Cytokinins: metabolism and function inplant adaptation to environmentalstressesSukbong Ha1*, Radomira Vankova2*, Kazuko Yamaguchi-Shinozaki3,Kazuo Shinozaki4 and Lam-Son Phan Tran5

1 Department of Plant Biotechnology, Chonnam National University, Buk-Gu, Gwangju 500-757, Korea2 Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany of the Academy of Sciences of the Czech Republic,

CZ-16502 Prague, Czech Republic3 Japan International Center of Agricultural Sciences, Ibaraki 305-8686, Japan4 Gene Discovery Research Group, Plant Science Center, RIKEN Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-

0045, Japan5 Signaling Pathway Research Unit, Plant Science Center, RIKEN Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-

0045, Japan

Review

In plants, the cytokinin (CK) phytohormones regulatenumerous biological processes, including responses toenvironmental stresses, via a complex network of CKsignaling. By an unknown mechanism, stress signals areperceived and transmitted through the His–Asp phos-phorelay, an important component of the CK signaltransduction pathway, triggering CK-responsive genes.Because of the intensive crosstalk between CKs andabscisic acid (ABA), modulation of CK levels and theirsignal transduction affects both ABA-dependent andABA-independent pathways, enabling plant adaptationto adverse conditions. This review presents our currentunderstanding of the functions of CKs and CK signalingin the regulation of plant adaptation to stress. Biotech-nological strategies based on the modulation of CKlevels have been examined with the aim of stabilizingagriculture yields.

Cytokinins (CKs) regulate plant adaptation to stressMembers of the CK family of plant hormones are consid-ered to be master regulators, strongly influencing plantgrowth and development. In the past 10 years, CK metab-olism and signaling pathways have become a major focusin plant biology research [1–3]. Increasing evidence hasdemonstrated that CKs play an important role in theregulation of environmental stress responses, involvingintensive interactions and crosstalk with abscisic acid(ABA) [4–9]. Environmental stresses, such as droughtand high salinity, decrease the production and transportof CKs from roots. Application of exogenous CKs to plantscan increase stomatal apertures and transpiration inmany plants [10–12] and can have a positive influenceon photosynthetic activity [13], indicating an importantimpact of CKs in the regulation of plant adaptation toenvironmental stresses. These findings point to potentialapplications in agricultural biotechnology. In this review,we briefly discuss recent advances in the understanding of

Corresponding author: Tran, L.-S. (tran@psc.riken.jp)* These authors contributed equally.

172 1360-1385/$ – see front matter � 2011 Elsevier Ltd. All rights reserv

CK metabolism and signaling and their roles in abioticstress responses, their interactions and crosstalk withABA and the implications of this knowledge for plantbiotechnological applications.

CK metabolismEndogenous CKs are adenine derivatives with either iso-prenoid or aromatic side chains. Depending on hydroxyl-ation and reduction of the side chain, isoprenoid CKs,which are widespread in nature, can be distinguished asisopentenyladenine (iP)-, trans-zeatin (tZ)-, cis-zeatin (cZ)-or dihydrozeatin-type derivatives. By contrast, aromaticCKs, such as N6-(meta-hydroxybenzyl)adenine, are foundin plants at a lower abundance [14,15].

