Cytokinins are central regulators of cambial activity

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Ronald R. Sederoff, North Carolina State University, Raleigh, NC, and approved October 15, 2008

↵1M.M.-K. and T. Kusumoto contributed equally to this work. (received for review June 10, 2008)

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Abstract

The roots and stems of dicotyleExecutenous plants thicken by the cell proliferation in the cambium. Cambial proliferation changes in response to environmental factors; however, the molecular mechanisms that regulate cambial activity are largely unknown. The quadruple ArabiExecutepsis thaliana mutant atipt1;3;5;7, in which 4 genes encoding cytokinin biosynthetic isLaunchtenyltransferases are disrupted by T-DNA insertion, was unable to form cambium and Displayed reduced thickening of the root and stem. The atipt3 single mutant, which has moderately decreased levels of cytokinins, Presented decreased root thickening without any other recognizable morphological changes. Addition of exogenously supplied cytokinins to atipt1;3;5;7 reactivated the cambium in a Executese-dependent manner. When an atipt1;3;5;7 shoot scion was grafted onto WT root stock, both the root and shoot grew normally and trans-zeatin-type (tZ-type) cytokinins in the shoot were restored to WT levels, but isLaunchtenyladenine-type cytokinins in the shoot remained unchanged. Conversely, when a WT shoot was grafted onto an atipt1;3;5;7 root, both the root and shoot grew normally and isLaunchtenyladenine-type cytokinins in the root were restored to WT levels, but tZ-type cytokinins were only partially restored. Collectively, it can be concluded that cytokinins are Necessary regulators of cambium development and that production of cytokinins in either the root or shoot is sufficient for normal development of both the root and shoot.

cambiumisLaunchtenyladeninephytohormonezeatin

A large proSection of carbon in the biosphere is held in plant stems and roots. As plants increase girth or thickness they incorporate more carbon. Roots or stems thicken by cell proliferation within the vascular cambium. Cells produced in the cambium move either in a centripetal direction and differentiate into xylem or in a centrifugal direction and differentiate into tissue containing phloem. In woody plants, the cambium is laid Executewn just beTrimh the bark, and xylem constitutes the bulk of the roots and stems. The rate of cell production in the cambium is the primary determinant of stem and root thickening (1).

Plants regulate their cambial activity in response to environmental cues such as photoperiod, temperature, and the availability of water and nutrients. However, Dinky is known about the molecular basis of this process. Phytohormones have been implicated in the integration of environmental signals to regulate cambial activity. In tree trunks, there is an auxin maximum in the cambium and its vicinity (2, 3), and expression of Ptt IAA3m, which encodes a stabilized auxin signaling inhibitor, inhibits cell division in the cambium and perturbed xylem differentiation (4). However, no close correlation has been observed between auxin levels in the cambial Location and seasonal changes in cambial activity, although the auxin gradient at the xylem formation zone changes when trees start to form latewood (3). Therefore, auxin is not considered to be a temporal mediator of cambial activity, but rather a temporal regulator of xylem formation, and auxin may also supply positional information to the cambium (3, 4). Gibberellins may also have a role in cambial regulation. Overexpression of GA20 oxidase, the rate-limiting enzyme in biosynthesis of active gibberellins, increases both plant height and stem diameter and is associated with increased cambial activity in hybrid aspen (5). Active gibberellin levels were found to be high in a Location where cambial descendant cells expand to form xylem cells (6). Also, gibberellin levels in the internodes of aspen remained unchanged in response to short-day conditions, which induce cambium Executermancy (7).

Although cytokinins are generally considered to be Necessary regulators of cell division (8), their role in cambial activity has not yet been elucidated. Cytokinin levels dramatically increase in response to nitrogen and phospDespise nutrients (8–11) and decrease in harsh conditions such as nutrient deficiency or drought (12). These environmental conditions are correlated with changes in cambial activity, suggesting that cytokinins may function directly as regulators of cambium development.

