A2-type cyclin is required for the asymmetric entry division in rice stomatal development

A2-type cyclin is required for the asymmetric entry division in rice stomatal development Abstract In rice, and other major cereal grass crops, stomata are arranged in linear files parallel to the long growth axis of leaves. Each stomatal unit comprises two dumbbell-shaped guard cells flanked by two subsidiary cells. These morphological and developmental characteristics enable grass stomata to respond to environmental changes more efficiently. Cyclin-dependent kinases (CDKs) and their cyclin partners co-ordinate cell proliferation and differentiation during the development of multicellular organisms. In contrast to animals, plants have many more types and members of cyclins. In Arabidopsis, four A2-type cyclins (CYCA2s) function redundantly in regulating CDKB1 activity to promote the asymmetric division for stomatal initiation and the symmetric division of guard mother cells (GMCs). In this study, we examine the function of the single A2-type cyclin in rice, OsCYCA2;1, as well the single B1-type CDK, OsCDKB1;1. Cross-species complementation tests demonstrated that OsCYCA2;1 and OsCDKB1;1 could complement the defective stomatal phenotypes of Arabidopsis cyca2 and cdkb1 mutants, but also could suppress DNA endoduplication and cell enlargement. The early asymmetric divisions that establish the stomatal lineages are often missing within the stomatal cell files of OsCYCA2;1-RNAi rice transgenic lines, leading to a significantly reduced stomatal production. However, GMC divisions are not disrupted either in OsCYCA2;1-RNAi or in OsCDKB1;1-RNAi rice transgenic lines as expected. Our results demonstrate a conserved but diverged function and behavior of rice A2-type cyclins, which might be associated with the distinct stomatal development pathways between rice and Arabidopsis. Cyclin-dependent kinases, cell differentiation, cell division, guard mother cells, cyclin, rice, stomata Introduction Stomata are microscopic valves on aerial surfaces of all land plants regulating the shoot–atmosphere gas exchange. Paleobotanical analyses revealed that stomata originated ~400 million years ago, a key evolutionary innovation formed in the early palaeozoic era (Raven, 2002). Despite the fact that the distribution pattern and morphology are highly diversified in different plants, stomata arise in the epidermis after a series of cell divisions, cell fate changes, and cell shape controls. In the past decades, results of molecular genetic studies demonstrated that stomatal development is an accessible system to reveal the evolution of genes and signals involved in plant development (Vatén and Bergmann, 2012; Ran et al., 2013; Chater et al., 2017; Qu et al., 2017). In the dicot model plant Arabidopsis, the earliest stomatal precursor, the meristemoid mother cell (MMC), divides asymmetrically (stomatal entry divisions) producing a smaller cell, the meristemoid, as well as a larger sister cell, the stomatal lineage ground cell (SLGC). Meristemoids normally undergo an additional 1–2 rounds of asymmetric divisions (amplifying divisions) to generate new meristemoids before converting into guard mother cells (GMCs). SLGCs can also undergo asymmetric divisions (spacing divisions) to generate satellite meristemoids. Meristemoids differentiate into GMCs after cell fate change. Then, GMCs divide symmetrically to produce paired young guard cells (GCs). During the final stage of stomatal development, GCs undergo cell differentiation, morphogenesis, and pore formation to form functional stomatal units (Bergmann and Sack, 2007). In contrast to the scattered pattern in dicot Arabidopsis leaves, monocot grass stomata are arranged within linear cell files that parallel the growth axis of the leaf. Stomatal lineage cells initiate at the base of the leaf, and divide asymmetrically to produce two daughter cells, a GMC, and a larger sister cell. At the final stage, GMCs divide symmetrically, producing paired dumbbell-shaped GCs. The stomatal subsidiary cells are produced from cell files flanking the stomatal lineage after asymmetric divisions (Franks and Farquhar, 2007; Liu et al., 2009; Serna, 2011; Raissig et al., 2016). Cyclins form complexes with specific cyclin-dependent kinases (CDKs) to co-ordinate the cell proliferation and differentiation during the development of multicellular organisms (Swenson et al., 1986; Obaya and Sedivy, 2002). Cyclins, acting as the regulator of CDK activity, contribute to the subcellular localization, substrate specificity, and protein stability of the CDK–cyclin complexes (Dewitte and Murray, 2003; Imai et al., 2006; Boudolf et al., 2009; Boruc et al., 2010). A-type cyclins, known as mitotic cyclins, are essential for the mitotic cell cycle. In contrast to animals, plants encode a large family of A-type cyclins that have been classed into A1, A2, and A3 groups (Vandepoele et al., 2002; Dewitte and Murray, 2003; Wang et al., 2004). The Arabidopsis genome has four genes encoding A2-type cyclins. AtCYCA2 genes display tissue- and cell type-specific and overlapping expression patterns, such as in vascular systems and stomatal lineage cells, which are associated with their redundant functions during plant development (Burssens et al., 2000; Imai et al., 2006; Vanneste et al., 2011; Donner and Scarpella, 2013). Mutants of AtCYCA2 genes frequently form unpaired single guard cells (SGCs), a similar defect of the terminal GMC division to that also observed in mutants of AtCDKB1 or AtCDKA;1 genes (Boudolf et al., 2004b; Vanneste et al., 2011; Yang et al., 2014). Overexpression of AtCYCA2:3 as well as AtCDKA;1 at the late stage of stomatal development induced excessive GC subdivisions (Yang et al., 2014). Arabidopsis cdkb1;1 1;2 double mutants and 35S:CDKB1;1.N161 dominant negative plants displayed decreased stomatal production and formation of SGCs, indicating that the activity of CDKB1 is required for both meristemoid asymmetric division and GMC symmetric division in stomatal development. In addition to the function in promoting mitosis, AtCYCA2s form functional complexes with CDKs to modulate the cell cycle transition from the mitotic cycle to the endocycle. Genetic suppression of AtCYCA2 or AtCDKB1 results in enhanced ploidy levels and enlarged pavement cells (PCs; Vanneste et al., 2011). Co-expression of CYCA2;3 and CDKB1;1 induces ectopic cell divisions, limits endoreduplication, and inhibits cell growth (Boudolf et al., 2009). There are at least 49 putative genes predicted to encode rice cyclins, which were classified into nine types based on evolutionary relationships. Eight of these nine types are common between rice and Arabidopsis (Umeda et al., 1999a; Cooper et al., 2003; La et al., 2006). The existence of numerous cyclins implies their diverse regulatory roles in modulating CDK activities during rice development and adaption in response to environmental changes (Cooper et al., 2003; Huang et al., 2008). For example, rice B2-type cyclins, OsCycB2;1 and OsCycB2;2, promote root cell divisions through an association with OsCDKB2;1 (Lee et al., 2003). OsCycH;1 specifically binds to R2 and positively controls CDK and CTD kinase activities to adjust the rate of cell proliferation (Fabian-Marwedel et al., 2002). In this study, we examine the function of the rice single A2-type cyclin OsCYCA2;1 and OsCDKB1;1 in rice development. Our results demonstrate a requirement for OsCYCA2;1 for stomatal and root development. In contrast to its homolog in Arabidopsis, OsCYCA2;1 is exclusively required for the asymmetric entry divisions to produce GMCs at the early stage of stomatal development. In addition, combined with phylogenetic analyses, we are providing new clues for further revealing the evolutionary correlation between cell cycle genes and developmental pathways. Materials and methods Plant materials and growth conditions The Col-0 ecotype of Arabidopsis thaliana L. was used as the wild-type control in the Arabidopsis study. The cdkb1;1 1;2 double mutants were confirmed by PCR-based approaches (Xie et al., 2010). The cyca2;34 double mutants were provided by Steffen Vanneste and Tom Beeckman (Vanneste et al., 2011). Seeds were surface sterilized (40 s) in an aqueous solution of 30% (w/v) hydrogen peroxide and 85% (v/v) ethanol in a volume ratio of 1:4 (v/v), and then sown on the surface of half-strength Murashige and Skoog (MS) medium supplemented with 0.8% agar and 1% sucrose. Plants were grown in a controlled temperature and photoperiod chamber at 22 ± 2 °C and 16 h/8 h light/dark illumination cycles. Oryza sativa L. spp. japonica cultivar Zhonghua 11 was used as the wild-type control and the transformation recipient in the rice study. Rice seeds were soaked in water at 28 °C for 2 d, and then grown in a controlled growth chamber with 30 °C/22 °C day/night temperature cycles, 12 h/12 h light/dark illumination cycles, and 60–70% relative humidity. Plasmid construction and generation of transgenic plants To obtain the construct of gene overexpression, cDNA of OsCDKB1;1 or OsCYCA2;1 was cloned into the pH7WG2D.1 vector by using gateway technology and LR Clonase™ II Enzyme Mix (Invitrogen). The recombinant plasmids were confirmed by DNA sequencing before the transformation into Arabidopsis wild type and mutants. To generate RNAi transgenic plants against OsCDKB1;1 and OsCYCA2;1, the conserved sequences from base pair 530 to 695 of OsCDKB1;1 cDNA and 747 to 979 of OsCYCA2;1 cDNA were amplified and cloned into pTCK303 vector, respectively. These constructs were electroporated into Agrobacterium tumefaciens EHA105 and transformed into rice Zhonghua 11 (Chen et al., 2011). T1 seeds were collected to screen positive transgenic plants by using 50 µg l−1 hygromycin B (Roche). Real-time quantitative PCR (RT-qPCR) was conducted to confirm the expression level of target genes in transgenic plants. The primer sequences used in this study are listed in Supplementary Table S1 at JXB online. Real-time quantitative PCR analysis Rice seedlings were harvested and immediately ground in liquid nitrogen, and the total RNA was isolated using TRNzol reagent (http://www.tiangen.com). The first-strand cDNA was synthesized using a Promega Reverse Transcription kit (http://www.promega.com). RT-qPCRs were performed by using SYBR Premix Ex Taq™ (TaKaRa) with a Corbett RG3000. The OsACTIN2 gene was used as an internal control. The primer sequences are listed in Supplementary Table S1. Yeast two-hybrid assay The full-length cDNA sequences of OsCYCA2;1 and OsCDKB1;1 were amplified using the primers listed in Supplementary Table S1 and cloned into pGBKT7 and pGADT7 vectors (Clontech), respectively. These constructs were transformed into Saccharomyces cerevisiae yeast strain AH109 and selected on SD/-Leu-Trp or SD/-Leu-Trp-His-Ade plates. X-Gal activity was then detected. Bimolecular fluorescence complementation assay For bimolecular fluorescence complementation (BiFC) assays, the full-length cDNAs of OsCYCA2;1 and OsCDKB1;1 were cloned into pSPYCE-35S and pSPYNE-35S vectors, respectively (Walter et al., 2004). These constructs were transformed into A. tumefaciens EH105 and co-injected into tobacco (Nicotiana benthamiana). Images were taken after 3 d using a laser scanning confocal microscope (FV1000-MPE, Olympus). Pull-down assay The OsCYCA2;1 and OsCDKB1;1 sequences were cloned into pET-28a and pGEX4T-1 vectors, respectively. The OsCYCA2;1-pET-28a and OsCDKB1;1-pGEX4T-1 constructs were transformed into the BL21 strain of Escherichia coli. The transformed strains were grown to OD600=0.5 under 37 °C and then placed at 18 °C for 30 min. Fusion proteins were induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 18 °C for 20 h. The harvested strains (5000 rpm, 10 min, 4 °C) were re-suspended with ice-cold phosphate-buffered saline (PBS) and lysed by sonication. The lysate was centrifuged at 10000 