The rate-limiting step of isoprenoid CK biosynthesis iscatalyzed by isopentenyltransferases (IPTs). Plant IPTsuse ADP, ATP and tRNA for isopentenylation of the ade-nine moiety [16,17]. Synthesized isopentenyladenine ribo-side phosphates and tZ riboside phosphates are convertedto active CKs by either gradual removal of the phosphategroup and then the ribosyl moiety or in a single step byLONELY GUY (LOG), the CK riboside 50-monophosphatephosphoribohydrolase [18,19]. tZ is formed from iP bycytochrome P450 monooxygenases (CYP735A1/A2), whichhydroxylate methyl groups on the iP side chain [20]. Themost biologically active tZ- and iP-type CKs are producedpredominantly in a chloroplast-localized pathway by ATP/ADP IPTs. The tZ and iP are differentially distributed dueto the differential expression patterns of correspondingIPT genes. tZ is likely to move acropetally through thexylem and iP is likely to move basipetally or systematicallythrough the phloem [21]. CK movements controlled byenvironmental and endogenous signals contribute to nu-trient translocation, sink strength and grain yield [22]. Theisoprenoid side chain of cZ is believed to be synthesized in acytoplasmic-localized pathway. At least part of cZ mayoriginate from degradation of (cis-hydroxy)isopentenyltRNAs [23,24]. The cZ-type CKs are highly abundant inmonocot species. The physiological role of cZ-type CKs is not

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Review Trends in Plant Science March 2012, Vol. 17, No. 3

clear, but there are indications that they might function asless active CKs under limited growth conditions [25].

In Arabidopsis (Arabidopsis thaliana), AtIPT1, AtIPT3and AtIPT4–AtIPT8 encode ATP/ADP IPTs, whereasAtIPT2 and AtIPT9 code for tRNA IPTs. Mutations ofthe AtIPT2 and AtIPT9 genes, particularly in combination,exhibit downregulation of isopentenyl- and (cis-hydroxy)i-sopentenyl-tRNAs as well as a significant reduction incZ-type CK levels [15,23]. Plants carrying mutant ATP/ADP AtIPT genes possess scarce amounts of iP- and tZ-typeCKs and exhibit shoot growth retardation, which can bepartially rescued by the addition of exogenous tZ [23].Conditional overexpression of AtLOGs results in decreasesin iP riboside monophosphate and increases in iP andcorresponding glucosides, leading to CK-induced pheno-types, such as delayed senescence and reduced apicaldominance. Multiple Arabidopsis log mutants show CKinsensitivity when iP riboside is applied exogenously [19].

Many studies have shown that CK degradation has animportant regulatory role. CK degradation requires CKdehydrogenases (CKXs), which remove CK unsaturatedisoprenyl side chains [26]. CKXs were reported to have ahigh affinity for iP and tZ and a much lower affinity fortheir ribosides [15,27]. However, a CKX with preference forcZ and CK N-glucoside was recently found in maize [28].There are seven CKX (CKX1 to CKX7) genes in Arabidopsis[15,27,29]. Each encoded isoenzyme has a different bio-chemical characteristic and expression pattern [25,29,30].CKXs are localized to vacuoles, the cytosol and the apo-plast [28,29] (Figure 1). Overexpression of CKX genescauses a decrease in CK levels and, to a lesser extent,auxin content, inhibiting shoot development, but enhanc-ing root development [31,32]. Overexpression of CKX1,CKX3 or CKX5 leads to stronger phenotypic changes thanthat of other CKX genes [32]. The ckx3 ckx5 double mutantexhibits larger inflorescences and floral meristems, in-creased size of the WUSCHEL expression domain in theorganizing center of the shoot apical meristem, supernu-merary ovules and increased seed yield [33], indicatingessential roles of CKs in the regulation of reproductivedevelopment and seed yield. In addition, glycosylation,predominantly with glucose, is a parallel and almost equal-ly important process of CK deactivation, which is necessaryfor maintenance or re-establishment of CK homeostasis.Conjugation with glucose to the N7- or N9-position of theadenine ring results in permanent deactivation of CKs,whereas conjugation to the N3-position of the adenine ringand to the hydroxyl group of the side chain is reversible,leading to CK storage forms [15,22,34].

