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This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online reduces the time to publication by several weeks. Inflorescence Meristem Identity in Rice Is Specified by Overlapping Functions of Three AP1/FUL-Like MADS Box Genes and PAP2, a SEPALLATA MADS Box Gene C W Kaoru Kobayashi,a Naoko Yasuno,a Yutaka Sato,b Masahiro Yoda,a Ryo Yamazaki,a Mayumi Kimizu,c Hitoshi Yoshida,d Yoshiaki Nagamura,b and Junko Kyozukaa,1 a Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan c Crop Development Division, National Agricultural Research Center, National Agriculture and Food Research Organization, Jo-etsu, Niigata 943-0193, Japan d Rice Research Division, National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan b National In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/ FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal. INTRODUCTION Plants progress through distinct developmental phases in their life cycles (reviewed in Huijser and Schmid, 2011). The transition from vegetative to reproductive growth, generally called flowering, is vital for reproductive success. Flowering occurs in response to environmental and developmental signals, such as daylength, plant age, and temperature (reviewed in Liu et al., 2009; Wellmer and Riechmann, 2010). Multiple flowering signals are eventually transmitted to the shoot apical meristem (SAM), a group of stem cells in the shoot apex, where they are converted to local cues that evoke the developmental phase transition. In Arabidopsis thaliana, SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and FLOWERING LOCUS T (FT) function as the main floral integrators that transmit multiple flowering signals to the meristem. SOC1 is one of the first genes to be induced in the SAM at this transition (Lee et al., 2000). FT is 1 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Junko Kyozuka ([email protected]). C Some figures in this article are displayed in color online but in black and white in the print edition. W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.112.097105 a major component of florigen, a long-distance flowering signal (Abe et al., 2005; Wigge et al., 2005). The FT protein is produced in leaves and then transported to the SAM, where it interacts with FLOWERING LOCUS D (FD). In the SAM, the SOC1 and FTFD complex promote expression of several downstream genes, including LEAFY, APETALA1 (AP1), and FRUITFULL (FUL), which specify floral meristem identity (Abe et al., 2005; Wigge et al., 2005; Corbesier et al., 2007). The aerial part of a plant is generated by the SAM, which produces leaves during vegetative development. Upon transition to reproductive development, the SAM converts into an inflorescence meristem (IM) and starts to generate components of the inflorescence. Development of the IM is variable depending on species, leading to formation of species-specific inflorescence architecture. The conversion from the vegetative SAM to the IM is less well understood at the molecular level than events such as flowering time control, floral meristem specification, and flower development. Several genes show a dramatic change in their expression patterns in the meristem at phase transition (Cardon et al., 1997; Hempel et al., 1997; Lee et al., 2000; Samach et al., 2000; Torti et al., 2012). This reflects a global change in meristem gene expression that is associated with the establishment of IM identity (Schmid et al., 2003; Park et al., 2012). In grasses, the shift of the meristem phase from the SAM to the IM is accompanied by clear changes in a number of The Plant Cell Preview, www.aspb.org ã 2012 American Society of Plant Biologists. All rights reserved. 1 of 12 2 of 12 The Plant Cell developmental and morphological traits. In rice (Oryza sativa), the first morphological change is a rapid increase in meristem size (Hoshikawa, 1989). The IM starts to initiate inflorescence branch meristem (BM) primordia. Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) function as major components of florigen in rice (Tamaki et al., 2007; Komiya et al., 2008, 2009). Recent findings show that Hd3a protein generated in leaves is transported to the cytoplasm of shoot apex cells where it interacts with 14-3-3 proteins (Taoka et al., 2011). The Hd3a/14-3-3 complex then enters the nucleus and activates the rice ortholog of FD through phosphorylation. The absence of both Hd3a and RFT1 functions completely blocks transition to reproductive growth (Komiya et al., 2009). This implies that, in the meristem, FD and possibly other downstream targets of Hd3a and RFT1 induce genes that promote meristem phase transition. Expression of both MADS14 and MADS15, which encode AP1/FUL-like MADS domain proteins, was suppressed in Hd3a and RFT1 double knockdown plants, suggesting that these genes function downstream of the florigen signal (Komiya et al., 2008, 2009). Moreover, it has been demonstrated that MADS15 transcription is induced by phosphorylated FD in the presence of Hd3a (Taoka et al., 2011). These reports suggest that MADS14 and MADS15 may be regulators of phase transition in the meristem; however, neither their functions nor their expression patterns in the meristem at phase transition have been demonstrated. In this study, we demonstrate that four MADS box genes, PANICLE PHYTOMER2 (PAP2) (Kobayashi et al., 2010), MADS14, MADS15, and MADS18 are activated in the meristem at phase transition and function redundantly in specifying IM identity in rice. RESULTS PAP2 Expression Is Induced in the Meristem upon Reproductive Transition Rice PAP2 encodes MADS34, a grass-specific SEPALLATA (SEP) subfamily MADS domain protein (see Supplemental Figure 1 online). PAP2 plays multiple roles, including the determination of spikelet meristem (SM) identity and the suppression of the extra growth of glumes (Gao et al., 2010; Kobayashi et al., 2010). To understand PAP2 function further, we analyzed its spatial and temporal expression throughout inflorescence development. A 2.2-kb fragment of the PAP2 promoter region was fused with the b-glucuronidase (GUS) gene and transformed into wild-type rice plants. T1 seedlings were grown for 7 d under long-day (LD) conditions and then transferred to short-day (SD) conditions, which promote the transition to the reproductive phase. GUS activity was not detected in the SAM during the vegetative phase (Figures 1A and 1B). Upon transition to reproductive development, clear induction of PAP2:GUS activity throughout the SAM was observed (Figure 1C). GUS activity became localized to the incipient SM at the spikelet initiation stage (Figures 1D and 1E). During spikelet organ development, GUS activity was observed in initiating glumes (Figure 1F). PAP2 expression in the SM and glumes was consistent with the known functions of PAP2, namely, promotion of spikelet identity and Figure 1. Spatial and Temporal Distribution of PAP2 Promoter Activity. (A) to (F) GUS staining in the shoot apex of PAP2:GUS plants at the vegetative stage (A), slightly before reproductive transition (B), at the transition stage (C), and at the spikelet initiation stage ([D] and [E]). A close-up view of the region enclosed with a square in (D) is shown in (E). GUS activity in the developing spikelet is shown in (F). (G) and (H) In situ hybridization analysis of PAP2 expression in the shoot apex at the transition stage (G) and in the inflorescence at spikelet initiation stage. Arrowhead, SAM; BM, branch meristem; SM, spikelet meristem; FM, floral meristem. (I) The relationship between meristem size and GUS activity is shown. suppression of extra growth of glumes (Kobayashi et al., 2010). Localization of PAP2 mRNA in the meristem at transition stage and the SMs was confirmed by in situ hybridization analysis (Figures 1G and 1H). To determine the timing of PAP2 induction precisely, the relationship between the meristem size and the onset of PAP2:GUS activity was examined (Figure 1I). Elongation of the SAM, a sign of the transition to reproductive growth, was accompanied by a dramatic increase in GUS activity. These results suggest that PAP2 is involved in reproductive transition and/or the establishment of the identity of the IM. Inflorescence Meristem Identity of Rice A null allele of the PAP2 locus, pap2-1, does not show any significant changes in the very early stages of inflorescence development (Kobayashi et al., 2010). Because the SEP MADS domain proteins usually function in complexes with other, related MADS domain proteins (reviewed in Gramzow and Theissen, 2010), we postulated that the effects of the loss of PAP2 activity during reproductive phase transition may be masked by redundancy with other gene(s). To test this idea, we searched for MADS box genes that show overlapping expression patterns with PAP2 in the SAM during the reproductive transition. For this purpose, we adopted a laser microdissection (LM) and microarray technique and specifically sampled SAMs at various developmental stages (Figure 2A). All samples were collected from plants