rpm for 60 min and the supernatant was collected. The glutathione S-transferase (GST)–OsCDKB1;1 supernatant was loaded on glutathione–Sepharose (GE Healthcare) and washed with PBS. The GST–OsCDKB1;1 fusion protein on glutathione–Sepharose was incubated with the His-OsCYCA2;1 supernatant at 4 °C for 2 h. Then the glutathione–Sepharose was washed with PBS and eluted with 10 mM reduced glutathione elution buffer. The samples were loaded on a 12% SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) by using a semi-dry blotting system (Bio-Rad), and then incubated with anti-His6 monoclonal antibodies followed by horseradish peroxidase (HRP)-conjugated anti-mouse antibodies. The color reaction was performed using the Pro-Light HRP Kit (Tiangen). Signals were exposed to X-ray films and developed. DAPI staining and DNA content measurement Ten-day-old rice roots were fixed in a mixture of 3:1 (v/v) ethanol and acetic acid for 30 min, then rinsed with distilled water. After staining for 30 min with 2 µg ml−1 DAPI (Roche) in a staining solution (0.1 M sodium phosphate, 1 mM EDTA, 0.1% Triton X-100, pH 7.0), roots were photographed by a fluorescence microscope. The relative fluorescence intensities were measured using ImageJ software (http://imagej.nih.gov/ij/). Flow cytometric analysis About 20–50 mg of fresh tissue were cut into 2–4 mm fragments and then chopped immediately using a razor blade in 1 ml of Galbraith’s buffer (45 mM MgCl2·6H2O, 30 mM sodium citrate, 20 mM MOPS, 0.1% Triton X-100, pH 7.0). The cell culture was collected by gentle pipetting and filtered through a cell strainer. The samples were stained with 2 µg ml−1 DAPI in an ice bath for 30 min before the analysis using a MoFlo-XDP flow cytometer (Beckman) (Dolezel et al., 2007). A total of ~10000 nuclei were measured for each sample. Root semi-thin sections About 1 cm long primary root tips from 6-day-old rice seedlings were harvested and immersed in cold formaldehyde solution. The samples were subjected to a vacuum for 10 min and placed at 4 °C overnight. The materials were washed with 0.1 M PBS (pH 7.2) four times and were fixed in 1% osmic acid for 1 h, followed by a series of dehydration steps, which were performed by using 30, 50, 70, 80, 90, 100, and 100% ethanol (each step lasted 20 min). Ethanol was substituted with 1:1 acetone and ethanol (v/v) as well as pure acetone twice (each step lasted 20 min). Permeation was performed by using a series of 2:1, 1:1, and 1:2 (v/v) of acetone and epoxy (SPURR) mixture solution. Each step lasted for 3 h. After adding pure epoxy for 8 h, samples were embedded and polymerized at 60 °C for 24 h. Semi-thin sections (thickness 1 µm) were obtained by using a Leica UC7 microtome. Before imaging, sections were stained with 0.1% toluidine blue O. Results Evolutionary analysis of A2 cyclins in plants Phylogenetic analysis indicates that homologs of A2-type cyclin are found in lineages that diverged early in the evolution of land plants, before the appearance of stomata. For example, the unicellular green alga Coccomyxa subellipsoidea has a CYCA2 gene in its genome. In the non-vascular land plant moss Physcomitrella patens, stomata are exclusively found on the diploid sporophyte (Chater et al., 2016); there are six putative orthologs of CYCA2. In the vascular dicot plants Arabidopsis, soybean (Glycine max), and alfalfa (Medicago truncatula), the number of CYCA2 genes was four, six, and four, respectively (Fig. 1). In contrast, the rice genome contains only one copy of the CYCA2 gene, Os012g31810, which is predicted to encode OsCYCA2;1 protein consisting of 490 amino acid residues. Multiple sequence alignment reveals that OsCYCA2;1 shows 40.5% amino acid sequence identity with Arabidopsis CYCA2s, and contains a CDK-binding cyclin box, which is highly conserved among mitotic cyclins (Supplementary Fig. S1) (Umeda et al., 1999a). Interestingly, similar to rice, Brachypodium stacei, Brachypodium distachyon, Zea mays (Fig. 1), and many other monocot grasses, Hordeum vulgare, Oropetium thomaeum, Panicum hallii, Sorghum bicolor, Setaria italica, and Setaria viridis, only have 1–2 CYCA2 genes (Supplementary Fig. S2). Fig. 1. View largeDownload slide Phylogenetic tree shows that A2-type cyclin-like proteins are conservatively present in green land plants. The phylogenetic tree was constructed using amino acid sequences of Arabidopsis CYCA2 family members based on Phytozome V12.1, using the Neighbor–Joining method in MEGA4. Bootstrap values for 1000 replicates are given in nodes as percentages. Amino acid sequences were used from Arabidopsis thaliana, Brachypodium distachyon, Brachypodium stacei, Coccomyxa subellipsoidea, Glycine max, Medicago truncatula, Oryza sativa, Physcomitrella patens, Zea mays, and Zostera marina. Fig. 1. View largeDownload slide Phylogenetic tree shows that A2-type cyclin-like proteins are conservatively present in green land plants. The phylogenetic tree was constructed using amino acid sequences of Arabidopsis CYCA2 family members based on Phytozome V12.1, using the Neighbor–Joining method in MEGA4. Bootstrap values for 1000 replicates are given in nodes as percentages. Amino acid sequences were used from Arabidopsis thaliana, Brachypodium distachyon, Brachypodium stacei, Coccomyxa subellipsoidea, Glycine max, Medicago truncatula, Oryza sativa, Physcomitrella patens, Zea mays, and Zostera marina. The seagrass Zostera marina belongs to basal monocots that returned to the sea. The absence of stomata in Z. marina is consistent with the evolutionary loss of entire genes that are required for stomatal development (Olsen et al., 2016). However, like the above grass plants, Z. marina possesses two CYCA2 genes, suggesting that the A2-type cyclin is fundamentally important for plant growth and development, and is not solely linked to stomatal development. The low number of CYCA2 genes in grasses indicates that CYCA2 gene duplication might not be necessary, which is associated with their unique developmental pathways and morphogenesis. Requirement of OsCYCA2;1 for stomatal initiation To elucidate the function of A2-type cyclin in rice development, RNAi transgenic rice lines targeting OsCYCA2;1 were generated. Transcript levels of OsCYCA2;1 in two lines, Ri1 and Ri3, were suppressed to 28% and 61%, respectively, in relation to the level in wild-type rice seedlings (Supplementary Fig. S3A). In rice leaf epidermis, stomata form within the stomatal lineage files following a gradual base to tip maturation pattern; the developing stomata can only be found at the proximal end (base) of the leaf. Unlike in Arabidopsis, GMCs in rice are produced directly by asymmetric entry divisions without the precursor stage of the meristemoid. Each undifferentiated cell close to the base of the leaf divides asymmetrically and generates one smaller GMC and one larger sister cell (Stage 2, upper panel of Fig. 2A). Subsidiary mother cells (SMCs) flanking the GMCs are produced by the cells in the neighboring cell files. The terminal division of GMC produces a pair of immature GCs (Stage 5, middle panel of Fig. 2A). Within the wild-type stomatal lineage cell files, stomatal complexes are spaced by one lobbed PC (Stage 6, lower panel of Fig. 2A). However, in OsCYCA2;1-RNAi transgenic plants, more than two spacing cells were often observed between two neighboring GMCs/stomata within the same cell file (Fig. 2B, C), leading to a decreased stomatal density and stomatal index (Fig. 2D, E). Mutations of Arabidopsis CYCA2 genes caused a failure of GMC division, leading to the formation of aberrant stomatal units (SGCs) (Vanneste et al., 2011). However, the structure and morphology of mature stomata in OsCYCA2;1-RNAi rice transgenic plants are indistinguishable from those of wild-type stomata, indicating that the subsequent GMC symmetric divisions as well the subsidiary cell asymmetric divisions are not interrupted by the suppression of OsCYCA2;1 (lower panels in Fig. 2A and B). Taken together, the above observations indicate that OsCYCA2;1 is essentially required for the asymmetric entry division during stomatal initiation at the early stage of stomatal development, but not for the terminal GMC symmetric divisions and subsidiary cell asymmetric divisions. Fig. 2. View largeDownload slide Suppression of OsCYCA2;1 causes defective cell division in rice. (A, B) Differential interference contrast micrographs of epidermal cells from 6-day-old rice seedlings grown in darkness. Stomata were initiated at the proximal end (base) of young leaves. Asymmetric cell divisions produce a smaller GMC and one larger sister cell (Stage 2, upper panels). The terminal symmetric division of the GMC produces a pair of immature GCs (Stage 5, middle panels). Mature stomatal complexes (a pair of dumbbell-shaped GCs and two flanking SCs) are spaced by one pavement cell (PC) (Stage 6, lower panels). Arrowheads indicate the GMCs or stomatal complexes. Asterisks indicate the PCs that separate stomata in the same cell file. Scale bar=20 µm. (C) The numbers of PCs between two adjacent stomata within the same cell file are often increased in RNAi lines. (D, E) Leaf stomatal density and index of two OsCYCA2;1-RNAi lines and the wild type (WT; n=12). Data represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01). (F–H) Flow cytometric analysis of nuclei in shoot cells. (I) Quantitative analysis of DAPI fluorescence. OsCYCA2;1-RNAi transgenic lines have a higher average 4C DNA content than the WT. For each line, ~10000 cell nuclei were measured. Fig. 2. View largeDownload slide Suppression of OsCYCA2;1 causes defective cell division in rice. (A, B) Differential interference contrast micrographs of epidermal cells from 6-day-old rice seedlings grown in darkness. Stomata were initiated at the proximal end (base) of young leaves. Asymmetric cell divisions produce a smaller GMC and one larger sister cell (Stage 2, upper panels). The terminal symmetric division of the GMC produces a pair of immature GCs (Stage 5, middle panels). Mature stomatal complexes (a pair of dumbbell-shaped GCs and two flanking SCs) are spaced by one pavement cell (PC) (Stage 6, lower panels). Arrowheads indicate the GMCs or stomatal complexes. Asterisks indicate the PCs that separate stomata in the same cell file. Scale bar=20 µm. (C) The numbers of PCs between two adjacent stomata within the same cell file are often increased in RNAi lines. (D, E) Leaf stomatal density and index of two OsCYCA2;1-RNAi lines and the wild type (WT; n=12). Data represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01). (F–H) Flow cytometric analysis of nuclei in shoot cells. (I) Quantitative analysis of DAPI fluorescence. OsCYCA2;1-RNAi transgenic lines have a higher average 4C DNA content than the WT. For each line, ~10000 cell nuclei were measured. It has been demonstrated that Arabidopsis CYCA2s not only promote cell proliferation but also negatively regulate endocycle onset (Imai et al., 2006; Yoshizumi et al., 2006; Vanneste et al., 2011). Flow cytometric analysis showed that in wild-type rice, only 6% of cells showed a 4C DNA content, whereas most cells were 2C (diploid). However, in OsCYCA2;1-RNAi transgenic rice lines Ri1 and Ri3, the fraction of 4C cells markedly increased to 36% and 18%, respectively. Higher ploidy levels, like in Arabidopsis cyca2 mutants (8C, 16C, 32C), were barely detectable in rice OsCYCA2;1-RNAi plants (Fig. 2F–I). OsCYCA2;1 complements epidermal defects of Arabidopsis cyca2 mutants The Arabidopsis epidermis is an ideal system to identify gene functions in plant development programs and morphogenetic patterns. To identify further the function of OsCYCA2;1 in epidermal development, OsCYCA2;1 coding sequences driven by 35S promoters were transformed into Arabidopsis cyca2;34 mutants. Compared with the wild type, cyca2;34 mutants display enlarged pavement cells and enhanced ploidy levels. Cross-species expression