His–Asp phosphorelay and CK signalingIn comparison with other signaling pathways, in whichproteasome-mediated degradation [35,36] or MAP kinasecascades are harnessed [37,38], signaling of CKs (as well asof ethylene) involves His–Asp phosphorelay, resemblingbacterial two-component phosphorelay pathways [39,40].In plants, CK receptors are autophosphorylated when CKsare perceived and the phosphoryl group is transferred tothe acceptor side of receptors, as CK receptors are hybridhistidine kinases (HKs) [41]. Histidine phosphotransferproteins receive the phosphoryl group from CK receptors

and transmit them to nuclear type-B response regulators(RRs), the positive CK signal transduction regulators,which turn on primary response genes, including type-ARRs [42]. New protein synthesis is not required for theseprompt cellular responses [42]. Type-A RRs are involved inCK signaling negative feedback loops. These negative RRshave a phosphorylation acceptor site and their expressionis rapidly induced by CK treatment [43] (Figure 1). Froman evolutionary perspective, CK signaling systems inplants appeared after the moss conquest of land, becausealgae do not possess CK receptors and negative RRs [44].

The Arabidopsis genome encodes six nonethylene HKs(AHK1, AHK2, AHK3, AHK4, AHK5/CKI2 and CKI1) [45].Loss-of function analyses have demonstrated that AHK2,AHK3 and AHK4 possess CK receptor functions and theseAHKs act redundantly in CK signal transduction [46,47].Individual CK receptors have distinct roles in the regula-tion of plant growth and development [46,48]. Recently, itwas reported that AHK2, AHK3 and AHK4 are predomi-nantly localized to the endoplasmic reticulum, suggestingthat CK signaling mainly initiates from this organelle [49–

51] (Figure 1). Biochemical assays showed that the CHASE(Cyclases/Histidine kinases Associated Sensory Extracel-lular) domain of CK receptors, which was proposed to beresponsible for CK perception, is essential and sufficientfor CK-binding [52]. The CHASE domain was found to beflanked by two transmembrane domains [52]. Membranedynamics seems to have important roles in triggering theautophosphorylation of the HKs. Mutations in the CHASEdomain, in the transmembrane domain or around thephosphorylation site render HKs constitutively active[53–55]. Interestingly, AHK4 possesses both kinase andphosphatase activity with and without CKs, respectively,which was not found in AHK2 or AHK3 [56].

Specificity of CK signaling is mediated by expressionpatterns of individual receptors as well as by their differentligand affinities. AHK2 and AHK4 have relatively similarligand specificity [57]. All three receptors have overlappingfunctions in shoot apical meristems and root cap columella,but AHK2 and AHK3 are specifically localized to leafparenchyma, whereas AHK4 is localized to the root vascu-lature [48]. Recent determination of the crystal structure ofthe AHK4 sensor domain has helped to elucidate the CK-binding specificity of this particular HK and has providedthe molecular basis for the recognition of natural andsynthetic CKs by the CK receptors [58]. Chemical inhibi-tors that target CK receptors could also be valuable tools toaid in the identification of the hormone-binding specificityof the CK receptors [59–61].

It has recently been shown that Arabidopsis histidinephosphotransfers (AHPs) are ubiquitous and neither theirtranscriptional level nor their subcellular localization ischanged upon CK treatment, in contrast to previousassumptions that they would be rapidly moved into thenucleus in response to CKs [43,62,63]. The AHP1–AHP5proteins have conserved phosphorylation sites and interactwith both AHKs and Arabidopsis response regulators(ARRs) [64]. Loss-of-function analyses have indicated apositive redundant function of authentic AHP1–AHP5 inCK signaling [65]. Unlike the authentic AHPs, the pseudoAHP6 lacks a phosphorylation site and is necessary for

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CK

CK

AHPPAHP

CK(iP, tZ)

CK(iPR, tZR)

CK

CK

ER

Permease(PUP)

Transporter (ENT)

HH

D D

AHK1

P

P

AHP6ARR22P

AHPPAHP

Cytosol

Nucleus

Type-A ARRsP

AH

K2,

AH

K3,

AH

K4

Type-B ARRsP

Target genes

Reduced CKs

Enhanced ABA sensitivitymorphological adjustment

Plant adaptation to stress

Preservation of photosyntheticactivity, delay of senescence

Increased CKs bygenetic engineering

AHKs

AHPs

ARRs

Vacuole

CKX1CKX3

CKX2, CKX4, CKX5, CKX6

CKX7

P

?

