Cytokinins are central regulators of cambial activityMiho Matsumoto-Kitanoa,1, Takami Kusumotoa,1, Petr Tarkowskib,c, Kaori Kinoshita-Tsujimuraa, Katerina Vaclavíkovab,c,Kaori Miyawakia,2, and Tatsuo Kakimotoa,3

aDepartment of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; bDepartment of Biochemistry, Facultyof Science, Palacky University, Slechtitelu 11, Olomouc 78371, Czech Republic; and cLaboratory of Growth Regulators, Palacky University and Institute ofExperimental Botany, Academy of Sciences of the Czech Republic, Slechtitelu 11, Olomouc 78371, Czech Republic

Edited by Ronald R. Sederoff, North Carolina State University, Raleigh, NC, and approved October 15, 2008 (received for review June 10, 2008)

The roots and stems of dicotyledonous plants thicken by the cellproliferation in the cambium. Cambial proliferation changes inresponse to environmental factors; however, the molecular mech-anisms that regulate cambial activity are largely unknown. Thequadruple Arabidopsis thaliana mutant atipt1;3;5;7, in which 4genes encoding cytokinin biosynthetic isopentenyltransferases aredisrupted by T-DNA insertion, was unable to form cambium andshowed reduced thickening of the root and stem. The atipt3 singlemutant, which has moderately decreased levels of cytokinins,exhibited decreased root thickening without any other recogniz-able morphological changes. Addition of exogenously suppliedcytokinins to atipt1;3;5;7 reactivated the cambium in a dose-dependent manner. When an atipt1;3;5;7 shoot scion was graftedonto WT root stock, both the root and shoot grew normally andtrans-zeatin-type (tZ-type) cytokinins in the shoot were restored toWT levels, but isopentenyladenine-type cytokinins in the shootremained unchanged. Conversely, when a WT shoot was graftedonto an atipt1;3;5;7 root, both the root and shoot grew normallyand isopentenyladenine-type cytokinins in the root were restoredto WT levels, but tZ-type cytokinins were only partially restored.Collectively, it can be concluded that cytokinins are importantregulators of cambium development and that production of cyto-kinins in either the root or shoot is sufficient for normal develop-ment of both the root and shoot.

cambium � isopentenyladenine � phytohormone � zeatin

A large proportion of carbon in the biosphere is held in plantstems and roots. As plants increase girth or thickness they

incorporate more carbon. Roots or stems thicken by cell prolif-eration within the vascular cambium. Cells produced in thecambium move either in a centripetal direction and differentiateinto xylem or in a centrifugal direction and differentiate intotissue containing phloem. In woody plants, the cambium is laiddown just beneath the bark, and xylem constitutes the bulk of theroots and stems. The rate of cell production in the cambium isthe primary determinant of stem and root thickening (1).

Plants regulate their cambial activity in response to envi-ronmental cues such as photoperiod, temperature, and theavailability of water and nutrients. However, little is knownabout the molecular basis of this process. Phytohormones havebeen implicated in the integration of environmental signals toregulate cambial activity. In tree trunks, there is an auxinmaximum in the cambium and its vicinity (2, 3), and expressionof Ptt IAA3m, which encodes a stabilized auxin signalinginhibitor, inhibits cell division in the cambium and perturbedxylem differentiation (4). However, no close correlation hasbeen observed between auxin levels in the cambial region andseasonal changes in cambial activity, although the auxingradient at the xylem formation zone changes when trees startto form latewood (3). Therefore, auxin is not considered to bea temporal mediator of cambial activity, but rather a temporalregulator of xylem formation, and auxin may also supplypositional information to the cambium (3, 4). Gibberellins mayalso have a role in cambial regulation. Overexpression ofGA20 oxidase, the rate-limiting enzyme in biosynthesis ofactive gibberellins, increases both plant height and stem

diameter and is associated with increased cambial activity inhybrid aspen (5). Active gibberellin levels were found to behigh in a region where cambial descendant cells expand to formxylem cells (6). Also, gibberellin levels in the internodes ofaspen remained unchanged in response to short-day condi-tions, which induce cambium dormancy (7).