grown in the field in 2008. SAMs from the Vegetative 1 (V1) stage were harvested on June 24th before the induction of RFT1, a primary determinant of rice flowering under natural LD conditions (Komiya et al., 2009). V2 SAMs were sampled on July 8th, several days after RFT1 induction was first observed in the leaves. Because slight variations in size were observed in the SAMs collected on July 15th, each SAM was identified as being at either the transition (V/R) or elongation (R1) stage, depending on its size as measured under the microscope. In situ hybridization analysis of ABERRANT PANICLE ORGANIZATION1 (APO1) expression in V/R stage SAMs revealed a mixture of APO1-expressing and nonexpressing SAMs (see Supplemental Figure 2 online). Because induction of APO1 is concurrent with reproductive transition (Ikeda-Kawakatsu et al., 2009), this indicates that the V/R stage is a critical time in the transition of the SAM to the IM. Inflorescences at the primary branch initiation, secondary branch initiation, and spikelet initiation stages were collected as R2, R3, and R4 stages (Figures 2A and 2B). Microarray analysis was performed with RNAs isolated from dissected meristems and very young inflorescences. The overall profile of gene expression during meristem phase transition is shown in Supplemental Figure 3 online. The levels of PAP2 RNA increased from the V2 to the V/R stage, and this was consistent with changes in PAP2:GUS activity (Figures 2B and 2C). In addition, expression of three MADS box genes (out of 34 MADS box genes on the microarray) showed increases in expression between the V1 and the V/R stages (Figures 2B and 2C). Intriguingly, these three genes are MADS14, MADS15, and MADS18, which belong to the AP1/FUL-like subfamily of MADS box genes (Litt and Irish 2003; Fornara et al., 2004; Preston and Kellogg, 2006; see Supplemental Figure 1 online). The only other member of this subfamily in rice, MADS20, showed no detectable expression in meristems at any of the stages examined in this study. Two of the three MADS box genes, MADS14 and MADS18, displayed similar expression patterns in which the mRNA level increased between the V1 and V2 stages (Figure 2C). Accumulation of MADS14 mRNA further increased from V2 to V/R, whereas the expression of MADS18 remained relatively constant after the V2 stage. MADS15 showed an expression pattern similar to PAP2; that is, its mRNA level increased from the V2 to the V/R stage, accompanying the transition to reproduction. The SEP subfamily of rice consists of five genes, including PAP2 (Malcomber and Kellogg, 2005; Zahn et al., 2005). As previously reported (Arora et al., 2007; Kobayashi et al., 2010), none of the other four SEP genes, MADS1, MADS5, MADS7, 3 of 12 Figure 2. LM Analysis of Genes Expressed in Shoot Meristems during Reproductive Transition. (A) Micrographs of the meristem stages that were analyzed. V1 and V2 are meristems collected before transition. V/R and R1 are meristems at the transition stage and elongation stage, respectively. R2 and R3 are the primary branch initiation and secondary branch initiation stages of the inflorescences, respectively. (B) Heat map representing the expression of rice MADS box genes during early stages of inflorescence development. Genes that show a significant level of expression in at least one of the 24 arrays performed are included. The intensity of the red color represents absolute expression of each gene. (C) Expression patterns of four MADS box genes that showed a significant increase in expression level at reproductive transition. The values represent mean expression levels 6 SD (n = 3 to 5) obtained from the microarray analysis of microdissected meristems. 4 of 12 The Plant Cell and MADS8, showed detectable expression at the early stages of inflorescence development studied here except that a low level of MADS5 expression was observed in the spikelet initiation stage (R4) (Figure 2B). Inflorescence Development Was Severely Perturbed in the MADS Quadruple Knockdown Plants To understand the role of PAP2 and the three AP1/FUL-like genes during reproductive transition and early stages of inflorescence development, we generated transgenic rice plants in which the expression levels of MADS14, MADS15, or MADS18 were reduced by RNA interference (RNAi). Despite a significant reduction in the levels of mRNA from each of these genes, neither inflorescence development nor flowering time was altered in single RNAi plants (see Supplemental Figure 4 online; Fornara et al., 2004). We then