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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 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 mor - phological and developmental characteristics enable grass stomata to respond to environmental changes more effi- ciently. 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 defec- tive 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. Keywords: Cyclin-dependent kinases, cell differentiation, cell division, guard mother cells, cyclin, rice, stomata. Introduction Stomata are microscopic valves on aerial surfaces of all distribution pattern and morphology are highly diversified in land plants regulating the shoot–atmosphere gas exchange. different plants, stomata arise in the epidermis after a series of Paleobotanical analyses revealed that stomata originated ~400 cell divisions, cell fate changes, and cell shape controls. In the million years ago, a key evolutionary innovation formed in the past decades, results of molecular genetic studies demonstrated early palaeozoic era (Raven, 2002). Despite the fact that the that stomatal development is an accessible system to reveal the Abbreviations: CYCA2, CYCLIN A2; CDKB1, CYCLIN-DEPENDENT KINASE B1; GC, guard cell; GMC, guard mother cell; PC, pavement cell; SGC, single guard cell. © The Author(s) 2018. 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. Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3588 | Qu et al. evolution of genes and signals involved in plant development GMC symmetric division in stomatal development. In addi- (Vatén and Bergmann, 2012; Ran et  al., 2013; Chater et  al., tion to the function in promoting mitosis, AtCYCA2s form 2017; Qu et al., 2017). functional complexes with CDKs to modulate the cell cycle In the dicot model plant Arabidopsis, the earliest stomatal transition from the mitotic cycle to the endocycle. Genetic precursor, the meristemoid mother cell (MMC), divides asym- suppression of AtCYCA2 or AtCDKB1 results in enhanced metrically (stomatal entry divisions) producing a smaller cell, ploidy levels and enlarged pavement cells (PCs;V anneste et al., the meristemoid, as well as a larger sister cell, the stomatal lin- 2011). Co-expression of CYCA2;3 and CDKB1;1 induces eage ground cell (SLGC). Meristemoids normally undergo ectopic cell divisions, limits endoreduplication, and inhibits cell an additional 1–2 rounds of asymmetric divisions (amplify- growth (Boudolf et al., 2009). ing divisions) to generate new meristemoids before convert- There are at least 49 putative genes predicted to encode rice ing into guard mother cells (GMCs). SLGCs can also undergo cyclins, which were classified into nine types based on evolution- asymmetric divisions (spacing divisions) to generate satellite ary relationships. Eight of these nine types are common between meristemoids. Meristemoids differentiate into GMCs after cell rice and Arabidopsis (Umeda et al., 1999a; Cooper et al., 2003; fate change. Then, GMCs divide symmetrically to produce La et al., 2006). The existence of numerous cyclins implies their paired young guard cells (GCs). During the final stage of sto- diverse regulatory roles in modulating CDK activities during matal development, GCs undergo cell differentiation, morpho- rice development and adaption in response to environmental genesis, and pore formation to form functional stomatal units changes (Cooper et al., 2003; Huang et al., 2008). For example, (Bergmann and Sack, 2007). rice B2-type cyclins, OsCycB2;1 and OsCycB2;2, promote root In contrast to the scattered pattern in dicot Arabidopsis cell divisions through an association with OsCDKB2;1 (Lee leaves, monocot grass stomata are arranged within linear cell et al., 2003). OsCycH;1 specifically binds to R2 and positively files that parallel the growth axis of the leaf. Stomatal lineage controls CDK and CTD kinase activities to adjust the rate of cell cells initiate at the base of the leaf, and divide asymmetrically to proliferation (Fabian-Marwedel et al., 2002). produce two daughter cells, a GMC, and a larger sister cell. At In this study, we examine the function of the rice single the final stage, GMCs divide symmetrically, producing paired A2-type cyclin OsCYCA2;1 and OsCDKB1;1 in rice develop- dumbbell-shaped GCs. The stomatal subsidiary cells are pro- ment. Our results demonstrate a requirement for OsCYCA2;1 duced from cell files flanking the stomatal lineage after asym- for stomatal and root development. In contrast to its homolog metric divisions (Franks and Farquhar, 2007; Liu et  al., 2009; in Arabidopsis, OsCYCA2;1 is exclusively required for the Serna, 2011; Raissig et al., 2016). asymmetric entry divisions to produce GMCs at the early stage Cyclins form complexes with specific cyclin-dependent of stomatal development. In addition, combined with phylo- kinases (CDKs) to co-ordinate the cell proliferation and dif- genetic analyses, we are providing new clues for further reveal- ferentiation during the development of multicellular organisms ing the evolutionary correlation between cell cycle genes and (Swenson et  al., 1986; Obaya and Sedivy, 2002). Cyclins, act- developmental pathways. ing as the regulator of CDK activity, contribute to the subcel- lular localization, substrate specificity, and protein stability of the CDK–cyclin complexes (Dewitte and Murray, 2003; Imai Materials and methods et  al., 2006; Boudolf et  al., 2009; Boruc et  al., 2010). A-type Plant materials and growth conditions cyclins, known as mitotic cyclins, are essential for the mitotic The Col-0 ecotype of Arabidopsis thaliana L. was used as the wild-type cell cycle. In contrast to animals, plants encode a large family control in the Arabidopsis study. The cdkb1;1 1;2 double mutants were of A-type cyclins that have been classed into A1, A2, and A3 confirmed by PCR-based approaches (Xie et  al., 2010). The cyca2;34 groups (Vandepoele et  al., 2002; Dewitte and Murray, 2003; double mutants were provided by Steffen Vanneste and Tom Beeckman Wang et al., 2004). (Vanneste et al., 2011). Seeds were surface sterilized (40 s) in an aqueous The Arabidopsis genome has four genes encoding A2-type 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 cyclins. AtCYCA2 genes display tissue- and cell type-specific Murashige and Skoog (MS) medium supplemented with 0.8% agar and and overlapping expression patterns, such as in vascular systems 1% sucrose. Plants were grown in a controlled temperature and pho- and stomatal lineage cells, which are associated with their redun- toperiod chamber at 22  ±  2  °C and 16  h/8  h light/dark illumination dant functions during plant development (Burssens et al., 2000; cycles. Oryza sativa L. spp. japonica cultivar Zhonghua 11 was used as the Imai et  al., 2006; Vanneste et  al., 2011; Donner and Scarpella, 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 con- 2013). Mutants of AtCYCA2 genes frequently form unpaired trolled growth chamber with 30 °C/22 °C day/night temperature cycles, single guard cells (SGCs), a similar defect of the terminal GMC 12 h/12 h light/dark illumination cycles, and 60–70% relative humidity. division to that also observed in mutants of AtCDKB1 or AtCDKA;1 genes (Boudolf et al., 2004b; Vanneste et al., 2011; Plasmid construction and generation of transgenic plants Yang et  al., 2014). Overexpression of AtCYCA2:3 as well as To obtain the construct of gene overexpression, cDNA of OsCDKB1;1 AtCDKA;1 at the late stage of stomatal development induced or OsCYCA2;1 was cloned into the pH7WG2D.1 vector by using gate- excessive GC subdivisions (Yang et  al., 2014). Arabidopsis way technology and LR Clonase™ II Enzyme Mix (Invitrogen). The cdkb1;1 1;2 double mutants and 35S:CDKB1;1.N161 domi- recombinant plasmids were confirmed by DNA sequencing before nant negative plants displayed decreased stomatal production the transformation into Arabidopsis wild type and mutants. To gener- and formation of SGCs, indicating that the activity of CDKB1 ate RNAi transgenic plants against OsCDKB1;1 and OsCYCA2;1, the conserved sequences from base pair 530 to 695 of OsCDKB1;1 cDNA is required for both meristemoid asymmetric division and Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3589 and 747 to 979 of OsCYCA2;1 cDNA were amplified and cloned into Flow cytometric analysis pTCK303 vector, respectively. These constructs were electroporated into About 20–50  mg of fresh tissue were cut into 2–4  mm fragments and Agrobacterium tumefaciens EHA105 and transformed into rice Zhonghua then chopped immediately using a razor blade in 1  ml of Galbraith’s 11 (Chen et  al., 2011). T seeds were collected to screen positive trans- buffer (45  mM MgCl ·6H O, 30  mM sodium citrate, 20  mM MOPS, 2 2 −1 genic plants by using 50 µg l hygromycin B (Roche). Real-time quan- 0.1% Triton X-100, pH 7.0). The cell culture was collected by gentle titative PCR (RT-qPCR) was conducted to confirm the expression level pipetting and filtered through a cell strainer. The samples were stained −1 of target genes in transgenic plants. The primer sequences used in this with 2 µg ml DAPI in an ice bath for 30 min before the analysis using study are listed in Supplementary Table S1 at JXB online. a MoFlo-XDP flow cytometer (Beckman) (Dolezel et al., 2007). A total of ~10 000 nuclei were measured for each sample. Real-time quantitative PCR analysis Rice seedlings were harvested and immediately ground in liquid nitro- Root semi-thin sections gen, and the total RNA was isolated using TRNzol reagent (http://www. About 1 cm long primary root tips from 6-day-old rice seedlings were tiangen.com). The first-strand cDNA was synthesized using a Promega harvested and immersed in cold formaldehyde solution. The samples Reverse Transcription kit (http://www.promega.com). RT-qPCRs were were subjected to a vacuum for 10 min and placed at 4 °C overnight. The performed by using SYBR Premix Ex Taq™ (TaKaRa) with a Corbett materials were washed with 0.1 M PBS (pH 7.2) four times and were RG3000. The OsACTIN2 gene was used as an internal control. The fixed in 1% osmic acid for 1 h, followed by a series of dehydration steps, primer sequences are listed in Supplementary Table S1. 