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Plant Physiology and Biochemistry 44 (2006) 248–252

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Exogenous cytokinin treatment maintains cyclinhomeostasis in rice seedlings that show changes of cyclin

expression when the photoperiod is rapidly changed

H. Lee a, C.-K. Auh b, D. Kim c, T.-K. Lee d, S. Lee a,*

aDepartment of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, South KoreabDivision of Applied Biotechnology, Mokpo National University, Chunnam 534-729, South Korea

cEnvironmental Biotechnology National Core Research Center and Division of Applied Life Science (BK21 Program),

Graduate School of Gyeongsang National University, Jinju 660-701, Korea

d Southern Coastal Environment Research Division, Korea Ocean Research and Development Institute, Geoje 656-830, South Korea

Received 1 November 2005Available online 25 April 2006

Abstract

Cyclin is a fundamental regulator of the plant cell cycle. Five different types of cyclin genes (the A-, B-, C-, D-, and H-types) have beenreported in Oryza sativa. However, except for Os;cycA1;1, Os;cycB2;1, and Os;cycB2;2, the mechanisms of expression of these cyclin geneshave not yet been studied. The interactions of cyclins with cytokinin, an important trigger for cell cycle regulation, have also not been wellstudied. Here we used semi-quantitative RT-PCR in rice seedlings to analyze the effect of cytokinin on photomorphogenesis and the expressionof six cyclin genes. Fifteen-day-old seedlings were grown in a 16/8 h light/dark cycle and then transferred to either constant light or constantdark. The expression of all the cyclin genes tested, except the C-type, decreased after 1 hour in the dark, but did not change after transfer to thelight or when kinetin was added to the medium. Similarly, seedlings grown in the dark had decreased expression of the cyclin genes, except Os;cycB2;2, after transfer to the light, a decrease that was prevented by kinetin treatment. Thus, exogenous cytokinin plays an important role inmaintaining homeostasis of cyclin gene expression following rapid changes of photoperiod.© 2006 Elsevier SAS. All rights reserved.

Keywords: Cyclin; Cytokinin; Homeostasis; Oryza sativa; Photoperiod

1. Introduction

Cell cycle regulation is an important factor in plant devel-opment and is mediated by the regulation of cyclins and cyclindependent kinase (CDK) [1]. Cyclins are essential factors foractivating CDK and have been isolated and characterized fromyeast, animals, and plants [2,3]. Five different types of cyclingenes have been reported in rice [4–6]; they are classified as A-type (Os;cycA1;1), B-type (Os;cycB2;1, Os;cycB2;2), C-type(Os;cycC;1), D-type (Os;cycD), or H-type (Os;cycH;1) basedon the similarity of their amino acid sequences to cyclins inother species. The different cyclins are expressed at differentphases of the cell cycle: cycA1;1 is expressed from G1 phaseto the early stage of M phase (determined through an in situ

author. Tel.: +82 31 290 7866; fax: +82 31 290 7870.: sukchan@skku.ac.kr (S. Lee).

front matter © 2006 Elsevier SAS. All rights reserved.y.2006.03.006

hybridization analysis), whereas Os;cycB2;1 and Os;cycB2;2are expressed until the late stage of M phase [5]. The transcrip-tion of Os;cycB2;1 and Os;cycB2;2 increases on the intercalarymeristem of the gibberellin-processed deepwater rice when celldivision is actively occurring [4]. In suspension culture, Os;cycH;1 is mostly expressed during S phase, and a yeast two-hybrid assay determined that Os;cycH;1 functions as a CDK-activating kinase (CAK) through interactions with rice R2 ki-nase [6,7]. The C-type cyclin and D-type cyclin genes werereported by comparison with amino acid sequences identifiedin animals; however, their definite functions and roles in ricehave not been reported yet.

Except for the A-type and B-type cyclins [5,8], no func-tional analyses of rice cyclins have been performed, and mostresearch about rice cyclins has involved gene isolation basedon sequence homology. In addition, the only study of the inter-action of rice cyclins with CDK involved transgenic rice over-

Fig. 1. Schematic of the experimental design to investigate the effect ofphotoperiod changes and kinetin treatment on cyclin gene expression.Seedlings were grown under a 16/8 h light/dark cycle (A, B) or in the dark (C),and were then transferred to continuous light or dark for 24 hours in thepresence or absence of kinetin.