Apoplast

Stress tolerance

HH

D

D

HH

D

D

TRENDS in Plant Science

Figure 1. Possible mechanisms leading to enhanced stress tolerance in Arabidopsis. Abiotic stress tolerance can be enhanced by a reduction of active CK levels or by repression

of CK signaling achieved by inactivation of the components of the CK signaling pathway (e.g. CK receptor AHKs). Synthesized CKs can be transported into the cytosol by purine

permeases (PUPs) and equilibrative nucleoside transporters (ENTs), or degraded by CKXs in the apoplast. After their synthesis, some CKXs (CKX2, CKX4, CKX5 and CKX6) are

secreted to the apoplast, whereas others are targeted to the vacuole (CKX1 and CKX3) or remain in the cytosol (CKX7). The majority of CK receptors (AHK2, AHK3 and AHK4,

shown in red) are localized to the endoplasmic reticulum (ER). CK perception is likely to occur inside the ER lumen, whereas autophosphorylation (P) of the His residue (H) and

transfer of phosphoryl group to the Asp residue (D) of the receptor receiver domain occurs in the cytosol. AHPs are phosphorylated at the conserved His residue by the AHKs, but

their localization at either the nucleus or the cytosol is independent of phosphorylation status. Type-B or type-A ARRs are final phosphoacceptor molecules in the His–Asp

phosphorelay. Type-B ARRs are transcription factors responsible for turning on expression of the type-A primary response genes and downstream CK-regulated target genes.

The type-A ARRs can repress CK signaling through negative feedback loops, perhaps at the level of AHP-mediated signaling (broken blue arrow). The atypical type-C ARR22,

with its phosphohistidine phosphatase activity on specific AHP2, AHP3 and AHP5 in the cytoplasm, can repress CK signaling. AHP6 lacking the conserved His residue can inhibit

the phosphoryl transfer to authentic AHPs. Repression of CK signaling is indicated by ‘X’. Activation of the osmosensor AHK1, which is localized to the plasma membrane, can

also improve stress tolerance in a CK-independent manner. The alternative strategy for an increase of stress tolerance is the elevation of CK levels after the initiation of stress

response, achieved by stimulation of CK biosynthetic genes driven by stress- and/or senescence-inducible promoters. Elevated CKs via genetic engineering may, at least

partially, alleviate negative effects of stress on photosynthetic activity and suppress stress-accelerated senescence of older and mature leaves.

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xylem development. AHP6 induces xylem developmentand simultaneously blocks CK signaling [66], which is inaccordance with the fact that CKs negatively control xylemformation and promote phloem development.

In Arabidopsis, multiple gene knockout analyses haveindicated that the type-B ARR1, ARR10 and ARR12 genesplay key roles in CK signaling [67,68]. Of the type-A ARRs,which interact with the AHPs but not with the type-BARRs [69], ARR3–ARR9 and ARR15 function as negativeregulators of CK signaling [70]. Type-A ARR genes areknown as primary response genes because their expressionis rapidly induced by CKs as a negative feedback loop [43].

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Mutual regulation exists between type-A and type-BARRs. Whereas type-A ARRs indirectly regulate theactivity of type-B ARRs through a negative feedback loop,type-B ARRs directly upregulate expression of certaintype-A ARR genes in a CK-dependent manner [71].

