An Arabidopsis Cell Cycle –Dependent Kinase-Related Gene, CDC2b, Plays a Role in Regulating Seedling Growth in Darkness

Correspondence to: Minami Matsui, minami{at}postman.riken.go.jp (E-mail), 81-48-462-9405 (fax)

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

The Arabidopsis CDC2b gene has been defined as a plant-specific cell cycle–dependent kinase-related gene, although it lacks the conserved cyclin binding motif, and its exact function is not known. Here, we report that in etiolated seedlings, the expression of the CDC2b gene is correlated with elongation rate of the hypocotyl. Inhibition of CDC2b gene expression by using an inducible antisense construct resulted in short-hypocotyl and open-cotyledon phenotypes when transgenic seedlings were grown in the dark. The severity of these phenotypes in dark-grown seedlings could be correlated with the level of the antisense gene expression. The short hypocotyl of seedlings underexpressing CDC2b was a result of inhibition of cell elongation rather than a reduction in cell number, whereas in cotyledons, inhibition of CDC2b expression resulted in large, open cotyledons with amyloplasts rather than etioplasts. Although the nuclear DNA was less compact in the antisense hypocotyl cells, DNA content and endoreduplication were not affected. Cell division of the shoot apical meristem also was not affected by antisense expression. The short-hypocotyl phenotype of these transgenic plants was partially rescued by the addition of brassinolide. Brassinolide can only induce CDC2b expression in darkness. These results suggest a role for the CDC2b gene in seedling growth via regulation of hypocotyl cell elongation and cotyledon cell development.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Depending on the light environment, Arabidopsis seedlings pursue two contrasting morphogenetic patterns (Chory 1993 ; McNellis and Deng 1995 ). In the light, Arabidopsis seedlings exhibit short hypocotyls and open, differentiated cotyledons, whereas in darkness, seedlings have an extremely elongated hypocotyl and closed undifferentiated cotyledons (Kendrick and Kronenberg 1994 ). In the hypocotyl, light primarily affects cell elongation and differentiation and has little effect on cell division (von Arnim and Deng 1996 ). However, an extra cycle of endoreduplication of the nuclear DNA in some of the cells has been observed in darkness (Gendreau et al. 1997 , Gendreau et al. 1998 ). In the cotyledons, light promotes cell enlargement, differentiation, and cell division (Gendreau et al. 1997 ). Although a number of regulatory components already have been identified that mediate this light control of seedling growth, less is known about the molecular connection between the cell cycle regulation and light-regulated growth.

It is well established that cell cycle–dependent kinases (CDKs) play a key role(s) in regulating cell cycle progression (reviewed in Forsburg and Nurse 1991 ; Reed 1992 ; Hunter and Pines 1994 ; Jacobs 1995 ). In mammalian cells, as well as in yeast, it is well known that CDK1 (CDC2) forms a complex with cyclin, whose activation controls mitosis (reviewed in Forsburg and Nurse 1991 ; Reed 1992 ; Heichman and Roberts 1994 ; Pines 1994 ). The activity of this complex is controlled by phosphorylation of the CDC2 protein and subsequent degradation of cyclin by proteasomes in a cell cycle–dependent manner (reviewed in Murray 1993 ; Solomon 1993 ; Heichman and Roberts 1994 ; Pines 1994 ; Jacobs 1995 ). However, some of the CDC2-related genes are reported to play a role in processes in which cell cycle control is not the key regulatory step. These processes include apoptosis (Lahti et al. 1995 ), phosphate metabolism (Kaffman et al. 1994 ), hemopoiesis (Lapidot-Lifson et al. 1992 ), and neural development (Lew and Wang 1995 ).

We and others previously reported the isolation of two Arabidopsis CDC2-related genes (Ferreira et al. 1991 ; Hirayama et al. 1991 ; Imajuku et al. 1992 ). CDC2a, an authentic homolog of the yeast and mammalian CDC2 gene, has a PSTAIRE motif and can complement the cdc2 mutation of Schizosaccharomyces pombe, whereas the CDC2b gene belongs to a non-PSTAIRE gene family and cannot complement the yeast cdc2 mutant. Both genes are reported to be expressed mainly in the meristematic tissues in light-grown plants, suggesting involvement in cell division (Hemerly et al. 1993 ; Fobert et al. 1996 ; Segers et al. 1996 ). Interestingly, however, the promoter–ß-glucuronidase (GUS) staining patterns of CDC2a and CDC2b in the apical meristem are different. In light-grown plants, CDC2a promoter–driven GUS expression is localized in all cells in the meristem, whereas CDC2b promoter–driven GUS expression is localized only in dividing cells in the meristem (Hemerly et al. 1993 ; Segers et al. 1996 ). In dark-grown seedlings, expression of CDC2a was observed at the apical meristem of etiolated seedlings, but CDC2b expression was observed at the hook region in which most actively growing cells are localized.

Four cdc2-related genes also have been isolated in Antirrhinum. From comparisons of both sequence similarity and expression pattern, two of these genes are closely related to Arabidopsis CDC2a, and two others are closely related to Arabidopsis CDC2b (Fobert et al. 1996 ). It has been suggested that the Arabidopsis CDC2b gene defines a subfamily of cell cycle–dependent kinase-related genes that is unique in plants (Fobert et al. 1996 ; Segers et al. 1996 ). However, the cellular function of this CDC2b subfamily is not known.

