[Frontiers in Bioscience E2, 752-763, January 1, 2010]
AtTFC B is involved in control of cell division
Yunlong Du1, Meiqiang Cui1, Dan Qian1, Lei Zhu3, Chunhong Wei1, Ming Yuan3, Zhongkai Zhang4, Yi Li1, 2
1
TABLE OF CONTENTS
1. ABSTRACT
Tubulin-folding cofactors play important roles in regulating plant development. Arabidopsis tubulin-folding cofactor B (AtTFC B) is an Arabidopsis homolog of mammalian tubulin-folding cofactor B, whose biological function in plant development remains poorly understood. Here we report that the homozygous attfc b (-/-) allele caused embryonic lethality. Embryogenesis was arrested at early embryo stage and the cells contained one or multiple nuclei. Plants carrying a heterozygous attfc b (+/-) allele exhibited enlarged mesophyll cells and leaf epidermal cells with bulged nuclei. Flow cytometry analysis showed increased ploidy in the leaves of the attfc b (+/-) mutant, as well as increased levels of Cdc2A and CycB1;1. In addition, immunofluorescence assay showed increased numbers of spindles and phragmoplasts in the attfc b (+/-) mutant. These results suggest that AtTFC B plays an important role in plant cell division.
2. INTRODUCTION
Microtubules control fundamental cellular processes, such as intracellular transport, cell division, cell expansion, cell polarity, and chromosome segregation. During cell cycle, plant cells display four kinds of microtubule arrays: the interphase cortical microtubule array, the preprophase band (PPB), the mitotic spindle, and the phragmoplast (1). Microtubules contain highly conserved alpha/beta-tubulin heterodimers, and different proteins are involved in the tubulin biogenesis, such as phosducin-like proteins required for beta-tubulin biogenesis (2, 3). The biogenesis of tubulin is a symmetrical process. First, the nascent alpha- and beta-polypeptide chains are captured and partially folded by prefoldin and chaperonins (4, 5). After release from the prefoldin and chaperonins, the partially folded alpha- and beta-tubulin chains are captured by tubulin-folding cofactors B and A, respectively. These cofactors are subsequently replaced by cofactors E and D, respectively, and form a quaternary complex containing alpha-tubulin/E and beta-tubulin/D. When cofactor C binds to the quaternary complex, the cofactors C, D, and E act as a GTPase-activating protein (GAP), triggering hydrolysis of GTP by beta-tubulin to release alpha/beta-tubulin heterodimers (6, 7).
Tubulin-folding cofactors not only regulate the folding of tubulin heterodimers, but are also involved in the regulation of cell division associated with microtubule function. In fission yeast, the alp1 and sto1 alleles, which encode the homologs of cofactor D and E, respectively, disturb the microtubule structure leading to condensed chromosomes or unequal segregation and mitotic division defects (8-11). In Arabidopsis, KIESEL (KIS) and PORCINO (POR), the orthologs of cofactor A and C, respectively, are required for the synthesis of microtubules; mutations in these two genes cause cell division defects (12, 13). Mutation in TITAN1 (TTN1), the ortholog of cofactor D, results in cell division defect attributable to the disruption of microtubule function and endosperm with condensed chromosomes (14, 15). The PILZ group genes include POR, CHO, and PFIFFERLING (PFI), which encode the Arabidopsis orthologs of tubulin-folding cofactors C, D, and E, respectively (16). In pilz mutants, embryos show an absence of microtubule arrays and condensed chromosomes (16). Although mitotic division and cytokinesis are disturbed, the process of cell cycle is not affected in the pilz mutants (16, 17).
The function of tubulin-folding cofactor B is dependent on microtubules. In most cases, mammalian cofactor B is considered as a reservoir to sequester and deliver the alpha-tubulin folding intermediate to cofactor E (18). However, cofactor B is also important to microtubule polymerization in mammalian cells. Overexpression of cofactor B can depolymerize microtubules (19-22). Mutation in Alp11, a fission yeast homolog of cofactor B, destroyed microtubule structure and delayed mitosis with condensed chromosomes (23, 24), and is also essential for cell viability in fission yeast (23, 24). Overexpression of Alf1, a budding yeast ortholog of mammalian cofactor B, caused depolymerization of microtubules; however, Alf1 is not essential for cell survival in budding yeast (25). Different roles of cofactor B in different species suggest a complexity of its function. AtTFC B (AT3G10220) is the Arabidopsis homolog of mammalian cofactor B (17), and it can interact with alpha-tubulin in vivo. Overexpression of AtTFC B in cowpea protoplasts reduced the number of microtubules (26). However, the function of AtTFC B in plant development remains unknown.