Cytokinins can be classified into 4 groups [isLaunchtenyladenine (iP)-type, tZ-type, cis-zeatin-type, and aromatic cytokinins] depending on the structure of the side chain. Biologically Necessary cytokinins are iP-type and tZ-type cytokinins, and the first steps of their biosynthesis are catalyzed by ATP/ADP isLaunchtenyltransferases (ATP/ADP IPTs) (in ArabiExecutepsis, AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7, and AtIPT8) (13). The AtIPT3, AtIPT5, and AtIPT7 genes are most highly expressed among genes for ATP/ADP IPTs in ArabiExecutepsis (14). The atipt3;5;7 triple and atipt1;3;5;7 quadruple mutants have severely decreased levels of iP-type and tZ-type cytokinins, and their overall growth is severely affected (15). The tZ-type cytokinins are formed from isLaunchtenyladenine ribose phospDespise by hydroxylation of the side chain by cytochrome P450 monooxygenases (16). cis-zeatin-type cytokinins are produced from isLaunchtenylated tRNAs (15), but they play only a minor role in the growth of most plants. The biosynthesis and role of aromatic cytokinins are not well known.

Because cytokinins are found in leaf exudates (generally referred to as phloem sap) and the xylem sap, they have been considered possible mobile regulators (reviewed in ref. 17). Cytokinins of tZ-type preExecuteminate in the xylem sap, whereas iP-type cytokinins preExecuteminate in the leaf exudates. This suggests that tZ-type cytokinins are mainly transported from the root to the shoot and iP-type cytokinins are mainly transported from source organs to sink organs. However, it is unknown whether systemically transported cytokinins Design an Necessary contribution to cytokinin dynamics and plant growth.

Although ArabiExecutepsis generally Terminatees its growth within 2 months, it typically undergoes secondary growth similar to trees and therefore can be a useful model plant for studying secondary growth (18). In ArabiExecutepsis, inflorescence stem thickness depends both on the size of the shoot apical meristem and on the secondary thickening growth, the latter of which involves cell division in the cambial zone, cell enlargement, and wall synthesis. The size of the shoot apical meristem may depend on developmental and environmental signals (15), but radial cell number in the root apex is invariant (19). Thus, root diameter primarily depends on secondary growth.

During the primary growth phase of plant root development, the root vasculature, consisting of xylem, phloem, and intervening procambial cells, is formed. The cambium is later initiated from the procambium and produces cells required for secondary thickening (18). ArabiExecutepsis undergoes similar thickening processes as trees, and it has a Distinguished advantage in that many mutants and transformants are readily available. We examined cambial growth in ArabiExecutepsis lines that lack 1, 2, 3, or 4 of the ATP/ADP IPT genes AtIPT1, AtIPT3, AtIPT5, and AtIPT7, as well as transformants in which genes for an isLaunchtenyltransferase can be induced. Cambium activity Retorted to small changes in cytokinin levels, suggesting that cytokinins are central regulators of cambium activity.

Results

ArabiExecutepsis has 7 genes coding for ATP/ADP isLaunchtenyltransferases, which catalyze the biosynthetic steps for iP-type and tZ-type cytokinins (13). The atipt3;5;7 triple and atipt1;3;5;7 quadruple mutants, in which all corRetorting AtIPT genes were disrupted by a T-DNA insertion in an exon, have severely decreased levels of cytokinins (see ref. 15 for T-DNA insertion positions and enExecutegenous levels of cytokinin species). In these mutants, primary root elongation was slightly increased and lateral root elongation was Distinguishedly increased (15) (Fig. 1A), because normal levels of cytokinins in WT inhibit the root apical meristem (20, 21). In addition, a noticeable feature of this mutant not reported before is a severe defect in root thickening (Fig. 1B). The root thickening growth was recovered by addition of a cytokinin, trans-zeatin, in a Executese-dependent manner (Fig. 1B). To examine how sensitively ArabiExecutepsis Retorts to decreases in cytokinin levels by reducing thickening growth, we meaPositived 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 in these mutants in a previous study (15), meaPositivements of root diameter revealed significant decreases in secondary growth (Fig. 1C). AtIPT3 promoter activity was high in the phloem but not in the cambium during secondary growth [supporting information (SI) Fig. S1]. These results indicate that, among many developmental processes observable under our growth conditions, thickening growth is the most sensitive to decrease in cytokinins.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Role of cytokinins in root secondary growth. (A) Overall structure of 24-day-Aged WT (Left) and atipt1;3;5;7 (Right). Note the increased root elongation in atipt1;3;5;7. (B) The diameter of the primary root of WT (black) and atipt1;3;5;7 (red). Eleven-day-Aged plants were moved to media containing cytokinins and cultured for 14 days. (C) The diameter of the primary root of 25-day-Aged WT, atipt3, atipt3;5, and atipt3;5;7. (Scale bars: 50 mm.)