The evident expression of CDC2b at the hook region of dark-grown Arabidopsis seedlings may imply a possible involvement of this gene in hypocotyl elongation. Although Arabidopsis hypocotyl elongation is controlled by light, little is known about the involvement of any specific cell cycle–related genes during its elongation. We thus examined expression of the CDC2b gene during Arabidopsis seedling growth to establish whether expression is correlated with the hypocotyl elongation rate. An inducible antisense CDC2b transgene also was stably introduced into Arabidopsis, and its phenotypic effect on development was analyzed. Our results suggest a role for CDC2b in regulating Arabidopsis seedling growth.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Expression of CDC2b Precedes the Change in the Hypocotyl Elongation Rate
To investigate a possible role of CDC2b in regulating hypocotyl elongation, we first examined its expression during seedling growth (Figure 1). In light-grown seedlings, the expression of both CDC2a and CDC2b peaked at days 2 and 3 after germination, just preceding the period during which the elongation rate of the hypocotyl was at a maximum (Figure 1A). In dark-grown seedlings, whereas expression of CDC2a was approximately the same from days 0 to 7 after germination, expression of CDC2b gene was much higher in the first 2 days and gradually decreased to an undetectable level by day 7. The peak of CDC2b expression just preceded the peak elongation rate of the hypocotyl, which occurs between days 2 and 3 (Figure 1B). This result would be consistent with a role of CDC2b in regulating hypocotyl elongation, especially in dark-grown seedlings. It is interesting that CDC2a gene expression still is maintained after day 7 in the dark-grown seedlings, even though the etiolated seedlings have stopped growing at this time. Its continuous expression is consistent with the notion that CDC2a expression is correlated with the potential for cell division rather than cell division per se (Hemerly et al. 1993 , Hemerly et al. 1995 ; Fobert et al. 1996 ), because etiolated seedlings can initiate active cell division once transferred to the light.

View larger version (44K): [in this window] [in a new window]   Figure 1. Expression of CDC2a and CDC2b Genes during Seedling Growth. Arabidopsis (Col-0) seeds were sown on GM plates and cold treated at 4°C for 2 days. After irradiation with red light (20 µmol-2 sec-1 for 10 min), plates were moved to white light (50 µmol m-2 sec-1 for 16 hr light and 8 hr dark at 22°C) or wrapped with aluminum foil and moved to a dark chamber (22°C; day 0). Seedlings were collected every day at a fixed time under a safe light for total RNA isolation and hypocotyl length measurements. (A) Equal amounts (10 µg) of total RNA were loaded in each lane and separated by electrophoresis. After transfer to a nylon membrane filter (Pall Biodyne A), the gel blot was hybridized with gene-specific probes. The 18S rRNA bands are shown at the bottom as an indication of equal loading. Numbers at top indicate days after the seeds were transferred from the cold to white light. Hypocotyl length (histogram, in millimeters) and elongation rate (line curve, in millimeters per day, indicating the difference between mean hypocotyl length of each day compared with that of the previous day) during the first 7 days of seedling growth are shown. A minimum of 30 seedlings was measured for each day. Error bars indicate ±SD. (B) The same conditions applied as in (A), except that plants were grown in the dark.

Expression of an Antisense CDC2b Gene under the Control of an Inducible Promoter
To further investigate the causal relationship between hypocotyl elongation and the expression of CDC2b, we used an antisense approach to inactivate the CDC2b gene. It has been reported that overexpression of a dominant-negative CDC2a gene in Arabidopsis is impossible, possibly due to lethality of inhibiting CDC2 activity (Hemerly et al. 1993 ). Therefore, we chose CDC2b for antisense expression in plants under an inducible promoter (Aoyama and Chua 1997 ). Specifically, the CDC2b cDNA was cloned into the pTA7002 plasmid in reverse orientation behind the GAL4-inducible promoter whose expression was only induced by the glucocorticoid hormone dexamethasone (DEX). This construct was transformed into Arabidopsis (ecotype Columbia [Col-0]). A total of 15 stable transgenic lines was produced and used for phenotypic analysis. There was no observable defect or lethality in transgenic lines when there was no induction of the antisense transgene. When expression of this gene was induced with DEX in dark-grown seedlings of the T₂ generation, however, 11 of the lines showed a clear phenotype. RNA gel blot analysis suggested clear induction of the antisense CDC2b RNA as well as inhibition of endogenous CDC2b mRNA expression (data not shown).

Morphological Phenotype of Antisense CDC2b Transgenic Seedlings
When antisense transgenic seedlings were grown on germination medium (GM) plates (Valvekens et al. 1988 ) containing 1 µM DEX, most transgenic lines segregated seedlings with short-hypocotyl and open-cotyledon phenotypes in darkness (cf. Figure 2D with 2B). This phenotype cosegregated with the presence of the antisense CDC2b gene as confirmed by polymerase chain reactions performed using CDC2b-specific and GAL4 promoter primers (data not shown). We noted that in the light, the antisense transgenic plants had smaller cotyledons in the presence of 1 µM DEX (Figure 2C), compared with control transgenic seedlings, which contained the inducible luciferase (LUC) transgene (Figure 2A).

View larger version (90K): [in this window] [in a new window]   Figure 2. CDC2b Antisense Transgenic Seedlings Exhibit Short-Hypocotyl and Open-Cotyledon Phenotypes in the Dark. (A) Control seedling grown in the light. (B) Control seedling grown in the dark. (C) CDC2b antisense seedling grown in the light. (D) CDC2b antisense seedling grown in the dark. Representative seedlings of a control transgenic line expressing the TA7002–LUC gene, which carries the same inducible promoter, and a CDC2b antisense transgenic line were grown on a plate containing 1 µM DEX. Seedlings were grown under white light (52 µmol m-2 sec-1 for 16 hr light and 8 hr dark at 22°C) or wrapped with aluminum foil and put into a dark chamber (22°C) and grown for 7 days. Bars in (A) to (D) = 1 mm.