produced pMADS14;15;18i, an RNAi construct that simultaneously suppresses expression of MADS14, MADS15, and MADS18. This construct was introduced into wild-type, pap2-1/+, and pap2-1 plants. Plants transformed with the empty RNAi vector were used as controls. Specificity of the RNAi target for the three genes was confirmed by quantitative real-time RT-PCR (see Supplemental Figure 5 online). The expression level of MADS20, another member of the AP1/FUL-like genes of rice, was not clearly affected in transgenic lines containing pMADS14;15;18i. Growth of all transgenic plants was normal during the vegetative phase, whereas inflorescence development was severely perturbed in the homozygous mutant pap2-1 plants transformed with pMADS14;15;18i (Figure 3). In normal rice development, reproductive transition is accompanied by the initiation of stem elongation. The stem gradually elongates during the early stages of inflorescence development, with rapid elongation starting a few days before heading. This results in the exposure of the inflorescence as the flowers open (Figures 3A and 3B). Although extensively delayed, stem elongation finally occurred and proceeded very slowly in the MADS14;15;18i pap2-1 quadruple knockdown lines, indicating that reproductive transition began in these plants. However, leaf initiation continued even after the start of stem elongation (Figure 3C). The leaves produced during the later stages showed various abnormalities, including serration and altered phyllotaxy (Figure 3C). The severity of defects in inflorescence development varied among transgenic lines. In weakly affected lines, although delayed and reduced in size, the inflorescence was eventually formed. In moderate lines, the vegetative SAM was observed in the growing inflorescence, indicating incomplete reproductive transition (see Supplemental Figure 6 online). In strong lines, the inflorescence was not formed (see below). The severity roughly correlates with the effectiveness of knockdown of the three AP1/ FUL-like genes (see Supplemental Figure 5 online). We chose two severe quadruple knockdown lines (q8 and q10) and analyzed their reproductive development by scanning electron microscopy (Figures 4A to 4J), sectioning (Figures 4K to 4R), in situ hybridization (Figures 4S and 4T), and observation under the microscope (Figures 4U to 4Z). In wild-type rice, primary inflorescence branches arise in the axils of bracts (extremely reduced leaves) in a spiral phyllotaxy (Figures 4A to 4C, 4L, 4U, Figure 3. Inflorescence Development of MADS14;15;18i/pap2-1 Quadruple Knockdown Plants. (A) Nontransgenic wild-type (left) and MADS14;15;18i pap2-1 quadruple knockdown (right) plants. (B) Inflorescences on a wild-type plant after heading. (C) Heading never occurred in the quadruple knockdown lines. Instead, leaf production continued after heading had occurred in the wild-type plants. A close-up view of the youngest leaf showing a serration (arrowhead) is shown in the inset. [See online article for color version of this figure.] and 4V). Each primary branch forms secondary branches and spikelets (Figures 4D, 4E, 4M, and 4N). Hair-like structures arise on the bracts (Figure 4C), and the developing inflorescence is covered with the hair-like structure by the spikelet initiation stage (Figures 4D, 4E, and 4V). Change to spiral phyllotaxy was observed in both q8 and q10, indicating the start of reproductive transition and consistent with the start of stem elongation (Figures 4F to 4I). However, unlike wild-type inflorescence development in which primary branches are formed, initiation of vegetative leaf primordia continued (Figures 4G to 4J, 4P, 4Q, and 4R). Axillary meristems were formed in the axils of these leaves and grew as shoots (Figures 4G, 4H, and 4W). This process was reiterated and led to the formation of multiple shoots on one stem (Figures 4I, 4J, 4Q, 4X, 4Y, and 4Z). Spikelets were produced in the wild-type inflorescence (Figures 4M and 4N), whereas shoot formation was repeated and each meristem continued to generate leaf primordia in the quadruple knockdown plants (Figures 4Q and 4R). To determine further the identity of meristems in the quadruple knockdown plants, we performed in situ hybridization analysis of APO1, which is normally expressed in the IM and the BM but not in the vegetative SAM (Ikeda-Kawakatsu et al., 2009). Absence of the APO1 signal in the quadruple knockdown plants confirmed that the axillary meristems retain the characteristics of the vegetative SAM (Figures 4S and 