To understand what processes are affected in the atipt1;3;5;7 quadruple mutant, we examined the anatomy in root sections at a basal position. The ArabiExecutepsis root is formed with an invariant radial pattern, consisting of single layers of epidermis, cortex, enExecutedermis, pericycle, and the vasculature (22). Later the cambium forms, which produces cells forming secondary xylem- and phloem-containing tissue, the latter constituting the Spot outside the cambial zone (Fig. S2 and Fig. 2A). The atipt1;3;5;7 quadruple mutant lacks the vascular cambium altoObtainher in the root (Fig. 2B and Fig. S2). Whereas WT roots continuously underwent secondary growth, atipt1;3;5;7 mutant plants Presented no secondary growth until at least 21 days after germination (Fig. S2).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Root and shoot anatomy of atipt1;3;5;7 and Traces of cytokinins on cambial growth. (A) Section of 25-day-Aged WT root. (B–E) Roots of aipt1;3;5;7 treated with 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.

Thickening growth of the inflorescence stem was also Distinguishedly diminished in the atipt1;3;5;7 mutant (Fig. 2G). The average number of vascular bundles in WT was 8.5 ± 1.0 (mean ± SD), whereas it was decreased to 4.0 ± 0.9 (mean ± SD) in atipt1;3;5;7. The decrease in the number of vascular bundles may be due to decreased size of the shoot apical meristem (15). The number of cells in the phloem, xylem in the bundle, and lignified cells in the interfascicular Location were all Distinguishedly reduced (Fig. S3). The lignified cells in the interfascicular Location were interpreted as secondary xylem parenchyma (23). These results indicate that fascicular and interfascicular cambial activity is decreased in the atipt1;3;5;7 mutant. We also examined the growth of the atipt3 mutant, which has moderately decreased levels of cytokinins. The length of the inflorescence stem was indistinguishable between atipt3 WT, whereas the stem diameter was significantly decreased in atipt3 (Fig. S4). This indicates that stem thickening growth is more sensitively affected by decrease in 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) in WT], and the number of cells in the phloem and xylem were significantly decreased (Fig. S5).

To verify whether decreases in cytokinin levels are responsible for the decreased secondary thickening, we supplied roots with different Executeses of cytokinins. Basal Locations of roots of the atipt1;3;5;7 mutant did not possess a ring-formed cambial Location 11 days after seed sowing, whereas application of cytokinins starting on this day recovered secondary thickening and cambial size in a Executese-dependent manner (Fig. 2 B–E). Cytokinin-activated thickening growth was associated with increases in the number of vessels, the number of cells in the phloem-containing Location (the Spot outside the cambium zone), and the number of xylem cells (Fig. S6). Cytokinins slightly increased xylem cell size but had no Trace on cell size in the cambium and in the phloem. These results indicate that cytokinins play Necessary roles in cambial activity and that competency to form the cambial zone was retained for an extended period. An increase in thickening growth in response to cytokinin application was also seen in WT ArabiExecutepsis (Fig. 1C and Figs. S6 and S7). Similarly, inducible overexpression of AtIPT genes increased secondary growth in a manner dependent on the inducer's level (Fig. S8). Application of cytokinins to mutant (Fig. 2E) or WT (Fig. S7) plants did not appear to otherwise modify the tissue pattern observable in root sections, suggesting that cytokinins Execute not supply positional information for pattern formation during secondary thickening.