Although cytokinins are generally considered to be importantregulators of cell division (8), their role in cambial activity hasnot yet been elucidated. Cytokinin levels dramatically increase inresponse to nitrogen and phosphate nutrients (8–11) and de-crease in harsh conditions such as nutrient deficiency or drought(12). These environmental conditions are correlated withchanges in cambial activity, suggesting that cytokinins mayfunction directly as regulators of cambium development.

Cytokinins can be classified into 4 groups [isopentenyladenine(iP)-type, tZ-type, cis-zeatin-type, and aromatic cytokinins] de-pending on the structure of the side chain. Biologically importantcytokinins are iP-type and tZ-type cytokinins, and the first stepsof their biosynthesis are catalyzed by ATP/ADP isopentenyl-transferases (ATP/ADP IPTs) (in Arabidopsis, AtIPT1, AtIPT3,AtIPT4, AtIPT5, AtIPT6, AtIPT7, and AtIPT8) (13). TheAtIPT3, AtIPT5, and AtIPT7 genes are most highly expressedamong genes for ATP/ADP IPTs in Arabidopsis (14). Theatipt3;5;7 triple and atipt1;3;5;7 quadruple mutants have severelydecreased levels of iP-type and tZ-type cytokinins, and theiroverall growth is severely affected (15). The tZ-type cytokininsare formed from isopentenyladenine ribose phosphate by hy-droxylation of the side chain by cytochrome P450 monooxygen-ases (16). cis-zeatin-type cytokinins are produced from isopen-tenylated tRNAs (15), but they play only a minor role in thegrowth of most plants. The biosynthesis and role of aromaticcytokinins are not well known.

Because cytokinins are found in leaf exudates (generallyreferred to as phloem sap) and the xylem sap, they have beenconsidered possible mobile regulators (reviewed in ref. 17).Cytokinins of tZ-type predominate in the xylem sap, whereasiP-type cytokinins predominate in the leaf exudates. This sug-gests that tZ-type cytokinins are mainly transported from theroot to the shoot and iP-type cytokinins are mainly transportedfrom source organs to sink organs. However, it is unknownwhether systemically transported cytokinins make an importantcontribution to cytokinin dynamics and plant growth.

Although Arabidopsis generally finishes its growth within 2months, it typically undergoes secondary growth similar to trees

Author contributions: M.M.-K., K.M., and T. Kakimoto designed research; M.M.-K., T.Kusumoto, P.T., K.K.-T., K.V., and T. Kakimoto performed research; M.M.-K., T. Kusumoto,and T. Kakimoto analyzed data; and M.M.-K. and T. Kakimoto wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1M.M.-K. and T. Kusumoto contributed equally to this work.

2Present address: National Institute for Basic Biology, Okazaki 444-8585, Japan.

3To whom correspondence should be addressed. E-mail: kakimoto@bio.sci.osaka-u.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0805619105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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and therefore can be a useful model plant for studying secondarygrowth (18). In Arabidopsis, inflorescence stem thickness de-pends both on the size of the shoot apical meristem and on thesecondary thickening growth, the latter of which involves celldivision in the cambial zone, cell enlargement, and wall synthesis.The size of the shoot apical meristem may depend on develop-mental and environmental signals (15), but radial cell number inthe root apex is invariant (19). Thus, root diameter primarilydepends on secondary growth.

During the primary growth phase of plant root development,the root vasculature, consisting of xylem, phloem, and interven-ing procambial cells, is formed. The cambium is later initiatedfrom the procambium and produces cells required for secondarythickening (18). Arabidopsis undergoes similar thickening pro-cesses as trees, and it has a great advantage in that many mutantsand transformants are readily available. We examined cambialgrowth in Arabidopsis lines that lack 1, 2, 3, or 4 of the ATP/ADPIPT genes AtIPT1, AtIPT3, AtIPT5, and AtIPT7, as well astransformants in which genes for an isopentenyltransferase canbe induced. Cambium activity responded to small changes incytokinin levels, suggesting that cytokinins are central regulatorsof cambium activity.