A close examination of the inhibition of hypocotyl elongation of dark-grown seedlings and the level of exogenous DEX hormone revealed a positive correlation in the concentration range 0 to 1 µM DEX (Figure 3A to 3D). However, inhibition of hypocotyl elongation was saturated with a DEX concentration higher than 1 µM (Figure 3C and Figure 3D). Among those lines that showed a phenotype, there were differences in their sensitivity in response to DEX induction. As shown in Figure 3B, some antisense lines only showed a slight short-hypocotyl phenotype when 0.1 µM DEX was applied but exhibited a much shorter hypocotyl and open-cotyledon phenotype at 1 µM or higher (Figure 3C and Figure 3D, right). However, some transgenic lines showed quite a short hypocotyl and open cotyledons at 0.1 µM DEX (Figure 3B, center). As a control, a LUC gene was expressed under the same inducible promoter, and its phenotypic response to DEX was examined. There was no observable phenotypic effect on control transgenic plants after induction with different concentrations of DEX (Figure 3A to 3D, left). Both the hypocotyl and cotyledon phenotypes of dark-grown seedlings were strictly dependent on the presence of the antisense transgene and DEX induction. We conclude that the short-hypocotyl and open-cotyledon phenotype observed in the dark-grown transgenic antisense lines is the result of the expression of antisense transgenes rather than toxicity to DEX. We could not detect inhibition of root elongation in the DEX-induced CDC2b transgenic seedlings either in the light or in the dark (data not shown).

View larger version (105K): [in this window] [in a new window]   Figure 3. The Dark-Grown Seedling Phenotype of the Antisense Lines Is Dependent on DEX Concentration. (A) Plants grown without DEX. (B) Plants grown with 0.1 µM DEX. (C) Plants grown with 1 µM DEX. (D) Plants grown with 10 µM DEX. Each panel contains three seedlings: left, TA7002–LUC control transgenic seedling; center, CDC2b antisense line 6; and right, CDC2b antisense line 7. The seeds were plated on a GM plate containing different concentrations of DEX. After cold treatment, plates were kept in complete darkness for 7 days, as described in the legend to Figure 1. Plants in (A) to (D) are shown at the same magnification x2.5 of the original.

The Short-Hypocotyl Phenotype in Antisense Lines Is Caused by a Reduction in Cell Size Rather than Cell Number
To investigate the cellular basis of the short-hypocotyl and large-cotyledon phenotype in dark-grown transgenic seedlings, scanning electron microscopy was used to evaluate epidermal cell morphology. As shown in Figure 4A and Figure 4B, the enlargement of the cotyledons in dark-grown antisense gene–expressing seedlings was due primarily to expansion of the epidermal cells. However, the extent of stomatal cell differentiation in the antisense seedling cotyledons was largely unchanged compared with the control transgenic seedlings under DEX induction conditions. To test whether the short hypocotyl of antisense lines was caused by a reduction in the number of cells or a reduction in cell size in the hypocotyl, we examined cell number in the hypocotyls of seedlings that had or had not been subjected to DEX induction of the antisense transgene. Arabidopsis hypocotyl epidermal cell files are composed of 22 to 26 cells on average both in darkness and light (Misera et al. 1994 ; Gendreau et al. 1997 ). There was almost no change in the number of hypocotyl cells in each cell file when CDC2b transgenic seedlings were grown at different DEX concentrations. A close examination of the hypocotyl cells under the scanning microscope revealed that the hypocotyl cells were reduced in their longitudinal length throughout the hypocotyls (cf. Figure 4C with 4E, and 4D with 4F and 4G). The transverse section of this hypocotyl indicated that there was some differentiation and enlargement of both hypocotyl epidermal cells and cortex cells, although the extent of differentiation is not so obvious as in light-grown seedlings (Figure 4H and Figure 4I).

View larger version (121K): [in this window] [in a new window]   Figure 4. Scanning Electron Microscopy and Light Imaging of Cotyledon and Hypocotyl Cells of the CDC2b Transgenic Antisense Seedlings. (A) Epidermal surface of the cotyledon of a transgenic control seedling. (B) Epidermal surface of the cotyledon of a CDC2b transgenic antisense seedling. (C) Epidermal surface of the top part (just beneath the apical hook) of the hypocotyl of a transgenic control seedling. (D) Epidermal surface of the central part of the hypocotyl of a transgenic control seedling. (E) Epidermal surface of the top part (just beneath the shoot apical meristem) of the hypocotyl of a transgenic antisense seedling. (F) Epidermal surface of the central part of the hypocotyl of a transgenic antisense seedling. (G) Epidermal surface of the bottom part (just above the hypocotyl–root junction) of the hypocotyl of a transgenic antisense seedling. (H) Light microscopy of a transverse section of the hypocotyl (central part) of a transgenic control seedling. (I) Light microscopy of a transverse section of the hypocotyl (central part) of a transgenic antisense seedling. The TA7002–LUC control transgenic lines and a representative antisense line (line 3; as in Figure 3) were grown on a plate containing 1 µM DEX in darkness for 7 days. After fixation, seedlings were examined with a scanning electron microscope, or sections were made and examined with a light microscope. Arrows in (C) to (G) indicate the representative cell junctions. (A) to (G) are shown at the same magnifications, as are (H) and (I). Magnification x130.

Endoreduplication in the Hypocotyl Cells Is Not Affected by Antisense CDC2b Expression
It has been reported that in dark-grown Arabidopsis seedlings, one more round of endoreduplication occurs compared with light-grown seedlings, and hypocotyl cells contain 2C, 4C, 8C, and 16C nuclei (Gendreau et al. 1997 ). It has been suggested, furthermore, that this endoreduplication is not correlated with cell size or hypocotyl elongation because mutants with short hypocotyls due to brassinolide deficiency are not affected in endoreduplication (Gendreau et al. 1998 ). Nevertheless, the fact that expression of the CDC2b gene has been observed at the apical hook, where endoreduplication occurs (Segers et al. 1996 ) in dark-grown seedlings, prompted us to investigate whether endoreduplication is reduced in the transgenic seedlings with a short-hypocotyl phenotype. As shown in Figure 5, normal 2C, 4C, and 8C nuclei in the light-grown wild-type hypocotyls and 2C, 4C, 8C, and 16C nuclei in the dark-grown wild-type hypocotyls were observed in untreated seedlings (Figure 5A and Figure 5B). However, the short-hypocotyl cells from dark-grown DEX-treated transgenic lines have patterns of endoreduplication similar to that of wild-type etiolated seedlings (Figure 5C). Therefore, we concluded that short-hypocotyl phenotypes caused by CDC2b antisense expression were not correlated with endoreduplication.