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). Yeast two-hybrid assay Permeation was performed by using a series of 2:1, 1:1, and 1:2 (v/v) of The full-length cDNA sequences of OsCYCA2;1 and OsCDKB1;1 were acetone and epoxy (SPURR) mixture solution. Each step lasted for 3 h. amplified using the primers listed in Supplementary Table S1 and cloned After adding pure epoxy for 8 h, samples were embedded and polymer- into pGBKT7 and pGADT7 vectors (Clontech), respectively. These con- ized at 60 °C for 24 h. Semi-thin sections (thickness 1 µm) were obtained structs were transformed into Saccharomyces cerevisiae yeast strain AH109 by using a Leica UC7 microtome. Before imaging, sections were stained and selected on SD/-Leu-Trp or SD/-Leu-Trp-His-Ade plates. X-Gal with 0.1% toluidine blue O. activity was then detected. Results Bimolecular fluorescence complementation assay For bimolecular fluorescence complementation (BiFC) assays, the full- Evolutionary analysis of A2 cyclins in plants length cDNAs of OsCYCA2;1 and OsCDKB1;1 were cloned into pSPYCE-35S and pSPYNE-35S vectors, respectively (Walter et al., 2004). Phylogenetic analysis indicates that homologs of A2-type cyclin These constructs were transformed into A.  tumefaciens EH105 and co- are found in lineages that diverged early in the evolution of land injected into tobacco (Nicotiana benthamiana). Images were taken after 3 plants, before the appearance of stomata. For example, the uni- d using a laser scanning confocal microscope (FV1000-MPE, Olympus). cellular green alga Coccomyxa subellipsoidea has a CYCA2 gene in its genome. In the non-vascular land plant moss Physcomitrella Pull-down assay patens, stomata are exclusively found on the diploid sporophyte The OsCYCA2;1 and OsCDKB1;1 sequences were cloned into pET- (Chater et al., 2016); there are six putative orthologs of CYCA2. 28a and pGEX4T-1 vectors, respectively. The OsCYCA2;1-pET-28a and In the vascular dicot plants Arabidopsis, soybean (Glycine max), OsCDKB1;1-pGEX4T-1 constructs were transformed into the BL21 strain and alfalfa (Medicago truncatula), the number of CYCA2 genes of Escherichia coli. The transformed strains were grown to OD =0.5 under was four, six, and four, respectively (Fig.  1). In contrast, the 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 rice genome contains only one copy of the CYCA2 gene, 20 h. The harvested strains (5000 rpm, 10 min, 4 °C) were re-suspended Os012g31810, which is predicted to encode OsCYCA2;1 pro- with ice-cold phosphate-buffered saline (PBS) and lysed by sonication. The tein consisting of 490 amino acid residues. Multiple sequence lysate was centrifuged at 10 000 rpm for 60 min and the supernatant was alignment reveals that OsCYCA2;1 shows 40.5% amino acid collected. The glutathione S-transferase (GST)–OsCDKB1;1 supernatant sequence identity with Arabidopsis CYCA2s, and contains a was loaded on glutathione–Sepharose (GE Healthcare) and washed with PBS. The GST–OsCDKB1;1 fusion protein on glutathione–Sepharose was CDK-binding cyclin box, which is highly conserved among incubated with the His-OsCYCA2;1 supernatant at 4 °C for 2 h. Then mitotic cyclins (Supplementary Fig. S1) (Umeda et al., 1999a). the glutathione–Sepharose was washed with PBS and eluted with 10 mM Interestingly, similar to rice, Brachypodium stacei, Brachypodium reduced glutathione elution buffer. The samples were loaded on a 12% distachyon, Zea mays (Fig. 1), and many other monocot grasses, SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride Hordeum vulgare, Oropetium thomaeum, Panicum hallii, Sorghum (PVDF) membrane (Millipore) by using a semi-dry blotting system (Bio- Rad), and then incubated with anti-His6 monoclonal antibodies followed bicolor, Setaria italica, and Setaria viridis, only have 1–2 CYCA2 by horseradish peroxidase (HRP)-conjugated anti-mouse antibodies. The genes (Supplementary Fig. S2). color reaction was performed using the Pro-Light HRP Kit (Tiangen). The seagrass Zostera marina belongs to basal monocots that Signals were exposed to X-ray films and developed. returned to the sea. The absence of stomata in Z.  marina is consistent with the evolutionary loss of entire genes that DAPI staining and DNA content measurement are required for stomatal development (Olsen et  al., 2016). Ten-day-old rice roots were fixed in a mixture of 3:1 (v/v) ethanol and However, like the above grass plants, Z.  marina possesses two acetic acid for 30 min, then rinsed with distilled water. After staining for CYCA2 genes, suggesting that the A2-type cyclin is funda- −1 30 min with 2 µg ml DAPI (Roche) in a staining solution (0.1 M sodium mentally important for plant growth and development, and phosphate, 1 mM EDTA, 0.1% Triton X-100, pH 7.0), roots were photo- is not solely linked to stomatal development. The low num- graphed by a fluorescence microscope. The relative fluorescence intensities ber of CYCA2 genes in grasses indicates that CYCA2 gene were measured using ImageJ software (http://imagej.nih.gov/ij/). Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3590 | Qu et al. Fig. 1. 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. duplication might not be necessary, which is associated with are produced by the cells in the neighboring cell files. The ter- their unique developmental pathways and morphogenesis. minal division of GMC produces a pair of immature GCs (Stage 5, middle panel of Fig. 2A). Within the wild-type stomatal line- age cell files, stomatal complexes are spaced by one lobbed PC Requirement of OsCYCA2;1 for stomatal initiation (Stage 6, lower panel of Fig.  2A). However, in OsCYCA2;1- To elucidate the function of A2-type cyclin in rice development, RNAi transgenic plants, more than two spacing cells were often RNAi transgenic rice lines targeting OsCYCA2;1 were gen- observed between two neighboring GMCs/stomata within the erated. Transcript levels of OsCYCA2;1 in two lines, Ri1 and same cell file (Fig. 2B , C), leading to a decreased stomatal density Ri3, were suppressed to 28% and 61%, respectively, in relation to and stomatal index (Fig. 2D, E). the level in wild-type rice seedlings (Supplementary Fig. S3A). Mutations of Arabidopsis CYCA2 genes caused a failure of In rice leaf epidermis, stomata form within the stomatal line- GMC division, leading to the formation of aberrant stomatal age files following a gradual base to tip maturation pattern; the units (SGCs) (Vanneste et al., 2011). However, the structure and developing stomata can only be found at the proximal end (base) morphology of mature stomata in OsCYCA2;1-RNAi rice of the leaf. Unlike in Arabidopsis, GMCs in rice are produced transgenic plants are indistinguishable from those of wild-type directly by asymmetric entry divisions without the precursor stomata, indicating that the subsequent GMC symmetric divi- stage of the meristemoid. Each undifferentiated cell close to sions as well the subsidiary cell asymmetric divisions are not the base of the leaf divides asymmetrically and generates one interrupted by the suppression of OsCYCA2;1 (lower panels smaller GMC and one larger sister cell (Stage 2, upper panel of in Fig. 2A and B). Taken together, the above observations indi- Fig.  2A). Subsidiary mother cells (SMCs) flanking the GMCs cate that OsCYCA2;1 is essentially required for the asymmetric Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3591 Fig. 2. 