AtDOF5.4/OBP4, a DOF Transcription Factor Gene that Negatively Regulates Cell Cycle Progression and Cell Expansion in Arabidopsis thaliana

In contrast to animals, plant development involves continuous organ formation, which requires strict regulation of cell proliferation. The core cell cycle machinery is conserved across plants and animals, but plants have developed new mechanisms that precisely regulate cell proliferation in response to internal and external stimuli. Here, we report that the DOF transcription factor OBP4 negatively regulates cell proliferation and expansion. OBP4 is a nuclear protein. Constitutive and inducible overexpression of OBP4 reduced the cell size and number, resulting in dwarf plants. Inducible overexpression of OBP4 in Arabidopsis also promoted early endocycle onset and inhibited cell expansion, while inducible overexpression of OBP4 fused to the VP16 activation domain in Arabidopsis delayed endocycle onset and promoted plant growth. Furthermore, gene expression analysis showed that cell cycle regulators and cell wall expansion factors were largely down-regulated in the OBP4 overexpression lines. Short-term inducible analysis coupled with in vivo ChIP assays indicated that OBP4 targets the CyclinB1;1, CDKB1;1 and XTH genes. These results strongly suggest that OBP4 is a negative regulator of cell cycle progression and cell growth. These findings increase our understanding of the transcriptional regulation of the cell cycle in plants.

The AP2 transcription factor ANT (AINTEGUMENTA) gene can maintain the meristematic competence of cells by regulating CYCD3;1 expression 37 . However, the mechanisms by which developmental signals interact with plant cell cycle progression remain unclear.
DNA binding with one finger (DOF) proteins are a group of plant-specific transcription factors. A conserved N-terminal DNA-binding domain is present in typical DOF proteins, and the N-terminal domain can bind to DNA sequences harbouring an AAAG core motif and interact with other proteins. A divergent transcription regulation domain is present at the C terminus, this divergent C terminus is used for transcriptional regulation 51,58 . In Arabidopsis, the DOF transcription factor family has 37 members that play diverse roles in plant development and in response to environmental stimuli. In a screen for proteins that interact with the bZIP transcription factor OCS element binding factor 4 (OBF4), the OBF binding protein 1 (OBP1) was the first to be reported. Furthermore, other DOF transcription family proteins, OBP2 and OBP3, were identified to have the ability to bind OBF4 and to enhance their binding to the OCS element in the downstream target genes 21 . OBP1 is involved in the control of cell division by targeting the core cell cycle regulator CYCB3;3 and the S phase-specific transcription factor DOF2. 3 47 . OBP2 plays a role in the regulation of indole glucosinolate metabolism 48 , and OBP3 has been characterized as a novel component of light signalling 54 .
This study identifies the function of DOF transcription factor OBP4/DOF5.4 in cell cycle regulation. OBP4 controlled plant growth by regulating core cell cycle genes involved in the replication machinery and cell expansion regulators. Correspondingly, OBP4 overexpression resulted in the early onset of endocycle progression. Based on the expression profile and genetic results, we propose that OBP4 is a novel regulator of cell cycle progression and cell expansion.

Materials and Methods
General methods. Arabidopsis seeds were sown in soil and grown in a growth chamber with a 16-h day length provided by fluorescent light at 120 μmol m −2 s −1 and a day:night temperature of 22 °C-18 °C and relative humidity of 60-75%. Agrobacterium tumefaciens strain GV3101 was used to transform Arabidopsis thaliana Col-0 (35S::OBP4, RNAi::OBP4, pOBP4::GUS). Chemicals were purchased from Roche (Basel, Switzerland) or Sigma (St. Louis, MO). We used NCBI (http://www.ncbi.nlm.nih.gov/) and TAIR (http://www.arabidopsis. org/) to analyze gene sequences. We used GraphPad primer 5 (GraphPad Software Inc., San Diego, CA) to make diagrams. For induction experiments, plants were sprayed with 20 μm estradiol solution or grown in MS medium containing 20 μm estradiol.