Here, we provide evidence that AtTFC B is essential for embryogenesis in Arabidopsis. Cellular and molecular data indicate that AtTFC B is also involved in the regulation of post-embryonic development. Our data suggest that AtTFC B is required for cell division.
3. MATERIALS AND METHODS
3.1. Plant growth, mutant screening, and plant transformation
All Arabidopsis materials used in this study are in the Columbia (Col) background. The T-DNA insertion lines attfc b (+/-) (SALK-019471) and attfc b-2 (SALK-002231) were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH). Seeds were surface-sterilized with 10% sodium hypochlorite, sown on Murashige and Skoog medium (27) supplemented with 0.8% agar and 3% sucrose, and cold-treated for one day at 4° C. Seedlings were then grown in the medium under a 16 h light photoperiod at 21° C. Adult plants were grown in environmental chambers under a 16 h photoperiod at 21° C.
We genotyped the attfc b (+/-) mutant plants by PCR using the following specific primers, LBa1 (5'-T G G T T C A C G T A G T G G G C C A T C G-3'), BLP (5'-T G G C T T A C A T T T G C A G G G T G A A-3'), and BRP (5'-G G T C T T C G G G T T G T C G C T C T T-3') designed from SALK (http://signal.salk.edu/tdnaprimers.2.html).
To generate transgenic plants ectopically expressing AtTFC B, we cloned the coding sequence of AtTFC B into the modified plasmid pCambia3301 under the control of CaMV 35S promoter, and transformed the construct into wild-type and attfc b (+/-) mutant plants using the floral dip method (28).
3.2. Cloning of AtTFC B coding sequence
Total RNA from wild-type Arabidopsis was extracted with RNeasy® Plant Mini kit (QIAGEN, Valencia CA). To obtain the first strand cDNA of the AtTFC B (AT3G10220) gene, total mRNA was used as a template for reverse transcription reaction with oligo-dT18 and SuperScript IITM RNase H-Reverse Transcriptase (Invitrogen, CA). The primers BFP (5'-C A C C C A T A T G A T G G C A A C T T C G C G T T T A C A G T T G-3') and BRP2 (5'-C G C G G A T C C T T A T A T T T C A T C T T C C T C G A A A G G-3') were used for RT-PCR. The purified PCR products were cloned into pENTR/D vector (Invitrogen, CA) following the manufacturer's instructions and confirmed by DNA sequencing.
3.3. Quantitative real-time PCR
To determine the expression levels of AtTFC B, Cdc2A, and CycB1;1, total RNA was isolated from different tissues using the RNA plant kit (Tiangen Biotech, Beijing). The RNA was quantified three times for each sample. To produce the first strand cDNA, 2 m g of total RNA pretreated with DNAase I (Promega, Madison, WI) was used as a template in reverse transcription reaction. At the end of the reaction, the cDNA was diluted with sterile distilled water. Real-time PCR was performed using the SYBR green-I qPCR kit (Finnzymes Oy), Fifty nanograms of the diluted cDNA and 0.75 pM gene-specific primers BFP450 (5'-C T A T A T G G A A G A T C T C T G C G C-3') and BRP732 (5'-T T A T A T T T C A T C T T C C T C G A A A G G-3'), Cdc2A FP (5'-A T G G G A A C T C C G T A C G A G G A T A C -3') and Cdc2A RP (5'-C T A A G G C A T G C C T C C A A G A T C C T T G-3'), CycB1;1 FP (5'-C T C A A G C A T C A C A C T G G C T A T T C-3') and CycB1;1 RP (5'-C T A A G C A G A T T C A G T T C C G G T C A A C-3') were used to amplify the AtTFC B, Cdc2A and CycB1;1, respectively, using the DNA Engine OpticonTM 2 machine (Bio-Rad, CA, USA). The Ubiquitin10 gene served as an internal control using specific primers UBQ10FP (5'-G A T C T T T G C C G G A A A A C A A T T G G A G G A T G G T-3') and UBQ10RP (5'-C G A C T T G T C A T T A G A A A G A A A G A G A T A A C A G G-3'). PCR conditions were as follows: 5 min at 94° C for the first denaturation step, 40 cycles followed with 10 s at 94° C, 20 s at 60° C, and 20 s at 72° C. Fluorescence of the PCR products was measured twice at the end of the extension step at 72° C and 84° C. Relative transcript levels of AtTFC B, Cdc2A, and CycB1;1 were calculated using the following equation: RE=2-D D Ct. For wild-type and attfc b (+/-) mutant plants, samples from three different individuals were measured.