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, ~10 000 cell nuclei were measured. entry division during stomatal initiation at the early stage of increased to 36% and 18%, respectively. Higher ploidy levels, stomatal development, but not for the terminal GMC symmet- like in Arabidopsis cyca2 mutants (8C, 16C, 32C), were barely ric divisions and subsidiary cell asymmetric divisions. detectable in rice OsCYCA2;1-RNAi plants (Fig. 2F–I). It has been demonstrated that Arabidopsis CYCA2s not only promote cell proliferation but also negatively regulate endocy- OsCYCA2;1 complements epidermal defects of cle onset (Imai et al., 2006; Yoshizumi et al., 2006; Vanneste et al., Arabidopsis cyca2 mutants 2011). Flow cytometric analysis showed that in wild-type rice, The Arabidopsis epidermis is an ideal system to identify gene only 6% of cells showed a 4C DNA content, whereas most cells functions in plant development programs and morphogenetic were 2C (diploid). However, in OsCYCA2;1-RNAi trans- patterns. To identify further the function of OsCYCA2;1 genic rice lines Ri1 and Ri3, the fraction of 4C cells markedly Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3592 | Qu et al. in epidermal development, OsCYCA2;1 coding sequences cross-species complement tests demonstrate the conserved driven by 35S promoters were transformed into Arabidopsis abilities of OsCYCA2;1 in limiting cell endoreduplication and cyca2;34 mutants. Compared with the wild type, cyca2;34 PC size, as well as in rescuing cyca2;34 defective asymmetric mutants display enlarged pavement cells and enhanced ploidy entry divisions (for stomatal initiation) and symmetric GMC levels. Cross-species expression of OsCYCA2;1 (line #7) divisions (for guard cell formation), despite OsCYCA2;1 being inhibits the abnormal PC enlargement in cyca2;34 epidermis, functionally required only for stomatal entry divisions in rice. 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 also required for cell division and OsCYCA2;1 is able to inhibit the high DNA ploidy levels in differentiation in roots cyca2;34 mutants (Fig. 3E; Supplementary Fig. S4). This result OsCYCA2;1 is preferentially expressed in proliferating tissues. is consistent with the previous findings that overexpression of Besides in the base dividing zone (proximal end) of leaves, a CYCA2 genes could restrain endoreduplication in Arabidopsis higher transcript level of OsCYCA2;1 is found in rice root (Imai et al., 2006; Boudolf et al., 2009). tips, implying that a similar OsCYCA2;1 regulatory mech- In cyca2;34 mutants, ~10% of GMCs failed to divide sym- anism exists in rice roots (Supplementary Fig. 3B). Therefore, metrically and formed into SGCs (Fig. 3B, arrow). Strikingly, we probed the impact of down-regulated expression of OsCYCA2;1 expression can fully rescue the defective GMC OsCYCA2;1 on root growth. As shown in Fig.  4, the over- division in cyca2;34 epider mis, suggesting that r ice OsCYCA2;1 all growth of shoots and roots in 10-day-old OsCYCA2;1- remains a conserved function in promoting the GMC sym- RNAi transgenic seedlings is much less than in the wild type metric divisions. In addition, expression of OsCYCA2;1 in (Fig.  4A–C). To determine whether the root growth defects the cyca2;34 mutant background induced formation of exces- arose from a defective cell proliferation, we compared the root sive stomata, reflected by an increased stomatal density and longitudinal sections of the wild type and the RNAi line. The stomatal index (Fig.  3F, G). In another 35S:OsCYCA2;1 shorter meristematic zone in OsCYCA2;1-RNAi is correlated cyca2;34 transgenic line (line #8), the relative transcript level with a considerably fewer number of cells within its meristem- of OsCYCA2;1 is much lower than in line #7; the defec- atic zone (Fig. 4D–F). Similarly, Arabidopsis cyca2;34 mutants tive GMC division and reduced stomatal production are exhibit a short meristematic zone and fewer cells than Col partially rescued, indicating that OsCYCA2;1 quantitatively wild type. Ectopic expression of OsCYCA2;1 restored the promotes stomatal development depending on its expression length of and cell number within the meristematic zone to level (Supplementary Fig.  S5). Taken together, our results of Fig. 3. 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). Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3593 Fig. 4. 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.) the wild-type level (Fig. 4G–I), supporting that OsCYCA2;1 Quantitative analysis of the DAPI fluorescence intensities fur- is an evolutionarily conserved regulator that is required for cell ther confirmed that a higher DNA level (~2-fold) is present in proliferation in roots. OsCYCA2;1-RNAi roots (Fig. 5I). The higher DNA content By means of flow cytometry approaches, we found that, in OsCYCA2;1-RNAi root cells might be due to delayed or in contrast to the 6% of 4C cells in the wild type, the frac- arrested G to M transition, a result supporting the idea that tions of cells with 4C DNA content in OsCYCA2;1-RNAi OsCYCA2;1 is required for cell mitosis. lines Ri1 and Ri3 are dramatically increased to 32% and 15%, respectively (Fig. 5A–D). Moreover, the relative expression lev- OsCYCA2;1 conservatively interacts with OsCDKB1;1 els of an S-phase-specific gene, PCNA, and a M-phase cyc- CYCA2s play their regulatory roles through interacting with lin gene, CYCB2;1, were suppressed in OsCYCA2;1-RNAi multiple CDKs, such as by forming CYCA2;3–CDKB1;1 plants (Fig.  5E). Consistently, epidermal cells in the matur- or CYCA2;3–CDKA;1 protein complexes. Arabidopsis ation zone of OsCYCA2;1-RNAi roots showed stronger AtCYCA2;3 interacts with AtCDKB1;1 to form a functional DAPI fluorescent signals than in the wild type (Fig.  5F–H). Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3594 | Qu et al. Fig. 5. 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.) complex which promotes the formation of a two-celled stoma Ri2 and Ri3, in which OsCDKB1;1 transcript levels were and prevents entry into the endocycle program (Boudolf significantly suppressed (Supplementary Fig.  S7A). However, et  al., 2009; Vanneste et  al., 2011). According to the sequence the overall growth of these two transgenic lines is compar- blasting results in the rice genome, Os01g67160 encodes the able with that of the untransformed controls (Supplementary putative OsCDKB1;1. The deduced amino acid sequence Fig. S7B–D). Longitudinal sections of roots demonstrate that of OsCDKB1;1 shares 88.5% sequence identity with the the suppression of OsCDKB1;1 has no significant impact on Arabidopsis CDKB1s. A  B1-type-specific cyclin interaction cell numbers of the root meristematic zone (Supplementary motif ‘PPTALRE’ is highly conserved in rice OsCDKB1;1 Fig.  S7E–G). In addition, we found that neither the stoma- (Supplementary Fig.  S6). Yeast two-hybrid assays showed tal production (stomatal density) nor the stomatal complex that OsCYCA2;1 can interact with OsCDKB1;1 (Fig.  6A). morphology has been affected in OsCDKB1;1-RNAi trans- Consistently, pull-down assays verified the direct protein inter- genic lines (Supplementary Fig. S7H–J). action between OsCYCA2;1 and OsCDKB1;1 (Fig.  6B). Flow cytometric assays also indicate that DNA ploidy levels To determine the subcellular localization, OsCYCA2;1 or were not changed in either the roots or shoots of OsCDKB1;1- OsCDKB1;1 fused with GFP were transiently expressed in RNAi (Supplementary Fig. S8A–H). Consistent with this, the tobacco (N.  benthamiana) leaves. The fluorescent signals from expression of S-phase PCNA and M-phase cyclin CYCB2;1 OsCYCA2;1–GFP were exclusively detected in nuclei, while was not different between wild-type and OsCDKB1;1-RNAi OsCDKB1;1–GFP was found in both the cytoplasm and transgenic plants (Supplementary Fig. S8I). Taken together, it nuclei (Fig.  6C). BiFC analysis confirmed that OsCYCA2;1 seems that cell division was not interrupted by down-regu- directly interacts with OsCDKB1;1 in nuclei (Fig. 6D). These lation of the OsCDKB1;1 transcript level in transgenic rice, results suggest that OsCYCA2;1 may act as a conserved activa- though we could not exclude the possibility that the remaining tor regulating the activity of OsCDKB1;1 kinase in rice. activity of OsCDKB1;1 protein is sufficient to function. Suppression of OsCDKB1;1 has no obvious effects on OsCYCA2;1 and OsCDKB1;1 enable complementation rice development of Arabidopsis cdkb1;1 1;2 To determine whether OsCDKB1;1, like its partner Arabidopsis cdkb1;1 1;2 mutants, like the cyca2;34 mutants, OsCYCA2;1, is required for rice development, we gener- display a decreased stomatal production, formation of SGCs, ated and selected two OsCDKB1;1-RNAi transgenic lines, enlarged PCs, and increased cell ploidy levels (Boudolf et  al., Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3595 Fig. 6. 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.) 2004a; Xie et  al., 2010). Introduction of OsCDKB1;1 fully genes and AtCYCA2 genes, even though the developmental complements the impaired GMC division in cdkb1;1 1;2, and pathways of the two species have been diverged. restores stomatal production, indicating that OsCDKB1;1 has the ability to promote both symmetric and asymmetric div- ision. Meanwhile, expression of OsCKDB1;1 could efficiently Discussion prevent the occurrence of enlarged PCs and increased DNA ploidy levels in cdkb1;1 1;2 mutants (Fig.  7; Supplementary The control of cell division and differentiation is the core of Figs S9, S10). the development and morphogenesis of multicellular organ- Interestingly, the defective stomatal production, impaired isms. Cyclins, known as conserved activators for the activity GMC division, and abnormal cell enlargement and DNA levels of CDKs, play a crucial regulatory role in cell cycle progres- in cdkb1;1 1;2 could be partially rescued by overexpression of sion in diverse species. The functional pathway of CYCA2s OsCYCA2;1 (Fig. 7; Supplementary Figs S9, S10). It is there- and CDKB1s has been well investigated in the model plant fore possible that OsCDKB1;1 and OsCYCA2;1 have evolved Arabidopsis (Boudolf et  al., 2004b, 2009; Imai et  al., 2006; from the common ancestor genes with Arabidopsis AtCDKB1 Xie et al., 2010; Vanneste et al., 2011; Yang et al., 2014). In this Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3596 | Qu et al. Fig. 7. 