Several proteins, such as those resembling RRs anddesignated atypical RRs, were also reported to be involvedin CK signaling. The mutant of an atypical RR encodinggene, ARR22, exhibits a similar dwarf phenotype as the CKreceptor ahk triple mutant [72]. Biochemical analysis hasindicated that ARR22 functions as a strong CK signalinginhibitor (Figure 1) [73]. Additionally, the expression levels

Table 1. Summary of CK metabolic and CK signaling genes involved in abiotic stress response based on published literature

Gene ID Description Regulatory function

in stress response

Type of stresses Regulatory function

in ABA response

Refs

AHK2 CK receptor kinase Negative Cold, drought, salt Negative [6,81]

AHK3 CK receptor kinase Negative Cold, drought, salt Negative [6,81]

AHK4 CK receptor kinase Negative regulator

with CKs

Salt Negative [81]

CKXs CK degradation Positive Drought, salt Positive [7,8]

IPTsa CK biosynthesis Negative Drought, salt Negative [7]

Type-B ARRs

ARR1, ARR12 Transcription factors Negative Salt Unknown [83]

Type-A ARRs

ARR3, ARR4,

ARR5, ARR6,

ARR8, ARR9

Primary response genes Positive Salt Positive [6,81–83]

ARR4, ARR7 Primary response genes Negative Cold, drought, osmotic Negative [6,81–83]

ARR3, ARR4 Primary response genes Positive Cold, drought, osmotic Positive [6,81–83]

ARR8, ARR9 Primary response genes Negative Osmotic Negative [6,81–83]

ARR5 Primary response genes Negative Cold None [6,81–83]

ARR6 Primary response genes Negative Cold Negative [6,81–83]

ARR7 Primary response genes Negative Cold Negative [6,81–83]

Abbreviations: AHK, Arabidopsis histidine kinase; ARR, Arabidopsis response regulator; CKXs, CK dehydrogenases; IPTs, isopentenyltransferases.

aConditional overexpression of the Agrobacterium tumefaciens IPT gene using, e.g. drought/senescence-dependent SARK promoter or senescence-inducible SAG12

promoter or stress-inducible RD29A promoter can enhance drought, heat and high-salinity tolerance of transgenic plants [78,90,91,93–95].

Review Trends in Plant Science March 2012, Vol. 17, No. 3

of several members of the AP2-EREBP transcription factorfamily were shown to be upregulated by CK treatment.These genes encode the so-called CK response factors(CRFs), which rapidly accumulate in the nucleus and theirdownstream targets significantly overlap with those oftype-B ARRs, suggesting that the CRFs function in parallelwith type-B ARRs to mediate CK response [74].

Modulation of endogenous CK levels in response toenvironmental stressesAn unfavorable environment was found to have an impacton CK content in plants and changes in endogenous CKlevels were reported to alter the stress tolerance of plants[5,7,8,75–78]. When evaluating the roles of CKs in stressresponses, several physiological functions of CKs need tobe taken into account; predominantly, strong promotion ofcell division, which is a highly energy demanding process; apositive effect on photosynthesis, the main energy sourcein plants; and stimulation of the sink strength. Thus, CKmetabolism is regulated in a highly dynamic way in re-sponse to stressful conditions. Short-term or mild stressesmight be associated with transient elevation of CK levelsand/or signaling [5]. Responses to more severe or prolongedstresses are associated with growth reductions and reloca-tion of limited energy resources towards defense againstharmful effects originating from exposure to environmen-tal stresses. Therefore, prolonged drought or extensive saltstress was reported to be associated with downregulationof active CK contents [7,75–77].

Recently, studies of stress responses in plants withdecreased CK levels (CK-deficient plants) have provideddirect evidence that CKs are negative regulators of stresssignaling. Constitutive downregulation of CK levels by theoverexpression of CKX genes or by inactivation of IPTgenes results in drought and salt stress-tolerant pheno-types [7,8] (Table 1). This may be partially due to a strongreduction of the growth rate, which is associated with lower

energy consumption. However, this assumption cannotfully explain the enhanced stress tolerance because someother dwarf mutants do not exhibit this feature; for in-stance, the Arabidopsis small ubiquitin-like modifier(SUMO) E3 ligase siz1 mutant, which is dwarf butsensitive to drought stress [79].