View larger version (55K): [in this window] [in a new window]   Figure 5. Nuclear Genome Copy Number Distribution and DNA Staining in the Hypocotyl Cells of Transgenic Seedlings. (A) Cell flow cytometric profile of hypocotyl cells of TA7002–LUC transgenic seedlings grown in the light. These cells contained 2C, 4C, and 8C nuclei. (B) Cell flow cytometric profile of hypocotyl cells of TA7002–LUC transgenic seedlings grown in complete darkness. These cells contained 2C, 4C, 8C, and 16C nuclei. (C) Cell flow cytometric profile of hypocotyl cells of the CDC2b antisense transgenic seedling (line 3) grown in complete darkness. These cells contained 2C, 4C, 8C, and 16C nuclei. (D) DAPI staining image of a TA7002–LUC transgenic seedling grown in complete darkness. (E) DAPI staining image of a CDC2b antisense transgenic seedling (line 3) grown in complete darkness. See Methods for details of experimental procedures. For (D) and (E), whole seedlings were stained with DAPI, as described in Methods, and directly photographed under a fluorescent microscope. Only those nuclei in sharp focus (indicated by arrows) are at the correct size and conformation; nuclei out of the focal plane seem larger than in reality. The antisense hypocotyl cells are also much shorter; thus, more nuclei and cells are present in (E). (D) and (E) are at the same magnification. Magnification x45 of the original.

It should be noted that when hypocotyl cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and observed by using a fluorescent microscope, the antisense transgenic seedlings grown in the presence DEX had a fuzzy and more diffuse staining pattern, whereas the normal nuclei exhibited very sharp fluorescent staining (Figure 5D and Figure 5E). This indicated that the inhibition of CDC2b expression in the antisense lines resulted in larger and less compact nuclei, although it does not affect DNA endoreduplication.

CDC2b Antisense Expression Does Not Have an Effect on Cell Division in the Shoot Apical Meristem
We examined DAPI staining patterns at the shoot apical meristem to determine whether CDC2b antisense transgenic plants have altered cell division (Figure 6). After making sections at the shoot apical meristem, nuclear DNA was stained with DAPI. When comparing antisense transgenic and control seedlings grown in the light, we could not observe any differences in the size or number of cells comprising the L1, L2, and L3 of the shoot apical meristem (Figure 6A and Figure 6C, and Figure 6B and Figure 6D with higher magnification). This observation is contrary to the report describing CDC2a dominant-negative transgenic tobacco plants. In these transgenic plants, the number of cells in each meristem layer was reduced, although adult plant morphology was not affected compared with the wild-type plant (Hemerly et al. 1995 ). In the dark-grown CDC2b antisense Col-0 seedlings, we observed vacuoles that occupied the greater part of the meristematic cells, the nuclei being pushed to the side of the cells (Figure 6H). When we compared the cell size and number composing the shoot apical meristem, we could not observe differences between the CDC2b antisense transgenic seedlings and the control transgenic seedlings (Figure 6E and Figure 6G, and Figure 6F and Figure 6H with higher magnification). These results indicate that CDC2b does not affect cell division in the shoot apical meristem.

View larger version (116K): [in this window] [in a new window]   Figure 6. DAPI Staining Image of the Shoot Apical Meristem of CDC2b Antisense Transgenic Seedlings. (A) Low-magnification image of the shoot apical meristem of a light-grown TA7002–LUC control seedling. (B) High magnification of (A). Cells of the shoot apical meristem are occupied by a nucleus, and the fluorescence of other organelles can be seen (small spots). (C) Low magnification of the shoot apical meristem of a light-grown CDC2b antisense transgenic seedling. Seedlings were grown with 1 µM DEX. At this stage, seedlings grow with a similar morphology, as represented in Figure 2C. (D) High magnification of (C). (E) Low magnification of the shoot apical meristem of a TA7002–LUC control seedling grown with 1 µM DEX in the dark. (F) High magnification of (E). (G) Low magnification of the shoot apical meristem of a dark-grown CDC2b antisense transgenic seedling grown with 1 µM DEX. At this stage, seedlings have a similar morphology to that represented in Figure 2D. (H) High magnification of (G). All seedlings were grown for 7 days. Bar in (A) = 20 µm for (A), (C), (E), and (G); bar in (B) = 10 µm for (B), (D), (F), and (H).

We also examined the DAPI staining pattern of root tips of CDC2b antisense transgenic seedlings and the TA7002–LUC control transgenic seedlings. We observed no differences in these root tip cells either when the seedlings were grown in the light or in the dark (data not shown).

CDC2b Antisense Lines Are Also Defective in Greening
Etiolated seedlings initiate greening immediately after transfer to light, and this process can be blocked if seedlings are pregrown in continuous far-red (FR) light. This block of greening is believed to be caused by the disappearance of the prolamellar body composed of protochlorophyllide oxidoreductase, which is responsible for the formation of chlorophyllide from protochlorophyllide (Barnes et al. 1996 ). In seedlings grown in continuous FR light, these prolamellar bodies are indeed absent in the cotyledons. As shown in Figure 7B, antisense seedlings grown in the dark in the presence of DEX (Figure 7A) also failed to green and became completely bleached after transfer to white light, whereas control seedlings in the presence of DEX or antisense seedlings grown in the absence of DEX all were able to green when transferred to white light.

View larger version (148K): [in this window] [in a new window]   Figure 7. CDC2b-Transformed Seedlings Are Defective in Greening during the Dark-to-Light Transition. (A) A control transgenic seedling (left) and two representative CDC2b antisense seedlings after 7 days of growth in the dark with 1 µM DEX. (B) The same seedlings as shown in (A) but after transfer to white light for 3 days.

