and Yamazaki, T.,Molecular Structure of the GARP Family of Plant Myb-Related DNA Binding Motifs of the ArabidopsisResponse Regulators, Plant Cell, 2002, vol. 14,pp. 2015–2029.

80. Taniguchi, M., Sasaki, N., Tsuge, T., Aoyama, T., andOka, A., ARR1 Directly Activates Cytokinin ResponseGenes That Encode Proteins with Diverse RegulatoryFunctions, Plant Cell Physiol., 2007, vol. 48, pp. 263–277.

81. Romanov, G.A., Getman, I.A., and Schmülling, T.,Investigation of Early Cytokinin Effects in a RapidAmaranthus Seedling Test, Plant Growth Regul., 2000,vol. 32, pp. 337–344.

82. Yu, C.H., Liu, S.Y., and Panagia, V., The Transphos-phatidylation Activity of Phospholipase D, Mol. Cell.Biochem., 1996, vol. 157, pp. 101–105.

83. Ella, K.M., Meier, K.E., Kumar, A., Zhang, Y., andMeier, G.P., Utilization of Alcohols by Plant and Mam-

malian Phospholipase D, Biochem. Mol. Biol. Int.,1997, vol. 41, pp. 715–724.

84. Wang, X., Plant Phospholipases, Annu. Rev. Plant Phys-iol. Plant Mol. Biol., 2001, vol. 52, pp. 211–231.

85. Munnik, T., Phosphatidic Acids: An Emerging PlantLipid Second Messenger, Trends Plant Sci., 2001,vol. 6, pp. 227–233.

86. Medvedev, S.S., Tankelyun, O.V., Batov, A.Yu., Voron-ina, O.V., Martinets, Ya., and Machá ková, I., Iono-phorous Functions of Phosphatidic Acid in the PlantCell, Russ. J. Plant Physiol., 2006, vol. 53, pp. 39–47.

87. Romanov, G.A., Getman, I.A., Bolyakina, Yu.P., Rak-ova, N.Yu., Kieber, J.J., and Schmülling, T., PrimaryAlcohols, Substrates for Phospholipase D-CatalyzedTransphosphatidylation, Suppress the CytokininAction, Phytohormones in Plant Biotechnology andAgriculture, Machá ková, I. and Romanov, G.A., Eds.,Dordrecht: Kluwer, 2003, pp. 129–139.

88. Ryu, S.B., Karlsson, B.H., Özgen, M., and Palta, J.P.,Inhibition of Phospholipase D by Lysophosphatidyleth-anolamine, a Lipid-Derived Senescence Retardant,Proc. Natl. Acad. Sci. USA, 1997, vol. 94, pp. 12 717–12 721.

89. Lomin, S.N., Bolyakina, Yu.P., Getman, I.A., Rak-ova, N.Yu., Martinec, J., and Romanov, G.A., Analysisof the Participation of Non-Canonic Intermediates inCytokinin Signaling, Abst. II Int. Symp. “Signaling Sys-tems of Plant Cells: Role in Adaptation and Immunity”,Kazan, 2007, pp. 193–194.

90. Kolesnikov, Ya.S., Kretinin, S.V., Kravets, V.S.,Romanov, G.A., Martinec, J., and Machá ková, I., Par-ticipation of PIP2–Phospholipase D in Cytokinin Sig-naling, Abst. II Int. Symp. “Plant Growth Substances:Intracellular Hormonal Signaling and Applying inAgriculture”, Kiev: Polytechnika, 2007, p. 49.

91. Kravets, V.S., Kolesnikov, Ya.S., Kuznetsov, Vl.V., andRomanov, G.A., The Second International Symposium“Plant Growth Substances: Intracellular Hormone Sig-naling and Applying in Agriculture” (Kiev, Ukraine,October 8–12, 2006), Russ. J. Plant Physiol., 2008,vol. 55, pp. 568–577.

92. Testerink, C., Dekker, H.L., Lim, Z.-Y., Johns, M.K.,Holmes, A.B., de Koster, C.G., Ktistakis, N.T., andMunnik, T., Isolation and Identification of PhosphatidicAcid Targets from Plants, Plant J., 2004, vol. 39,pp. 527–536.