When the regulation of water loss was followed in CK-deficient plants (ipt1 ipt3 ipt5 ipt7 quadruple mutant andCKX overexpressors), neither reductions in stomatal aper-ture nor changes in stomatal density were observed. Im-proved drought tolerance of the CK-deficient plants wasexplained by the stimulation of protective mechanismscoinciding with improved maintenance of cell membraneintegrity and stability under water deficit. The increasedABA sensitivity found in CK-deficient plants is also favor-able to adaptation to adverse conditions as it leads toinduced expression of stress- and ABA-responsive genes[7]. In short, reduction of active CK content by either stimu-lation of CK degradation or by inactivation of IPT biosyn-thetic genes contributes to increased salt and drought stresstolerance, which is in good accordance with the finding thatsevere stress conditions are generally associated with therepression of CK biosynthesis (Figure 1).

CK signaling and plant adaptation to environmentalstressesApart from dynamic changes in CK content during stressresponses, there is also tightly regulated control of CKsignal transduction during unfavorable conditions. Sincethe discovery of AHK1 as an osmotic sensor, which posi-tively regulates stress responses [80], the participations ofother nonethylene receptor HKs in stress perception havebeen identified [6,81] (Table 1). Although AHK2, AHK3 andAHK4 are rapidly induced by stress, as has been found forAHK1 [81], there is nevertheless an important differencebetween the role of AHK1 and the CK receptor HKs in thisresponse. AHK1 was recognized as a positive regulator of

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osmotic stress signaling because AHK1 overexpressorsenhanced stress tolerance, whereas the ahk1 mutantwas sensitive to osmotic stresses. Furthermore, the ahk1mutation reduced ABA sensitivity and downregulated theexpression of stress-inducible and/or ABA-responsivegenes, suggesting that AHK1 positively regulates stressresponses through both ABA-dependent and ABA-inde-pendent pathways [81,82]. By contrast, the CK receptorsAHK2, AHK3 and AHK4 are negative regulators of ABAand stress signaling, because loss-of-function mutants ex-hibit strong sensitivity to exogenous ABA and improvedtolerance to cold, salt and drought stresses [6,81]. Further-more, unlike the ahk1 mutant, the ahk2 ahk3 doublemutant exhibits upregulation of stress-inducible and/orABA-responsive genes. Together, it has been suggestedthat one of the mechanisms used by plants to adapt toadverse conditions is the downregulation of CK signalingby the repression of CK metabolism (Figure 1).

Other components of the CK signaling pathway havebeen shown to be involved in the regulation of stressresponses, including several members of type-A andtype-B ARR subfamilies (Table 1). Multiple mutant analyseshave suggested a complex function for type-A ARRs [82].Individual mutations in arr5, arr6 or arr7 resulted inenhanced cold tolerance, suggesting that these genes nega-tively regulate cold stress responses [6]. Recently, functionalanalyses of the arr1 arr12 double mutant indicated thatthe type-B ARR1 and ARR12 act as negative regulatorsduring a salt stress response. These two proteins redun-dantly regulate sodium accumulation through AtHKT1;1,which encodes a high affinity potassium transporterresponsible for removing sodium ions from the root xylem[83].

Interaction and crosstalk between CKs and ABAABA is the key hormone involved in the regulation of bothstress and non-stress-related processes associated with de-hydration [84,85]. It is generally postulated that CKs act asantagonists to ABA in various growth and physiologicalprocesses, including environmental stress responses. Re-cent findings have suggested the existence of intensiveinteractions and crosstalk between CKs and ABA and theirsignaling pathways. A reduction in CK content was reportedto lead to hypersensitivity to ABA and upregulation ofstress- and/or ABA-responsive genes, whereas the reverseprocess was observed with elevation of CK levels [7,9].Hypersensitivity to ABA was suggested as one of the con-tributors to the increased stress tolerance of CK-deficientand CK signaling mutants. The faster and more profoundstress responses of these mutant plants partially resultedfrom the activation of the ABA-dependent AREB (ABA-responsive-element – ABRE – binding) pathway [4,7,81],which governs the majority of ABA-mediated ABRE-depen-dent gene expression in response to osmotic stresses [86].