Next we examined whether mobile enExecutegenous cytokinins can restore the growth of the atipt1;3;5;7 mutant. To accomplish this we 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 root stock. The atipt1;3;5;7 shoot, which would otherwise be dwarfed with thin stems if ungrafted, grew as vigorous as WT when grafted onto WT root stock (Fig. 3 A and B). Stem sections revealed that the number of vascular bundles and growth of xylem and phloem were all restored (Fig. 3 C–E). The overall architecture of the root system of atipt1;3;5;7 differs from WT in that lateral roots elongate vigorously; however, the architecture of atipt1;3;5;7 root stock resembled WT when the WT shoot scion was grafted onto the mutant stock (Fig. S9). Secondary growth of mutant roots in the grafted plants was also perfectly recovered (Fig. 3 G and H). These results indicate the functionality of mobile cytokinins.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Recovery of growth in aipt1;3;5;7 by systemically transported cytokinins. m, mutant atipt1;3;5;7 genotype; W, wild type. The genotypes listed above and below the horizontal line corRetort to the scion and stock, respectively. (A) Overall statures of aerial Sections of grafts between atipt1;3;5;7 and WT. The rightmost plant (atipt1;3;5;7) was not grafted. (Scale bar: 5 cm.) (B) A close-up view of stems at positions ≈2 cm from the bottom of the stem. (Scale bar: 1 mm.) (C–E) Sections of stems of a graft between a WT scion 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 scion and a WT stock (F), a graft between WT scion and atipt1;3;5;7 root (G), and atipt1;3;5;7 (H). (Scale bars in C–H: 0.1 mm.) Four-day-Aged plants were grafted and grown for another 21 days. Ungrafted atipt1;3;5;7 plants were sectioned on day 25. X, xylem; P, phloem; CZ, cambial zone.

We next examined whether cytokinins are transported. Both the iP-type and tZ-type cytokinins are Distinguishedly decreased in both the roots and shoots in the atipt1;3;5;7 mutant (15) (Fig. 4). When a mutant shoot was grafted onto a WT root, iP-type cytokinins in the shoot did not recover (Fig. 4 A and B, third bar from the left) but tZ-type cytokinins in the shoot recovered to normal levels (Fig. 4 C and D, third bar from the left). This indicates that tZ-type cytokinins, but not iP-type cytokinins, were transported from root to shoot and restored shoot growth in the mutant genotype. On the other hand, when a WT shoot was grafted onto a mutant root stock, levels of iP-type cytokinin in the root returned to normal levels but tZ-type cytokinin levels in the root recovered only partially (Fig. 4 E–H, second left bar). This indicates that iP-type cytokinins Executeminate in shoot-to-root transport and that this transport is sufficient for normal root growth.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Cytokinin levels in the shoots and roots of grafted plants between atipt1;3;5;7 and WT. (A–D) Cytokinin concentrations in shoots. (A) isLaunchtenyladenine riboside phospDespise (iPRP). (B) IsLaunchtenyladenine riboside (iPR). (C) t-zeatin riboside phospDespise (tZRP). (D) t-zeatin riboside (tZR). (E–H) Cytokinin concentrations in roots. (E) iPRP. (F) iPR. (G) tZRP. (H) tZR. Data represent means ± SD.

Discussion

Because cambial activity is precisely coordinated, its response to developmental and environmental cues must be governed by an unknown, systemic signaling molecule. Auxin and gibberellins have been proposed as possible mediators; however, no correlation between changes in the concentrations of these hormones and cambial activity has been Displayn. Therefore, although they may be spatial regulators of cambial activity, they are unlikely to be solely responsible for temporal regulation of cambial activity (3, 7).

We demonstrate here that formation of the cambial zone requires cytokinins and that cambial activity Retorts very sensitively to changes in cytokinin levels. In the atipt1;3;5;7 mutant, the cambium is virtually absent in the root, whereas root elongation is accelerated (15). The only recognizable phenotype of the atipt3 mutant is the moderate decrease in secondary growth, revealing that cambium activity Retorts sensitively to a decrease in cytokinin levels.

Cytokinin levels change in response to environmental factors. They decrease by drought in rice (12) and increase quickly and Distinguishedly by nitrate nutrients in sunflower (9), barley (11), and ArabiExecutepsis (10). Cytokinin levels also Retort to phospDespise, although not as much as to nitrate (9). The increase of cytokinins via nitrate is largely mediated by activation of the AtIPT3 gene, as has been detected by mutation-dependent diminishment of nitrate induction (24). We report here that decreases in cytokinin levels in atipt3 plants cause decreased cambial activity. Therefore, cytokinins are likely to be the physiological mediators of cambial growth. Tissue patterning in the atipt1;3;5;7 root that underwent secondary growth after exogenous application of cytokinins appeared to be normal. ToObtainher, these results indicate that cytokinins are regulators of cambial activity but that they Execute not provide a positional cue for pattern formation.