H. Lee et al. / Plant Physiology and Biochemistry 44 (2006) 248–252 249

expressing the B-type cyclin gene [8]. There has been only onereport on the definite expression mechanism of most cyclingenes and for the regulation of cyclin expression by bioticand abiotic factors [8].

In plant tissue culture, the plant hormone cytokinin workstogether with auxin to regulate cell division. Cytokinin particu-larly affects the expression of the cdc2 gene in Arabidopsis [9],where it is also involved in the expression of a D-type cyclinthat regulates the transition from S phase to G1 phase [10]. Theexpression of A-, B-, and C-type cyclin genes increased in calliand leaf blades of rice grown on cytokinin-containing media[11]. Few studies have examined the expression of cyclins inthe context of cell cycle regulation and the relationship withcytokinin. In particular, few studies have examined the effectof plant growth hormones on the expression of genes related tothe cell cycle in rice. The functions of light and plant growthregulators in plant development partially overlap, but it is notclear whether light and plant growth regulators affect the de-velopmental responses independently, or plant growth regula-tors interact with physiologically active photoreceptors. Re-cently there was a report about the direct interactions betweencytokinin reception and far-red light sensitivity in Arabidopsisdevelopment [12]. Mutant seeds of two Arabidopsis thalianasensor histidine kinases (AHK2 and AHK3), known to be cy-tokinin receptors had more rapid germination, reduced require-ment for light, and decreased far-red light sensitivity, unravel-ing cytokinin functions in seed germination control. Thesestudies demonstrated that AHK2 and AHK3 play a role inquantitative control of organ growth in plants, with oppositeregulatory functions in roots and shoots under the light control.

Most research about the role of cytokinin in photomorpho-genesis has used seedlings of wild-type and photomorphogenicmutants of Arabidopsis. Dark-grown wild-type seedlings havedevelopmental changes that are similar to the phenotypic char-acteristics of constitutive photomorphogenic mutants of det1,det2, and amp1 [13]. The concentration of cytokinin increasessixfold in amp1 mutants relative to the wild-type [14]; how-ever, the concentration of cytokinin does not change in det1and det2 mutants, although the sensitivity to exogenous cyto-kinin does increase, as found in tissue culture and leaf senes-cence bioassays [15]. The cytokinin concentration also in-creases in seedlings grown in the light following their transferto dark conditions, indicating that there is a relationship be-tween endogenous cytokinin and photomorphogenesis [16,17].

To examine the mutual relationships between cell cycle con-trol, cytokinin, and the photoperiod of plants, we transferredseedlings from light conditions to constant light- or dark-cul-ture or from dark- to light-culture in the presence or absence ofcytokinin. We then measured the expression levels of six cy-clin genes that had been isolated from rice [4–6,8].

2. Results and discussion

We analyzed the transcript levels of cyclin genes in riceseedlings following treatment with exogenous cytokinin and achange in photoperiod. Fifteen-day-old seedlings that had beencultivated in a 16/8 h photoperiod were transferred to constant

light or dark for 24 hours in the presence or absence of kinetin,a cytokinin (Fig. 1). The differential expression of cyclin wasanalyzed with semi-quantitative RT-PCR performed on totalRNA isolated from the leaf blades of the cultivated seedlings.Northern blot hybridization is often used to investigate specificgene expression patterns at different developmental stages orafter applying specific growth hormones or stresses; however,semi-quantitative RT-PCR or Southern hybridization after RT-PCR are now being used more commonly because cell cyclerelated genes such as cyclins and CDKs have relatively highsequence homology [3]. Sequence homology both between andwithin specific types of cyclin or CDK genes is over 60% [18–20]. Transcript levels of the cyclin genes used for this experi-ment did not change when 15-day-old seedlings that had beencultivated in a 16/8 photoperiod were placed in the light for24 hours without kinetin supplement (Fig. 2A). However,transferring 15-day-old seedlings grown in the same conditionsto the dark for 24 hours caused the transcript levels of a fewcyclin genes to decrease (Fig. 2B). The transcripts of the B-type, D-type, and H-type cyclins decreased rapidly 2 hoursafter transfer to the dark condition, and the decreased transcriptlevels were maintained thereafter. The transcript level ofcycA1;1 decreased slightly after transfer to the dark condition,but for the C-type cyclin, no change of transcript level occurredin the dark, similar to the result in continuous light. When ki-netin was added to the media, the transcript levels of the cyclingenes did not change when the 15-day-old seedlings weretransferred to continuous dark or light for 24 hours (Fig. 3A–B).