During drought stress, ABA content is increased, where-as the active CK content is decreased. It remains to beresolved whether increased ABA content plays a role in thedecrease of CK content. Stress responses are usually asso-ciated with downregulation of the expression of the IPTgenes, which results in a decrease of CK content andconcomitantly leads to reduced CK degradation associated

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with suppression of CKX gene expression. Measurementsof CK levels and expression of CK metabolic genes in plantstreated with exogenous ABA suggested that stress-inducedABA might be involved in downregulation of CK levelsthrough repression of CK metabolic genes [7,87]. However,further experiments in ABA-deficient or ABA-overprodu-cing plants should provide more precise information.

Stress tolerance of CK-deficient and signaling mutantplants was shown to be associated with the downregulationof CK signaling. Positive regulators of the CK signal trans-duction pathway, the type-B RRs, were found to be negativeregulators in salt stress responses [80]. Consistently, anincrease in CK content led to the downregulation of ABA-and stress-responsive genes, including ABI5, and dimin-ished ABA-mediated repression of the type-A ARR4, ARR5and ARR6. Expression of ABI5 was negatively controlled byARR4, ARR5 and ARR6, and these four proteins were foundto interact directly [9]. Thus, ABI5 and ARR4, ARR5 andARR6 may form a protein complex to control the interplaybetween CK and ABA signaling in response to stresses.

Lateral root (LR) development is considered a morpho-logical adjustment to nutrient shortage. By contrast, thedrought stress response is associated with suppression ofLR formation and with promotion of primary root growth,which allows plants to reach the water in deeper soillayers. Both ABA and CKs, which are elevated indrought-stressed roots [5], act as antagonists of auxin, aplant hormone necessary for the initiation of LR develop-ment. CKs and ABA were shown to inhibit LR initiation byupregulation of the expression of ABI4 which in turnrepressed polar auxin transport, resulting in a decreasein auxin maxima formation and consequently in a reduc-tion in LR development [88]. This evidence suggests that insome biological processes, ABA and CKs may act synergis-tically. This observation is in accordance with the findingthat a reduction of CK content in CK-deficient plants led to areduction, not an increase, in endogenous ABA levels nec-essary for maintaining the appropriate ABA/CK ratio toavoid unfavorable consequences, e.g. on photosynthesis [7].

Biotechnological manipulation of CK contents forimprovement of stress toleranceEven though detailed molecular players and their specificroles in the individual abiotic stress responses have notbeen thoroughly identified, it is now becoming clear thatmodulation of CK levels and CK signal transduction repre-sents a tool to enhance abiotic stress tolerance. Possibleand conceivable biotechnological applications can be car-ried out based on the knowledge of regulatory mechanismsthat are involved in CK metabolism and CK signaling(Figure 2). With the availability of whole sequence dataof crop species, such as rice (Oryza sativa), maize (Zeamays), soybean (Glycine max) and others, more field-oriented applications are expected.

Desirable outcomes of biotechnological approaches havealready come to fruition. Although constitutive overexpres-sion of genes for CK degradation is associated with anenlarged root system, it also results in stunted shoot growth.A reduction of CK levels specifically in roots with the use ofroot-targeted overexpression of CKX genes results in plantswith increased root biomass. This response results in

Normal Stressed

Increase of CK levels byconditional overexpression

of IPTs

Inactivation of CK signalingthroughAHKsAHPs

type-A ARRstype-B ARRs

AHP6ARR22 etc.