4T). Our combined data suggest that progression to reproductive transition was severely blocked in MADS14;15;18i pap2-1 plants; thus, IM identity was not established. We propose that PAP2 and three AP1/FUL-like genes act redundantly to promote IM identity and are thus IM identity genes. AP1/FUL-Like MADS Box Genes Are Involved in the Regulation of Flowering In contrast with the severe defects observed in the quadruple knockdown plants, few abnormalities was observed in inflorescence development of the MADS14;15;18i wild-type and Inflorescence Meristem Identity of Rice 5 of 12 Figure 4. Impaired Inflorescence Development in MADS14;15;18i pap2-1 Quadruple Knockdown Plants. (A) to (J) Scanning electron microscopy analysis of wild-type ([A] to [E]) and MADS14;15;18i pap2-1 ([F] to [J]) apices. Each shoot and leaf like structure are indicated with an arrow and yellow dot, respectively ([F] and [G]). (K) to (N) Sections of wild-type inflorescences immediately after transition (K), at the inflorescence branch initiation stage (L), and at the spikelet initiation stage (M). A close-up view of a developing spikelet from (M) is shown in (N). (O) to (R) Sectioning of an MADS14;15;18i pap2-1 apex. Shoot apexes at an early stage (O) and the later stage ([P] to [Q]) are shown. The apical meristem continued to initiate leaf primordia. Multiple shoots derived from axillary meristems of the leaves are observed (Q), and each meristem appears as a vegetative SAM producing leaf primordia. Each shoot is indicated with a star ([P] and [Q]). A close-up view of a meristem initiating leaf primordia in (Q) is shown in (R). (S) and (T) In situ hybridization showing APO1 expression (dark stain) in a developing inflorescence at the secondary branch initiation stage in the wildtype apex (S). APO1 expression is not observed in the apex of the MADS14;15;18i pap2 plant (T). (U) and (V) Developing inflorescences in wild-type plants at the primary branch initiation stage (U) and secondary branch initiation to spikelet initiation stage (V). (W) to (Z) Continuous shoot production in MADS14;15;18i pap2-1 quadruple knockdown plants. Shoots and axillary buds are indicated by circles and arrowheads, respectively. Leaves were removed to show the shoot apices in (U) to (Z). B, bract; FM, floral meristem; LP, leaf primordia; PB, primary branch; PH, panicle hair; S, spikelet. Bars = 200 mm in (K) to (T), 100 mm in (U) to (W), 1 mm in (X) and (Y), and 1 cm in (Z). 6 of 12 The Plant Cell MADS14;15;18i pap2-1/+ plants (see Supplemental Figure 7 online). This implies that, among the four genes, PAP2 function alone is sufficient for normal inflorescence development. However, because upregulation of PAP2 expression was observed in some of the triple knockdown lines (see Supplemental Figure 5 online), we cannot rule out a possibility that the increased PAP2 activity compensated for the reduction of AP1/FUL expression in triple knockdown lines. We observed a slight delay in flowering in the MADS14;15;18i wild-type and MADS14;15;18i pap2-1/+ plants (Figure 5A). To quantify the delay, transgenic plants of the T0 generation were grown under LD conditions in a growth chamber until emergence of the eighth leaf and then transferred to SD conditions. We counted the number of leaves produced after transfer to SD conditions in six and seven independent transgenic plants of MADS14;15;18i wild type and MADS14;15;18i pap2-1/+, respectively. The number of leaves in a single plant from each line and the average leaf number in each genetic background are shown in Figure 5A. Although the effects varied among independent transgenic lines, a noticeable increase in leaf number was observed in plants from transgenic lines containing MADS14;15;18i. These results indicate a possibility that the three AP1/FUL-like genes of rice are involved in the regulation of flowering time. PAP2 and AP1/FUL-Like Genes Function Upstream of Hd3a and RFT1 in Leaves To reveal the cause of the delay in flowering time, the expression levels of the rice flowering genes Hd3a and RFT1 were examined (Figures 5B and 5C). Quadruple knockdown and pap2-1 plants were grown under natural LD conditions and then transferred to a growth chamber under SD conditions. Expression of Hd3a and RFT1 were induced after the transfer to SD conditions in wild- type control plants. By contrast, extremely low levels of Hd3a induction were observed in the quadruple knockdown lines. A slight decrease in Hd3a induction rate was observed in pap2-1. Expression levels of RFT1 under the SD