of OsCYCA2;1 (line #7) inhibits the abnormal PC enlargement in cyca2;34 epidermis, to a cell size even smaller than in the wild type (Fig. 3A–D). Moreover, revealed by flow cytometric analysis, expression of OsCYCA2;1 is able to inhibit the high DNA ploidy levels in cyca2;34 mutants (Fig. 3E; Supplementary Fig. S4). This result is consistent with the previous findings that overexpression of CYCA2 genes could restrain endoreduplication in Arabidopsis (Imai et al., 2006; Boudolf et al., 2009). Fig. 3. View largeDownload slide Cross-species expression of OsCYCA2;1 complements the epidermal defects of Arabidopsis cyca2;34 mutants. (A–C) Differential interference contrast micrographs of cotyledon epidermal cells of 14-day-old Arabidopsis seedlings of the Col, cyca2;34, and cyca2;34 harboring 35S:OsCYCA2;1, Line #7. An arrow points to a single guard cell (SGC). Representative pavement cells (PCs) are traced with dashed lines. Scale bar=50 µm. (D) Comparison of PC area (n=30). (E) Proportions of cells with different ploidies. (F and G) Stomatal density and index. The diagonal line-filled box indicates the SGCs. Data in (D, F, G) represent the mean ±SD. Asterisks indicate a significant difference from Col wild-type controls (Student’s t-test, **P<0.01). Fig. 3. View largeDownload slide Cross-species expression of OsCYCA2;1 complements the epidermal defects of Arabidopsis cyca2;34 mutants. (A–C) Differential interference contrast micrographs of cotyledon epidermal cells of 14-day-old Arabidopsis seedlings of the Col, cyca2;34, and cyca2;34 harboring 35S:OsCYCA2;1, Line #7. An arrow points to a single guard cell (SGC). Representative pavement cells (PCs) are traced with dashed lines. Scale bar=50 µm. (D) Comparison of PC area (n=30). (E) Proportions of cells with different ploidies. (F and G) Stomatal density and index. The diagonal line-filled box indicates the SGCs. Data in (D, F, G) represent the mean ±SD. Asterisks indicate a significant difference from Col wild-type controls (Student’s t-test, **P<0.01). In cyca2;34 mutants, ~10% of GMCs failed to divide symmetrically and formed into SGCs (Fig. 3B, arrow). Strikingly, OsCYCA2;1 expression can fully rescue the defective GMC division in cyca2;34 epidermis, suggesting that rice OsCYCA2;1 remains a conserved function in promoting the GMC symmetric divisions. In addition, expression of OsCYCA2;1 in the cyca2;34 mutant background induced formation of excessive stomata, reflected by an increased stomatal density and stomatal index (Fig. 3F, G). In another 35S:OsCYCA2;1 cyca2;34 transgenic line (line #8), the relative transcript level of OsCYCA2;1 is much lower than in line #7; the defective GMC division and reduced stomatal production are partially rescued, indicating that OsCYCA2;1 quantitatively promotes stomatal development depending on its expression level (Supplementary Fig. S5). Taken together, our results of cross-species complement tests demonstrate the conserved abilities of OsCYCA2;1 in limiting cell endoreduplication and PC size, as well as in rescuing cyca2;34 defective asymmetric entry divisions (for stomatal initiation) and symmetric GMC divisions (for guard cell formation), despite OsCYCA2;1 being functionally required only for stomatal entry divisions in rice. OsCYCA2;1 is also required for cell division and differentiation in roots OsCYCA2;1 is preferentially expressed in proliferating tissues. Besides in the base dividing zone (proximal end) of leaves, a higher transcript level of OsCYCA2;1 is found in rice root tips, implying that a similar OsCYCA2;1 regulatory mechanism exists in rice roots (Supplementary Fig. 3B). Therefore, we probed the impact of down-regulated expression of OsCYCA2;1 on root growth. As shown in Fig. 4, the overall growth of shoots and roots in 10-day-old OsCYCA2;1-RNAi transgenic seedlings is much less than in the wild type (Fig. 4A–C). To determine whether the root growth defects arose from a defective cell proliferation, we compared the root longitudinal sections of the wild type and the RNAi line. The shorter meristematic zone in OsCYCA2;1-RNAi is correlated with a considerably fewer number of cells within its meristematic zone (Fig. 4D–F). Similarly, Arabidopsis cyca2;34 mutants exhibit a short meristematic zone and fewer cells than Col wild type. Ectopic expression of OsCYCA2;1 restored the length of and cell number within the meristematic zone to the wild-type level (Fig. 4G–I), supporting that OsCYCA2;1 is an evolutionarily conserved regulator that is required for cell proliferation in roots. Fig. 4. View largeDownload slide Suppression of CYCA2 expression causes defective cell proliferation within the meristematic zone of roots. (A) Ten-day-old OsCYCA2;1-RNAi and wild-type (WT) rice seedlings. Scale bar=1 cm. (B, C) The length of shoots and primary roots (n=24). (D) Longitudinal sections of the primary root tips. Scale bar=100 µm. (E, F) Length and cell number of the meristematic zone in roots (n=20). (G) Propidium iodide-stained images of Arabidopsis root tips. Scale bar=100 µm. (H, I) Length and cell number of the meristematic zone in Arabidopsis roots (n=20). Double-headed arrows in (D, G) indicate the extent of the meristematic zone. Data in (B, C, E, F, H, I) represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01, *P<0.05). (This figure is available in colour at JXB online.) Fig. 4. View largeDownload slide Suppression of CYCA2 expression causes defective cell proliferation within the meristematic zone of roots. (A) Ten-day-old OsCYCA2;1-RNAi and wild-type (WT) rice seedlings. Scale bar=1 cm. (B, C) The length of shoots and primary roots (n=24). (D) Longitudinal sections of the primary root tips. Scale bar=100 µm. (E, F) Length and cell number of the meristematic zone in roots (n=20). (G) Propidium iodide-stained images of Arabidopsis root tips. Scale bar=100 µm. (H, I) Length and cell number of the meristematic zone in Arabidopsis roots (n=20). Double-headed arrows in (D, G) indicate the extent of the meristematic zone. Data in (B, C, E, F, H, I) represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01, *P<0.05). (This figure is available in colour at JXB online.) By means of flow cytometry approaches, we found that, in contrast to the 6% of 4C cells in the wild type, the fractions of cells with 4C DNA content in OsCYCA2;1-RNAi lines Ri1 and Ri3 are dramatically increased to 32% and 15%, respectively (Fig. 5A–D). Moreover, the relative expression levels of an S-phase-specific gene, PCNA, and a M-phase cyclin gene, CYCB2;1, were suppressed in OsCYCA2;1-RNAi plants (Fig. 5E). Consistently, epidermal cells in the maturation zone of OsCYCA2;1-RNAi roots showed stronger DAPI fluorescent signals than in the wild type (Fig. 5F–H). Quantitative analysis of the DAPI fluorescence intensities further confirmed that a higher DNA level (~2-fold) is present in OsCYCA2;1-RNAi roots (Fig. 5I). The higher DNA content in OsCYCA2;1-RNAi root cells might be due to delayed or arrested G2 to M transition, a result supporting the idea that OsCYCA2;1 is required for cell mitosis. Fig. 5. View largeDownload slide OsCYCA2;1 is required for rice root cell mitosis. (A–C) Profiles of distribution of cells with different DNA content after flow cytometric analysis. Roots of OsCYCA2;1-RNAi lines Ri1 (B) and Ri3 (C) have more 4C cells than wild-type (WT) roots (A). (D) Quantitative analysis of the cell DNA ploidy levels. (E) Relative expression levels of PCNA and CYCB2;1 in OsCYCA2;1-RNAi lines and WT roots. (F–H) DAPI staining of the epidermal cells in the maturation zone of the WT (F), OsCYCA2;1-RNAi line Ri1 (G), and Ri3 (H). Scale bar=50 µm. (I) Quantitative analysis of DAPI fluorescence revealed that OsCYCA2;1-RNAi transgenic lines have a higher average DNA content than the WT. Data represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01). (This figure is available in colour at JXB online.) Fig. 5. View largeDownload slide OsCYCA2;1 is required for rice root cell mitosis. (A–C) Profiles of distribution of cells with different DNA content after flow cytometric analysis. Roots of OsCYCA2;1-RNAi lines Ri1 (B) and Ri3 (C) have more 4C cells than wild-type (WT) roots (A). (D) Quantitative analysis of the cell DNA ploidy levels. (E) Relative expression levels of PCNA and CYCB2;1 in OsCYCA2;1-RNAi lines and WT roots. (F–H) DAPI staining of the epidermal cells in the maturation zone of the WT (F), OsCYCA2;1-RNAi line Ri1 (G), and Ri3 (H). Scale bar=50 µm. (I) Quantitative analysis of DAPI fluorescence revealed that OsCYCA2;1-RNAi transgenic lines have a higher average DNA content than the WT. Data represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01). (This figure is available in colour at JXB online.) OsCYCA2;1 conservatively interacts with OsCDKB1;1 CYCA2s play their regulatory roles through interacting with multiple CDKs, such as by forming CYCA2;3–CDKB1;1 or CYCA2;3–CDKA;1 protein complexes. Arabidopsis AtCYCA2;3 interacts with AtCDKB1;1 to form a functional complex which promotes the formation of a two-celled stoma and prevents entry into the endocycle program (Boudolf et al., 2009; Vanneste et al., 2011). According to the sequence blasting results in the rice genome, Os01g67160 encodes the putative OsCDKB1;1. The deduced amino acid sequence of OsCDKB1;1 shares 88.5% sequence identity with the Arabidopsis CDKB1s. A B1-type-specific cyclin interaction motif ‘PPTALRE’ is highly conserved in rice OsCDKB1;1 (Supplementary Fig. S6). Yeast two-hybrid assays showed that OsCYCA2;1 can interact with OsCDKB1;1 (Fig. 6A). Consistently, pull-down assays verified the direct protein interaction between OsCYCA2;1 and OsCDKB1;1 (Fig. 6B). To determine the subcellular localization, OsCYCA2;1 or OsCDKB1;1 fused with GFP were transiently expressed in tobacco (N. benthamiana) leaves. The fluorescent signals from OsCYCA2;1–GFP were exclusively detected in nuclei, while OsCDKB1;1–GFP was found in both the cytoplasm and nuclei (Fig. 6C). BiFC analysis confirmed that OsCYCA2;1 directly interacts with OsCDKB1;1 in nuclei (Fig. 6D). These results suggest that OsCYCA2;1 may act as a conserved activator regulating the activity of OsCDKB1;1 kinase in rice. Fig. 6. View largeDownload slide OsCYCA2;1 directly interacts with OsCDKB1;1. (A) Yeast two-hybrid assay. (B) Protein pull-down assay. (C) Transient expression of OsCDKB1;1–GFP and OsCYCA2;1–GFP in tobacco leaves. Scale bar=20 µm. (D) Bimolecular fluorescence complementation assay shows that OsCDKB1;1 interacts with OsCYCA2;1 in nuclei. Scale bar=20 µm. (This figure is available in colour at JXB online.) Fig. 6. View largeDownload slide OsCYCA2;1 directly interacts with OsCDKB1;1. (A) Yeast two-hybrid assay. (B) Protein pull-down assay. (C) Transient expression of OsCDKB1;1–GFP and OsCYCA2;1–GFP in tobacco leaves. Scale bar=20 µm. (D) Bimolecular fluorescence complementation assay shows that OsCDKB1;1 interacts with OsCYCA2;1 in nuclei. Scale bar=20 µm. (This figure is available in colour at JXB online.) Suppression of OsCDKB1;1 has no obvious effects on rice development To determine whether OsCDKB1;1, like its partner OsCYCA2;1, is required for rice development, we generated and selected two OsCDKB1;1-RNAi transgenic lines, Ri2 and Ri3, in which OsCDKB1;1 transcript levels were significantly suppressed (Supplementary Fig. S7A). However, the overall growth of these two transgenic lines is comparable with that of the untransformed controls (Supplementary Fig. S7B–D). Longitudinal sections of roots demonstrate that the suppression of OsCDKB1;1 has no significant impact on cell numbers of the root meristematic zone (Supplementary Fig. S7E–G). In addition, we found that neither the stomatal production (stomatal density) nor the stomatal complex morphology has been affected in OsCDKB1;1-RNAi transgenic lines (Supplementary Fig. S7H–J). Flow cytometric assays also indicate that DNA ploidy levels were not changed in either the