3.4. Examination of ovules
Siliques at different developmental stages were harvested and dissected in Hoyer's solution (7.5 g of arabic gum, 100 g of trichloroacetaldehyde hydrate, and 5 mL of glycerol diluted with water to 100 mL) under a stereomicroscope. The ovules were examined immediately under a Zeiss Axioskop2 plus Microscope and digital images were acquired using Carl Zeiss imaging systems (Carl Zeiss, Jena, Germany).
3.5. Leaf morphology and structure
Leaf epidermal cells were examined with a Field Emission Environmental Scanning Electron Microscopy (model QUANTA 200F, FEI, OR). To study the mesophyll structure, leaves were fixed overnight in a solution of 3% glutaraldehyde (Sigma-Aldrich, St. Louis, MO), washed, dehydrated in a series of ethanol, and embedded in LR White resin (London Resin Co., London, United Kingdom). Five micrometer-thick sections were obtained with a glass knife on a microtome (Model 1512, Leitz), mounted onto slides, and stained with 0.1% (w/v) toluidine blue-O. The slides were examined with a Leica microscope (Type 020-525.025, Leica Microsystems, Wetzlar, Germany) and photographed with KX Series Imaging System (Model KX32E, Apogee Instruments Inc., Roseville, CA). The nuclei of leaf epidermal cells were detected as previously described by Boudolf and his colleagues (29). The nuclei and cell sizes of leaf epidermal cells and mesophyll cells were measured using Image J 1.36b software (NIH, Bethesda, MD).
3.6. Analysis of microtubule
Microtubule arrays were detected in roots based on previously described (30). The root tips were dissected from seven-day-old seedlings grown under light conditions and fixed in 4% (v/v) paraformaldehyde. For immunolabeling, the root samples were first incubated with a mouse anti-alpha-tubulin monoclonal antibody (Sigma-Aldrich) at a 1:200 dilution, washed and then incubated with FITC-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) at a 1:500 dilution. The samples were examined under a Leica fluorescence microscope (Type 020-525.025, Leica Microsystems, Wetzlar, Germany).
3.7. Flow cytometry
The third leaf was chopped with a razor blade in 0.5 mL of cold Galbraith buffer (31). The extracts were filtered through a 60 m m pre-wet nylon mesh and collected in a 1.5 mL eppendorf tube. The filtrate was incubated with 10 m L of 10 mg/mL RNaseA for 20 min at 37° C, and then stained with 2 m L of 5 mg/mL propidium iodide (Sigma-Aldrich) and gently dispersed the nuclei. The sample was kept at 4° C in dark conditions for 10 min. Approximately 3500-5500 nuclei were analyzed with FACS Calibur flow cytometer, and DNA histograms and proportions were generated using Cellquest software (Becton, Dickinson and Company, Franklin Lakes, NJ).
3.8. Statistical analyses
Data were analyzed using student t-test for statistical significance and all images were processed with PhotoshopTM 7.0.1 (Adobe Systems Inc., San Jose, CA).
4. RESULTS
4.1. The homozygous attfc b (-/-) allele caused embryonic lethality
To understand the function of AtTFC B in plant development, a T-DNA insertion line (SALK-019471) was obtained from ABRC (Figure 1A). The T-DNA was inserted at position +1846 of the 3' region of AtTFC B genomic sequence (Figure 1B). To test if this insertion affects transcription of AtTFC B gene, the level of AtTFC B mRNA was determined using quantitative real-time PCR. As shown in Figure 1C, AtTFC B mRNA expression was reduced in the roots, stems, leaves and siliques in the T-DNA insertion line.
In screening for attfc b mutant plants, only heterozygous mutant plants were obtained in this T-DNA insertion line (Figure 1D). Progeny analysis using the X2 test for heterozygous attfc b (+/-) plants showed a segregation ratio of 1:2 for wild-type (n = 188) to heterozygous plants (n = 321), suggesting the potential embryo lethality of the homozygous attfc b (-/-) plants. To test this hypothesis, we examined the phenotypes of the embryos using differential interference contrast microscope. In contrast to the wild-type embryos (Figures 2A, 2C, 2E and 2G), approximately 20% of mutant embryos were arrested at early two or four cells developmental stage, carrying multiple nuclei (Figures 2B, 2D, 2F and 2H). Given the possibility that some embryos may have stopped development or degenerated at an earlier stage, the percentage of defective embryos was close to what was expected for attfc b (-/-) genotype.