Plasmid constructs.
To construct the 35S::OBP4 vector, polymerase chain reaction (PCR) was used to amplify the OBP4 CDS using Col-0 leaf cDNA as the template. The OBP4 cDNA was cloned into a modified pHB plant transformation vector via the PstI and SacI sites. To construct the pER8::OBP4 and pER8::OBP4::HA vectors, OBP4 cDNA or OBP4 cDNA fused to an HA tag were cloned into the pER8 vector via the SpeI and XhoI sites. To construct the pER8::OBP4::VP16 vector, OBP4 cDNA was fused to the VP16 domain and cloned into the pER8 vector via the SpeI and XhoI sites. To construct the RNAi::OBP4 vector, the forward and reverse fragments of a 3′-UTR OBP4 sequence that does not have similarity with the UTRs of the other genes were linked to the pCAMBIA1301-RNAi vector. To construct the Promoter::GUS fusion vector, an approximately 2.0 kb 5′-UTR upstream of the ATG start codon was fused to the Escherichia coli GUS reporter gene in the pCAMBIA1300 vector. The primer sequences are shown in Supplementary Table S1. RNA isolation and Quantitative real-time PCR. The roots, stems, leaves, flowers, and siliques of five-week-old plant were harvested for RNA isolation using TRIzol reagent (Invitrogen, Grand Island, NY). 2 μg RNA was digested with DNase and reverse-transcribed by Superscript III reverse transcriptase (Invitrogen) to generate first-strand cDNA in a 20 μl reaction volume. Quantitative RT-PCR was performed in 96-well plates with 1 μl of a 1:5 dilution of the first-strand cDNA reaction and in a 10 μl volume on a LightCycler 96 Sequence Detection System (Roche). Transcript levels were measured based on SYBR Green technology using SYBR Green reagent (Applied Biosystems Applera, Darmstadt, Germany) according to the manufacturer's instructions. Data were analyzed using the ΔΔC T method. The qPCR results were analyzed using LightCycler 96 analysis software 1.1 (Roche). The Arabidopsis ACTIN2 gene was used as a control. Primer sequences are shown in Supplementary  Table S2. Subcellular localization of OBP4. OBP4 sequences were cloned into PBI121 vector to construct fusion plasmid by using primers containing BamH1 and Sal1 sites. PBI121 plasmid was used as a control. The fusion construct and the control plasmid were transformed into Arabidopsis and tobacco leaf epidermal cells. Transformed cells were observed under a OLYMPUS FV1000 microscope.
Culture and synchronization of Arabidopsis suspension cell. Col Arabidopsis Seeds were surface-sterilized and vernalized. Callus formation was induced under continuous darkness at 23 °C by placing root explants excised from 2-week-old seedlings onto callus induction medium. The rapidly dividing callus was inoculated into MS medium containing 3% w/v sucrose, 0.5 mg/L 2,4-D, 0.05 mg/L kinetin to establish suspension cultures,, and incubated on a rotary shaker at 110 rpm. The method to achieve synchronization was as previous described 62 .
Flow Cytometry of Cell Cycle Progression. WT, pER8::OBP4 and pER8::OBP4::VP16 transgenic Arabidopsis seeds were sterilized with 0.15% mercuric chloride and germinated on a plate. After induction with estradiol, nuclears were isolated from Arabidopsis leaves at different time points 55 . The DNA content of individual transgenic cells was determined by flow cytometry. Each sample was prepared three times and subjected to Beckman Coulter MoFlo XDP. A total of 10,000 nuclei were measured per analysis.
Scientific RepoRts | 6:27705 | DOI: 10.1038/srep27705 ChIP assays. An HA-coding sequence was fused in frame to the end of the OBP4 gene, and the gene fusion was sub-cloned under the estradiol-inducible promoter in the pER8 vector. The expression construct was then transformed into Col-0 plants. Two-week-old pER8::OBP4::HA transgenic plants that were grown on MS agar plates that expressed OBP4::HA via induction by estradiol for a period were used to conduct ChIP experiments according to a previously described method using anti-HA polyclonal antibodies (Roche:118674231) 64 . The qRT-PCR primers used are listed in Supplemental Table 2.