To understand further the basis for this defect in greening, the plastid structure of the cotyledons of 7-day-old DEX-treated dark-grown antisense seedlings was analyzed by transmission electron microscopy. At this stage, transgenic seedlings have open cotyledons and short hypocotyls (Figure 2D). As shown in Figure 8A and Figure 8B, normal etioplasts were observed in untreated antisense plants and in control transgenic plants treated with DEX. However, no etioplasts were observed in >100 cells that we examined from dark-grown antisense seedlings treated with DEX (1 µM). Surprisingly, we observed amyloplast-like structures in all cells (Figure 8C and Figure 8D). Amyloplasts usually are observed in nonphotosynthetic cell types, such as root cells, and also in the cotyledons of some dicotyledonous seeds, where they are used for starch storage. Some of the amyloplast-like structures also seem to have oil drop–like vesicles (plastoglobuli) of various sizes (Figure 8E). As observed in FR light–treated seedlings, the defect in greening correlated with the absence of etioplasts (Barnes et al. 1996 ).

View larger version (131K): [in this window] [in a new window]   Figure 8. CDC2b Transgenic Seedlings Contain Amyloplasts Instead of Etioplasts. (A) Etioplasts with prolamellar bodies from dark-grown TA7002–LUC control transgenic seedlings. (B) Higher magnification of (A) to reveal the structure of the prolamellar body. (C) Representative plastids from a cotyledon cell of a 7-day-old dark-grown CDC2b antisense seedling (line 3). (D) Representative plastids from a cotyledon cell of a 7-day-old dark-grown CDC2b antisense seedling (line 6). (E) Higher magnification of an amyloplast from the same cotyledon section shown in (D). (F) Chloroplast from a cotyledon cell of a light-grown TA7002–LUC control transgenic seedling. (G) Representative chloroplast from a cotyledon of a 7-day-old light-grown CDC2b antisense seedling (line 3). All seedlings were grown on GM plates with 1 µM DEX. After 7 days, the seedlings were harvested in the light or under a green safe light, fixed immediately, and examined with a transmission electron microscope. Bar in (C) = 1 µm for (A), (C), and (D); bar in (B) = 200 nm for (B) and (E); bar in (F) = 1 µm for (F) and (G).

In the light, development of chloroplasts was observed in cotyledon cells of antisense transgenic seedlings, just like those of the control plants (Figure 8F). We also observed accumulation of starch in almost all the chloroplasts of cotyledons of antisense transgenic seedlings in the presence of DEX (Figure 8G).

Plant Hormones Can Partially Restore Hypocotyl Cell Elongation in CDC2b Antisense Seedlings
Hypocotyl elongation is known to be controlled by several plant hormones, such as gibberellins and brassinosteroids (Chory and Li 1997 ; Peng et al. 1997 ). These hormones did not affect the hypocotyl length of CDC2b antisense transgenic seedlings grown without DEX (Figure 9A to 9C). To understand further possible relationships of CDC2b and hormonal regulation, we applied those hormones together with 1 µM DEX to a plate of CDC2b antisense transgenic seedlings. As shown in Figure 9F, the shortened hypocotyl cells were quite responsive to brassinolide and to a lesser degree to the gibberellin GA₄ (Figure 9E). However, neither hormone could completely rescue the phenotype caused by CDC2b antisense expression (Figure 9D), whereas under the same conditions, a brassinolide mutant could be completely rescued by brassinolide application (data not shown). The number of hypocotyl cells was not changed, and we also observed amyloplast-like structures in the cotyledons of CDC2b antisense transgenic plants, in which the hypocotyl was partially recovered by the addition of brassinolide (data not shown).

View larger version (143K): [in this window] [in a new window]   Figure 9. Effect of Phytohormones on the Phenotype of the CDC2b Transgenic Antisense Seedlings. (A) 7-day-old etiolated seedlings grown on a GM plate. From left to right, transgenic control (TA7002–LUC) seedling and four representative CDC2b transgenic antisense seedlings (lines 6, 7, 22, and 28, respectively). (B) Same seedling lines as shown in (A), with 1 µM GA4. (C) Same seedling lines as shown in (A), with 0.1 µM brassinolide. (D) Same seedling lines as shown in (A), with 1 µM DEX. (E) Same seedling lines as shown in (B), with 1 µM DEX. (F) Same seedling lines as shown in (C), with 1 µM DEX. Phytohormones at the concentrations indicated were added to GM plates with or without 1 µM DEX. Seedlings were grown for 7 days in complete darkness. All seedlings were photographed at the same magnification. Magnification x1.8 of the original.

Brassinolide Induces CDC2b Expression in the Dark
To understand the relationship between brassinolide and CDC2b, we investigated further the effect of brassinolide on CDC2b gene expression. CDC2b promoter–GUS transgenic plants were grown with 0.1 µM brassinolide in both dark and light conditions, and histochemical and quantitative analyses of CDC2b expression were performed. In light-grown seedlings, CDC2b expression was observed at the shoot apical meristem in both seedlings grown with and without brassinolide (Figure 10A and Figure 10B). However, in the dark-grown seedlings, CDC2b expression was observed in the hook region, as reported previously (Figure 10C; Segers et al. 1996 ). Although the location of CDC2b expression was not changed in dark-grown seedlings grown with 0.1 µM brassinolide, apparent enhancement of expression was observed by brassinolide (Figure 10D).

View larger version (92K): [in this window] [in a new window]   Figure 10. Histochemical Analysis of the Effect of Brassinolide on the CDC2b Gene Expression Pattern. (A) GUS staining pattern of CDC2b promoter–GUS transgenic seedlings grown under white light for 7 days. (B) Same as (A), with 0.1 µM brassinolide. (C) GUS staining pattern of CDC2b promoter–GUS transgenic seedlings grown in darkness for 7 days. (D) Same as (C), with 0.1 µM brassinolide.