roots or shoots of OsCDKB1;1-RNAi (Supplementary Fig. S8A–H). Consistent with this, the expression of S-phase PCNA and M-phase cyclin CYCB2;1 was not different between wild-type and OsCDKB1;1-RNAi transgenic plants (Supplementary Fig. S8I). Taken together, it seems that cell division was not interrupted by down-regulation of the OsCDKB1;1 transcript level in transgenic rice, though we could not exclude the possibility that the remaining activity of OsCDKB1;1 protein is sufficient to function. OsCYCA2;1 and OsCDKB1;1 enable complementation of Arabidopsis cdkb1;1 1;2 Arabidopsis cdkb1;1 1;2 mutants, like the cyca2;34 mutants, display a decreased stomatal production, formation of SGCs, enlarged PCs, and increased cell ploidy levels (Boudolf et al., 2004a; Xie et al., 2010). Introduction of OsCDKB1;1 fully complements the impaired GMC division in cdkb1;1 1;2, and restores stomatal production, indicating that OsCDKB1;1 has the ability to promote both symmetric and asymmetric division. Meanwhile, expression of OsCKDB1;1 could efficiently prevent the occurrence of enlarged PCs and increased DNA ploidy levels in cdkb1;1 1;2 mutants (Fig. 7; Supplementary Figs S9, S10). Fig. 7. View largeDownload slide Ectopic expression of OsCYCA2;1 and OsCDKB1;1 complements Arabidopsis cdkb1;1 1;2 mutant phenotypes. (A–D) DIC images of the epidermis of 14-day-old Arabidopsis cotyledons. Arrows indicate the formation of SGCs. Representative PCs are traced with dashed lines. Scale bar=50 µm. (E–G) Comparison of stomatal density, stomatal index, and area of PCs from the cotyledons. The diagonal line-filled box indicates the fraction of SGCs. Data represent the mean ±SD (n=24). Asterisks indicate a significant difference from Col after Student’s t-test, **P<0.01. (H) Proportions of cells with different ploidies. (I–L) Results from the flow cytometric analysis; ~10000 cell nuclei were measured for each sample. Fig. 7. View largeDownload slide Ectopic expression of OsCYCA2;1 and OsCDKB1;1 complements Arabidopsis cdkb1;1 1;2 mutant phenotypes. (A–D) DIC images of the epidermis of 14-day-old Arabidopsis cotyledons. Arrows indicate the formation of SGCs. Representative PCs are traced with dashed lines. Scale bar=50 µm. (E–G) Comparison of stomatal density, stomatal index, and area of PCs from the cotyledons. The diagonal line-filled box indicates the fraction of SGCs. Data represent the mean ±SD (n=24). Asterisks indicate a significant difference from Col after Student’s t-test, **P<0.01. (H) Proportions of cells with different ploidies. (I–L) Results from the flow cytometric analysis; ~10000 cell nuclei were measured for each sample. Interestingly, the defective stomatal production, impaired GMC division, and abnormal cell enlargement and DNA levels in cdkb1;1 1;2 could be partially rescued by overexpression of OsCYCA2;1 (Fig. 7; Supplementary Figs S9, S10). It is therefore possible that OsCDKB1;1 and OsCYCA2;1 have evolved from the common ancestor genes with Arabidopsis AtCDKB1 genes and AtCYCA2 genes, even though the developmental pathways of the two species have been diverged. Discussion The control of cell division and differentiation is the core of the development and morphogenesis of multicellular organisms. Cyclins, known as conserved activators for the activity of CDKs, play a crucial regulatory role in cell cycle progression in diverse species. The functional pathway of CYCA2s and CDKB1s has been well investigated in the model plant Arabidopsis (Boudolf et al., 2004b, 2009; Imai et al., 2006; Xie et al., 2010; Vanneste et al., 2011; Yang et al., 2014). In this study, we generated RNAi transgenic rice lines and performed cross-species complement tests to explore the function of the single rice A2-type cyclin, OsCYCA2;1, as well the single rice B1-type CDK, OsCDKB1;1. Cross-species expression of OsCYCA2;1 or OsCDKB1;1 enables rescue of the defective asymmetric entry divisions for stomatal initiation and GMC symmetric divisions for GC production in Arabidopsis cyca2;34 and/or cdkb1;1 1;2 mutants, suggesting that both OsCYCA2;1 and OsCDKB1;1 might have evolved from the common ancestor genes with Arabidopsis. In Arabidopsis, asymmetric divisions generated the early stomatal precursor cells, meristemoids. Meristemoids then differentiate into GMCs after a cell fate change. In grasses, GMCs are created directly by stomata initiating asymmetric divisions (entry division) without the prior precursor stage of meristemoid. Orthologs of Arabidopsis stomatal transcriptional regulators SPCH, MUTE, FAMA, ICE1, and SCRM2 have been identified in grasses (Liu et al., 2009; Vatén and Bergmann, 2012; Ran et al., 2013; Chen et al., 2017). Instead of a single copy in Arabidopsis, the rice genome has duplicated SPCH genes, OsSPCH1 and OsSPCH2. Similar to the weak allele of Arabidopsis spch, the rice mutant osspch2 exhibits a reduced number of stomata (Liu et al., 2009). In Arabidopsis, SPCH heterodimerizes with SCRM/ICE1 or AtSCRM2 to promote stomatal lineage initiation (Kanaoka et al., 2008; Horst et al., 2015). In contrast, in the grass B. distachyon, BdICE1 and BdSCRM2 show a functional diversity in regulating stomatal pattern and morphology (Raissig et al., 2016). It is already known that Arabidopsis SPCH activity or stability is modulated by multiple kinases, including MPKs, GSK3/BIN2, and CDKs (Lampard et al., 2009; Gudesblat et al., 2012; Kim et al., 2012; Le et al., 2014). Phosphorylation of Ser186 of SPCH, which might be the target residue of CDKs, positively regulates stomata production (Yang et al., 2015). Thus, it will be interesting to establish if there is a conserved regulatory mechanism between CDK–cyclin and SPCH-ICE1/SCRM2 in grasses. Besides the involvement in stomatal initiation, Arabidopsis AtCYCA2s and CDKB1s are synergistically required for the GMC symmetric division that is a prerequisite for the final stomatal development (Vanneste et al., 2011). Arabidopsis and rice share common GMC–GC processing; GMCs divide symmetrically to produce the paired GCs of stomata, though the GC shapes are distinct. Thus, a role for OsCDKB1–OsCYCA2 in rice GMC divisions has been highly expected. However, suppression of OsCYCA2;1 transcription in rice by RNAi does not affect the rice GMC symmetric division. It has been identified that transcription of CDKB1;1, CYCA2;3, and CDKA;1 in Arabidopsis is repressed by FOUR LIPS(FLP)/MYB88 MYB transcription factors during the GMC–GC transition stages (Xie et al., 2010; Vanneste et al., 2011; Yang et al., 2014). FAMA, like FLP/MYB, also binds to the CDKB1;1 promoter (Hachez et al., 2011) to limit the GMC divisions to one. In contrast to the tumor-like phenotype in Arabidopsis fama-1 mutants, the loss-of-function rice allele osfama-1 did not undergo excessive division except the appearance of misshaped GCs and showing a lack of stomatal pores (Liu et al., 2009). These observations suggest that GMC–GC differentiation is uncoupled from GMC division, in which the putative downstream FAMA/FLP/MYB88, CDKB1;1, and CYCA2;1 are not essential. According to the phylogenetic analysis, CYCA2 and CDKB1 widely exist in diverse plant species, both in plants bearing stomata and in plants lacking stomata, indicating that CYCA2 and CDKB1 might function as fundamental regulators of the mitotic cell cycle, as well as outside stomatal development. High expression of OsCYCA2;1 is associated with a high activity of cell proliferation, such as in the proximal end of leaves or root tips (Supplementary Fig. S3B). Endoreduplication often occurs in cell types that undergo specialized differentiation. In Arabidopsis, the highly differentiated epidermal cells, such as mature PCs and trichomes, usually undergo multiple rounds of DNA replication without mitosis, resulting in polyploid cells (Burssens et al., 2000). In contrast to the differentiated cells in Arabidopsis, polyploid cells in rice can only be found in the endosperm (Sabelli and Larkins, 2009). In Drosophila, it has been reported that cyclin A is one of the key components of chromosomal DNA replication that prevents re-initiation of DNA replication. Overexpression of Drosophila cyclin A caused a reduction in ploidy levels and inhibition of the endocycle (Hayashi and Yamaguchi, 1999). Here we found that the fraction of 4C cells remarkably increased in OsCYCA2;1-RNAi transgenic plants, while most cells keep a 2C DNA content. However, the expression levels of an S-phase gene PCNA and a M-phase gene CYCB2;1 were suppressed in OsCYCA2;1-RNAi plants. Therefore, we speculated that the increase of 4C cells might be caused by the arrested G2 to M transition, similar to the observation in the rice knockdown lines of OsCDKB2;1 (Endo et al., 2012). However, the OsCDKB1;1-RNAi transgenic rice plants, in which OsCDKB1;1 transcript levels were significantly decreased, display phenotypes comparable with wild-type rice seedlings regarding the stomatal density, root cell division, and DNA content. CDKBs are plant-specific cyclin-dependent kinases that can be subdivided into two groups according to the different cyclin-binding motifs, namely ‘PPTALRE’ in CDKB1s and ‘PPTTLRE’ in CDKB2s (Joubès et al., 2001). In Arabidopsis, each CDKB1 and CDKB2 subgroup contains two members (Vandepoele et al., 2002). It has been predicted that the rice genome has a single CDKB1 gene and a single CDKB2 gene, encoding OsCDKB1;1 and OsCDKB2;1, respectively (Supplementary Fig. S11). However, the amino acid sequence alignment revealed that rice OsCDKB1;1 and OsCDKB2;1 share the same ‘PPTALRE’ cyclin-binding motif (Supplementary Fig. S12). Expression of OsCDKB2;1 has been detected in the dividing region of the rice root apex (Umeda et al., 1999a). Transcription of rice OsCDKB2;1 is abundant during the G2 to M phase. Knockdown of the OsCDKB2;1 gene in rice induces an increase of the 4C cell population (Umeda et al., 1999b; Endo et al., 2012). In addition, OsCDKB2;1 promotes cell division in the root meristem probably through the association with OsCYCB2s (Lee et al., 2003). Thus, we cannot rule out that OsCDKB1;1 might function redundantly with OsCDKB2;1, such as forming active CDK–cyclin complexes via binding to the same type cyclins (i.e. OsCYCA2;1). Previous in situ hybridization results showed that both OsCDKA;1 and OsCDKA;2 are expressed in dividing root cells of rice (Umeda et al., 1999b). Thus, further characterization of rice CDK–cyclin pairing and activity can help to reveal the regulatory mechanisms of cell division and differentiation during rice development. Supplementary data Supplementary data are available at JXB online. Table S1. List of primers used in this study. Fig. S1. Amino acid sequence comparison of A2-type cyclins from rice and Arabidopsis. Fig. S2. In contrast to dicot Arabidopsis, only one or two copies of genes encoding CYCA2 are found in monocot grasses. Fig. S3. Relative expression of OsCYCA2;1 in rice RNAi transgenic plants and in different tissues of wild-type plants. Fig. S4. Overexpression of rice OsCYCA2;1 suppresses the enhanced endoreduplication levels in Arabidopsis cyca2;34. Fig. S5. Correlation between stomatal phenotypes and OsCYCA2;1 expression levels in Arabidopsis cyca2;34 mutants harboring OsCYCA2;1. Fig. S6. Comparison of the amino acid sequence of OsCDKB1;1 with that of Arabidopsis CDKB1;1 and CDKB1;2. Fig. S7. Suppression of OsCDKB1;1 has no obvious impact on rice root and stomatal development. Fig. S8. Suppression of OsCDKB1;1 has no obvious impact on the distribution of DNA ploidy. Fig. S9. Expression analysis of OsCDKB1;1-OE and OsCYCA2;1-OE transgenic plants in Arabidopsis cdkb1 mutants. Fig. S10. 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Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

A2-type cyclin is required for the asymmetric entry division in rice stomatal development

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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0022-0957