For scanning electron microscopy. The leaves and hypocotyls were cut and sputter-coated with gold and further visualized by using a Hitachi JEOL JSM-6360LV SEM (scanning electron microscope).
Microscopy and GUS staining. After removal of chlorophyll using 100% ethanol, epidermal cells were observed under a Olympus light microscope (Tokyo, Japan) and the size of cells were analyzed by cell^P Software, about thirty cells were observed of each genotype. β-Glucuronidase activity was determined histochemically as described 20 and ten lines of transgenic plants were observed.

Results
OBP4 is a DOF transcription factor family protein. The OBP1 protein was previously isolated 47,61 .
Other members of the OBP family were identified using the sequence of the OBP1 DOF domain to screen an Arabidopsis cDNA library. OBP4 is a previously uncharacterized, new member of the DOF family of proteins in Arabidopsis. The amino acid sequences of the OBPs (OBP1, OBP2, OBP3, and OBP4) are shown in Fig. S1. The OBP4 protein has 308 amino acids, and its DOF domain is located at the N terminus (boxed) 32,38 . The homology among the OBP proteins is restricted to the DOF domain. The specific characteristics of the OBPs are indicated in Fig. S1. In OBP1 and OBP2, a serine-rich domain is observed, while in OBP2, an asparagine-rich domain is present. We further compared the OBPs to DOF proteins from other species in the database. We did not identify any significant homology between the OBP proteins and other DOF members other than the DOF domain. As shown in Fig. 1A, OBP4 is closely related to the Brassica napus BnaC02g41820D protein.
OBP1 was first isolated through interactions with OBF proteins by an ocs element 61 . Ocs elements respond to salicylic acid (SA), and OBP1-3 expression has been reported to be induced by SA. Furthermore, we assessed whether OBP4 is affected by hormone treatment. After IAA and SA treatment, OBP4 expression in the leaves increased substantially at 3 h and remained high at 24 h (Fig. 1B,E), while after 8 h of GA treatment, OBP4 expression increased only slightly (Fig. 1D).

OBP4 gene expression and OBP4 protein localization. To investigate the expression pattern of OBP4
during development, qPCR was used to examine the OBP4 mRNA levels in different plant organs, including seedlings, roots, shoots, flowers, rosette leaves, cauline leaves, buds and siliques. Our results showed that OBP4 was ubiquitously expressed at different levels in all of the examined organs ( Fig. 2A). Then, approximately 2.0 kb of the 5′-UTR promoter region of OBP4 was fused to the β-glucuronidase (GUS) reporter gene to investigate OBP4 expression at the tissue level. The GUS signal was observed in the silique, trichomes, flower organs and leaves ( Fig. 2F-H). GUS was also detected in the seedlings, hypocotyls and roots ( Fig. 2B-E). Furthermore, the subcellular localization of the OBP4 protein was analyzed. Figure 2I shows that the GFP control protein was observed in both the nuclei and the cytoplasm, whereas the 35 S::OBP4::GFP protein was localized in the nuclei when transiently expressed in tobacco epidermal cells. The same results were observed in transgenic roots stably expressing OBP4::GFP. These results indicate that OBP4 is a nuclear protein.

Constitutive and inducible overexpression of OBP4 alters cell proliferation and results in a dwarf phenotype.