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; ~10 000 cell nuclei were measured for each sample. study, we generated RNAi transgenic rice lines and performed (entry division) without the prior precursor stage of meriste- cross-species complement tests to explore the function of moid. Orthologs of Arabidopsis stomatal transcriptional regu- the single rice A2-type cyclin, OsCYCA2;1, as well the sin- lators SPCH, MUTE, FAMA, ICE1, and SCRM2 have been gle rice B1-type CDK, OsCDKB1;1. Cross-species expres- identified in grasses (Liu et  al., 2009; Vatén and Bergmann, sion of OsCYCA2;1 or OsCDKB1;1 enables rescue of the 2012; Ran et  al., 2013; Chen et  al., 2017). Instead of a single defective asymmetric entry divisions for stomatal initiation and copy in Arabidopsis, the rice genome has duplicated SPCH GMC symmetric divisions for GC production in Arabidopsis genes, OsSPCH1 and OsSPCH2. Similar to the weak allele cyca2;34 and/or cdkb1;1 1;2 mutants, suggesting that both of Arabidopsis spch, the rice mutant osspch2 exhibits a reduced OsCYCA2;1 and OsCDKB1;1 might have evolved from the number of stomata (Liu et  al., 2009). In Arabidopsis, SPCH common ancestor genes with Arabidopsis. heterodimerizes with SCRM/ICE1 or AtSCRM2 to pro- In Arabidopsis, asymmetric divisions generated the early sto- mote stomatal lineage initiation (Kanaoka et  al., 2008; Horst matal precursor cells, meristemoids. Meristemoids then differ- et al., 2015). In contrast, in the grass B. distachyon, BdICE1 and entiate into GMCs after a cell fate change. In grasses, GMCs BdSCRM2 show a functional diversity in regulating stoma- are created directly by stomata initiating asymmetric divisions tal pattern and morphology (Raissig et al., 2016). It is already Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3597 known that Arabidopsis SPCH activity or stability is modulated M transition, similar to the observation in the rice knockdown by multiple kinases, including MPKs, GSK3/BIN2, and CDKs lines of OsCDKB2;1 (Endo et al., 2012). (Lampard et al., 2009; Gudesblat et al., 2012; Kim et al., 2012; However, the OsCDKB1;1-RNAi transgenic rice plants, Le et  al., 2014). Phosphorylation of Ser186 of SPCH, which in which OsCDKB1;1 transcript levels were significantly might be the target residue of CDKs, positively regulates sto- decreased, display phenotypes comparable with wild-type mata production (Yang et  al., 2015). Thus, it will be interest- rice seedlings regarding the stomatal density, root cell divi- ing to establish if there is a conserved regulatory mechanism sion, and DNA content. CDKBs are plant-specific cyclin- between CDK–cyclin and SPCH-ICE1/SCRM2 in grasses. dependent kinases that can be subdivided into two groups Besides the involvement in stomatal initiation, Arabidopsis according to the different cyclin-binding motifs, namely AtCYCA2s and CDKB1s are synergistically required for the ‘PPTALRE’ in CDKB1s and ‘PPTTLRE’ in CDKB2s GMC symmetric division that is a prerequisite for the final (Joubès et  al., 2001). In Arabidopsis, each CDKB1 and stomatal development (Vanneste et al., 2011). Arabidopsis and CDKB2 subgroup contains two members (Vandepoele rice share common GMC–GC processing; GMCs divide sym- et al., 2002). It has been predicted that the rice genome has metrically to produce the paired GCs of stomata, though the a single CDKB1 gene and a single CDKB2 gene, encoding GC shapes are distinct. Thus, a role for OsCDKB1–OsCYCA2 OsCDKB1;1 and OsCDKB2;1, respectively (Supplementary in rice GMC divisions has been highly expected. However, Fig.  S11). However, the amino acid sequence alignment suppression of OsCYCA2;1 transcription in rice by RNAi revealed that rice OsCDKB1;1 and OsCDKB2;1 share the does not affect the rice GMC symmetric division. It has been same ‘PPTALRE’ cyclin-binding motif (Supplementary identified that transcription of CDKB1;1, CYCA2;3, and Fig.  S12). Expression of OsCDKB2;1 has been detected CDKA;1 in Arabidopsis is repressed by FOUR LIPS(FLP)/ in the dividing region of the rice root apex (Umeda et al., MYB88 MYB transcription factors during the GMC–GC 1999a). Transcription of rice OsCDKB2;1 is abundant dur- transition stages (Xie et  al., 2010; Vanneste et  al., 2011; Yang ing the G to M phase. Knockdown of the OsCDKB2;1 et al., 2014). FAMA, like FLP/MYB, also binds to the CDKB1;1 gene in rice induces an increase of the 4C cell popula- promoter (Hachez et al., 2011) to limit the GMC divisions to tion (Umeda et  al., 1999b; Endo et  al., 2012). In addition, one. In contrast to the tumor-like phenotype in Arabidopsis OsCDKB2;1 promotes cell division in the root meristem fama-1 mutants, the loss-of-function rice allele osfama-1 did probably through the association with OsCYCB2s (Lee et al., not undergo excessive division except the appearance of mis- 2003). Thus, we cannot rule out that OsCDKB1;1 might shaped GCs and showing a lack of stomatal pores (Liu et  al., function redundantly with OsCDKB2;1, such as forming 2009). These observations suggest that GMC–GC differentia- active CDK–cyclin complexes via binding to the same type tion is uncoupled from GMC division, in which the putative cyclins (i.e. OsCYCA2;1). Previous in situ hybridization downstream FAMA/FLP/MYB88, CDKB1;1, and CYCA2;1 results showed that both OsCDKA;1 and OsCDKA;2 are are not essential. expressed in dividing root cells of r ice (Umeda et al., 1999b). According to the phylogenetic analysis, CYCA2 and Thus, further characterization of rice CDK–cyclin pairing CDKB1 widely exist in diverse plant species, both in plants and activity can help to reveal the regulatory mechanisms bearing stomata and in plants lacking stomata, indicating that of cell division and differentiation during rice development. CYCA2 and CDKB1 might function as fundamental regula- tors of the mitotic cell cycle, as well as outside stomatal devel- Supplementary data opment. High expression of OsCYCA2;1 is associated with a Supplementary data are available at JXB online. high activity of cell proliferation, such as in the proximal end Table S1. List of primers used in this study. of leaves or root tips (Supplementary Fig. S3B). Fig. S1. Amino acid sequence comparison of A2-type cyc- Endoreduplication often occurs in cell types that undergo lins from rice and Arabidopsis. specialized differentiation. In Arabidopsis, the highly differenti- Fig.  S2. In contrast to dicot Arabidopsis, only one or two ated epidermal cells, such as mature PCs and trichomes, usually copies of genes encoding CYCA2 are found in monocot undergo multiple rounds of DNA replication without mitosis, grasses. resulting in polyploid cells (Burssens et  al., 2000). In contrast Fig. S3. Relative expression of OsCYCA2;1 in rice RNAi to the differentiated cells in Arabidopsis, polyploid cells in transgenic plants and in different tissues of wild-type plants. rice can only be found in the endosperm (Sabelli and Larkins, Fig. S4. Overexpression of rice OsCYCA2;1 suppresses the 2009). In Drosophila, it has been reported that cyclin A is one enhanced endoreduplication levels in Arabidopsis cyca2;34. of the key components of chromosomal DNA replication that Fig.  S5. Correlation between stomatal phenotypes and prevents re-initiation of DNA replication. Overexpression of OsCYCA2;1 expression levels in Arabidopsis cyca2;34 mutants Drosophila cyclin A caused a reduction in ploidy levels and inhi- harboring OsCYCA2;1. bition of the endocycle (Hayashi and Yamaguchi, 1999). Here Fig.  S6. Comparison of the amino acid sequence of we found that the fraction of 4C cells remarkably increased in OsCDKB1;1 with that of Arabidopsis CDKB1;1 and OsCYCA2;1-RNAi transgenic plants, while most cells keep a CDKB1;2. 2C DNA content. However, the expression levels of an S-phase Fig. S7. Suppression of OsCDKB1;1 has no obvious impact gene PCNA and a M-phase gene CYCB2;1 were suppressed on rice root and stomatal development. in OsCYCA2;1-RNAi plants. Therefore, we speculated that the increase of 4C cells might be caused by the arrested G to 2 Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3598 | Qu et al. Endo M, Nakayama S, Umeda-Hara C, Ohtsuki N, Saika H, Umeda M, Fig. S8. Suppression of OsCDKB1;1 has no obvious impact Toki S. 2012. CDKB2 is involved in mitosis and DNA damage response in on the distribution of DNA ploidy. rice. The Plant Journal 69, 967–977. Fig.  S9. 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A2-type cyclin is required for the asymmetric entry division in rice stomatal development