This observation was confirmed further by measuring GUS enzyme activity. Using 4-methylumbelliferyl ß-D-gluc-uronide as a substrate, we observed a clear increase in enzyme activity when seedlings were grown in darkness with 0.1 µM brassinolide (Figure 11). On the other hand, there was almost no difference in activity when seedlings were grown in the light with or without brassinolide (Figure 11). These results indicated that brassinolide can induce CDC2b expression only in darkness. This induction of CDC2b gene by brassinolide can titrate out the CDC2b antisense RNA in DEX-induced CDC2b antisense transgenic seedlings. Thus, the short-hypocotyl phenotype of DEX-induced dark-grown transgenic seedlings was rescued by the addition of brassinolide. However, this induction level is not sufficient to fully rescue the short-hypocotyl phenotype of these transgenic plants.

View larger version (16K): [in this window] [in a new window]   Figure 11. Fluorometric Analysis of the Effect of Brassinolide on the CDC2b Gene Expression Pattern. GUS activity was measured to quantify the expression level of the CDC2b gene by using 4-methylumbelliferyl ß-D-glucuronide as a substrate. The experiment was repeated three times, and the mean values are represented with error bars indicating ±SD. -BR WL, a CDC2b promoter–GUS transgenic seedling grown under white light for 7 days; +BR WL, same with 0.1 µM brassinolide; -BR D, a CDC2b promoter–GUS transgenic seedling grown in the dark for 7 days; +BR D, same with 0.1 µM brassinolide.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Our studies provide substantial evidence of a role for CDC2b in the regulation of hypocotyl cell elongation and cotyledon development in dark-grown Arabidopsis seedlings. First, we determined that the expression pattern of CDC2b is consistent with a role in regulating hypocotyl elongation in darkness, because its temporal expression just precedes the changes in the elongation rate of the seedlings. Second, antisense inhibition of CDC2b expression resulted in short hypocotyl cells and open, enlarged cotyledons. Third, inhibition of CDC2b expression also resulted in a block of greening in dark-grown seedlings, likely due to the abnormal development of amyloplasts instead of etioplasts. Therefore, our data indicate a physiological function for CDC2b, a novel subfamily of cell cycle–dependent kinase-related genes unique to plants.

CDC2b and Arabidopsis Seedling Growth
It has been reported that hypocotyls of Arabidopsis are composed of 22 to 26 cells when grown in both light and darkness (Misera et al. 1994 ; Gendreau et al. 1997 ). During hypocotyl elongation, only a few cell divisions occur, and therefore growth is essentially due to the elongation of hypocotyl cells. Previous extensive studies have clearly established that hypocotyl cell elongation is regulated by many factors, including light and plant hormones (von Arnim and Deng 1996 ; Chory and Li 1997 ). Furthermore, mutational studies of signaling components in light as well as hormonal pathways have revealed their specific involvement in regulating hypocotyl cell elongation, but the role of cell cycle–related genes has hardly been investigated. Overexpression of the CDC2a gene in both Arabidopsis and tobacco does not have any detectable influence on hypocotyl cell size (Hemerly et al. 1995 ). A dominant-negative construct of the CDC2a gene resulted in enlarged epidermal cells in transgenic tobacco, but overall plant morphology was largely unaffected. It therefore was suggested that CDC2a may be strictly involved in the control of the cell cycle and may not regulate specific morphological or developmental programs. However, our results indicate a role of CDC2b in regulating hypocotyl elongation, cotyledon enlargement, and apical hook formation.

CDC2b-related genes are highly expressed in dividing cells of meristems and also in the hook region of etiolated seedlings (Fobert et al. 1996 ; Segers et al. 1996 ). Whereas CDC2b expression in dividing cells indicates a possible role in some aspect of cell cycle regulation, its expression in hypocotyl cells would imply a role in hypocotyl development. The hypocotyl cell rarely divides, although most of its cells go through one to three cycles of DNA endoreduplication (Gendreau et al. 1998 ). The lack of effect of CDC2b antisense expression on the endoreduplication of hypocotyl cells (Figure 5C) indicates that CDC2b is not responsible for the promotion of DNA endoreduplication. There are at least two explanations of how CDC2b might regulate hypocotyl cell elongation and cotyledon development. The first is that CDC2b plays a direct role in regulating hypocotyl cell elongation or cotyledon cell expansion, which is independent of its putative role in cell cycle regulation. The fact that CDC2b lacks the conserved PSTAIRE domain responsible for cyclin association and that it has been found only in plants would be consistent with this possibility. The other possibility is that CDC2b is involved in regulating a specific phase of the cell cycle and/or overall chromosome spatial organization, which is critical for hypocotyl cell elongation and cotyledon development. The fact that CDC2b antisense lines exhibited less compact nuclear DNA staining supports this notion. Clearly, future investigation is required to differentiate between these possibilities.

CDC2b and Light-Regulated Growth
Interestingly, seedlings expressing antisense CDC2b also displayed open and enlarged cotyledons and no apical hook in darkness. Scanning electron microscopy indicated that dark-grown antisense seedlings grown in the presence of DEX have more enlarged epidermal cells, and there seems to be no notable increase in cell numbers as a consequence of cell division. Although these alterations in some way can be described as a partial photomorphogenetic development in darkness, there are, however, a few key features different from photomorphogenetic development. First, there is very little difference in the stomatal cell maturation of the antisense seedlings grown in the presence of DEX compared with those of the wild type or uninduced antisense seedlings. However, in light-grown seedlings, the development of the stomatal cells is much more advanced. Second, the development of amyloplasts in the cotyledons of dark-grown antisense seedlings grown in the presence of DEX is different from chloroplast development in light-grown seedlings. The latter result implies a role for CDC2b in maintaining a proper plastid differentiation pattern in developing plants. Therefore, the cotyledon phenotype in the antisense lines provides a better indicator than the hypocotyl phenotype for showing the difference in CDC2b function and light-triggered development. Our data seem to indicate that the consequence of inhibiting CDC2b expression in dark-grown seedlings is distinct from photomorphogenetic development triggered by light.