ResultsArabidopsis has 7 genes coding for ATP/ADP isopentenyltrans-ferases, which catalyze the biosynthetic steps for iP-type andtZ-type cytokinins (13). The atipt3;5;7 triple and atipt1;3;5;7quadruple mutants, in which all corresponding AtIPT genes weredisrupted by a T-DNA insertion in an exon, have severelydecreased levels of cytokinins (see ref. 15 for T-DNA insertionpositions and endogenous levels of cytokinin species). In thesemutants, primary root elongation was slightly increased andlateral root elongation was greatly increased (15) (Fig. 1A),because normal levels of cytokinins in WT inhibit the root apicalmeristem (20, 21). In addition, a noticeable feature of thismutant not reported before is a severe defect in root thickening(Fig. 1B). The root thickening growth was recovered by additionof a cytokinin, trans-zeatin, in a dose-dependent manner (Fig.1B). To examine how sensitively Arabidopsis responds to de-creases in cytokinin levels by reducing thickening growth, wemeasured the diameter of roots in the atipt3 and atipt3;5 mutants,which have moderately decreased levels of cytokinins (15).

Although we could not detect any morphological changes inthese mutants in a previous study (15), measurements of rootdiameter revealed significant decreases in secondary growth(Fig. 1C). AtIPT3 promoter activity was high in the phloem butnot in the cambium during secondary growth [supporting infor-mation (SI) Fig. S1]. These results indicate that, among manydevelopmental processes observable under our growth condi-tions, thickening growth is the most sensitive to decrease incytokinins.

To understand what processes are affected in the atipt1;3;5;7quadruple mutant, we examined the anatomy in root sections ata basal position. The Arabidopsis root is formed with an invariantradial pattern, consisting of single layers of epidermis, cortex,endodermis, pericycle, and the vasculature (22). Later thecambium forms, which produces cells forming secondary xylem-and phloem-containing tissue, the latter constituting the areaoutside the cambial zone (Fig. S2 and Fig. 2A). The atipt1;3;5;7quadruple mutant lacks the vascular cambium altogether in theroot (Fig. 2B and Fig. S2). Whereas WT roots continuouslyunderwent secondary growth, atipt1;3;5;7 mutant plants exhib-ited no secondary growth until at least 21 days after germination(Fig. S2).

Thickening growth of the inflorescence stem was also greatlydiminished in the atipt1;3;5;7 mutant (Fig. 2G). The averagenumber of vascular bundles in WT was 8.5 � 1.0 (mean � SD),whereas it was decreased to 4.0 � 0.9 (mean � SD) inatipt1;3;5;7. The decrease in the number of vascular bundles maybe due to decreased size of the shoot apical meristem (15). Thenumber of cells in the phloem, xylem in the bundle, and lignifiedcells in the interfascicular region were all greatly reduced (Fig.S3). The lignified cells in the interfascicular region were inter-preted as secondary xylem parenchyma (23). These resultsindicate that fascicular and interfascicular cambial activity isdecreased in the atipt1;3;5;7 mutant. We also examined thegrowth of the atipt3 mutant, which has moderately decreasedlevels of cytokinins. The length of the inflorescence stem wasindistinguishable between atipt3 WT, whereas the stem diameterwas significantly decreased in atipt3 (Fig. S4). This indicates thatstem thickening growth is more sensitively affected by decreasein cytokinins than stem elongation growth. In the atipt3 mutant,the average number of vascular bundles was slightly decreased[6.3 � 0.9 (mean � SD) in atipt3 and 7.1 � 0.8 (mean � SD) inWT], and the number of cells in the phloem and xylem weresignificantly decreased (Fig. S5).