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1460-2431
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10.1093/jxb/ery158
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Abstract

Abstract In rice, and other major cereal grass crops, stomata are arranged in linear files parallel to the long growth axis of leaves. Each stomatal unit comprises two dumbbell-shaped guard cells flanked by two subsidiary cells. These morphological and developmental characteristics enable grass stomata to respond to environmental changes more efficiently. Cyclin-dependent kinases (CDKs) and their cyclin partners co-ordinate cell proliferation and differentiation during the development of multicellular organisms. In contrast to animals, plants have many more types and members of cyclins. In Arabidopsis, four A2-type cyclins (CYCA2s) function redundantly in regulating CDKB1 activity to promote the asymmetric division for stomatal initiation and the symmetric division of guard mother cells (GMCs). In this study, we examine the function of the single A2-type cyclin in rice, OsCYCA2;1, as well the single B1-type CDK, OsCDKB1;1. Cross-species complementation tests demonstrated that OsCYCA2;1 and OsCDKB1;1 could complement the defective stomatal phenotypes of Arabidopsis cyca2 and cdkb1 mutants, but also could suppress DNA endoduplication and cell enlargement. The early asymmetric divisions that establish the stomatal lineages are often missing within the stomatal cell files of OsCYCA2;1-RNAi rice transgenic lines, leading to a significantly reduced stomatal production. However, GMC divisions are not disrupted either in OsCYCA2;1-RNAi or in OsCDKB1;1-RNAi rice transgenic lines as expected. Our results demonstrate a conserved but diverged function and behavior of rice A2-type cyclins, which might be associated with the distinct stomatal development pathways between rice and Arabidopsis. Cyclin-dependent kinases, cell differentiation, cell division, guard mother cells, cyclin, rice, stomata Introduction Stomata are microscopic valves on aerial surfaces of all land plants regulating the shoot–atmosphere gas exchange. Paleobotanical analyses revealed that stomata originated ~400 million years ago, a key evolutionary innovation formed in the early palaeozoic era (Raven, 2002). Despite the fact that the distribution pattern and morphology are highly diversified in different plants, stomata arise in the epidermis after a series of cell divisions, cell fate changes, and cell shape controls. In the past decades, results of molecular genetic studies demonstrated that stomatal development is an accessible system to reveal the evolution of genes and signals involved in plant development (Vatén and Bergmann, 2012; Ran et al., 2013; Chater et al., 2017; Qu et al., 2017). In the dicot model plant Arabidopsis, the earliest stomatal precursor, the meristemoid mother cell (MMC), divides asymmetrically (stomatal entry divisions) producing a smaller cell, the meristemoid, as well as a larger sister cell, the stomatal lineage ground cell (SLGC). Meristemoids normally undergo an additional 1–2 rounds of asymmetric divisions (amplifying divisions) to generate new meristemoids before converting into guard mother cells (GMCs). SLGCs can also undergo asymmetric divisions (spacing divisions) to generate satellite meristemoids. Meristemoids differentiate into GMCs after cell fate change. Then, GMCs divide symmetrically to produce paired young guard cells (GCs). During the final stage of stomatal development, GCs undergo cell differentiation, morphogenesis, and pore formation to form functional stomatal units (Bergmann and Sack, 2007). In contrast to the scattered pattern in dicot Arabidopsis leaves, monocot grass stomata are arranged within linear cell files that parallel the growth axis of the leaf. Stomatal lineage cells initiate at the base of the leaf, and divide asymmetrically to produce two daughter cells, a GMC, and a larger sister cell. At the final stage, GMCs divide symmetrically, producing paired dumbbell-shaped GCs. The stomatal subsidiary cells are produced from cell files flanking the stomatal lineage after asymmetric divisions (Franks and Farquhar, 2007; Liu et al., 2009; Serna, 2011; Raissig et al., 2016). Cyclins form complexes with specific cyclin-dependent kinases (CDKs) to co-ordinate the cell proliferation and differentiation during the development of multicellular organisms (Swenson et al., 1986; Obaya and Sedivy, 2002). Cyclins, acting as the regulator of CDK activity, contribute to the subcellular localization, substrate specificity, and protein stability of the CDK–cyclin complexes (Dewitte and Murray, 2003; Imai et al., 2006; Boudolf et al., 2009; Boruc et al., 2010). A-type cyclins, known as mitotic cyclins, are essential for the mitotic cell cycle. In contrast to animals, plants encode a large family of A-type cyclins that have been classed into A1, A2, and A3 groups (Vandepoele et al., 2002; Dewitte and Murray, 2003; Wang et al., 2004). The Arabidopsis genome has four genes encoding A2-type cyclins. AtCYCA2 genes display tissue- and cell type-specific and overlapping expression patterns, such as in vascular systems and stomatal lineage cells, which are associated with their redundant functions during plant development (Burssens et al., 2000; Imai et al., 2006; Vanneste et al., 2011; Donner and Scarpella, 2013). Mutants of AtCYCA2 genes frequently form unpaired single guard cells (SGCs), a similar defect of the terminal GMC division to that also observed in mutants of AtCDKB1 or AtCDKA;1 genes (Boudolf et al., 2004b; Vanneste et al., 2011; Yang et al., 2014). Overexpression of AtCYCA2:3 as well as AtCDKA;1 at the late stage of stomatal development induced excessive GC subdivisions (Yang et al., 2014). Arabidopsis cdkb1;1 1;2 double mutants and 35S:CDKB1;1.N161 dominant negative plants displayed decreased stomatal production and formation of SGCs, indicating that the activity of CDKB1 is required for both meristemoid asymmetric division and GMC symmetric division in stomatal development. In addition to the function in promoting mitosis, AtCYCA2s form functional complexes with CDKs to modulate the cell cycle transition from the mitotic cycle to the endocycle. Genetic suppression of AtCYCA2 or AtCDKB1 results in enhanced ploidy levels and enlarged pavement cells (PCs; Vanneste et al., 2011). Co-expression of CYCA2;3 and CDKB1;1 induces ectopic cell divisions, limits endoreduplication, and inhibits cell growth (Boudolf et al., 2009). There are at least 49 putative genes predicted to encode rice cyclins, which were classified into nine types based on evolutionary relationships. Eight of these nine types are common between rice and Arabidopsis (Umeda et al., 1999a; Cooper et al., 2003; La et al., 2006). The existence of numerous cyclins implies their diverse regulatory roles in modulating CDK activities during rice development and adaption in response to environmental changes (Cooper et al., 2003; Huang et al., 2008). For example, rice B2-type cyclins, OsCycB2;1 and OsCycB2;2, promote root cell divisions through an association with OsCDKB2;1 (Lee et al., 2003). OsCycH;1 specifically binds to R2 and positively controls CDK and CTD kinase activities to adjust the rate of cell proliferation (Fabian-Marwedel et al., 2002). In this study, we examine the function of the rice single A2-type cyclin OsCYCA2;1 and OsCDKB1;1 in rice development. Our results demonstrate a requirement for OsCYCA2;1 for stomatal and root development. In contrast to its homolog in Arabidopsis, OsCYCA2;1 is exclusively required for the asymmetric entry divisions to produce GMCs at the early stage of stomatal development. In addition, combined with phylogenetic analyses, we are providing new clues for further revealing the evolutionary correlation between cell cycle genes and developmental pathways. Materials and methods Plant materials and growth conditions The Col-0 ecotype of Arabidopsis thaliana L. was used as the wild-type control in the Arabidopsis study. The cdkb1;1 1;2 double mutants were confirmed by PCR-based approaches (Xie et al., 2010). The cyca2;34 double mutants were provided by Steffen Vanneste and Tom Beeckman (Vanneste et al., 2011). Seeds were surface sterilized (40 s) in an aqueous solution of 30% (w/v) hydrogen peroxide and 85% (v/v) ethanol in a volume ratio of 1:4 (v/v), and then sown on the surface of half-strength Murashige and Skoog (MS) medium supplemented with 0.8% agar and 1% sucrose. Plants were grown in a controlled temperature and photoperiod chamber at 22 ± 2 °C and 16 h/8 h light/dark illumination cycles. Oryza sativa L. spp. japonica cultivar Zhonghua 11 was used as the wild-type control and the transformation recipient in the rice study. Rice seeds were soaked in water at 28 °C for 2 d, and then grown in a controlled growth chamber with 30 °C/22 °C day/night temperature cycles, 12 h/12 h light/dark illumination cycles, and 60–70% relative humidity. Plasmid construction and generation of transgenic plants To obtain the construct of gene overexpression, cDNA of OsCDKB1;1 or OsCYCA2;1 was cloned into the pH7WG2D.1 vector by using gateway technology and LR Clonase™ II Enzyme Mix (Invitrogen). The recombinant plasmids were confirmed by DNA sequencing before the transformation into Arabidopsis wild type and mutants. To generate RNAi transgenic plants against OsCDKB1;1 and OsCYCA2;1, the conserved sequences from base pair 530 to 695 of OsCDKB1;1 cDNA and 747 to 979 of OsCYCA2;1 cDNA were amplified and cloned into pTCK303 vector, respectively. These constructs were electroporated into Agrobacterium tumefaciens EHA105 and transformed into rice Zhonghua 11 (Chen et al., 2011). T1 seeds were collected to screen positive transgenic plants by using 50 µg l−1 hygromycin B (Roche). Real-time quantitative PCR (RT-qPCR) was conducted to confirm the expression level of target genes in transgenic plants. The primer sequences used in this study are listed in Supplementary Table S1 at JXB online. Real-time quantitative PCR analysis Rice seedlings were harvested and immediately ground in liquid nitrogen, and the total RNA was isolated using TRNzol reagent (http://www.tiangen.com). The first-strand cDNA was synthesized using a Promega Reverse Transcription kit (http://www.promega.com). RT-qPCRs were performed by using SYBR Premix Ex Taq™ (TaKaRa) with a Corbett RG3000. The OsACTIN2 gene was used as an internal control. The primer sequences are listed in Supplementary Table S1. Yeast two-hybrid assay The full-length cDNA sequences of OsCYCA2;1 and OsCDKB1;1 were amplified using the primers listed in Supplementary Table S1 and cloned into pGBKT7 and pGADT7 vectors (Clontech), respectively. These constructs were transformed into Saccharomyces cerevisiae yeast strain AH109 and selected on SD/-Leu-Trp or SD/-Leu-Trp-His-Ade plates. X-Gal activity was then detected. Bimolecular fluorescence complementation assay For bimolecular fluorescence complementation (BiFC) assays, the full-length cDNAs of OsCYCA2;1 and OsCDKB1;1 were cloned into pSPYCE-35S and pSPYNE-35S vectors, respectively (Walter et al., 2004). These constructs were transformed into A. tumefaciens EH105 and co-injected into tobacco (Nicotiana benthamiana). Images were taken after 3 d using a laser scanning confocal microscope (FV1000-MPE, Olympus). Pull-down assay The OsCYCA2;1 and OsCDKB1;1 sequences were cloned into pET-28a and pGEX4T-1 vectors, respectively. The OsCYCA2;1-pET-28a and OsCDKB1;1-pGEX4T-1 constructs were transformed into the BL21 strain of Escherichia coli. The transformed strains were grown to OD600=0.5 under 37 °C and then placed at 18 °C for 30 min. Fusion proteins were induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 18 °C for 20 h. The harvested strains (5000 rpm, 10 min, 4 °C) were re-suspended with ice-cold phosphate-buffered saline (PBS) and lysed by sonication. The lysate