To elucidate the mechanisms underlying the effects of OBP4 on cell cycle progression and cell expansion, which influence plant development, we performed a phenotypic analysis of plants that were constitutively overexpressing OBP4. OBP4 transgenic lines were screened and eight transgenic lines overexpressing OBP4 were obtained, with the highest expression level approximately 32-fold to that of the WT at mRNA level (Fig. S2). We further attempted to increase the OBP4 expression in transgenic Arabidopsis without success. Constitutive OBP4 overexpression resulted in obvious defects in the plant development. Mature 35S::OBP4 plants had a greatly reduced plant size with small and curly rosette leaves (Fig. 3A,B). To investigate the reason for the arrested plant development, leaf cells and hypocotyl cells were counted and measured. In the one-week-old 35S::OBP4 seedling hypocotyls and the fourth leaves from three-week-old 35S::OBP4 plants, the cell numbers were decreased, and epidermal cell size was also reduced compared to that of the wild-type plants ( Fig. 3C-F,O,P). The petals were shorter, the siliques produced were shorter and fewer, and seed setting did not occur ( Fig. 3G-L). Furthermore, transgenic plants were characterized by shorter plant height and reduced flower number (Fig. 3M,N).
We also observed a similar seedling phenotype by inducing OBP4 expression in pER8::OBP4 transgenic plants. pER8::OBP4 plants treated with 20 μM estradiol were smaller than WT and showed growth arrest 4 days after induction in dark ( Fig. 3Q,R,S). At the cellular level, the hypocotyl cells of pER8::OBP4 plants were smaller than those of WT (Fig. 3T). The situation was similar for the 35S::OBP4 plants which were smaller than WT. Unfortunately, OBP4 loss-of-function mutant was not available in the public seed collections and RNAi approaches were unsuccessful. To convert OBP4 into a transcriptional activator, we fused it with the strong activation domain VP16. The resulting OBP4::VP16 causes strong transcriptional activation. Therefore, we transformed pER8::OBP4::VP16 as a potential negative OBP4 allele into plants. Further we obtained pER8::OBP4::VP16 transgenic plants, and after four days of estradiol induction, the leaves were obviously larger than those of the control (Fig. S4). These results demonstrated that OBP4 can regulate plant development. OBP4 negatively regulates G2-to-M phase transition and promotes endocycle onset. We hypothesized that G2-to-M phase transition is defective in pER8::OBP4 plants. To investigate the mechanism underlying OBP4 regulation of G2-to-M phase transition, we determined whether the transgenic plant cells showed defects during cell cycle progression, particularly G2-to-M phase transition, such as G2 phase arrest 2,39 and early endocycle entry 49 . Overexpression of OBP4 affected cell proliferation in suspension cells (Fig. S5). To assess whether G2-to-M phase transition is defective in pER8::OBP4 plants, we measured the DNA content in pER8::OBP4 plant cells by flow cytometry. Before induction, at 7 days after germination (DAG), the ploidy level distribution was similar to that of wild-type (Fig. 4D,H); Nuclei isolated from both WT and pER8::OBP4 seedlings displayed four primary peaks that corresponded to 2 C, 4 C, 8 C, and 16 C DNA content. After 1-day induction with estradiol, pER8::OBP4 plants showed an increased proportion of 8 C cells and a decreased proportion of 2 C and 4 C cells (Fig. 4E,I). After 4-day induction, the proportion of 8 C cells greatly increased in the pER8::OBP4 plants, and the proportion of 4 C and 2 C cells further decreased. Additionally, 32 C cells appeared in the β-estradiol-induced plants (Fig. 4F,J). These results suggest that more pER8::OBP4 plant cells escaped from the cell cycle compared to control plants and that early endocycle onset occurred.
We further measured the DNA content in pER8::OBP4::VP16 transgenic plant cells by flow cytometry. Before induction, at 9 days after germination (DAG), the ploidy level distribution was similar to that of the control. After the plants were induced for 4 days, pER8::OBP4::VP16 transgenic plants had a decreased proportion of 8 C and 32 C cells. In contrast, the vector control and the wild-type plants showed similar DNA content before and after estradiol treatment (Fig. S4 D-G). Thus, Delayed endocycle onset was observed in the OBP4::VP16 plants.