The development of the amyloplasts in dark-grown antisense seedlings in the presence of DEX also implied that plastid differentiation could be uncoupled from the other development processes. To our knowledge, enlargement of cotyledons in dark-grown seedlings accompanied by amyloplast development in the cotyledon cells has not been reported previously. Clearly, CDC2b plays a critical role in ensuring the coordination of the development programs of plastid and overall cell growth. In some dicotyledonous plants, amyloplasts, used for starch storage, are present in the cotyledons of mature seeds, but they are quickly converted to etioplasts in darkness or chloroplasts in the light during seedling growth. It thus is possible that inhibition of CDC2b expression prevented the amyloplasts from being converted into etioplasts during seedling growth in darkness. Furthermore, because only amyloplasts are found in dark-grown antisense CDC2b-expressing seedlings, it is expected that some of the amyloplasts also probably are derived from proplastids in the cotyledons. As mentioned above, it would be interesting to find out whether this is a CDC2b function independent of cell cycle regulation or an indirect consequence of its role in regulating a specific aspect of cell cycle progression in dark-grown seedlings.

Relationship between CDC2b and Plant Hormone Action
Brassinolides and gibberellins are phytohormones effective in regulating hypocotyl elongation (von Arnim and Deng 1996 ; Chory and Li 1997 ). Several mutants have been isolated with a short-hypocotyl phenotype in darkness. Some of these mutants can be rescued by the addition of brassinolide, the end product of brassinolide biosynthesis, and have been found to be affected in brassinolide biosynthesis (Chory and Li 1997 ). Gibberellin mutants also have a dwarf phenotype (Peng et al. 1997 ). Although application of gibberellin had very little effect, brassinolide can cause elongation of the hypocotyl of the CDC2b antisense seedlings in the presence of DEX. Furthermore, induction of CDC2b expression by brassinolide in dark-grown seedlings indicates that CDC2b is acting downstream of brassinolide. However, because brassinolide cannot fully rescue the short-hypocotyl phenotype of DEX-treated transgenic seedlings by titrating out the antisense RNA, it is possible that brassinolide is only part of the regulatory system controlling CDC2b expression. From the observation that brassinolide induces CDC2b gene expression only in the darkness, there may be some regulatory pathways that make it possible to induce CDC2b gene in the light. Nevertheless, this would appear to be a novel report of a gene whose expression is controlled by brassinolide. It would be interesting to discover how CDC2b is regulated by brassinolide and by light.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Plant Material and Growth Conditions
All Arabidopsis thaliana lines were in a Columbia (Col-0) background and were grown on germination medium (GM) (Valvekens et al. 1988 ). For hormone experiments, hormones were added to the medium with or without dexamethasone (DEX; Sigma). Gibberellin (GA₄) was used at a final concentration of 1 µM, and brassinolide (kindly provided by S. Fujioka, RIKEN, Saitama, Japan) was used at a final concentration of 0.1 µM.

All seedlings were grown in a temperature-controlled growth chamber (Koito Co. Ltd., Tokyo, Japan) at 22°C. Hypocotyl length was measured under a stereomicroscope (Plan Apo; Leica, Heerbrugg, Switzerland), and photographs were taken using the same microscope.

Transgene Constructs and Arabidopsis Transformation
To make transgenic plants expressing the cell cycle–dependent CDC2b antisense RNA, cDNA of the CDC2b gene was cloned into the pTA7002 vector downstream of the inducible promoter with GAL4 binding sites. This plasmid also contains the DNA binding domain of the GAL4 protein fused to a transcription activator, VP16, and glucocorticoid receptor binding domain (Aoyama and Chua 1997 ). After induction with glucocorticoid, this fusion protein interacts with GAL4 binding sites in the promoter and induces downstream gene expression (Aoyama and Chua 1997 ).

This plasmid was introduced into Agrobacterium tumefaciens GV2260 and transformed into Arabidopsis by using vacuum infiltration (Bechtold et al. 1993 ). Transgenic plants were selected on GM plates containing 20 µg/mL hygromycin and 100 µg/mL claforan. Hygromycin-resistant plants were grown to maturity, and seeds were harvested. Homozygous transgenic lines were produced for selected transgenic lines and used for the detailed studies reported in this article. The transgenic lines that showed a phenotype under DEX induction exhibited 100% inheritance in the progeny. The control transgenic line (harboring TA7002–LUC, i.e., the luciferase-encoding gene under the control of the same inducible promoter as used for the CDC2b antisense gene) was kindly provided by T. Aoyama (Aoyama and Chua 1997 ).

DEX Treatment
DEX was dissolved in dimethylsulfoxide at a concentration of 3.3 mM and kept in a freezer (-30°C). DEX at the indicated concentration was added to the medium. For DEX treatment, seeds were sown on a plate containing 0.1 to 10 µM DEX and cold treated at 4°C for 2 days to enhance germination. After irradiation with red light (20 µmol m^-2 sec^-1 for 10 min), plates were moved to white light (50 mmol m^-2 sec^-1, 16 hr light and 8 hr dark, 22°C) or wrapped with aluminum foil and kept in a dark chamber at 22°C.

After 7 days, we observed the seedling phenotype caused by DEX addition. A wild-type plant or transgenic plant containing the DEX-inducible LUC gene (TA7002–LUC) was used as a control.

Electron Microscopy
Whole dark-grown seedlings were used for scanning electron microscopy. Seedlings were fixed with 4% (v/v) glutaraldehyde solution and washed several times with 0.1 M phosphate-buffered saline, pH 6.8. They were dehydrated with a series of ethanol solutions, exchanged with CO₂ by using a Polazon critical point device (model HCP-2; Hitachi, Tokyo, Japan), and spatter coated with carbon ions (E-102; Hitachi). Carbon-coated seedlings were observed with scanning electron microscopy (ISI-SS40; Hitachi).