The transcript levels of the B-type, D-type, and H-type ricecyclin genes were thus affected by rapid changes in photoper-

Fig. 2. The transcript level of cyclins in leaf blades under different conditions.Transcript levels of six cyclins were analyzed for 24 h after (A) constant light or(B) dark without kinetin. The fold induction/repression for the selected cyclinswas calculated as described in Section 3.

Fig. 3. The transcript level of cyclins in leaf blades under different conditions.Transcript levels of seven cyclins were analyzed for 24 h in (A) constant lightor (B) dark in medium containing 1 mg/l kinetin.

H. Lee et al. / Plant Physiology and Biochemistry 44 (2006) 248–252250

iod, and not by continuous light or dark. It is known that bluelight illumination delays cell division of the unicellular greenalga, Chlamydomonas reinhardtii [21], and it was reported thatthis delay in cell division is due to delayed DNA synthesis anddelayed increase in CDK-like activity [22]. However, this isthe first report indicating that the transcript levels of cyclingenes are changed by rapid changes in the photoperiod. It ap-pears that the rapid change in the light conditions acted as anabiotic stress on the rice seedlings, resulting in a change to theplant cell cycle control. The exogenous cytokinin supplementin the media presumably minimized changes in plant cell cyclecontrol even when the photoperiod changed rapidly. Cytokininis involved in cell cycle control at both the G1/S and G2/Mphase transitions. Plant cell cultivation research has shown thatD-type cyclins accumulate at the entry to S phase through ahormone-dependent mechanism. It has also been reported thatD-type cyclin accumulation induces CDK activation for therelease of a transcription factor involved in DNA replication[23]. G2/M progression is controlled by cytokinins as well. Cy-tokinin activates CDK by removing inhibitory phosphorylationduring the G2/M transition of tobacco [24]. Posttranslationalactivation of CDK affects plant mitosis through the posttransla-tional CDK activator and CDK-Tyr phosphatase Cdc25, whichinduces cell division in the leaf primordium in tobacco [25].Both B-type cyclin and D-type cyclin, together with CDK,are important regulators of the G1/S phase transition [1]. Exo-

genous cytokinin does not affect the transcript level of the H-type cyclin because it functions as a cyclin-dependent kinaseactivating kinase (CAK) in rice [6].

To investigate whether the expression of cyclin genes wasaffected by rapid changes of photoperiod and exogenous cyto-kinin supplementation in etiolated seedlings, we cultivated riceseedlings in the dark for 15 days after germination on a med-ium with no added growth regulator. Then we transferred thedark-grown seedlings to the light for 24 hours; half were culti-vated in medium containing kinetin and half in kinetin-freemedium (Fig. 1). Semi-quantitative RT-PCR was performedwith total RNA isolated from leaf blades (Fig. 4A–B). Whendark-grown seedlings were transferred to the light on kinetin-free media, the transcript levels of all the cyclin genes exceptcycB2;2 rapidly decreased by 2 hours after the transfer. Afterthat, no changes in gene expression were seen (Fig. 4A). Whenthe medium contained kinetin, however, only the cycB2;2 tran-script changed (Fig. 4B).

The rapid change of photoperiod affected the transcript le-vels of all the cyclin genes except cycB2;2 in the leaf blades ofseedlings. A similar observation about the functions of lightand cytokinins on plant development was made in a previousstudy. Exogenous cytokinins replaced the role of light in theinduction of deetiolation [26]. In the dark, cytokinins inducethe expression of genes that are usually induced by light [27],and they are partially involved in chloroplast development[27]. We found that the expression of cyclin genes in seedlings

Fig. 4. The transcript level of cyclins in leaf blades under different conditions.Transcript levels of seven cyclins were analyzed from dark-grown seedlingsexposed for 0, 1, 2, 5, 10, or 24 h to continuous light in Gamborg B5 solutioncontaining (A) 0.1 mg/l kinetin or (B) without kinetin.