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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 mor - phological and developmental characteristics enable grass stomata to respond to environmental changes more effi- ciently. 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 defec- tive 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. Keywords: Cyclin-dependent kinases, cell differentiation, cell division, guard mother cells, cyclin, rice, stomata. Introduction Stomata are microscopic valves on aerial surfaces of all distribution pattern and morphology are highly diversified in land plants regulating the shoot–atmosphere gas exchange. different plants, stomata arise in the epidermis after a series of Paleobotanical analyses revealed that stomata originated ~400 cell divisions, cell fate changes, and cell shape controls. In the million years ago, a key evolutionary innovation formed in the past decades, results of molecular genetic studies demonstrated early palaeozoic era (Raven, 2002). Despite the fact that the that stomatal development is an accessible system to reveal the Abbreviations: CYCA2, CYCLIN A2; CDKB1, CYCLIN-DEPENDENT KINASE B1; GC, guard cell; GMC, guard mother cell; PC, pavement cell; SGC, single guard cell. © The Author(s) 2018. 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. Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3588 | Qu et al. evolution of genes and signals involved in plant development GMC symmetric division in stomatal development. In addi- (Vatén and Bergmann, 2012; Ran et  al., 2013; Chater et  al., tion to the function in promoting mitosis, AtCYCA2s form 2017; Qu et al., 2017). functional complexes with CDKs to modulate the cell cycle In the dicot model plant Arabidopsis, the earliest stomatal transition from the mitotic cycle to the endocycle. Genetic precursor, the meristemoid mother cell (MMC), divides asym- suppression of AtCYCA2 or AtCDKB1 results in enhanced metrically (stomatal entry divisions) producing a smaller cell, ploidy levels and enlarged pavement cells (PCs;V anneste et al., the meristemoid, as well as a larger sister cell, the stomatal lin- 2011). Co-expression of CYCA2;3 and CDKB1;1 induces eage ground cell (SLGC). Meristemoids normally undergo ectopic cell divisions, limits endoreduplication, and inhibits cell an additional 1–2 rounds of asymmetric divisions (amplify- growth (Boudolf et al., 2009). ing divisions) to generate new meristemoids before convert- There are at least 49 putative genes predicted to encode rice ing into guard mother cells (GMCs). SLGCs can also undergo cyclins, which were classified into nine types based on evolution- asymmetric divisions (spacing divisions) to generate satellite ary relationships. Eight of these nine types are common between meristemoids. Meristemoids differentiate into GMCs after cell rice and Arabidopsis (Umeda et al., 1999a; Cooper et al., 2003; fate change. Then, GMCs divide symmetrically to produce La et al., 2006). The existence of numerous cyclins implies their paired young guard cells (GCs). During the final stage of sto- diverse regulatory roles in modulating CDK activities during matal development, GCs undergo cell differentiation, morpho- rice development and adaption in response to environmental genesis, and pore formation to form functional stomatal units changes (Cooper et al., 2003; Huang et al., 2008). For example, (Bergmann and Sack, 2007). rice B2-type cyclins, OsCycB2;1 and OsCycB2;2, promote root In contrast to the scattered pattern in dicot Arabidopsis cell divisions through an association with OsCDKB2;1 (Lee leaves, monocot grass stomata are arranged within linear cell et al., 2003). OsCycH;1 specifically binds to R2 and positively files that parallel the growth axis of the leaf. Stomatal lineage controls CDK and CTD kinase activities to adjust the rate of cell cells initiate at the base of the leaf, and divide asymmetrically to proliferation (Fabian-Marwedel et al., 2002). produce two daughter cells, a GMC, and a larger sister cell. At In this study, we examine the function of the rice single the final stage, GMCs divide symmetrically, producing paired A2-type cyclin OsCYCA2;1 and OsCDKB1;1 in rice develop- dumbbell-shaped GCs. The stomatal subsidiary cells are pro- ment. Our results demonstrate a requirement for OsCYCA2;1 duced from cell files flanking the stomatal lineage after asym- for stomatal and root development. In contrast to its homolog metric divisions (Franks and Farquhar, 2007; Liu et  al., 2009; in Arabidopsis, OsCYCA2;1 is exclusively required for the Serna, 2011; Raissig et al., 2016). asymmetric entry divisions to produce GMCs at the early stage Cyclins form complexes with specific cyclin-dependent of stomatal development. In addition, combined with phylo- kinases (CDKs) to co-ordinate the cell proliferation and dif- genetic analyses, we are providing new clues for further reveal- ferentiation during the development of multicellular organisms ing the evolutionary correlation between cell cycle genes and (Swenson et  al., 1986; Obaya and Sedivy, 2002). Cyclins, act- developmental pathways. ing as the regulator of CDK activity, contribute to the subcel- lular localization, substrate specificity, and protein stability of the CDK–cyclin complexes (Dewitte and Murray, 2003; Imai Materials and methods et  al., 2006; Boudolf et  al., 2009; Boruc et  al., 2010). A-type Plant materials and growth conditions cyclins, known as mitotic cyclins, are essential for the mitotic The Col-0 ecotype of Arabidopsis thaliana L. was used as the wild-type cell cycle. In contrast to animals, plants encode a large family control in the Arabidopsis study. The cdkb1;1 1;2 double mutants were of A-type cyclins that have been classed into A1, A2, and A3 confirmed by PCR-based approaches (Xie et  al., 2010). The cyca2;34 groups (Vandepoele et  al., 2002; Dewitte and Murray, 2003; double mutants were provided by Steffen Vanneste and Tom Beeckman Wang et al., 2004). (Vanneste et al., 2011). Seeds were surface sterilized (40 s) in an aqueous The Arabidopsis genome has four genes encoding A2-type 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 cyclins. AtCYCA2 genes display tissue- and cell type-specific Murashige and Skoog (MS) medium supplemented with 0.8% agar and and overlapping expression patterns, such as in vascular systems 1% sucrose. Plants were grown in a controlled temperature and pho- and stomatal lineage cells, which are associated with their redun- toperiod chamber at 22  ±  2  °C and 16  h/8  h light/dark illumination dant functions during plant development (Burssens et al., 2000; cycles. Oryza sativa L. spp. japonica cultivar Zhonghua 11 was used as the Imai et  al., 2006; Vanneste et  al., 2011; Donner and Scarpella, 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 con- 2013). Mutants of AtCYCA2 genes frequently form unpaired trolled growth chamber with 30 °C/22 °C day/night temperature cycles, single guard cells (SGCs), a similar defect of the terminal GMC 12 h/12 h light/dark illumination cycles, and 60–70% relative humidity. division to that also observed in mutants of AtCDKB1 or AtCDKA;1 genes (Boudolf et al., 2004b; Vanneste et al., 2011; Plasmid construction and generation of transgenic plants Yang et  al., 2014). Overexpression of AtCYCA2:3 as well as To obtain the construct of gene overexpression, cDNA of OsCDKB1;1 AtCDKA;1 at the late stage of stomatal development induced or OsCYCA2;1 was cloned into the pH7WG2D.1 vector by using gate- excessive GC subdivisions (Yang et  al., 2014). Arabidopsis way technology and LR Clonase™ II Enzyme Mix (Invitrogen). The cdkb1;1 1;2 double mutants and 35S:CDKB1;1.N161 domi- recombinant plasmids were confirmed by DNA sequencing before nant negative plants displayed decreased stomatal production the transformation into Arabidopsis wild type and mutants. To gener- and formation of SGCs, indicating that the activity of CDKB1 ate RNAi transgenic plants against OsCDKB1;1 and OsCYCA2;1, the conserved sequences from base pair 530 to 695 of OsCDKB1;1 cDNA is required for both meristemoid asymmetric division and Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3589 and 747 to 979 of OsCYCA2;1 cDNA were amplified and cloned into Flow cytometric analysis pTCK303 vector, respectively. These constructs were electroporated into About 20–50  mg of fresh tissue were cut into 2–4  mm fragments and Agrobacterium tumefaciens EHA105 and transformed into rice Zhonghua then chopped immediately using a razor blade in 1  ml of Galbraith’s 11 (Chen et  al., 2011). T seeds were collected to screen positive trans- buffer (45  mM MgCl ·6H O, 30  mM sodium citrate, 20  mM MOPS, 2 2 −1 genic plants by using 50 µg l hygromycin B (Roche). Real-time quan- 0.1% Triton X-100, pH 7.0). The cell culture was collected by gentle titative PCR (RT-qPCR) was conducted to confirm the expression level pipetting and filtered through a cell strainer. The samples were stained −1 of target genes in transgenic plants. The primer sequences used in this with 2 µg ml DAPI in an ice bath for 30 min before the analysis using study are listed in Supplementary Table S1 at JXB online. a MoFlo-XDP flow cytometer (Beckman) (Dolezel et al., 2007). A total of ~10 000 nuclei were measured for each sample. Real-time quantitative PCR analysis Rice seedlings were harvested and immediately ground in liquid nitro- Root semi-thin sections gen, and the total RNA was isolated using TRNzol reagent (http://www. About 1 cm long primary root tips from 6-day-old rice seedlings were tiangen.com). The first-strand cDNA was synthesized using a Promega harvested and immersed in cold formaldehyde solution. The samples Reverse Transcription kit (http://www.promega.com). RT-qPCRs were were subjected to a vacuum for 10 min and placed at 4 °C overnight. The performed by using SYBR Premix Ex Taq™ (TaKaRa) with a Corbett materials were washed with 0.1 M PBS (pH 7.2) four times and were RG3000. The OsACTIN2 gene was used as an internal control. The fixed in 1% osmic acid for 1 h, followed by a series of dehydration steps, primer sequences are listed in Supplementary Table S1. 