We further identified genes that were immediately expressed upon activation of OBP4. The estradiol induction system is successful at inducing plant transcription factors 47 . We generated a homozygous transgenic plant (pER8:: Pro OBP4::OBP4) with the OBP4 promoter driving a fusion of OBP4 cDNA under the β-estradiol-inducible promoter in the Col-0 background. The transgenic plant was similar to wild-type plants under normal conditions. In contrast, estradiol spray clearly inhibited leaf development; upward curling leaves were observed (Fig. 6A). Similar to stimulation of steroid receptors in animals, estradiol treatment led to transport of the OBP4 protein from the cytoplasm to the nucleus, resulting in downstream events. Furthermore, we performed qPCR analysis using RNA samples within 20 h of induction. Figure 6B indicates that the 16 isolated genes were clearly down-regulated after estradiol treatment for 20 h. The large number of expression changes at 20 h after estradiol induction suggests that secondary targets of OBP4 might exist. Genes with reduced expression were further examined for their induction kinetics. We profiled the selected genes at 3 h and 8 h after induction to identify direct targets. Among these genes, we identified 5 genes that were consistently down-regulated at 3 h and 8 h after induction: two core cell cycle proteins (CYCB1;1 and CDKB1;1) and three cell wall expansion factors (XTH3, XTH9, and XTH17). Moreover, their expression levels were higher in the OBP4::VP16 transgenic plants (Fig. S3). These results show that CYCB1;1, CDKB1;1, XTH3, XTH9, and XTH17 are putative genes immediately downstream of OBP4.
The OBP4 transcription factor belongs to the DOF subfamily, whose predicted DNA binding sites are [A/T] AAAG and CTTT[A/T] [57][58][59] . Based on these DNA sequences, we found many DOF binding sites in the promoter regions of the CYCB1;1, CDKB1;1, XTH3, XTH9, and XTH17 genes (Fig. 7A). To verify whether OBP4 can bind to these consensus sequences in planta, we performed ChIP analysis to test the relative enrichment of OBP4 in the selected fragments. An antibody directed against HA was used to precipitate OBP4::HA and its crosslinked DNA, which was isolated from 2-week-old pER8::OBP4::HA seedlings after estradiol induction for 36 h, and pER8::HA seedlings without induction served as a negative control. Immunoprecipitated DNA crosslinked with OBP4::HA was analyzed by real-time PCR using the gene-specific primers listed in Supplemental Table 2. OBP4 protein enrichment was indeed detected at the promoter regions of the CYCB1;1, CDKB1;1, XTH9, and XTH17 genes containing the predicted DOF binding sites (Fig. 7B). In sum, we conclude that OBP4 can transcriptionally regulate both cell cycle progression and cell expansion by regulating key cell cycle genes and cell expansion factors (Fig. 8).

Discussion
In most eukaryotic cells, cell cycle progression is an ordered series of events during which the chromosomes are duplicated and one copy of each duplicated chromosome segregates into each of the two daughter cells. Cell cycle regulation is critical for the normal development of multicellular organisms. The cell cycle is controlled by numerous mechanisms that ensure correct cell division, i.e., regulation of cyclin-dependent kinases (CDKs) by cyclins, CDK inhibitors and phosphorylation 50 . Fundamentally, the molecular mechanisms controlling the primary events in cell cycle progression are similar to those in eukaryotic cells. Thus, research on diverse organisms has contributed to the growing understanding of how these events are coordinated and controlled in plants 10 . However, the plant cell has a cell wall, and the mechanisms of plant cell proliferation that are coordinated with cell wall expansion and contribute to plant growth remain unclear. A number of DOF proteins have been reported to participate in plant development and to respond to environmental stimuli. Previous results have shown that OBP1 regulates cell cycle re-entry by targeting CYCD3;3 and DOF2.3 47 . Here, we report that another plant-specific OBP family protein, OBP4, is a negative regulator of cell division and plant growth. This study demonstrates the OBP4-mediated inhibition of plant growth via defective cell cycle progression and cell wall expansion.