To verify whether decreases in cytokinin levels are responsiblefor the decreased secondary thickening, we supplied roots withdifferent doses of cytokinins. Basal regions of roots of theatipt1;3;5;7 mutant did not possess a ring-formed cambial region11 days after seed sowing, whereas application of cytokininsstarting on this day recovered secondary thickening and cambialsize in a dose-dependent manner (Fig. 2 B–E). Cytokinin-activated thickening growth was associated with increases in thenumber of vessels, the number of cells in the phloem-containingregion (the area outside the cambium zone), and the number ofxylem cells (Fig. S6). Cytokinins slightly increased xylem cell sizebut had no effect on cell size in the cambium and in the phloem.These results indicate that cytokinins play important roles incambial activity and that competency to form the cambial zonewas retained for an extended period. An increase in thickeninggrowth in response to cytokinin application was also seen in WTArabidopsis (Fig. 1C and Figs. S6 and S7). Similarly, inducibleoverexpression of AtIPT genes increased secondary growth in amanner dependent on the inducer’s level (Fig. S8). Applicationof cytokinins to mutant (Fig. 2E) or WT (Fig. S7) plants did notappear to otherwise modify the tissue pattern observable in rootsections, suggesting that cytokinins do not supply positionalinformation for pattern formation during secondary thickening.

Fig. 1. Role of cytokinins in root secondary growth. (A) Overall structure of24-day-old WT (Left) and atipt1;3;5;7 (Right). Note the increased root elon-gation in atipt1;3;5;7. (B) The diameter of the primary root of WT (black) andatipt1;3;5;7 (red). Eleven-day-old plants were moved to media containingcytokinins and cultured for 14 days. (C) The diameter of the primary root of25-day-old WT, atipt3, atipt3;5, and atipt3;5;7. (Scale bars: 50 mm.)

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Next we examined whether mobile endogenous cytokinins canrestore the growth of the atipt1;3;5;7 mutant. To accomplish thiswe reciprocally grafted an atipt1;3;5;7 shoot scion onto a wild-type root stock and a WT shoot scion onto an atipt1;3;5;7 rootstock. The atipt1;3;5;7 shoot, which would otherwise be dwarfedwith thin stems if ungrafted, grew as vigorous as WT whengrafted onto WT root stock (Fig. 3 A and B). Stem sectionsrevealed that the number of vascular bundles and growth ofxylem and phloem were all restored (Fig. 3 C–E). The overallarchitecture of the root system of atipt1;3;5;7 differs from WT inthat lateral roots elongate vigorously; however, the architectureof atipt1;3;5;7 root stock resembled WT when the WT shootscion was grafted onto the mutant stock (Fig. S9). Secondarygrowth of mutant roots in the grafted plants was also perfectlyrecovered (Fig. 3 G and H). These results indicate the function-ality of mobile cytokinins.

We next examined whether cytokinins are transported. Boththe iP-type and tZ-type cytokinins are greatly decreased in boththe roots and shoots in the atipt1;3;5;7 mutant (15) (Fig. 4).When a mutant shoot was grafted onto a WT root, iP-typecytokinins in the shoot did not recover (Fig. 4 A and B, third barfrom the left) but tZ-type cytokinins in the shoot recovered tonormal levels (Fig. 4 C and D, third bar from the left). Thisindicates that tZ-type cytokinins, but not iP-type cytokinins,were transported from root to shoot and restored shoot growthin the mutant genotype. On the other hand, when a WT shootwas grafted onto a mutant root stock, levels of iP-type cytokininin the root returned to normal levels but tZ-type cytokinin levels inthe root recovered only partially (Fig. 4 E–H, second left bar). Thisindicates that iP-type cytokinins dominate in shoot-to-root trans-port and that this transport is sufficient for normal root growth.

DiscussionBecause cambial activity is precisely coordinated, its response todevelopmental and environmental cues must be governed by anunknown, systemic signaling molecule. Auxin and gibberellinshave been proposed as possible mediators; however, no corre-lation between changes in the concentrations of these hormonesand cambial activity has been shown. Therefore, although they

may be spatial regulators of cambial activity, they are unlikely tobe solely responsible for temporal regulation of cambial activity(3, 7).

We demonstrate here that formation of the cambial zonerequires cytokinins and that cambial activity responds verysensitively to changes in cytokinin levels. In the atipt1;3;5;7mutant, the cambium is virtually absent in the root, whereas rootelongation is accelerated (15). The only recognizable phenotypeof the atipt3 mutant is the moderate decrease in secondarygrowth, revealing that cambium activity responds sensitively toa decrease in cytokinin levels.