was centrifuged at 10000 rpm for 60 min and the supernatant was collected. The glutathione S-transferase (GST)–OsCDKB1;1 supernatant was loaded on glutathione–Sepharose (GE Healthcare) and washed with PBS. The GST–OsCDKB1;1 fusion protein on glutathione–Sepharose was incubated with the His-OsCYCA2;1 supernatant at 4 °C for 2 h. Then the glutathione–Sepharose was washed with PBS and eluted with 10 mM reduced glutathione elution buffer. The samples were loaded on a 12% SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) by using a semi-dry blotting system (Bio-Rad), and then incubated with anti-His6 monoclonal antibodies followed by horseradish peroxidase (HRP)-conjugated anti-mouse antibodies. The color reaction was performed using the Pro-Light HRP Kit (Tiangen). Signals were exposed to X-ray films and developed. DAPI staining and DNA content measurement Ten-day-old rice roots were fixed in a mixture of 3:1 (v/v) ethanol and acetic acid for 30 min, then rinsed with distilled water. After staining for 30 min with 2 µg ml−1 DAPI (Roche) in a staining solution (0.1 M sodium phosphate, 1 mM EDTA, 0.1% Triton X-100, pH 7.0), roots were photographed by a fluorescence microscope. The relative fluorescence intensities were measured using ImageJ software (http://imagej.nih.gov/ij/). Flow cytometric analysis About 20–50 mg of fresh tissue were cut into 2–4 mm fragments and then chopped immediately using a razor blade in 1 ml of Galbraith’s buffer (45 mM MgCl2·6H2O, 30 mM sodium citrate, 20 mM MOPS, 0.1% Triton X-100, pH 7.0). The cell culture was collected by gentle pipetting and filtered through a cell strainer. The samples were stained with 2 µg ml−1 DAPI in an ice bath for 30 min before the analysis using a MoFlo-XDP flow cytometer (Beckman) (Dolezel et al., 2007). A total of ~10000 nuclei were measured for each sample. Root semi-thin sections About 1 cm long primary root tips from 6-day-old rice seedlings were harvested and immersed in cold formaldehyde solution. The samples were subjected to a vacuum for 10 min and placed at 4 °C overnight. The materials were washed with 0.1 M PBS (pH 7.2) four times and were fixed in 1% osmic acid for 1 h, followed by a series of dehydration steps, which were performed by using 30, 50, 70, 80, 90, 100, and 100% ethanol (each step lasted 20 min). Ethanol was substituted with 1:1 acetone and ethanol (v/v) as well as pure acetone twice (each step lasted 20 min). Permeation was performed by using a series of 2:1, 1:1, and 1:2 (v/v) of acetone and epoxy (SPURR) mixture solution. Each step lasted for 3 h. After adding pure epoxy for 8 h, samples were embedded and polymerized at 60 °C for 24 h. Semi-thin sections (thickness 1 µm) were obtained by using a Leica UC7 microtome. Before imaging, sections were stained with 0.1% toluidine blue O. Results Evolutionary analysis of A2 cyclins in plants Phylogenetic analysis indicates that homologs of A2-type cyclin are found in lineages that diverged early in the evolution of land plants, before the appearance of stomata. For example, the unicellular green alga Coccomyxa subellipsoidea has a CYCA2 gene in its genome. In the non-vascular land plant moss Physcomitrella patens, stomata are exclusively found on the diploid sporophyte (Chater et al., 2016); there are six putative orthologs of CYCA2. In the vascular dicot plants Arabidopsis, soybean (Glycine max), and alfalfa (Medicago truncatula), the number of CYCA2 genes was four, six, and four, respectively (Fig. 1). In contrast, the rice genome contains only one copy of the CYCA2 gene, Os012g31810, which is predicted to encode OsCYCA2;1 protein consisting of 490 amino acid residues. Multiple sequence alignment reveals that OsCYCA2;1 shows 40.5% amino acid sequence identity with Arabidopsis CYCA2s, and contains a CDK-binding cyclin box, which is highly conserved among mitotic cyclins (Supplementary Fig. S1) (Umeda et al., 1999a). Interestingly, similar to rice, Brachypodium stacei, Brachypodium distachyon, Zea mays (Fig. 1), and many other monocot grasses, Hordeum vulgare, Oropetium thomaeum, Panicum hallii, Sorghum bicolor, Setaria italica, and Setaria viridis, only have 1–2 CYCA2 genes (Supplementary Fig. S2). Fig. 1. View largeDownload slide Phylogenetic tree shows that A2-type cyclin-like proteins are conservatively present in green land plants. The phylogenetic tree was constructed using amino acid sequences of Arabidopsis CYCA2 family members based on Phytozome V12.1, using the Neighbor–Joining method in MEGA4. Bootstrap values for 1000 replicates are given in nodes as percentages. Amino acid sequences were used from Arabidopsis thaliana, Brachypodium distachyon, Brachypodium stacei, Coccomyxa subellipsoidea, Glycine max, Medicago truncatula, Oryza sativa, Physcomitrella patens, Zea mays, and Zostera marina. Fig. 1. View largeDownload slide Phylogenetic tree shows that A2-type cyclin-like proteins are conservatively present in green land plants. The phylogenetic tree was constructed using amino acid sequences of Arabidopsis CYCA2 family members based on Phytozome V12.1, using the Neighbor–Joining method in MEGA4. Bootstrap values for 1000 replicates are given in nodes as percentages. Amino acid sequences were used from Arabidopsis thaliana, Brachypodium distachyon, Brachypodium stacei, Coccomyxa subellipsoidea, Glycine max, Medicago truncatula, Oryza sativa, Physcomitrella patens, Zea mays, and Zostera marina. The seagrass Zostera marina belongs to basal monocots that returned to the sea. The absence of stomata in Z. marina is consistent with the evolutionary loss of entire genes that are required for stomatal development (Olsen et al., 2016). However, like the above grass plants, Z. marina possesses two CYCA2 genes, suggesting that the A2-type cyclin is fundamentally important for plant growth and development, and is not solely linked to stomatal development. The low number of CYCA2 genes in grasses indicates that CYCA2 gene duplication might not be necessary, which is associated with their unique developmental pathways and morphogenesis. Requirement of OsCYCA2;1 for stomatal initiation To elucidate the function of A2-type cyclin in rice development, RNAi transgenic rice lines targeting OsCYCA2;1 were generated. Transcript levels of OsCYCA2;1 in two lines, Ri1 and Ri3, were suppressed to 28% and 61%, respectively, in relation to the level in wild-type rice seedlings (Supplementary Fig. S3A). In rice leaf epidermis, stomata form within the stomatal lineage files following a gradual base to tip maturation pattern; the developing stomata can only be found at the proximal end (base) of the leaf. Unlike in Arabidopsis, GMCs in rice are produced directly by asymmetric entry divisions without the precursor stage of the meristemoid. Each undifferentiated cell close to the base of the leaf divides asymmetrically and generates one smaller GMC and one larger sister cell (Stage 2, upper panel of Fig. 2A). Subsidiary mother cells (SMCs) flanking the GMCs are produced by the cells in the neighboring cell files. The terminal division of GMC produces a pair of immature GCs (Stage 5, middle panel of Fig. 2A). Within the wild-type stomatal lineage cell files, stomatal complexes are spaced by one lobbed PC (Stage 6, lower panel of Fig. 2A). However, in OsCYCA2;1-RNAi transgenic plants, more than two spacing cells were often observed between two neighboring GMCs/stomata within the same cell file (Fig. 2B, C), leading to a decreased stomatal density and stomatal index (Fig. 2D, E). Mutations of Arabidopsis CYCA2 genes caused a failure of GMC division, leading to the formation of aberrant stomatal units (SGCs) (Vanneste et al., 2011). However, the structure and morphology of mature stomata in OsCYCA2;1-RNAi rice transgenic plants are indistinguishable from those of wild-type stomata, indicating that the subsequent GMC symmetric divisions as well the subsidiary cell asymmetric divisions are not interrupted by the suppression of OsCYCA2;1 (lower panels in Fig. 2A and B). Taken together, the above observations indicate that OsCYCA2;1 is essentially required for the asymmetric entry division during stomatal initiation at the early stage of stomatal development, but not for the terminal GMC symmetric divisions and subsidiary cell asymmetric divisions. Fig. 2. View largeDownload slide Suppression of OsCYCA2;1 causes defective cell division in rice. (A, B) Differential interference contrast micrographs of epidermal cells from 6-day-old rice seedlings grown in darkness. Stomata were initiated at the proximal end (base) of young leaves. Asymmetric cell divisions produce a smaller GMC and one larger sister cell (Stage 2, upper panels). The terminal symmetric division of the GMC produces a pair of immature GCs (Stage 5, middle panels). Mature stomatal complexes (a pair of dumbbell-shaped GCs and two flanking SCs) are spaced by one pavement cell (PC) (Stage 6, lower panels). Arrowheads indicate the GMCs or stomatal complexes. Asterisks indicate the PCs that separate stomata in the same cell file. Scale bar=20 µm. (C) The numbers of PCs between two adjacent stomata within the same cell file are often increased in RNAi lines. (D, E) Leaf stomatal density and index of two OsCYCA2;1-RNAi lines and the wild type (WT; n=12). Data represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01). (F–H) Flow cytometric analysis of nuclei in shoot cells. (I) Quantitative analysis of DAPI fluorescence. OsCYCA2;1-RNAi transgenic lines have a higher average 4C DNA content than the WT. For each line, ~10000 cell nuclei were measured. Fig. 2. View largeDownload slide Suppression of OsCYCA2;1 causes defective cell division in rice. (A, B) Differential interference contrast micrographs of epidermal cells from 6-day-old rice seedlings grown in darkness. Stomata were initiated at the proximal end (base) of young leaves. Asymmetric cell divisions produce a smaller GMC and one larger sister cell (Stage 2, upper panels). The terminal symmetric division of the GMC produces a pair of immature GCs (Stage 5, middle panels). Mature stomatal complexes (a pair of dumbbell-shaped GCs and two flanking SCs) are spaced by one pavement cell (PC) (Stage 6, lower panels). Arrowheads indicate the GMCs or stomatal complexes. Asterisks indicate the PCs that separate stomata in the same cell file. Scale bar=20 µm. (C) The numbers of PCs between two adjacent stomata within the same cell file are often increased in RNAi lines. (D, E) Leaf stomatal density and index of two OsCYCA2;1-RNAi lines and the wild type (WT; n=12). Data represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01). (F–H) Flow cytometric analysis of nuclei in shoot cells. (I) Quantitative analysis of DAPI fluorescence. OsCYCA2;1-RNAi transgenic lines have a higher average 4C DNA content than the WT. For each line, ~10000 cell nuclei were measured. It has been demonstrated that Arabidopsis CYCA2s not only promote cell proliferation but also negatively regulate endocycle onset (Imai et al., 2006; Yoshizumi et al., 2006; Vanneste et al., 2011). Flow cytometric analysis showed that in wild-type rice, only 6% of cells showed a 4C DNA content, whereas most cells were 2C (diploid). However, in OsCYCA2;1-RNAi transgenic rice lines Ri1 and Ri3, the fraction of 4C cells markedly increased to 36% and 18%, respectively. Higher ploidy levels, like in Arabidopsis cyca2 mutants (8C, 16C, 32C), were barely detectable in rice OsCYCA2;1-RNAi plants (Fig. 2F–I). OsCYCA2;1 complements epidermal defects of Arabidopsis cyca2 mutants The Arabidopsis epidermis is an ideal system to identify gene functions in plant development programs and morphogenetic patterns. To identify further the function of OsCYCA2;1 in epidermal development, OsCYCA2;1 coding sequences driven by 35S promoters were transformed into Arabidopsis cyca2;34 mutants. Compared with the wild type, cyca2;34 mutants display enlarged pavement cells and enhanced ploidy