93. Testerink, C. and Munnik, T., Phosphatidic Acid: AMultifunctional Stress Signaling Lipid in Plants, TrendsPlant Sci., 2005, vol. 10, pp. 368–375.

94. Andrsen, B.T., Rizzo, M.A., Shome, K., andRomero, G., The Role of Phosphatidic Acid in the Reg-ulation of the RAS/MEK/Erk Signaling Cascade, FEBSLett., 2002, vol. 531, pp. 65–68.

95. Romanov, G.A., Rakova, N.Yu., and Vanyushin, B.F.,Polyamines Inhibit Expression in Cytokinin-DependentTransgenic Arabidopsis, Dokl. Akad. Nauk, 2004,vol. 398, pp. 415–418.

96. Rakova, N.Yu. and Romanov, G.A., Polyamines Sup-press Manifestation of Cytokinin Primary Effects, Russ.J. Plant Physiol., 2005, vol. 52, pp. 50–57.

c

c

c

290


ROMANOV

97. Schmülling, T., Schäfer, S., and Romanov, G., Cytoki-nins as Regulators of Gene Expression, Physiol. Plant.,1997, vol. 100, pp. 505–517.

98. Klyachko, N.L., Post-Transcriptional Hormonal Regu-lation of Protein Synthesis, Doctoral (Biol.) Disserta-tion, Moscow: Timiryazev Inst. Plant Physiol. Russ.Acad. Sci., 1986.

99. Sherameti, I., Shahollari, B., Landsberger, M., Wester-mann, M., Cherepneva, G., Kusnetsov, V., andOelmüller, R., Cytokinin Stimulates PolyribosomeLoading of Nuclear-Encoded mRNAs for the PlastidATP Synthase in Etioplasts of Lupinus luteus: TheComplex Accumulates in the Inner-Envelope Mem-brane with the CF1 Moiety Located towards the StromalSpace, Plant J., 2004, vol. 38, pp. 578–593.

100. Chae, H.S., Faure, F., and Kieber, J.J., The eto1, eto2,and eto3 Mutations and Cytokinin Treatment IncreaseEthylene Biosynthesis in Arabidopsis by Increasing theStability of ACS Protein, Plant Cell, 2003, vol. 15,pp. 545–559.

101. Romanov, G.A. and Medvedev, S.S., Auxins and Cyto-kinins in Plant Development. Advances in Phytohor-mone Studies. The Second International Symposium(Prague, Czech Republic, July 7–12, 2005), Russ.J. Plant Physiol., 2006, vol. 53, pp. 278–286.

102. Li, C.H., Yu, N., Jiang, S.M., Shangguan, X.X.,Wang, L.J., and Chen, X.Y., Down-Regulation ofS-Adenosyl-L-Homocysteine Hydrolase Reveals aRole of Cytokinin in Promoting TransmethylationReactions, Planta, 2008, vol. 228, pp. 125–136.

103. Vanyushin, B.F., DNA Methylation in Plants, Curr. Top-ics Microbiol. Immunol., 2006, vol. 301, pp. 67–122.

104. Deikman, J. and Hammer, P.E., Induction of Anthocya-nin Accumulation by Cytokinins in Arabidopsisthaliana, Plant Physiol., 1995, vol. 108, pp. 47–57.

105. Dewitte, W. and Murray, J.A.H., The Plant Cell Cycle,Annu. Rev. Plant Biol., 2003, vol. 54, pp. 235–264.

106. Roef, L. and van Onckelen, H., Cytokinin Regulation ofthe Cell Division Cycle, Plant Hormones. Biosynthesis,Signal Transduction, Action, Davies, P.J., Ed., Dor-drecht: Kluwer, 2003, pp. 241–261.

107. Li, X., Mo, X., Shou, H., and Wu, P., Cytokinin-Medi-ated Cell Cycling Arrest of Pericycle Founder Cells inLateral Root Initiation of Arabidopsis, Plant Cell Phys-iol., 2006, vol. 47, pp. 1112–1123.