Cytokinin levels change in response to environmental factors.They decrease by drought in rice (12) and increase quickly andgreatly by nitrate nutrients in sunflower (9), barley (11), andArabidopsis (10). Cytokinin levels also respond to phosphate,although not as much as to nitrate (9). The increase of cytokininsvia nitrate is largely mediated by activation of the AtIPT3 gene,as has been detected by mutation-dependent diminishment ofnitrate induction (24). We report here that decreases in cytoki-nin levels in atipt3 plants cause decreased cambial activity.Therefore, cytokinins are likely to be the physiological mediatorsof cambial growth. Tissue patterning in the atipt1;3;5;7 root thatunderwent secondary growth after exogenous application ofcytokinins appeared to be normal. Together, these results indi-cate that cytokinins are regulators of cambial activity but thatthey do not provide a positional cue for pattern formation.

The role of systemically transported cytokinins has beencontroversial. The presence of cytokinins in xylem sap and leafexudates suggests the possibility of a systemic role for cytokinins(17); however, no study has unequivocally demonstrated abiological role of systemically transported cytokinins. In recip-rocal grafts between WT and Agrobacterium IPT-expressingtobacco, only tissues with transgenic genotype exhibited pheno-types typical of cytokinin overproduction, disfavoring the idea ofa role for systemically transported cytokinins (25). However, inthat experiment, only the effects of cytokinins at concentrationsover normal levels could be examined. Our study stronglysuggests that physiological levels of mobile cytokinins can conveyinformation and regulate growth, including cambium. It has also

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Fig. 2. Root and shoot anatomy of atipt1;3;5;7 and effects of cytokinins on cambial growth. (A) Section of 25-day-old WT root. (B–E) Roots of aipt1;3;5;7 treatedwith 0 (B), 20 (C), 200 (D), and 2,000 (E) ng/mL trans-zeatin during the last 14 days of the 25-day growth period. (F) WT inflorescence stem. (G) atipt1;3;5;7 stem.(Scale bars: 0.1 mm.) CZ, cambial zone.

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been controversial whether cytokinins produced in the root arerequired for growth of the shoot and vice versa. The graftingexperiment also demonstrates that the shoot can grow normallywithout root-derived cytokinins and that the root can grownormally without shoot-derived cytokinins.

External application of cytokinins or overexpression of IPTgenes increased secondary growth in WT as well as in atiptmutants. This suggests potential for molecular breeding ofgreater thickening growth in trees and crops by activation of thecytokinin signal. However, systemic application of cytokininswould affect many developmental processes in addition tocambium activity, which could be deleterious in practical use.Thus, tissue-specific modulation of cytokinin signaling wouldhave to be tested for practical purposes.

Materials and MethodsPlant Materials and Culture. atipt1-1, atipt3-2, atipt5-2, atipt7-1, and theirmultiple mutants were used in this study. Detailed descriptions of geneticinformation and concentrations of cytokinin species of these mutants werereported before (15).

For plant growth we used vertically placed plates containing modifiedMurashige and Skoog (MS) medium (26) [1� MS salts, 1% sucrose, 0.05%2-[N-morpholino]-ethanesulfonic acid-KOH (Mes-KOH, pH 5.7), 100 mg/mLinositol, 10 mg/mL thiamine-HCl, 1 mg/mL pyridoxine HCl, and 1 mg/mLnicotinic acid] solidified with 3 mM MgCl2 and 0.6% Phytagel (Sigma–Aldrich), over which we placed a cellophane sheet. Soon after germinationplants were placed on the cellophane sheets. The cellophane sheets hadbeen autoclaved in 5 mM EDTA (pH 8.0) and thoroughly washed withdistilled water. The use of cellophane sheets prevented penetration of theroots into the solidified plates and thus allowed us to move the plants ontoother plates without damage. Plants were grown under continuous light at22 °C. After 11 days, plants were moved onto vertically placed solidified MSmedium (without cellophane) with or without cytokinin and cultured for14 days. The roots were then examined. Root diameter was measured 5 mmfrom the root-hypocotyl junction. For examination of the atipt1;3;5;7inflorescence stems, WT and atipt1;3;5;7 plants were grown on MS mediumwith 1% sucrose and 0.3% Phytagel for 25 days. For atipt3 stem examina-tion, plants were grown on MS medium with 1% sucrose and 0.6% Phytagelfor 13 days and then moved onto vermiculite with half-strength MS me-dium and grown for 15 days.