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). Yeast two-hybrid assay Permeation was performed by using a series of 2:1, 1:1, and 1:2 (v/v) of The full-length cDNA sequences of OsCYCA2;1 and OsCDKB1;1 were acetone and epoxy (SPURR) mixture solution. Each step lasted for 3 h. amplified using the primers listed in Supplementary Table S1 and cloned After adding pure epoxy for 8 h, samples were embedded and polymer- into pGBKT7 and pGADT7 vectors (Clontech), respectively. These con- ized at 60 °C for 24 h. Semi-thin sections (thickness 1 µm) were obtained structs were transformed into Saccharomyces cerevisiae yeast strain AH109 by using a Leica UC7 microtome. Before imaging, sections were stained and selected on SD/-Leu-Trp or SD/-Leu-Trp-His-Ade plates. X-Gal with 0.1% toluidine blue O. activity was then detected. Results Bimolecular fluorescence complementation assay For bimolecular fluorescence complementation (BiFC) assays, the full- Evolutionary analysis of A2 cyclins in plants length cDNAs of OsCYCA2;1 and OsCDKB1;1 were cloned into pSPYCE-35S and pSPYNE-35S vectors, respectively (Walter et al., 2004). Phylogenetic analysis indicates that homologs of A2-type cyclin These constructs were transformed into A.  tumefaciens EH105 and co- are found in lineages that diverged early in the evolution of land injected into tobacco (Nicotiana benthamiana). Images were taken after 3 plants, before the appearance of stomata. For example, the uni- d using a laser scanning confocal microscope (FV1000-MPE, Olympus). cellular green alga Coccomyxa subellipsoidea has a CYCA2 gene in its genome. In the non-vascular land plant moss Physcomitrella Pull-down assay patens, stomata are exclusively found on the diploid sporophyte The OsCYCA2;1 and OsCDKB1;1 sequences were cloned into pET- (Chater et al., 2016); there are six putative orthologs of CYCA2. 28a and pGEX4T-1 vectors, respectively. The OsCYCA2;1-pET-28a and In the vascular dicot plants Arabidopsis, soybean (Glycine max), OsCDKB1;1-pGEX4T-1 constructs were transformed into the BL21 strain and alfalfa (Medicago truncatula), the number of CYCA2 genes of Escherichia coli. The transformed strains were grown to OD =0.5 under was four, six, and four, respectively (Fig.  1). In contrast, the 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 rice genome contains only one copy of the CYCA2 gene, 20 h. The harvested strains (5000 rpm, 10 min, 4 °C) were re-suspended Os012g31810, which is predicted to encode OsCYCA2;1 pro- with ice-cold phosphate-buffered saline (PBS) and lysed by sonication. The tein consisting of 490 amino acid residues. Multiple sequence lysate was centrifuged at 10 000 rpm for 60 min and the supernatant was alignment reveals that OsCYCA2;1 shows 40.5% amino acid collected. The glutathione S-transferase (GST)–OsCDKB1;1 supernatant sequence identity with Arabidopsis CYCA2s, and contains a was loaded on glutathione–Sepharose (GE Healthcare) and washed with PBS. The GST–OsCDKB1;1 fusion protein on glutathione–Sepharose was CDK-binding cyclin box, which is highly conserved among incubated with the His-OsCYCA2;1 supernatant at 4 °C for 2 h. Then mitotic cyclins (Supplementary Fig. S1) (Umeda et al., 1999a). the glutathione–Sepharose was washed with PBS and eluted with 10 mM Interestingly, similar to rice, Brachypodium stacei, Brachypodium reduced glutathione elution buffer. The samples were loaded on a 12% distachyon, Zea mays (Fig. 1), and many other monocot grasses, SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride Hordeum vulgare, Oropetium thomaeum, Panicum hallii, Sorghum (PVDF) membrane (Millipore) by using a semi-dry blotting system (Bio- Rad), and then incubated with anti-His6 monoclonal antibodies followed bicolor, Setaria italica, and Setaria viridis, only have 1–2 CYCA2 by horseradish peroxidase (HRP)-conjugated anti-mouse antibodies. The genes (Supplementary Fig. S2). color reaction was performed using the Pro-Light HRP Kit (Tiangen). The seagrass Zostera marina belongs to basal monocots that Signals were exposed to X-ray films and developed. returned to the sea. The absence of stomata in Z.  marina is consistent with the evolutionary loss of entire genes that DAPI staining and DNA content measurement are required for stomatal development (Olsen et  al., 2016). Ten-day-old rice roots were fixed in a mixture of 3:1 (v/v) ethanol and However, like the above grass plants, Z.  marina possesses two acetic acid for 30 min, then rinsed with distilled water. After staining for CYCA2 genes, suggesting that the A2-type cyclin is funda- −1 30 min with 2 µg ml DAPI (Roche) in a staining solution (0.1 M sodium mentally important for plant growth and development, and phosphate, 1 mM EDTA, 0.1% Triton X-100, pH 7.0), roots were photo- is not solely linked to stomatal development. The low num- graphed by a fluorescence microscope. The relative fluorescence intensities ber of CYCA2 genes in grasses indicates that CYCA2 gene were measured using ImageJ software (http://imagej.nih.gov/ij/). Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3590 | Qu et al. Fig. 1. 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. duplication might not be necessary, which is associated with are produced by the cells in the neighboring cell files. The ter- their unique developmental pathways and morphogenesis. minal division of GMC produces a pair of immature GCs (Stage 5, middle panel of Fig. 2A). Within the wild-type stomatal line- age cell files, stomatal complexes are spaced by one lobbed PC Requirement of OsCYCA2;1 for stomatal initiation (Stage 6, lower panel of Fig.  2A). However, in OsCYCA2;1- To elucidate the function of A2-type cyclin in rice development, RNAi transgenic plants, more than two spacing cells were often RNAi transgenic rice lines targeting OsCYCA2;1 were gen- observed between two neighboring GMCs/stomata within the erated. Transcript levels of OsCYCA2;1 in two lines, Ri1 and same cell file (Fig. 2B , C), leading to a decreased stomatal density Ri3, were suppressed to 28% and 61%, respectively, in relation to and stomatal index (Fig. 2D, E). the level in wild-type rice seedlings (Supplementary Fig. S3A). Mutations of Arabidopsis CYCA2 genes caused a failure of In rice leaf epidermis, stomata form within the stomatal line- GMC division, leading to the formation of aberrant stomatal age files following a gradual base to tip maturation pattern; the units (SGCs) (Vanneste et al., 2011). However, the structure and developing stomata can only be found at the proximal end (base) morphology of mature stomata in OsCYCA2;1-RNAi rice of the leaf. Unlike in Arabidopsis, GMCs in rice are produced transgenic plants are indistinguishable from those of wild-type directly by asymmetric entry divisions without the precursor stomata, indicating that the subsequent GMC symmetric divi- stage of the meristemoid. Each undifferentiated cell close to sions as well the subsidiary cell asymmetric divisions are not the base of the leaf divides asymmetrically and generates one interrupted by the suppression of OsCYCA2;1 (lower panels smaller GMC and one larger sister cell (Stage 2, upper panel of in Fig. 2A and B). Taken together, the above observations indi- Fig.  2A). Subsidiary mother cells (SMCs) flanking the GMCs cate that OsCYCA2;1 is essentially required for the asymmetric Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3591 Fig. 2. 