levels. Cross-species expression of OsCYCA2;1 (line #7) inhibits the abnormal PC enlargement in cyca2;34 epidermis, to a cell size even smaller than in the wild type (Fig. 3A–D). Moreover, revealed by flow cytometric analysis, expression of OsCYCA2;1 is able to inhibit the high DNA ploidy levels in cyca2;34 mutants (Fig. 3E; Supplementary Fig. S4). This result is consistent with the previous findings that overexpression of CYCA2 genes could restrain endoreduplication in Arabidopsis (Imai et al., 2006; Boudolf et al., 2009). Fig. 3. View largeDownload slide Cross-species expression of OsCYCA2;1 complements the epidermal defects of Arabidopsis cyca2;34 mutants. (A–C) Differential interference contrast micrographs of cotyledon epidermal cells of 14-day-old Arabidopsis seedlings of the Col, cyca2;34, and cyca2;34 harboring 35S:OsCYCA2;1, Line #7. An arrow points to a single guard cell (SGC). Representative pavement cells (PCs) are traced with dashed lines. Scale bar=50 µm. (D) Comparison of PC area (n=30). (E) Proportions of cells with different ploidies. (F and G) Stomatal density and index. The diagonal line-filled box indicates the SGCs. Data in (D, F, G) represent the mean ±SD. Asterisks indicate a significant difference from Col wild-type controls (Student’s t-test, **P<0.01). Fig. 3. View largeDownload slide Cross-species expression of OsCYCA2;1 complements the epidermal defects of Arabidopsis cyca2;34 mutants. (A–C) Differential interference contrast micrographs of cotyledon epidermal cells of 14-day-old Arabidopsis seedlings of the Col, cyca2;34, and cyca2;34 harboring 35S:OsCYCA2;1, Line #7. An arrow points to a single guard cell (SGC). Representative pavement cells (PCs) are traced with dashed lines. Scale bar=50 µm. (D) Comparison of PC area (n=30). (E) Proportions of cells with different ploidies. (F and G) Stomatal density and index. The diagonal line-filled box indicates the SGCs. Data in (D, F, G) represent the mean ±SD. Asterisks indicate a significant difference from Col wild-type controls (Student’s t-test, **P<0.01). In cyca2;34 mutants, ~10% of GMCs failed to divide symmetrically and formed into SGCs (Fig. 3B, arrow). Strikingly, OsCYCA2;1 expression can fully rescue the defective GMC division in cyca2;34 epidermis, suggesting that rice OsCYCA2;1 remains a conserved function in promoting the GMC symmetric divisions. In addition, expression of OsCYCA2;1 in the cyca2;34 mutant background induced formation of excessive stomata, reflected by an increased stomatal density and stomatal index (Fig. 3F, G). In another 35S:OsCYCA2;1 cyca2;34 transgenic line (line #8), the relative transcript level of OsCYCA2;1 is much lower than in line #7; the defective GMC division and reduced stomatal production are partially rescued, indicating that OsCYCA2;1 quantitatively promotes stomatal development depending on its expression level (Supplementary Fig. S5). Taken together, our results of cross-species complement tests demonstrate the conserved abilities of OsCYCA2;1 in limiting cell endoreduplication and PC size, as well as in rescuing cyca2;34 defective asymmetric entry divisions (for stomatal initiation) and symmetric GMC divisions (for guard cell formation), despite OsCYCA2;1 being functionally required only for stomatal entry divisions in rice. OsCYCA2;1 is also required for cell division and differentiation in roots OsCYCA2;1 is preferentially expressed in proliferating tissues. Besides in the base dividing zone (proximal end) of leaves, a higher transcript level of OsCYCA2;1 is found in rice root tips, implying that a similar OsCYCA2;1 regulatory mechanism exists in rice roots (Supplementary Fig. 3B). Therefore, we probed the impact of down-regulated expression of OsCYCA2;1 on root growth. As shown in Fig. 4, the overall growth of shoots and roots in 10-day-old OsCYCA2;1-RNAi transgenic seedlings is much less than in the wild type (Fig. 4A–C). To determine whether the root growth defects arose from a defective cell proliferation, we compared the root longitudinal sections of the wild type and the RNAi line. The shorter meristematic zone in OsCYCA2;1-RNAi is correlated with a considerably fewer number of cells within its meristematic zone (Fig. 4D–F). Similarly, Arabidopsis cyca2;34 mutants exhibit a short meristematic zone and fewer cells than Col wild type. Ectopic expression of OsCYCA2;1 restored the length of and cell number within the meristematic zone to the wild-type level (Fig. 4G–I), supporting that OsCYCA2;1 is an evolutionarily conserved regulator that is required for cell proliferation in roots. Fig. 4. View largeDownload slide Suppression of CYCA2 expression causes defective cell proliferation within the meristematic zone of roots. (A) Ten-day-old OsCYCA2;1-RNAi and wild-type (WT) rice seedlings. Scale bar=1 cm. (B, C) The length of shoots and primary roots (n=24). (D) Longitudinal sections of the primary root tips. Scale bar=100 µm. (E, F) Length and cell number of the meristematic zone in roots (n=20). (G) Propidium iodide-stained images of Arabidopsis root tips. Scale bar=100 µm. (H, I) Length and cell number of the meristematic zone in Arabidopsis roots (n=20). Double-headed arrows in (D, G) indicate the extent of the meristematic zone. Data in (B, C, E, F, H, I) represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01, *P<0.05). (This figure is available in colour at JXB online.) Fig. 4. View largeDownload slide Suppression of CYCA2 expression causes defective cell proliferation within the meristematic zone of roots. (A) Ten-day-old OsCYCA2;1-RNAi and wild-type (WT) rice seedlings. Scale bar=1 cm. (B, C) The length of shoots and primary roots (n=24). (D) Longitudinal sections of the primary root tips. Scale bar=100 µm. (E, F) Length and cell number of the meristematic zone in roots (n=20). (G) Propidium iodide-stained images of Arabidopsis root tips. Scale bar=100 µm. (H, I) Length and cell number of the meristematic zone in Arabidopsis roots (n=20). Double-headed arrows in (D, G) indicate the extent of the meristematic zone. Data in (B, C, E, F, H, I) represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01, *P<0.05). (This figure is available in colour at JXB online.) By means of flow cytometry approaches, we found that, in contrast to the 6% of 4C cells in the wild type, the fractions of cells with 4C DNA content in OsCYCA2;1-RNAi lines Ri1 and Ri3 are dramatically increased to 32% and 15%, respectively (Fig. 5A–D). Moreover, the relative expression levels of an S-phase-specific gene, PCNA, and a M-phase cyclin gene, CYCB2;1, were suppressed in OsCYCA2;1-RNAi plants (Fig. 5E). Consistently, epidermal cells in the maturation zone of OsCYCA2;1-RNAi roots showed stronger DAPI fluorescent signals than in the wild type (Fig. 5F–H). Quantitative analysis of the DAPI fluorescence intensities further confirmed that a higher DNA level (~2-fold) is present in OsCYCA2;1-RNAi roots (Fig. 5I). The higher DNA content in OsCYCA2;1-RNAi root cells might be due to delayed or arrested G2 to M transition, a result supporting the idea that OsCYCA2;1 is required for cell mitosis. Fig. 5. View largeDownload slide OsCYCA2;1 is required for rice root cell mitosis. (A–C) Profiles of distribution of cells with different DNA content after flow cytometric analysis. Roots of OsCYCA2;1-RNAi lines Ri1 (B) and Ri3 (C) have more 4C cells than wild-type (WT) roots (A). (D) Quantitative analysis of the cell DNA ploidy levels. (E) Relative expression levels of PCNA and CYCB2;1 in OsCYCA2;1-RNAi lines and WT roots. (F–H) DAPI staining of the epidermal cells in the maturation zone of the WT (F), OsCYCA2;1-RNAi line Ri1 (G), and Ri3 (H). Scale bar=50 µm. (I) Quantitative analysis of DAPI fluorescence revealed that OsCYCA2;1-RNAi transgenic lines have a higher average DNA content than the WT. Data represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01). (This figure is available in colour at JXB online.) Fig. 5. View largeDownload slide OsCYCA2;1 is required for rice root cell mitosis. (A–C) Profiles of distribution of cells with different DNA content after flow cytometric analysis. Roots of OsCYCA2;1-RNAi lines Ri1 (B) and Ri3 (C) have more 4C cells than wild-type (WT) roots (A). (D) Quantitative analysis of the cell DNA ploidy levels. (E) Relative expression levels of PCNA and CYCB2;1 in OsCYCA2;1-RNAi lines and WT roots. (F–H) DAPI staining of the epidermal cells in the maturation zone of the WT (F), OsCYCA2;1-RNAi line Ri1 (G), and Ri3 (H). Scale bar=50 µm. (I) Quantitative analysis of DAPI fluorescence revealed that OsCYCA2;1-RNAi transgenic lines have a higher average DNA content than the WT. Data represent the mean ±SD. Asterisks indicate a significant difference from WT controls (Student’s t-test, **P<0.01). (This figure is available in colour at JXB online.) OsCYCA2;1 conservatively interacts with OsCDKB1;1 CYCA2s play their regulatory roles through interacting with multiple CDKs, such as by forming CYCA2;3–CDKB1;1 or CYCA2;3–CDKA;1 protein complexes. Arabidopsis AtCYCA2;3 interacts with AtCDKB1;1 to form a functional complex which promotes the formation of a two-celled stoma and prevents entry into the endocycle program (Boudolf et al., 2009; Vanneste et al., 2011). According to the sequence blasting results in the rice genome, Os01g67160 encodes the putative OsCDKB1;1. The deduced amino acid sequence of OsCDKB1;1 shares 88.5% sequence identity with the Arabidopsis CDKB1s. A B1-type-specific cyclin interaction motif ‘PPTALRE’ is highly conserved in rice OsCDKB1;1 (Supplementary Fig. S6). Yeast two-hybrid assays showed that OsCYCA2;1 can interact with OsCDKB1;1 (Fig. 6A). Consistently, pull-down assays verified the direct protein interaction between OsCYCA2;1 and OsCDKB1;1 (Fig. 6B). To determine the subcellular localization, OsCYCA2;1 or OsCDKB1;1 fused with GFP were transiently expressed in tobacco (N. benthamiana) leaves. The fluorescent signals from OsCYCA2;1–GFP were exclusively detected in nuclei, while OsCDKB1;1–GFP was found in both the cytoplasm and nuclei (Fig. 6C). BiFC analysis confirmed that OsCYCA2;1 directly interacts with OsCDKB1;1 in nuclei (Fig. 6D). These results suggest that OsCYCA2;1 may act as a conserved activator regulating the activity of OsCDKB1;1 kinase in rice. Fig. 6. View largeDownload slide OsCYCA2;1 directly interacts with OsCDKB1;1. (A) Yeast two-hybrid assay. (B) Protein pull-down assay. (C) Transient expression of OsCDKB1;1–GFP and OsCYCA2;1–GFP in tobacco leaves. Scale bar=20 µm. (D) Bimolecular fluorescence complementation assay shows that OsCDKB1;1 interacts with OsCYCA2;1 in nuclei. Scale bar=20 µm. (This figure is available in colour at JXB online.) Fig. 6. View largeDownload slide OsCYCA2;1 directly interacts with OsCDKB1;1. (A) Yeast two-hybrid assay. (B) Protein pull-down assay. (C) Transient expression of OsCDKB1;1–GFP and OsCYCA2;1–GFP in tobacco leaves. Scale bar=20 µm. (D) Bimolecular fluorescence complementation assay shows that OsCDKB1;1 interacts with OsCYCA2;1 in nuclei. Scale bar=20 µm. (This figure is available in colour at JXB online.) Suppression of OsCDKB1;1 has no obvious effects on rice development To determine whether OsCDKB1;1, like its partner OsCYCA2;1, is required for rice development, we generated and selected two OsCDKB1;1-RNAi transgenic lines, Ri2 and Ri3, in which OsCDKB1;1 transcript levels were significantly suppressed (Supplementary Fig. S7A). However, the overall growth of these two transgenic lines is comparable with that of the untransformed controls (Supplementary Fig. S7B–D). Longitudinal sections of roots demonstrate that the suppression of OsCDKB1;1 has no significant impact on cell numbers of the root meristematic zone (Supplementary Fig. S7E–G). In addition, we found that neither the stomatal production (stomatal density) nor the stomatal complex morphology has been affected in OsCDKB1;1-RNAi transgenic lines (Supplementary Fig. S7H–J). Flow cytometric