4.2. Plants carrying a heterozygous attfc b (+/-) allele had enlarged cell size
To test whether AtTFC B plays a role in plant growth beyond embryogenesis, we examined the phenotypes of heterozygous attfc b (+/-) plants. Heterozygous plants had different phenotypes from the wild-type: the plants were taller with bigger rosettes (Figures 3A, 3B), and the third true leaf was significantly longer and wider (Figure 3C) (Table 1). Consistent with enlarged leaf, the mesophyll cells in attfc b (+/-) mutant were significantly larger than those in the wild-type (Figures 3D, 3E; Table 1). We found that the leaf epidermal cells of attfc b (+/-) mutant were also larger than those in the wild-type (Figures 4A, 4B). The axial length in the mutant (52.2 m m) was significantly longer than that of the wild-type (44.8 m m) (Table 1), and the leaf epidermal cells contained enlarged nuclei (Figure 4D, Table 1).
To confirm that mutations in AtTFC B caused leaf morphological defects, we analyzed the phenotype of another attfc b mutant (SALK-002231) named attfc b-2. The attfc b-2 mutant also exhibited enlarged leaf sizes (Figure 5). These results indicate that AtTFC B play an important role in post-embryonic growth in Arabidopsis.
4.3. Overexpression of AtTFC B restored the attfc b (+/-) phenotype and inhibited growth in the wild-type background
We further tested if the expression of AtTFC B in attfc b (+/-) mutant background would rescue the attfc b mutant phenotype. We expressed the AtTFC B coding sequence (CDS) under the control of CaMV 35S promoter (Figure 6A). Phenotypic analysis showed that the expression of AtTFC B restored attfc b (+/-) phenotype to the wild-type phenotype (Figures 6B, 6C, Table 2).
The phenotypes of attfc b (+/-) plants suggested that AtTFC B functions to inhibit cell and organ growth in post-embryonic development. In this scenario, we would expect that overexpression of AtTFC B in the wild-type background would lead to inhibition of plant growth. Indeed, overexpression of AtTFC B in the wild-type background displayed reduction in both rosette size and leaf size (Figures 6B, 6D, Table 2).
4.4. Mutation in AtTFC B altered ploidy levels and mRNA levels of Cyclin B1;1 and Cdc2A
Homozygous attfc b (-/-) embryos displayed impaired nuclear division (Figures 2B, 2D, 2F, 2H) and the heterozygous attfc b (+/-) plants showed enlarged nuclei in leaf epidermal cells (Figure 4D). To further test the karyokinesis defect in the attfc b mutant, we measured DNA ploidy levels in the leaves of 21-day-old attfc b (+/-) mutants, AtTFC B-overexpressing plants and wild-type plants leaves using flow cytometry analysis. In contrast to the 1.6-fold increase observed in the ratio of 4C leaf cells to 2C leaf cells in wild-type (2C = 34.7% and 4C = 54.7%), there was an approximately 3-fold increase in the attfc b (+/-) mutant (2C = 15.1% and 4C = 45.4%) (Figures 7A, 7B, Table 3). Furthermore, mutation in AtTFC B increased the polyploidy levels. The fraction of 8C and 16C cells significantly increased in the attfc b (+/-) mutant leaf cells (Figure 7B, Table 3). It also had a small fraction of 32C cells (0.13%) that was absent from the wild-type leaf cells (Figures 7A, 7B, Table 3). However, the fraction of 8C and 16C cells were slightly decreased in the AtTFC B-overexpressing plants compared with the wild-type (Figure 7C, Table 3).
In the pilz group mutants, GUS staining of embryos revealed the expression of Cdc2A and CycB1;1 (16). To determine if these two genes were affected in attfc b (+/-) mutant and AtTFC B-overexpressing plants, we analyzed the levels of CyclinB1;1 and Cdc2A mRNA. Consistent with the ploidy level, the levels of Cdc2A and CycB1;1 mRNA were upregulated in attfc b (+/-) mutant and reduced in AtTFC B transgenic plants (Figure 8).
4.5. Microtubule arrays were affected in the attfc b (+/-) mutant
We further tested if microtubule arrays were affected in the attfc b (+/-) mutant. We performed immunostaining for alpha-tubulin in root tip cells of attfc b (+/-) mutant and the wild-type. As shown in Table 4, the percentage of spindle and phragmoplasts in attfc b (+/-) mutant increased by 30% and 77%, respectively, relative to the wild-type. The PPB was slightly increased (2.5%) and the cortical microtubule arrays were reduced (1.5%) in the attfc b (+/-) mutant compared with those in wild-type plants (Table 4). These data suggested that cytokinesis might be defective in the attfc b (+/-) mutant.