H. Lee et al. / Plant Physiology and Biochemistry 44 (2006) 248–252 251

was strongly correlated with changes in the light condition andexogenous cytokinin. Thus, not only are exogenous cytokininand light involved in the expansion of cotyledons in dicotplants [15,28], they also seem to be involved in cell cycle con-trol in seedling leaf blades of rice, a monocot plant. A fewreports have described a relationship between cell cycle controland a change in light conditions or between cell cycle controland cytokinin treatment, but it has not previously been reportedthat a cytokinin and the light condition have a mutual effect oncell cycle control, especially with regard to cyclin gene expres-sion in the course of plant development. Hence, this experi-ment indicates that cytokinin is involved in cyclin homeostasisto prevent rapid changes in cyclin gene expression in plantsundergoing rapid changes of photoperiod.

3. Materials and methods

3.1. Plant materials and treatments

Seeds of rice (Oryza sativa L. cv. Il-Poum) were obtainedfrom the Rural Development Administration (RDA, SouthKorea). For the time–course analyses of the expression of ricecyclin genes, seeds were germinated at 30 °C for 2 days. Theywere grown in distilled water at 25 °C for 15 days in a growthchamber set to a 16/8 h light/dark cycle. The seedlings werethen exposed for 0, 1, 2, 5, 10, or 24 h to continuous light

treatment in Gamborg B5 [29] solution with or without0.1 mg/l kinetin or continuous dark treatment in Gamborg B5solution with or without 0.1 mg/l kinetin. Conversely, dark-grown seedlings were exposed for 0, 1, 2, 5, 10, or 24 h tocontinuous light treatment in Gamborg B5 solution with orwithout 0.1 mg/l kinetin (Fig. 1). After these treatments, theseedling leaves were pruned to a height of 8–10 cm.

3.2. Semi-quantitative RT-PCR

Total RNA was extracted from leaf blades of rice plantstreated with the various conditions mentioned above using anRNeasy kit (Qiagen, USA). Poly (A)+ RNA was purified withOligoTex dT30. The cDNA library was constructed as pre-viously described in [11]. In order to determine the relativetranscript levels of each cDNA specifically, semi-quantitativeRT-PCR assays were performed as previously described in[11]. Specific primers for the Os;cycA1;1(GI:6331694), Os;cycB2;1(GI:6331703), Os;cycB2;2(GI:1694891), Os;cycC;1(GI:1695697), Os;cycD (GI:18916915) and Os;cycH;1(GI:9796395) were as follows: cycA1;1 forward primer, 5′-taactacattgatcgttatctttctg-3′; cycA1;1 reverse primer, 5′-tctctgaagtatgtgttgtcagttat-3′; cycB2;1 forward primer, 5′-agttc-tatagagaaaatgaggaaatg-3′ cycB2;1 reverse primer, 5′-tttccaa-gaatctgtctactatgtta-3′; cycB2;2 forward primer, 5′-ctgcatacaaaa-tactctgaagaac-3′; cycB2;1 reverse primer, 5′-taaatgtgctgtactttctatgaact-3′; cycC forward primer, 5′-aggcactt-gactattatttagttgtt-3′; cycC reverse primer, 5′-atgagaataagatc-catcttgtaagt-3′; cycD forward primer, 5′-atagctagaggaaca-gaatgcttg-3′; cycD reverse primer, 5′-gacatcctctccttatttacatgg-3′; cycH1 forward primer, 5′-acttgtatttattcttcttgcaaagt-3′, cycH1reverse primer, 5′-cataaacaatcagatcaaaatctaaa-3′. Each reactionwas performed at least three times. The signals of amplifiedcyclin gene products were calculated with the Image analyzerdensitometer (ACI Auto Chemi System, UK).

Acknowledgements

Donggiun Kim was supported by the Environmental Bio-technology National Core Research Center (R15-2003-012-01002-0) and post-BK21 Program.

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