OBP4 is a positive regulator of the endocycle during plant development. To characterize the function of OBP4, we examined transgenic plants with up-regulated or reduced OBP4 activity. The 35S constitutive and inducible overexpression transgenic plants presented obvious growth defects (Fig. 3A,B). While overexpression of OBP4 fused to an activation domain promoted plant growth (Fig. S3). OBP4 was expressed in all of the examined organs but at different levels ( Fig. 2B-H). The OBP4 expression data from qPCR analysis were consistent with the GUS staining results (Fig. 2A). The timing and localization of OBP4 expression suggest that OBP4 has a role in cell cycle regulation. In inducible OBP4 transgenic plants, the cell size and other characteristics related to cell division were inhibited. OBP4 overexpression caused defective G2-to-M phase transition. Furthermore, we hypothesized that OBP4 plays a role in the onset of the endocycle 35 . Indeed, OBP4 activation resulted in a longer G2 phase and early endoreduplication onset (Fig. 4). These findings support the conclusion that endoreplication entry involves the engagement of B-type cyclins and cyclin-dependent kinases 19 . Cell proliferation was negatively controlled by OBP4 activation, and subsequently, the rate of cell proliferation was lower than that of the control, eventually resulting in fewer and smaller cells. The expression pattern of OBP4 together with its effect on cell cycle regulators, is a novel mechanism by which this protein controls cell proliferation.
OBP4 inhibits cell expansion by negatively regulating cell wall expansion factors. In contrast to animal cells, plant cells have cell walls. Cell morphogenesis involves the spatially controlled remodeling of the cell wall. Cell expansion is believed to be controlled by a balance between the drive for expansion and localized wall loosening 43 . There are two types of proteins that are involved in this process. Xyloglucan endotransglycosylases cleave the glycan backbone and transfer the cut end to an acceptor molecule. Expansins appear to loosen hydrogen bonds between cellulose microfibrils and cellulose fibers 11,26 . XTH9 may be associated with cell expansion in Arabidopsis 18 . A T-DNA insertion mutant of XTH17 showed low XET activity and had moderately shorter roots than wild-type plants 66 . Plants overexpressing AtEXP10 have large organs with enlarged cells, supporting the role of expansion in cell enlargement. Arabidopsis has 26 expansin genes, and more than 5 expansin genes may act redundantly to influence plant growth 8 . To determine how OBP4 affects cell expansion, we examined the expression levels of several characterized Arabidopsis genes involved in cell expansion. The expression levels of the expansion genes AtEXPA3, AtEXPA5, AtEXPA8, AtEXPA9 and AtEXPB3 were significantly lower in 35S::OBP4 plants than in wild-type plants. AtEXP3 has been proposed to promote cell expansion 24 , and the atexp5 mutant has smaller leaves than wild-type 40 . These results suggest that reduced cell size in OBP4 overexpression lines might result from decreased expression of particular expansion genes. OBP4 may negatively regulate cell wall expansion factors to modify cell wall plasticity during the cell growth stage.
The well-accepted "compensated cell enlargement theory" indicates that a reduced cell number could be compensated by increased cell size and vice versa 50 . Constitutive and inducible activation of OBP4 in plants resulted in a significant reduction in both cell size and cell number. The leaves of 35S::OBP4 plants were characterized by fewer cells and a reduced cell size, which could not be explained by the compensation theory and which might suggest that OBP4 targets not only cell proliferation but also cell expansion. In contrast, the reduced cell size correlated well with the reduced level of the endocycle (fewer cells entering the endocycle), which has often been reported previously. To identify possible molecular mechanisms that could explain the changes in cell size, we examined the expression of genes encoding growth-related proteins among transcripts that were down-regulated in our profiling data and identified a large number of genes encoding cell wall-loosening enzymes, which are essential for cell growth and expansion 30 . Therefore, OBP4 can also affect growth by targeting the expression of genes encoding cell wall loosening enzymes. In conclusion, OBP4, as a DOF family transcription factor, can modify the onset of the endocycle and cell growth during plant development. This characterization of the function of OBP4 extends our understanding of the molecular mechanisms underlying cell cycle progression and cell expansion.