For transmission electron microscopy of cotyledon cells, seedlings were fixed with 2% (v/v) glutaraldehyde in cacodylate buffer (20 mM sodium cacodylate, pH 7.0) and 1% (w/v) osmium tetroxide in cacodylate buffer, dehydrated in a graded ethanol series, and embedded in Spurr's resin (Taab, Berkshire, UK). Sections were made using an ultramicrotome (model Ultracut UCT; Leica, Vienna, Austria). Thin sections were poststained with 1% (w/v) uranyl acetate and counterstained with 3% (w/v) lead nitrate. Observations of plastids were made with transmission electron microscopy (model JEM-2000 FXII; JEOL, Tokyo, Japan).

Cell Flow Cytometric Analysis
The nuclear DNA copy number was analyzed with a flow cytometer (Partec PA, Tokyo, Japan), according to the manufacturer's recommended procedure, with a modification according to a previous report (Galbraith et al. 1983 ). Approximately 10 to 15 hypocotyls of seedlings were chopped with a razor blade into small pieces and put into a 4',6-diamidino-2-phenylindole (DAPI) solution for 30 sec. The solution was filtered through a 30-µm nylon mesh tube to isolate plant nuclei, and the filtered solution was analyzed with the flow cytometer.

DAPI Staining
To observe DAPI-stained nuclei, whole seedlings were fixed in 2% (v/v) glutaraldehyde in cacodylate buffer for 20 hr at 4°C, dehydrated with a series of ethanol solutions, and then embedded in Technovit 7100 resin (Kulzer and Co., Wehrheim, Germany). To observe fluorescence images, thin sections (0.7 µm thick) were made with a glass knife on an Ultracut UCT ultramicrotome (Leica). Sections were stained with 1 µg/mL DAPI and 1 mg/mL n-propyl gallate in 50% glycerol. The samples were left for 5 min at room temperature and observed using an Olympus IX70 microscope (Tokyo, Japan).

RNA Gel Blot Analysis
Extraction of total RNA and RNA gel blotting were performed as described previously (Matsui et al. 1995 ). Approximately 10 µg of total RNA was used in each lane, and the RNA was transferred to nylon membrane filters (0.2 µm; Pall Biodyne A, East Hills, NY) after electrophoresis. To detect antisense RNA, the pGEM2bF plasmid, which contains a full-length CDC2b cDNA, was digested with BglII and transcribed with T7 RNA polymerase in the presence of ³²P-UTP (3000 Ci/mmol; Amersham, Buckinghamshire, UK). To detect CDC2b mRNA, the same digested plasmid was used but it was transcribed with the SP6 polymerase. For CDC2a mRNA detection, pCDC2aAT containing the full-length CDC2a cDNA was digested with XbaI and transcribed with SP6 polymerase.

Light Images
To observe transverse sections of hypocotyls, whole seedlings were fixed and embedded in resin, as described in DAPI Staining. The sections (2.5 µm thick) were prepared with a glass knife on an Ultracut UCT ultramicrotome (Leica), placed on cover slips, and dried. They were stained with 0.5% (v/v) toluidine blue–O in 0.1 M phosphate-buffered saline, pH 7.0, for 30 sec and then washed in distilled water for 10 sec. The samples were observed with an Olympus IX70 microscope.

Fluorometric and Histochemical Analyses of ß-Glucuronidase
Fluorometric and histochemical analyses of ß-glucuronidase (GUS) were performed as described previously with modifications (Jefferson et al. 1987 ). CDC2b promoter–GUS transgenic plants were grown for 5 days with or without 0.1 µM brassinolide on a GM plate.

For histochemical assays, seedlings were fixed with 2% (v/v) paraformaldehyde in 50 mM sodium phosphate buffer, pH 7.0, for 10 min, and then washed twice with 50 mM sodium phosphate buffer. Fixed seedlings were stained in 0.5 mg/mL 5-bromo-4-chloro-3-indolyl ß-D–glucuronic acid solution containing 50 mM sodium phosphate, pH 7.0, and 0.5% (v/v) Triton X-100 at 37°C for 24 hr in the dark. Stained seedlings were dehydrated in an ethanol series and stored in 98% (v/v) ethanol at 4°C.

For fluorometric analyses, seedlings were extracted with 200 µL of an extraction buffer composed of 50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.1% (v/v) Triton X-100, 0.1% (v/v) sarkosyl, and 10 mM ß-mercaptoethanol. Each sample (100 µg) was incubated with 20 mM 4-methylumbelliferyl glucuronide at 37°C in the dark for 2 hr. The incubation solution (200 µL) was transferred into 800 µL of 0.2 M Na₂CO₃ to stop the reaction. After excitation at 366 nm, fluorescence was measured at 455 nm by using a fluorescence spectrometer (model F-4010; Hitachi) and quantified using 4-methylumbelliferone as a standard.

	ACKNOWLEDGMENTS

We thank Dr. D. Inzé for providing CDC2a and CDC2b genes and CDC2b–GUS transgenic seeds and Dr. T. Aoyama for kindly supplying the pTA7002 plasmid and TA7002–LUC transgenic seeds. We thank R. Ishizuka (Ikedarika, Tsukuba, Japan) and K. Ichinose for their excellent help with flow cytometry. We thank Dr. S. Fujioka for providing brassinolide and helpful discussions. We also thank Drs. R.E. Kendrick and K. Bishop for critically reading the manuscript and Dr. Y.Y. Yamamoto for helpful discussions. We also thank Dr. X.-W. Deng for his help during the preparation of the manuscript. This work is supported by the Research for the Future Program from Japan Society for the Promotion of Science (Grant No. JSPS-RFTF96L00601) and a Human Frontier long-term grant (No. RG43/97) to M.M. T.Y. is a Junior Research Associate Fellow supported by RIKEN.

Received May 10, 1999; accepted July 21, 1999.

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