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, ~10 000 cell nuclei were measured. entry division during stomatal initiation at the early stage of increased to 36% and 18%, respectively. Higher ploidy levels, stomatal development, but not for the terminal GMC symmet- like in Arabidopsis cyca2 mutants (8C, 16C, 32C), were barely ric divisions and subsidiary cell asymmetric divisions. detectable in rice OsCYCA2;1-RNAi plants (Fig. 2F–I). It has been demonstrated that Arabidopsis CYCA2s not only promote cell proliferation but also negatively regulate endocy- OsCYCA2;1 complements epidermal defects of cle onset (Imai et al., 2006; Yoshizumi et al., 2006; Vanneste et al., Arabidopsis cyca2 mutants 2011). Flow cytometric analysis showed that in wild-type rice, The Arabidopsis epidermis is an ideal system to identify gene only 6% of cells showed a 4C DNA content, whereas most cells functions in plant development programs and morphogenetic were 2C (diploid). However, in OsCYCA2;1-RNAi trans- patterns. To identify further the function of OsCYCA2;1 genic rice lines Ri1 and Ri3, the fraction of 4C cells markedly Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3592 | Qu et al. in epidermal development, OsCYCA2;1 coding sequences cross-species complement tests demonstrate the conserved driven by 35S promoters were transformed into Arabidopsis abilities of OsCYCA2;1 in limiting cell endoreduplication and cyca2;34 mutants. Compared with the wild type, cyca2;34 PC size, as well as in rescuing cyca2;34 defective asymmetric mutants display enlarged pavement cells and enhanced ploidy entry divisions (for stomatal initiation) and symmetric GMC levels. Cross-species expression of OsCYCA2;1 (line #7) divisions (for guard cell formation), despite OsCYCA2;1 being inhibits the abnormal PC enlargement in cyca2;34 epidermis, functionally required only for stomatal entry divisions in rice. 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 also required for cell division and OsCYCA2;1 is able to inhibit the high DNA ploidy levels in differentiation in roots cyca2;34 mutants (Fig. 3E; Supplementary Fig. S4). This result OsCYCA2;1 is preferentially expressed in proliferating tissues. is consistent with the previous findings that overexpression of Besides in the base dividing zone (proximal end) of leaves, a CYCA2 genes could restrain endoreduplication in Arabidopsis higher transcript level of OsCYCA2;1 is found in rice root (Imai et al., 2006; Boudolf et al., 2009). tips, implying that a similar OsCYCA2;1 regulatory mech- In cyca2;34 mutants, ~10% of GMCs failed to divide sym- anism exists in rice roots (Supplementary Fig. 3B). Therefore, metrically and formed into SGCs (Fig. 3B, arrow). Strikingly, we probed the impact of down-regulated expression of OsCYCA2;1 expression can fully rescue the defective GMC OsCYCA2;1 on root growth. As shown in Fig.  4, the over- division in cyca2;34 epider mis, suggesting that r ice OsCYCA2;1 all growth of shoots and roots in 10-day-old OsCYCA2;1- remains a conserved function in promoting the GMC sym- RNAi transgenic seedlings is much less than in the wild type metric divisions. In addition, expression of OsCYCA2;1 in (Fig.  4A–C). To determine whether the root growth defects the cyca2;34 mutant background induced formation of exces- arose from a defective cell proliferation, we compared the root sive stomata, reflected by an increased stomatal density and longitudinal sections of the wild type and the RNAi line. The stomatal index (Fig.  3F, G). In another 35S:OsCYCA2;1 shorter meristematic zone in OsCYCA2;1-RNAi is correlated cyca2;34 transgenic line (line #8), the relative transcript level with a considerably fewer number of cells within its meristem- of OsCYCA2;1 is much lower than in line #7; the defec- atic zone (Fig. 4D–F). Similarly, Arabidopsis cyca2;34 mutants tive GMC division and reduced stomatal production are exhibit a short meristematic zone and fewer cells than Col partially rescued, indicating that OsCYCA2;1 quantitatively wild type. Ectopic expression of OsCYCA2;1 restored the promotes stomatal development depending on its expression length of and cell number within the meristematic zone to level (Supplementary Fig.  S5). Taken together, our results of Fig. 3. 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). Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3593 Fig. 4. 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.) the wild-type level (Fig. 4G–I), supporting that OsCYCA2;1 Quantitative analysis of the DAPI fluorescence intensities fur- is an evolutionarily conserved regulator that is required for cell ther confirmed that a higher DNA level (~2-fold) is present in proliferation in roots. OsCYCA2;1-RNAi roots (Fig. 5I). The higher DNA content By means of flow cytometry approaches, we found that, in OsCYCA2;1-RNAi root cells might be due to delayed or in contrast to the 6% of 4C cells in the wild type, the frac- arrested G to M transition, a result supporting the idea that tions of cells with 4C DNA content in OsCYCA2;1-RNAi OsCYCA2;1 is required for cell mitosis. lines Ri1 and Ri3 are dramatically increased to 32% and 15%, respectively (Fig. 5A–D). Moreover, the relative expression lev- OsCYCA2;1 conservatively interacts with OsCDKB1;1 els of an S-phase-specific gene, PCNA, and a M-phase cyc- CYCA2s play their regulatory roles through interacting with lin gene, CYCB2;1, were suppressed in OsCYCA2;1-RNAi multiple CDKs, such as by forming CYCA2;3–CDKB1;1 plants (Fig.  5E). Consistently, epidermal cells in the matur- or CYCA2;3–CDKA;1 protein complexes. Arabidopsis ation zone of OsCYCA2;1-RNAi roots showed stronger AtCYCA2;3 interacts with AtCDKB1;1 to form a functional DAPI fluorescent signals than in the wild type (Fig.  5F–H). Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3594 | Qu et al. Fig. 5. 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.) complex which promotes the formation of a two-celled stoma Ri2 and Ri3, in which OsCDKB1;1 transcript levels were and prevents entry into the endocycle program (Boudolf significantly suppressed (Supplementary Fig.  S7A). However, et  al., 2009; Vanneste et  al., 2011). According to the sequence the overall growth of these two transgenic lines is compar- blasting results in the rice genome, Os01g67160 encodes the able with that of the untransformed controls (Supplementary putative OsCDKB1;1. The deduced amino acid sequence Fig. S7B–D). Longitudinal sections of roots demonstrate that of OsCDKB1;1 shares 88.5% sequence identity with the the suppression of OsCDKB1;1 has no significant impact on Arabidopsis CDKB1s. A  B1-type-specific cyclin interaction cell numbers of the root meristematic zone (Supplementary motif ‘PPTALRE’ is highly conserved in rice OsCDKB1;1 Fig.  S7E–G). In addition, we found that neither the stoma- (Supplementary Fig.  S6). Yeast two-hybrid assays showed tal production (stomatal density) nor the stomatal complex that OsCYCA2;1 can interact with OsCDKB1;1 (Fig.  6A). morphology has been affected in OsCDKB1;1-RNAi trans- Consistently, pull-down assays verified the direct protein inter- genic lines (Supplementary Fig. S7H–J). action between OsCYCA2;1 and OsCDKB1;1 (Fig.  6B). Flow cytometric assays also indicate that DNA ploidy levels To determine the subcellular localization, OsCYCA2;1 or were not changed in either the roots or shoots of OsCDKB1;1- OsCDKB1;1 fused with GFP were transiently expressed in RNAi (Supplementary Fig. S8A–H). Consistent with this, the tobacco (N.  benthamiana) leaves. The fluorescent signals from expression of S-phase PCNA and M-phase cyclin CYCB2;1 OsCYCA2;1–GFP were exclusively detected in nuclei, while was not different between wild-type and OsCDKB1;1-RNAi OsCDKB1;1–GFP was found in both the cytoplasm and transgenic plants (Supplementary Fig. S8I). Taken together, it nuclei (Fig.  6C). BiFC analysis confirmed that OsCYCA2;1 seems that cell division was not interrupted by down-regu- directly interacts with OsCDKB1;1 in nuclei (Fig. 6D). These lation of the OsCDKB1;1 transcript level in transgenic rice, results suggest that OsCYCA2;1 may act as a conserved activa- though we could not exclude the possibility that the remaining tor regulating the activity of OsCDKB1;1 kinase in rice. activity of OsCDKB1;1 protein is sufficient to function. Suppression of OsCDKB1;1 has no obvious effects on OsCYCA2;1 and OsCDKB1;1 enable complementation rice development of Arabidopsis cdkb1;1 1;2 To determine whether OsCDKB1;1, like its partner Arabidopsis cdkb1;1 1;2 mutants, like the cyca2;34 mutants, OsCYCA2;1, is required for rice development, we gener- display a decreased stomatal production, formation of SGCs, ated and selected two OsCDKB1;1-RNAi transgenic lines, enlarged PCs, and increased cell ploidy levels (Boudolf et  al., Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3595 Fig. 6. 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.) 2004a; Xie et  al., 2010). Introduction of OsCDKB1;1 fully genes and AtCYCA2 genes, even though the developmental complements the impaired GMC division in cdkb1;1 1;2, and pathways of the two species have been diverged. restores stomatal production, indicating that OsCDKB1;1 has the ability to promote both symmetric and asymmetric div- ision. Meanwhile, expression of OsCKDB1;1 could efficiently Discussion prevent the occurrence of enlarged PCs and increased DNA ploidy levels in cdkb1;1 1;2 mutants (Fig.  7; Supplementary The control of cell division and differentiation is the core of Figs S9, S10). the development and morphogenesis of multicellular organ- Interestingly, the defective stomatal production, impaired isms. Cyclins, known as conserved activators for the activity GMC division, and abnormal cell enlargement and DNA levels of CDKs, play a crucial regulatory role in cell cycle progres- in cdkb1;1 1;2 could be partially rescued by overexpression of sion in diverse species. The functional pathway of CYCA2s OsCYCA2;1 (Fig. 7; Supplementary Figs S9, S10). It is there- and CDKB1s has been well investigated in the model plant fore possible that OsCDKB1;1 and OsCYCA2;1 have evolved Arabidopsis (Boudolf et  al., 2004b, 2009; Imai et  al., 2006; from the common ancestor genes with Arabidopsis AtCDKB1 Xie et al., 2010; Vanneste et al., 2011; Yang et al., 2014). In this Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3596 | Qu et al. Fig. 7. 