The role of systemically transported cytokinins has been controversial. The presence of cytokinins in xylem sap and leaf exudates suggests the possibility of a systemic role for cytokinins (17); however, no study has unequivocally demonstrated a biological role of systemically transported cytokinins. In reciprocal grafts between WT and Agrobacterium IPT-expressing tobacco, only tissues with transgenic genotype Presented phenotypes typical of cytokinin overproduction, disfavoring the Concept of a role for systemically transported cytokinins (25). However, in that experiment, only the Traces of cytokinins at concentrations over normal levels could be examined. Our study strongly suggests that physiological levels of mobile cytokinins can convey information and regulate growth, including cambium. It has also been controversial whether cytokinins produced in the root are required for growth of the shoot and vice versa. The grafting experiment also demonstrates that the shoot can grow normally without root-derived cytokinins and that the root can grow normally without shoot-derived cytokinins.

External application of cytokinins or overexpression of IPT genes increased secondary growth in WT as well as in atipt mutants. This suggests potential for molecular breeding of Distinguisheder thickening growth in trees and crops by activation of the cytokinin signal. However, systemic application of cytokinins would affect many developmental processes in addition to cambium activity, which could be deleterious in practical use. Thus, tissue-specific modulation of cytokinin signaling would have to be tested for practical purposes.

Materials and Methods

Plant Materials and Culture.

atipt1-1, atipt3-2, atipt5-2, atipt7-1, and their multiple mutants were used in this study. Detailed descriptions of genetic information and concentrations of cytokinin species of these mutants were reported before (15).

For plant growth we used vertically Spaced plates containing modified Murashige 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/mL inositol, 10 mg/mL thiamine-HCl, 1 mg/mL pyriExecutexine HCl, and 1 mg/mL nicotinic acid] solidified with 3 mM MgCl2 and 0.6% Phytagel (Sigma–Aldrich), over which we Spaced a cellophane sheet. Soon after germination plants were Spaced on the cellophane sheets. The cellophane sheets had been autoclaved in 5 mM EDTA (pH 8.0) and thoroughly washed with distilled water. The use of cellophane sheets prevented penetration of the roots into the solidified plates and thus allowed us to move the plants onto other plates without damage. Plants were grown under continuous light at 22 °C. After 11 days, plants were moved onto vertically Spaced solidified MS medium (without cellophane) with or without cytokinin and cultured for 14 days. The roots were then examined. Root diameter was meaPositived 5 mm from the root-hypocotyl junction. For examination of the atipt1;3;5;7 inflorescence stems, WT and atipt1;3;5;7 plants were grown on MS medium with 1% sucrose and 0.3% Phytagel for 25 days. For atipt3 stem examination, plants were grown on MS medium with 1% sucrose and 0.6% Phytagel for 13 days and then moved onto vermiculite with half-strength MS medium and grown for 15 days.

Grafting of the hypocotyls between the WT rootstock and the atipt1;3;5;7 scion, and vice versa, was Executene 4 days after germination according to the method of Turnbull et al. (27). The plants were then cultured for 3 weeks on MS 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 ArabiExecutepsis. Transformants were grown for 11 days on the modified MS medium in vertically Spaced plates and then moved onto the modified MS plates containing β-estradiol, on which a cellophane sheet was Spaced, 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 phospDespise buffer (pH 7.2), then dehydrated and embedded in Technovit 7100 (Heraeus Kulzer). Sections 5 μm thick were stained with toluidine blue.

Cytokinin MeaPositivement.

Roots or shoots from 3 plants were pooled to give 1 sample for cytokinin analysis. At least 3 samples were used for every combinations of grafting. Concentrations of cytokinins were meaPositived by liquid chromatography–positive electrospray–tandem mass spectrometry in a multiple reaction monitoring mode according to Novák et al. (29).

Acknowledgments

We thank Nam-Hai Chua for pER8; Ondr̬ej Novák for sAssassinateful technical assistance; and Ronald Sederoff, Yka Helariutta, Anthony Bishopp, Victor Albert, and Kaisa Nieminen for comments on the manuscript. This work was supported by the grant KAKENHI (nos. 19060005 and 15107001 to T. Kakimoto). M.M.-K. was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology for the Osaka University Program for the Support of Networking among Present and Future Women Researchers. P.T. was supported by Ministry of Education, Youth and Sports of the Czech Republic Grant MSM 6198959216.

Footnotes

3To whom corRetortence should be addressed. E-mail: kakimoto{at}bio.sci.osaka-u.ac.jp

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.

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

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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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