conditions were also lowered in pap2-1 and the quadruple lines compared with that observed in wild-type plants. Interaction of MADS Domain Proteins MADS domain proteins dimerize and may form multimeric complexes to function (reviewed in Gramzow and Theissen, 2010). Therefore, we examined the interactions among PAP2, MADS14, MADS15, and MADS18 using the yeast two-hybrid method (Figure 6A). The entire coding regions of the cDNAs were fused to the activation domain (pGAD) and binding domain (pGBD) and tested for interactions. As shown in Figure 6A, a strong interaction was observed between MADS14 and MADS15. Weak but significant interactions also occurred between PAP2 and PAP2, PAP2 and MADS14, and PAP2 and MADS15. This indicates that PAP2 is able to form heterodimers with MADS14 and MADS15, in addition to forming a homodimer. To pursue further the possibility that PAP2 interacts with MADS14 and MADS15, we performed transient coexpression assays of epitope-tagged PAP2 and MADS14 or MADS15 in Nicotiana benthamiana leaves. The presence of MADS14-FLAG, MADS15-FLAG, and PAP2-Myc proteins in total protein samples was confirmed by immunoblotting using anti-Myc antibodies (Figures 6B and 6C). After coimmunoprecipitation using anti-FLAG antibodies, interactions between MADS14 and PAP2 (Figure 6B) and MADS15 and PAP2 (Figure 6C) were detected by immunoblotting analysis with an anti-Myc antibody. These biochemical data indicate that PAP2 has the ability to interact with MADS14 and MADS15 in vivo. Figure 5. Delay of Flowering Time in Triple Knockdown Plants. (A) The numbers of leaves produced by individual plants between transfer to SD conditions and heading. Plants were transferred to SD conditions at the eight-leaf stage, when the eighth leaf was fully expanded. Data are shown for wild-type (wt) and pap2-1/+ and pap2-1 mutant lines transformed with either the empty vector or the MADS14;15;18i construct. Each bar in the left panel indicates an independent transgenic line. The average number of leaves in each genotype is shown in the right panel. Bars and asterisks in the right panel represent SD and significant difference from wild-type determined by the Student’s t test at P < 0.05, respectively. (B) and (C) Relative expression levels of Hd3a (B) and RFT1 (C) in leaves of pap2-1 and MADS14;15;18i pap2-1 (quad) plants. RNA was isolated from plants under LD conditions and 3 d after transfer to SD conditions. Bars represent SD for three biological replications. Inflorescence Meristem Identity of Rice 7 of 12 7B). In severe cases, spikelets developed directly from callus (data not shown). By contrast, when the same rolC-Hd3a:GFP construct was introduced into a pap2-1 background, the plants exhibited a novel phenotype (Figures 7C and 7D). Precocious stem elongation was observed, suggesting transition to reproductive development; however, inflorescence development did not initiate. Instead, vegetative shoots were repeatedly generated resulting in a bushy appearance. Roots initiated from each tiller shoot, and tillers were connected with very thin stringlike stems (Figure 7C). Inflorescences eventually initiated in the rolC-Hd3a:GFP pap2-1 lines. Spikelets in these lines contained increased numbers of glumes (specialized leaves subtending flowers), indicating retarded specification of SM identity. These results indicate that PAP2 function is required to initiate the precocious inflorescence development induced by Hd3a misexpression, supporting our hypothesis that PAP2 and the three AP1/FUL-like genes act downstream of Hd3a to promote IM identity. DISCUSSION Regulation of IM Identity in Rice Figure 6. Interaction among Four MADS Box Proteins. (A) Yeast two-hybrid interaction assays. Full-length coding regions of PAP2, MADS14, MADS15, and MADS18 were cloned in pGBKT7. PAP2 or MADS15 were also cloned in pGADT7. After cotransformation of pGBKT7 and pGAD17 vectors, yeast was grown on nonselective (-LT), weak selective (-LTH), and strong selective (-LTHA) media. The empty activation domain vector (empty) was included as a negative control. (B) and (C) Interaction assay in N. benthamiana leaves using MADS14FLAG, PAP2-Myc, and MADS15-Myc (B) and MADS15-FLAG, PAP2-Myc, and MADS14-Myc (C). Proteins were coexpressed in N. benthamiana leaves, and the total protein extracts were subjected to immunoprecipitation (IP) using an anti-FLAG antibody. The resultant immune complexes were analyzed on immunoblots with anti-Myc antibodies. Genetic Interaction between Hd3a and PAP2 Although it has been