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; ~10 000 cell nuclei were measured for each sample. study, we generated RNAi transgenic rice lines and performed (entry division) without the prior precursor stage of meriste- cross-species complement tests to explore the function of moid. Orthologs of Arabidopsis stomatal transcriptional regu- the single rice A2-type cyclin, OsCYCA2;1, as well the sin- lators SPCH, MUTE, FAMA, ICE1, and SCRM2 have been gle rice B1-type CDK, OsCDKB1;1. Cross-species expres- identified in grasses (Liu et  al., 2009; Vatén and Bergmann, sion of OsCYCA2;1 or OsCDKB1;1 enables rescue of the 2012; Ran et  al., 2013; Chen et  al., 2017). Instead of a single defective asymmetric entry divisions for stomatal initiation and copy in Arabidopsis, the rice genome has duplicated SPCH GMC symmetric divisions for GC production in Arabidopsis genes, OsSPCH1 and OsSPCH2. Similar to the weak allele cyca2;34 and/or cdkb1;1 1;2 mutants, suggesting that both of Arabidopsis spch, the rice mutant osspch2 exhibits a reduced OsCYCA2;1 and OsCDKB1;1 might have evolved from the number of stomata (Liu et  al., 2009). In Arabidopsis, SPCH common ancestor genes with Arabidopsis. heterodimerizes with SCRM/ICE1 or AtSCRM2 to pro- In Arabidopsis, asymmetric divisions generated the early sto- mote stomatal lineage initiation (Kanaoka et  al., 2008; Horst matal precursor cells, meristemoids. Meristemoids then differ- et al., 2015). In contrast, in the grass B. distachyon, BdICE1 and entiate into GMCs after a cell fate change. In grasses, GMCs BdSCRM2 show a functional diversity in regulating stoma- are created directly by stomata initiating asymmetric divisions tal pattern and morphology (Raissig et al., 2016). It is already Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 CYCA2 and rice stomatal development | 3597 known that Arabidopsis SPCH activity or stability is modulated M transition, similar to the observation in the rice knockdown by multiple kinases, including MPKs, GSK3/BIN2, and CDKs lines of OsCDKB2;1 (Endo et al., 2012). (Lampard et al., 2009; Gudesblat et al., 2012; Kim et al., 2012; However, the OsCDKB1;1-RNAi transgenic rice plants, Le et  al., 2014). Phosphorylation of Ser186 of SPCH, which in which OsCDKB1;1 transcript levels were significantly might be the target residue of CDKs, positively regulates sto- decreased, display phenotypes comparable with wild-type mata production (Yang et  al., 2015). Thus, it will be interest- rice seedlings regarding the stomatal density, root cell divi- ing to establish if there is a conserved regulatory mechanism sion, and DNA content. CDKBs are plant-specific cyclin- between CDK–cyclin and SPCH-ICE1/SCRM2 in grasses. dependent kinases that can be subdivided into two groups Besides the involvement in stomatal initiation, Arabidopsis according to the different cyclin-binding motifs, namely AtCYCA2s and CDKB1s are synergistically required for the ‘PPTALRE’ in CDKB1s and ‘PPTTLRE’ in CDKB2s GMC symmetric division that is a prerequisite for the final (Joubès et  al., 2001). In Arabidopsis, each CDKB1 and stomatal development (Vanneste et al., 2011). Arabidopsis and CDKB2 subgroup contains two members (Vandepoele rice share common GMC–GC processing; GMCs divide sym- et al., 2002). It has been predicted that the rice genome has metrically to produce the paired GCs of stomata, though the a single CDKB1 gene and a single CDKB2 gene, encoding GC shapes are distinct. Thus, a role for OsCDKB1–OsCYCA2 OsCDKB1;1 and OsCDKB2;1, respectively (Supplementary in rice GMC divisions has been highly expected. However, Fig.  S11). However, the amino acid sequence alignment suppression of OsCYCA2;1 transcription in rice by RNAi revealed that rice OsCDKB1;1 and OsCDKB2;1 share the does not affect the rice GMC symmetric division. It has been same ‘PPTALRE’ cyclin-binding motif (Supplementary identified that transcription of CDKB1;1, CYCA2;3, and Fig.  S12). Expression of OsCDKB2;1 has been detected CDKA;1 in Arabidopsis is repressed by FOUR LIPS(FLP)/ in the dividing region of the rice root apex (Umeda et al., MYB88 MYB transcription factors during the GMC–GC 1999a). Transcription of rice OsCDKB2;1 is abundant dur- transition stages (Xie et  al., 2010; Vanneste et  al., 2011; Yang ing the G to M phase. Knockdown of the OsCDKB2;1 et al., 2014). FAMA, like FLP/MYB, also binds to the CDKB1;1 gene in rice induces an increase of the 4C cell popula- promoter (Hachez et al., 2011) to limit the GMC divisions to tion (Umeda et  al., 1999b; Endo et  al., 2012). In addition, one. In contrast to the tumor-like phenotype in Arabidopsis OsCDKB2;1 promotes cell division in the root meristem fama-1 mutants, the loss-of-function rice allele osfama-1 did probably through the association with OsCYCB2s (Lee et al., not undergo excessive division except the appearance of mis- 2003). Thus, we cannot rule out that OsCDKB1;1 might shaped GCs and showing a lack of stomatal pores (Liu et  al., function redundantly with OsCDKB2;1, such as forming 2009). These observations suggest that GMC–GC differentia- active CDK–cyclin complexes via binding to the same type tion is uncoupled from GMC division, in which the putative cyclins (i.e. OsCYCA2;1). Previous in situ hybridization downstream FAMA/FLP/MYB88, CDKB1;1, and CYCA2;1 results showed that both OsCDKA;1 and OsCDKA;2 are are not essential. expressed in dividing root cells of r ice (Umeda et al., 1999b). According to the phylogenetic analysis, CYCA2 and Thus, further characterization of rice CDK–cyclin pairing CDKB1 widely exist in diverse plant species, both in plants and activity can help to reveal the regulatory mechanisms bearing stomata and in plants lacking stomata, indicating that of cell division and differentiation during rice development. CYCA2 and CDKB1 might function as fundamental regula- tors of the mitotic cell cycle, as well as outside stomatal devel- Supplementary data opment. High expression of OsCYCA2;1 is associated with a Supplementary data are available at JXB online. high activity of cell proliferation, such as in the proximal end Table S1. List of primers used in this study. of leaves or root tips (Supplementary Fig. S3B). Fig. S1. Amino acid sequence comparison of A2-type cyc- Endoreduplication often occurs in cell types that undergo lins from rice and Arabidopsis. specialized differentiation. In Arabidopsis, the highly differenti- Fig.  S2. In contrast to dicot Arabidopsis, only one or two ated epidermal cells, such as mature PCs and trichomes, usually copies of genes encoding CYCA2 are found in monocot undergo multiple rounds of DNA replication without mitosis, grasses. resulting in polyploid cells (Burssens et  al., 2000). In contrast Fig. S3. Relative expression of OsCYCA2;1 in rice RNAi to the differentiated cells in Arabidopsis, polyploid cells in transgenic plants and in different tissues of wild-type plants. rice can only be found in the endosperm (Sabelli and Larkins, Fig. S4. Overexpression of rice OsCYCA2;1 suppresses the 2009). In Drosophila, it has been reported that cyclin A is one enhanced endoreduplication levels in Arabidopsis cyca2;34. of the key components of chromosomal DNA replication that Fig.  S5. Correlation between stomatal phenotypes and prevents re-initiation of DNA replication. Overexpression of OsCYCA2;1 expression levels in Arabidopsis cyca2;34 mutants Drosophila cyclin A caused a reduction in ploidy levels and inhi- harboring OsCYCA2;1. bition of the endocycle (Hayashi and Yamaguchi, 1999). Here Fig.  S6. Comparison of the amino acid sequence of we found that the fraction of 4C cells remarkably increased in OsCDKB1;1 with that of Arabidopsis CDKB1;1 and OsCYCA2;1-RNAi transgenic plants, while most cells keep a CDKB1;2. 2C DNA content. However, the expression levels of an S-phase Fig. S7. Suppression of OsCDKB1;1 has no obvious impact gene PCNA and a M-phase gene CYCB2;1 were suppressed on rice root and stomatal development. in OsCYCA2;1-RNAi plants. Therefore, we speculated that the increase of 4C cells might be caused by the arrested G to 2 Downloaded from https://academic.oup.com/jxb/article/69/15/3587/4986277 by DeepDyve user on 19 July 2022 3598 | Qu et al. Endo M, Nakayama S, Umeda-Hara C, Ohtsuki N, Saika H, Umeda M, Fig. S8. Suppression of OsCDKB1;1 has no obvious impact Toki S. 2012. CDKB2 is involved in mitosis and DNA damage response in on the distribution of DNA ploidy. rice. The Plant Journal 69, 967–977. Fig.  S9. 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Journal of Experimental Botany – Oxford University Press

Published: Jun 27, 2018

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