Decrease of CK levels byoverexpression of CKXs or

disruption of IPTs

Shoot

Root

TRENDS in Plant Science

Figure 2. Strategies for the development of stress-tolerant plants. Transgenic plants that are tolerant to abiotic stresses can be developed with the manipulation of CK

metabolism and repression of CK signaling. Stress-associated and senescence-specific overexpression of CK biosynthesis genes can delay plant senescence and render

plants stress-tolerant. A reduction in CK levels in the root can enhance root architecture and increase stress tolerance without negative impacts on shoot growth that

requires CKs for prosperity. Interference at the CK signaling level also bears significant potential for the development of improved stress-tolerant plants.

Review Trends in Plant Science March 2012, Vol. 17, No. 3

increased drought tolerance and a higher accumulation ofnutrients, without a penalty in shoot growth. These engi-neered plants have better survival chances in stress orlimited nutrient conditions, because of their improved waterand nutrient use efficiency [8].

By contrast, although constitutive IPT expression causesreduced root growth and enhanced sensitivity to waterstress [89], the stimulation of CK biosynthesis by stress-inducible promoter(s) may diminish stress-induced acceler-ation of leaf senescence, enhance reactive oxygen speciesscavenging capability and allow the maintenance of photo-synthetic activity under stress conditions [78,90–93]. Thisinducible overexpression strategy also enables to maintainthe balance between the levels of CKs and other hormones,such as ABA, for normal growth under unstressed condi-tions. Increasing evidence suggests that increasing CKcontent just prior to the onset of senescence, which helpsavoid the negative impact of excessively overproduced CKson plant growth and delay in the stimulation of defense, mayimprove leaf longevity and photosynthetic capacity underdrought and heat stresses, leading to enhanced stress toler-ance without yield penalties [78,90–92,94,95] (Figure 1). Intransgenic tobacco (Nicotiana tabacum) plants with stress-induced CK biosynthesis, carotenoid pathway genesinvolved in ABA biosynthesis are repressed, but genes inbrassinosteroid biosynthetic pathways are highly expressed[96]. These transgenic plants also show better photosyn-thetic performance under stress [97]. Stress-induced CKsynthesis in rice was reported to result in changes in hor-mone homeostasis and consequently in a stronger sinkcapacity [94]. Inducible or artificially activated promoters

driving gene expression in specific stress conditions could beuseful for overcoming negative stress effects, minimizingyield loss [98,99] (Figure 2).

Concluding remarksAlthough recent research has shown clear evidence thatCKs are involved in the regulation of abiotic stressresponses through CK signaling, uncovering the precisemechanisms of stress signal perception by the CK recep-tors remains a task for the future. It would be interestingto examine the specificity of the phosphorelay in itsmodulation by different AHKs with opposite regulatoryfunctions. Therefore, it is of importance to preciselydetermine the specific interactions of AHPs with individ-ual AHKs by genetic means and the specific functions ofall of the AHPs and ARRs in individual stress responses.Identification of downstream components of the phos-phorelay in stress signaling is also an important andchallenging task. Moreover, recent discovery of a His–

Asp phosphorelay-independent route, by which CKs con-trol auxin flux, provides an open question on whether CKsalso regulate stress responses via this alternative path[100].

Growing world population and climate challenges haveset a tough challenge for the research community. CKhomeostasis and signaling components have emerged asengineered targets for the development of stress-tolerantcrops. Recent results on transgenic research indicate thatCK biology can provide multiple biotechnological strate-gies, contributing to the maintenance of agriculture in asustainable manner.

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AcknowledgmentsThis work was supported by the Grants-in-Aid (Start-up) for ScientificResearch (21870046) from the Ministry of Education, Culture, Sports,Science and Technology of Japan to L-S.P.T. and by the Czech ScienceFoundation (522/09/2058 and 206/09/2062) to R.V. We apologize to allcolleagues whose relevant work we were not able to cite owing to spacelimitations.

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