suggested that MADS14 and MADS15 act downstream of Hd3a/RFT1 in the meristem, their functions in this pathway have not been clearly elucidated (Komiya et al., 2008, 2009; Taoka et al., 2011). Misexpression of Hd3a causes extremely early flowering in rice and a number of other plant species (Izawa et al., 2002; Tamaki et al., 2007). We took advantage of this phenomenon to test the hypothesis that PAP2 functions downstream of Hd3a in the meristem to establish IM identity. Reproductive transition was remarkably accelerated by introduction of the rolC-Hd3a:GFP (for green fluorescent protein) gene (Tamaki et al., 2007) in wild-type plants (Figures 7A and Our results show that expression of PAP2, a MADS box gene belonging to a grass-specific subclade of the SEP subfamily, and three AP1/FUL-like genes (MADS14, MADS15, and MADS18) is activated in the meristem at the transition to the reproductive phase. The knockdown of the three AP1/FUL-like genes did not significantly affect inflorescence development. On the other hand, the elimination of PAP2 function in the triple knockdown plants severely impeded transition of the SAM to the IM. Because IM identity is established normally in the pap2-1 mutant (Kobayashi et al., 2010), the most likely interpretation of the results with the quadruple knockdown plants is that IM identity in rice is determined by the combined actions of the three AP1/FUL-like genes with PAP2. The negative correlation between total expression levels of three AP1/FUL-like genes and the severity observed in inflorescence development probably indicates the redundancy in the functions of the three genes. However, currently it is unknown whether all four genes contribute equally in specifying IM identity. The individual contributions of each gene to the regulation of IM identity needs to be elucidated. It has been proposed that MADS14 and MADS15 function downstream of Hd3a and RFT1 to transduce the florigen signal (Komiya et al., 2008, 2009). More recently, transient assays in cultured cells were used to show that the transcription of MADS15 is indeed induced by a complex containing Hd3a, a 14-3-3 protein, and FD (Taoka et al., 2011). Here, we show that not only MADS14 and MADS15 but also MADS18 and PAP2 exhibit clear induction in the SAM before any visible change in meristem morphology. We also confirmed that PAP2, MADS14, and MADS15 interact with one another in vivo. The alleviation of the precocious flowering phenotype caused by ectopic overexpression of Hd3a in the pap2-1 background also supports the notion that PAP2 functions downstream of Hd3a in the meristem. On the basis of these results, we conclude that the four genes analyzed in this study act in the SAM as downstream 8 of 12 The Plant Cell Figure 7. Genetic Interaction between PAP2 and Hd3a. (A) A wild-type plant regenerated from callus with no inflorescences. (B) A wild-type plant transformed with prolC-Hd3a:GFP, of the same age as that shown in (A), with two inflorescences (yellow arrowheads). (C) A pap2-1 plant transformed with rolC-Hd3a:GFP, of the same age as those shown in (A) and (B). Stem elongation is observed but no inflorescences have formed. The thin, elongated stems are marked with white arrowheads. (D) Frequencies of phenotypes observed in rolC-Hd3a:GFP/wild type (wt) and rolC-Hd3a:GFP/pap2-1 transgenic plants. Twenty and 16 independent lines for rolC-Hd3a:GFP/wild type and rolC-Hd3a:GFP/pap21, respectively, were analyzed. (E) An inflorescence on a rolC:Hd3a/wild-type plant. A few normal spikelets are present. (F) An inflorescence on a rolC:Hd3a/pap2-1 plant. Each spikelet contains an increased number of elongated glumes (white arrowheads). regulators of the florigen signal and give rise to the phase change of the SAM to the IM (Figure 8). Meanwhile, the extensive decline of the Hd3a and RFT1 expression levels in leaves of the knockdown plants under SD conditions suggests the possibility that the AP1/FUL-like genes of rice and PAP2 function upstream of Hd3a and RFT1 in leaves. An attractive scenario to explain these results is that AP1/FULlike genes work in distinct ways in leaves and the SAM to promote reproductive transition (Figure 8). First, AP1/FUL-like genes induce expression of Hd3a and RFT1 in leaves. Once the florigen is transported from the leaves to the SAM, transcription of the AP1/FUL-like genes and PAP2 is induced in the SAM itself, resulting in the transition of the meristem phase to the IM. This two-step regulation might help to accelerate meristem phase change by amplifying