5. DISCUSSION
Tubulin-folding cofactors are involved in the control of cell division. In this study, we showed that AtTFC B, the homolog of mammalian cofactor B, is required for cell division in Arabidopsis development. Specifically, embryos of homozygous attfc b (-/-) plants were arrested at early developmental stage and cells have multiple nuclei. In the PILZ group genes, mutant embryos contain varying numbers of nuclei and lack microtubules such as the spindle and phragmoplast (16, 17). Mutation in the EDE1 gene caused defective endosperm, which was consistent with the absence of mitotic spindles and phragmoplasts (32). In the attfc b (+/-) mutant, the number of spindles and phragmoplasts was increased in root cells (Table 4). Given the direct role of the phragmoplast in the formation of new cell plate during cytokinesis (33), the possibility that the cytokinesis arrest resulted from the phragmoplast defect in the attfc b (-/-) mutant and caused improper cell division can not be excluded.
Besides cell division defect in embryogenesis, the heterozygous attfc b (+/-) mutant caused larger leaf epidermal cells (Figure 4B, Table 1), while AtTFC B-overexpressing plants were smaller in leaf size (Table 2). The distinct leaf phenotypes between attfc b (+/-) mutant and AtTFC B-overexpressing plants, suggest that AtTFC B functions as a negative regulator of cell division, at least in the post-embryonic organs.
Cell division is tightly related to cell cycle. Although function of tubulin-folding cofactors could affect cell division, the roles of tubulin-folding cofactors in cell cycle are different. Mammalian cells transfected with cofactor A siRNA were arrested at G1 phase (34), and overexpression of Alf1, the budding yeast homolog of cofactor B, arrested cell cycle causing defects in microtubule function (25). However, in pilz group mutant plants, cell cycle was not arrested even when mitotic division was arrested (16). In this study, the formation of PPB was still present in the root meristem cells (Table 4), indicating that G2-to-M transition point of the cell cycle was not arrested in attfc b (+/-) mutant. However, the number of phragmoplasts was obviously increased in attfc b (+/-) mutant (Table 4), suggesting that cell cycle might be arrested at the mitotic telophase, which resulted in cytokinesis defect.
Mutation of AtTFC B showed enlarged nuclei in leaf epidermal cells and was consistent with higher ploidy levels in the leaves (Figures 4D, 7B). A previous study showed that enhanced levels of a non-destructible Cyclin B1 (also named CycB1;1) leads to doubled DNA content resulting from endomitosis. Weingartner and his colleagues concluded that the mitotic cyclins were required for reorganization of the phragmoplast and for proper cytokinesis (35). In this study, mutation in AtTFC B upregulated levels of Cdc2A and CycB1;1 (Figure 8), as well as the number of phragmoplasts (Table 4). In contrast, overexpression of AtTFC B decreased the levels of Cdc2A and CycB1;1 (Figure 8) and the polyploidy levels (Figure 7C). Thus, endomitosis might have occurred in attfc b (+/-) mutants when cytokinesis was disturbed. Mutation in AtTFC B also increased the number of spindles (Table 4), and increased mRNA levels of Cdc2A (Figure 8A). Cdc2 activity is required for microtubule recruitment. It is located at the nucleus and is associated with spindles and phragmoplasts during mitosis and cytokinesis (36). Taking these phenotypes into account, our data suggest that AtTFC B plays a critical role in ensuring a normal cell division process, and AtTFC B mutation might promote cells to enter into the process of endomitosis when cell division is arrested.
6. ACKNOWLEDGEMENTS
This project was supported by grants from the National Science Foundation of China and 985 programs to Yi. Li. We thank Professor Liying Du (Peking University) for assistance with the flow cytometry analysis. We thank Dr. Biao Ding (Ohio State University), Dr. Zhenbiao Yang (University of California, Riverside), Dr. Gang Wu (University of Pennsylvania) and Dr. Lieven De Veylder (Ghent University) for their critical reading and suggestions. We thank the Arabidopsis Biological Resource Center (Columbus, Ohio) for providing the attfc b (+/-) (SALK-019471) and attfc b-2 (SALK-002231) mutant plants.
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Abbreviations: AtTFC B: Arabidopsis tubulin-folding cofactor B; PPB: preprophase band; GAP: GTPase-activating protein; Col: Columbia; ABRC: Arabidopsis Biological Resource Center; KIS: KIESEL; POR: PORCINO; TTN1: TITAN1; PFI: PFIFFERLING
Key Words: AtTFC B, Cell Division, Embryo, Microtubule, Nucleus
Send correspondence to: Yi Li, Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Science, The National Plant Gene Research Center, Peking University, Beijing 100871, China. National Center for Plant Gene Research, Beijing 100101, China, Tel: 86-10-62759690, Fax: 86-10-62756903, E-mail:liyi@ pku.edu.cn