Grafting of the hypocotyls between the WT rootstock and the atipt1;3;5;7scion, and vice versa, was done 4 days after germination according to themethod of Turnbull et al. (27). The plants were then cultured for 3 weeks onMS solidified with 0.3% Phytagel.

Inducible Overexpression of AtIPT Genes. Coding sequences of AtIPT1, AtIPT3,AtIPT4, and AtIPT7, which have no introns, were cloned in the pER8 vector (29)and transformed into Arabidopsis. Transformants were grown for 11 days on

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Fig. 3. Recovery of growth in aipt1;3;5;7 by systemically transported cyto-kinins. m, mutant atipt1;3;5;7 genotype; W, wild type. The genotypes listedabove and below the horizontal line correspond to the scion and stock,respectively. (A) Overall statures of aerial portions of grafts betweenatipt1;3;5;7 and WT. The rightmost plant (atipt1;3;5;7) was not grafted. (Scalebar: 5 cm.) (B) A close-up view of stems at positions �2 cm from the bottom ofthe stem. (Scale bar: 1 mm.) (C–E) Sections of stems of a graft between a WTscion and a WT stock (C), a graft between an atipt1;3;5;7 scion and a WT root(D), and atipt1;3;5;7 (E). (F–H) Sections of roots of a graft between a WT scionand a WT stock (F), a graft between WT scion and atipt1;3;5;7 root (G), andatipt1;3;5;7 (H). (Scale bars in C–H: 0.1 mm.) Four-day-old plants were graftedand grown for another 21 days. Ungrafted atipt1;3;5;7 plants were sectionedon day 25. X, xylem; P, phloem; CZ, cambial zone.

Fig. 4. Cytokinin levels in the shoots and roots of grafted plants betweenatipt1;3;5;7 and WT. (A–D) Cytokinin concentrations in shoots. (A) isopente-nyladenine riboside phosphate (iPRP). (B) Isopentenyladenine riboside (iPR).(C) t-zeatin riboside phosphate (tZRP). (D) t-zeatin riboside (tZR). (E–H) Cyto-kinin concentrations in roots. (E) iPRP. (F) iPR. (G) tZRP. (H) tZR. Data representmeans � SD.

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the modified MS medium in vertically placed plates and then moved onto themodified MS plates containing �-estradiol, on which a cellophane sheet wasplaced, and grown for 14 days.

Sectioning. Plant samples (roots at �5 mm from the root–hypocotyl junction;stems immediately above the node of the first cauline leaf) were fixed in 1%glutaraldehyde, 3% formaldehyde, and 50 mM sodium phosphate buffer (pH7.2), then dehydrated and embedded in Technovit 7100 (Heraeus Kulzer).Sections 5 �m thick were stained with toluidine blue.

Cytokinin Measurement. Roots or shoots from 3 plants were pooled to give 1sample for cytokinin analysis. At least 3 samples were used for every combi-nations of grafting. Concentrations of cytokinins were measured by liquid

chromatography–positive electrospray–tandem mass spectrometry in a mul-tiple reaction monitoring mode according to Novak et al. (29).

ACKNOWLEDGMENTS. We thank Nam-Hai Chua for pER8; Ondr�ej Novak forskillful technical assistance; and Ronald Sederoff, Yka Helariutta, AnthonyBishopp, Victor Albert, and Kaisa Nieminen for comments on the manuscript.This work was supported by the grant KAKENHI (nos. 19060005 and 15107001to T. Kakimoto). M.M.-K. was supported by Special Coordination Funds forPromoting Science and Technology from the Ministry of Education, Culture,Sports, Science and Technology for the Osaka University Program for theSupport of Networking among Present and Future Women Researchers. P.T.was supported by Ministry of Education, Youth and Sports of the CzechRepublic Grant MSM 6198959216.

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