assays also indicate that DNA ploidy levels were not changed in either the roots or shoots of OsCDKB1;1-RNAi (Supplementary Fig. S8A–H). Consistent with this, the expression of S-phase PCNA and M-phase cyclin CYCB2;1 was not different between wild-type and OsCDKB1;1-RNAi transgenic plants (Supplementary Fig. S8I). Taken together, it seems that cell division was not interrupted by down-regulation of the OsCDKB1;1 transcript level in transgenic rice, though we could not exclude the possibility that the remaining activity of OsCDKB1;1 protein is sufficient to function. OsCYCA2;1 and OsCDKB1;1 enable complementation of Arabidopsis cdkb1;1 1;2 Arabidopsis cdkb1;1 1;2 mutants, like the cyca2;34 mutants, display a decreased stomatal production, formation of SGCs, enlarged PCs, and increased cell ploidy levels (Boudolf et al., 2004a; Xie et al., 2010). Introduction of OsCDKB1;1 fully complements the impaired GMC division in cdkb1;1 1;2, and restores stomatal production, indicating that OsCDKB1;1 has the ability to promote both symmetric and asymmetric division. Meanwhile, expression of OsCKDB1;1 could efficiently prevent the occurrence of enlarged PCs and increased DNA ploidy levels in cdkb1;1 1;2 mutants (Fig. 7; Supplementary Figs S9, S10). Fig. 7. View largeDownload slide Ectopic expression of OsCYCA2;1 and OsCDKB1;1 complements Arabidopsis cdkb1;1 1;2 mutant phenotypes. (A–D) DIC images of the epidermis of 14-day-old Arabidopsis cotyledons. Arrows indicate the formation of SGCs. Representative PCs are traced with dashed lines. Scale bar=50 µm. (E–G) Comparison of stomatal density, stomatal index, and area of PCs from the cotyledons. The diagonal line-filled box indicates the fraction of SGCs. Data represent the mean ±SD (n=24). Asterisks indicate a significant difference from Col after Student’s t-test, **P<0.01. (H) Proportions of cells with different ploidies. (I–L) Results from the flow cytometric analysis; ~10000 cell nuclei were measured for each sample. Fig. 7. View largeDownload slide Ectopic expression of OsCYCA2;1 and OsCDKB1;1 complements Arabidopsis cdkb1;1 1;2 mutant phenotypes. (A–D) DIC images of the epidermis of 14-day-old Arabidopsis cotyledons. Arrows indicate the formation of SGCs. Representative PCs are traced with dashed lines. Scale bar=50 µm. (E–G) Comparison of stomatal density, stomatal index, and area of PCs from the cotyledons. The diagonal line-filled box indicates the fraction of SGCs. Data represent the mean ±SD (n=24). Asterisks indicate a significant difference from Col after Student’s t-test, **P<0.01. (H) Proportions of cells with different ploidies. (I–L) Results from the flow cytometric analysis; ~10000 cell nuclei were measured for each sample. Interestingly, the defective stomatal production, impaired GMC division, and abnormal cell enlargement and DNA levels in cdkb1;1 1;2 could be partially rescued by overexpression of OsCYCA2;1 (Fig. 7; Supplementary Figs S9, S10). It is therefore possible that OsCDKB1;1 and OsCYCA2;1 have evolved from the common ancestor genes with Arabidopsis AtCDKB1 genes and AtCYCA2 genes, even though the developmental pathways of the two species have been diverged. Discussion The control of cell division and differentiation is the core of the development and morphogenesis of multicellular organisms. Cyclins, known as conserved activators for the activity of CDKs, play a crucial regulatory role in cell cycle progression in diverse species. The functional pathway of CYCA2s and CDKB1s has been well investigated in the model plant Arabidopsis (Boudolf et al., 2004b, 2009; Imai et al., 2006; Xie et al., 2010; Vanneste et al., 2011; Yang et al., 2014). In this study, we generated RNAi transgenic rice lines and performed cross-species complement tests to explore the function of the single rice A2-type cyclin, OsCYCA2;1, as well the single rice B1-type CDK, OsCDKB1;1. Cross-species expression of OsCYCA2;1 or OsCDKB1;1 enables rescue of the defective asymmetric entry divisions for stomatal initiation and GMC symmetric divisions for GC production in Arabidopsis cyca2;34 and/or cdkb1;1 1;2 mutants, suggesting that both OsCYCA2;1 and OsCDKB1;1 might have evolved from the common ancestor genes with Arabidopsis. In Arabidopsis, asymmetric divisions generated the early stomatal precursor cells, meristemoids. Meristemoids then differentiate into GMCs after a cell fate change. In grasses, GMCs are created directly by stomata initiating asymmetric divisions (entry division) without the prior precursor stage of meristemoid. Orthologs of Arabidopsis stomatal transcriptional regulators SPCH, MUTE, FAMA, ICE1, and SCRM2 have been identified in grasses (Liu et al., 2009; Vatén and Bergmann, 2012; Ran et al., 2013; Chen et al., 2017). Instead of a single copy in Arabidopsis, the rice genome has duplicated SPCH genes, OsSPCH1 and OsSPCH2. Similar to the weak allele of Arabidopsis spch, the rice mutant osspch2 exhibits a reduced number of stomata (Liu et al., 2009). In Arabidopsis, SPCH heterodimerizes with SCRM/ICE1 or AtSCRM2 to promote stomatal lineage initiation (Kanaoka et al., 2008; Horst et al., 2015). In contrast, in the grass B. distachyon, BdICE1 and BdSCRM2 show a functional diversity in regulating stomatal pattern and morphology (Raissig et al., 2016). It is already known that Arabidopsis SPCH activity or stability is modulated by multiple kinases, including MPKs, GSK3/BIN2, and CDKs (Lampard et al., 2009; Gudesblat et al., 2012; Kim et al., 2012; Le et al., 2014). Phosphorylation of Ser186 of SPCH, which might be the target residue of CDKs, positively regulates stomata production (Yang et al., 2015). Thus, it will be interesting to establish if there is a conserved regulatory mechanism between CDK–cyclin and SPCH-ICE1/SCRM2 in grasses. Besides the involvement in stomatal initiation, Arabidopsis AtCYCA2s and CDKB1s are synergistically required for the GMC symmetric division that is a prerequisite for the final stomatal development (Vanneste et al., 2011). Arabidopsis and rice share common GMC–GC processing; GMCs divide symmetrically to produce the paired GCs of stomata, though the GC shapes are distinct. Thus, a role for OsCDKB1–OsCYCA2 in rice GMC divisions has been highly expected. However, suppression of OsCYCA2;1 transcription in rice by RNAi does not affect the rice GMC symmetric division. It has been identified that transcription of CDKB1;1, CYCA2;3, and CDKA;1 in Arabidopsis is repressed by FOUR LIPS(FLP)/MYB88 MYB transcription factors during the GMC–GC transition stages (Xie et al., 2010; Vanneste et al., 2011; Yang et al., 2014). FAMA, like FLP/MYB, also binds to the CDKB1;1 promoter (Hachez et al., 2011) to limit the GMC divisions to one. In contrast to the tumor-like phenotype in Arabidopsis fama-1 mutants, the loss-of-function rice allele osfama-1 did not undergo excessive division except the appearance of misshaped GCs and showing a lack of stomatal pores (Liu et al., 2009). These observations suggest that GMC–GC differentiation is uncoupled from GMC division, in which the putative downstream FAMA/FLP/MYB88, CDKB1;1, and CYCA2;1 are not essential. According to the phylogenetic analysis, CYCA2 and CDKB1 widely exist in diverse plant species, both in plants bearing stomata and in plants lacking stomata, indicating that CYCA2 and CDKB1 might function as fundamental regulators of the mitotic cell cycle, as well as outside stomatal development. High expression of OsCYCA2;1 is associated with a high activity of cell proliferation, such as in the proximal end of leaves or root tips (Supplementary Fig. S3B). Endoreduplication often occurs in cell types that undergo specialized differentiation. In Arabidopsis, the highly differentiated epidermal cells, such as mature PCs and trichomes, usually undergo multiple rounds of DNA replication without mitosis, resulting in polyploid cells (Burssens et al., 2000). In contrast to the differentiated cells in Arabidopsis, polyploid cells in rice can only be found in the endosperm (Sabelli and Larkins, 2009). In Drosophila, it has been reported that cyclin A is one of the key components of chromosomal DNA replication that prevents re-initiation of DNA replication. Overexpression of Drosophila cyclin A caused a reduction in ploidy levels and inhibition of the endocycle (Hayashi and Yamaguchi, 1999). Here we found that the fraction of 4C cells remarkably increased in OsCYCA2;1-RNAi transgenic plants, while most cells keep a 2C DNA content. However, the expression levels of an S-phase gene PCNA and a M-phase gene CYCB2;1 were suppressed in OsCYCA2;1-RNAi plants. Therefore, we speculated that the increase of 4C cells might be caused by the arrested G2 to M transition, similar to the observation in the rice knockdown lines of OsCDKB2;1 (Endo et al., 2012). However, the OsCDKB1;1-RNAi transgenic rice plants, in which OsCDKB1;1 transcript levels were significantly decreased, display phenotypes comparable with wild-type rice seedlings regarding the stomatal density, root cell division, and DNA content. CDKBs are plant-specific cyclin-dependent kinases that can be subdivided into two groups according to the different cyclin-binding motifs, namely ‘PPTALRE’ in CDKB1s and ‘PPTTLRE’ in CDKB2s (Joubès et al., 2001). In Arabidopsis, each CDKB1 and CDKB2 subgroup contains two members (Vandepoele et al., 2002). It has been predicted that the rice genome has a single CDKB1 gene and a single CDKB2 gene, encoding OsCDKB1;1 and OsCDKB2;1, respectively (Supplementary Fig. S11). However, the amino acid sequence alignment revealed that rice OsCDKB1;1 and OsCDKB2;1 share the same ‘PPTALRE’ cyclin-binding motif (Supplementary Fig. S12). Expression of OsCDKB2;1 has been detected in the dividing region of the rice root apex (Umeda et al., 1999a). Transcription of rice OsCDKB2;1 is abundant during the G2 to M phase. Knockdown of the OsCDKB2;1 gene in rice induces an increase of the 4C cell population (Umeda et al., 1999b; Endo et al., 2012). In addition, OsCDKB2;1 promotes cell division in the root meristem probably through the association with OsCYCB2s (Lee et al., 2003). Thus, we cannot rule out that OsCDKB1;1 might function redundantly with OsCDKB2;1, such as forming active CDK–cyclin complexes via binding to the same type cyclins (i.e. OsCYCA2;1). Previous in situ hybridization results showed that both OsCDKA;1 and OsCDKA;2 are expressed in dividing root cells of rice (Umeda et al., 1999b). Thus, further characterization of rice CDK–cyclin pairing and activity can help to reveal the regulatory mechanisms of cell division and differentiation during rice development. Supplementary data Supplementary data are available at JXB online. Table S1. List of primers used in this study. Fig. S1. Amino acid sequence comparison of A2-type cyclins from rice and Arabidopsis. Fig. S2. In contrast to dicot Arabidopsis, only one or two copies of genes encoding CYCA2 are found in monocot grasses. Fig. S3. Relative expression of OsCYCA2;1 in rice RNAi transgenic plants and in different tissues of wild-type plants. Fig. S4. Overexpression of rice OsCYCA2;1 suppresses the enhanced endoreduplication levels in Arabidopsis cyca2;34. Fig. S5. Correlation between stomatal phenotypes and OsCYCA2;1 expression levels in Arabidopsis cyca2;34 mutants harboring OsCYCA2;1. Fig. S6. Comparison of the amino acid sequence of OsCDKB1;1 with that of Arabidopsis CDKB1;1 and CDKB1;2. Fig. S7. Suppression of OsCDKB1;1 has no obvious impact on rice root and stomatal development. Fig. S8. Suppression of OsCDKB1;1 has no obvious impact on the distribution of DNA ploidy. Fig. S9. Expression analysis of OsCDKB1;1-OE and OsCYCA2;1-OE transgenic plants in Arabidopsis cdkb1 mutants. Fig. S10. 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Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Experimental BotanyOxford University Press

Published: Apr 26, 2018

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