108. Corbesier, L., Prinsen, E., Jackmard, A., Lejeune, P.,van Onckelen, H., Perilleux, C., and Bernier, G., Cyto-kinin Levels in Leaves, Leaf Exudate and Shoot ApicalMeristem of Arabidopsis thaliana during Floral Transi-tion, J. Exp. Bot., 2003, vol. 54, pp. 2511–2517.

109. Romanov, G.A. and Sukhoverov, V.S., A Study ofGrowth Kinetics and Hormonal Gradient Dynamics in

Computer Plants, Simulated Plant Multicellular Struc-tures, Dokl. Biol. Sci., 1997, vol. 352, pp. 87–89.

110. Aloni, R., Aloni, E., Langhans, M., and Ullrich, C.I.,Role of Cytokinin and Auxin in Shaping Root Architec-ture: Regulating Vascular Differentiation, Lateral RootInitiation, Root Apical Dominance and Root Gravitro-pism, Ann. Bot., 2006, vol. 97, pp. 883–893.

111. Dodueva, I.E., Frolova, N.V., and Lutova, L.A., PlantTumorigenesis: Different Ways for Shifting SystemicControl of Plant Cell Division and Differentiation,Transgen. Plant J., 2007, vol. 1, pp. 17–38.

112. Kusnetsov, V.V., Herrmann, R.G., Kulaeva, O.N., andOelmüller, R., Cytokinin Stimulates and Abscisic AcidInhibits Greening of Etiolated Lupinus luteus Cotyle-dons by Affecting the Expression of the Light-SensitiveProtochlorophyllide Oxidoreductase, Mol. Gen. Genet.,1998, vol. 259, pp. 21–28.

113. Buchanan-Wollaston, V., The Molecular Biology ofLeaf Senescence, J. Exp. Bot., 1997, vol. 48, pp. 181–199.

114. Gan, S. and Amasino, R.M., Making Sense of Senes-cence, Plant Physiol., 1997, vol. 113, pp. 313–319.

115. Chailakhyan, M.Kh. and Khryanin, V.N., Pol rastenii iego gormonal’naya regulyatsiya (Plant Sex and Its Hor-monal Regulation), Moscow: Nauka, 1982.

116. Jasinski, S., Piazza, P., Craft, J., Hay, A., Woolley, L.,Rieu, I., Phillips, A., Hedden, P., and Tsiantis, M.,KNOX Action in Arabidopsis Is Mediated by Coordi-nate Regulation of Cytokinin and Gibberellin Activities,Curr. Biol., 2005, vol. 15, pp. 1560–1565.

117. Greenboim-Wainberg, Y., Maymon, I., Borochov, R.,Alvarez, J., Olszewski, N., Ori, N., Eshed, Y., andWeiss, D., Cross Talk between Gibberellin and Cytoki-nin: The Arabidopsis GA Response Inhibitor SPINDLYPlays a Positive Role in Cytokinin Signaling, PlantCell, 2005, vol. 17, pp. 92–102.

118. Aksenova, N.P., Konstantinova, T.N., Gukasyan, I.A.,Golyanovskaya, S.A., and Romanov, G.A., KinetinEliminates an Enhanced Photoperiodic Sensitivity ofPHYB Transgenic Potato Plants, Dokl. Biol. Sci, 2003,vol. 391, pp. 340–342.

119. Aksenova, N.P., Konstantinova, T.N., Lozhnikova, V.N.,Golyanovskaya, S.A., Gukasyan, I.A., Gatz, C., andRomanov, G.A., Photoperiodic and Hormonal Controlof Tuberization in Potato Plants Transformed with thePHYB Gene from Arabidopsis, Russ. J. Plant Physiol.,2005, vol. 52, pp. 623–628.

120. Romanov, G.A., Plant Hormone-Binding Proteins andPhytohormone Perception, Sov. Plant Physiol., 1989,vol. 36, pp. 136–146.

121. Stepanova, A.N. and Ecker, J.R., Ethylene Signaling:From Mutants to Molecules, Curr. Opin. Plant Biol.,2000, vol. 3, pp. 353–360.

Documents

How do cytokinins affect the cell?