the florigen signal. Currently, it is not clear whether the delay in flowering observed in the triple and quadruple knockdown plants is caused by the loss of AP1/FUL-like activities in leaves, in the meristem, or both. Similarly, we cannot rule out a possibility that the failure in establishing IM identity in the quadruple knockdown plants is a consequence of the reduced activity of Hd3a and RFT1, florigen genes, in leaves. However, we favor an interpretation that the meristem defects were caused by the reduced function of the three AP1/FUL-like and PAP2 genes in the meristem based on the clear induction of these genes in the SAM upon the transition. Results of the genetic analysis between PAP2 and Hd3a also support a model in which Hd3a works upstream of the four MADS box genes in the control of meristem phase transition. To obtain a conclusive view, roles of the four MADS box genes in leaves and the meristem should be experimentally distinguished by more detailed analysis in the near future. Our findings that AP1/FUL-like genes work upstream of Hd3a and RFT1 in leaves are consistent with the report that MADS14 and MADS15 work downstream of Early heading date1, a positive regulator of rice flowering, in leaves (Doi et al., 2004). However, these results are inconsistent with a previous report describing that the expression of MADS14 and MADS15 in leaves is regulated by RFT1 (Komiya et al., 2008). A possible explanation for this discrepancy is that RFT1 and AP1/FUL-like genes mutually activate each other’s expression. Temperate grasses, including wheat (Triticum aestivum) and barley (Hordeum vulgare), require a period of cold temperature to initiate reproductive transition. In these species, VERNALIZATION1 (VRN1), encoding an AP1/FUL-like subfamily MADS domain protein, is a critical regulator of the vernalization response and flowering (Danyluk et al., 2003; Trevaskis et al., 2003; Yan et al., 2003; Shitsukawa et al., 2007). In wheat leaves, VRN1 functions upstream of FT (Shimada et al., 2009). It has been proposed that VRN1 triggers flowering through activation of the daylength response in leaves and acceleration of phase transition in the meristem, although the function of VRN1 in the meristem has not been clearly demonstrated (Greenup et al., 2009). This suggests that the functions of the AP1/FUL-like genes might be conserved in grass species irrespective of any vernalization requirement for flowering. Contribution of Grass-Specific Genes to the Evolution of Grass-Specific Forms Gene duplication is a source of novel genes. Duplicated genes acquire new functions by changes in their coding regions and/or their regulatory sequences. The SEP and AP1/FUL subfamilies of the MADS box genes probably originated and underwent multiple duplication events before the diversification of the extant angiosperms (Litt and Irish, 2003; Malcomber and Kellogg, 2005; Malcomber et al., 2006; Preston and Kellogg 2006; Zahn et al., 2005). The evolution of the SEP and AP1/FUL homologs is conceivably associated with the subsequent dramatic success of angiosperms. Further duplication events occurred in the ancestors of grasses, resulting in grass-specific SEP and AP1/FUL Inflorescence Meristem Identity of Rice 9 of 12 Figure 8. A Model of the Genetic Interaction between MADS Box Genes Regulating the Specification of IM Identity in Rice. AP1/FUL-like genes and probably PAP2 induce expression of Hd3a and RFT1 in leaves. Once the florigen is transported from the leaves to the SAM, transcription of the AP1/FUL-like genes and PAP2 is induced in the SAM. This results in the transition of the vegetative meristem phase to the IM. genes. Among the five SEP genes in rice, PAP2 is unique in being transcribed in the SAM upon phase transition. This is in addition to its expression in the SM and glumes, in which abnormalities are observed in pap2-1 mutants. The importance of the SEP proteins to mediate multimerization of MADS proteins has been demonstrated. In Arabidopsis, SEP genes influence floral meristem identity and floral organ development through formation of multimeric complexes with other MADS domain proteins that are expressed in a spatially and temporally regulated manner (de Folter et al., 2005; Immink et al., 2009). In addition to biochemical analysis, the interaction between SEP and AP1/FUL MADS box genes has also been demonstrated by genetic analysis in a variety of species (Ditta et al., 2004; Pelaz et al., 2001). This indicates that SEP and AP1/FUL proteins potentially interact when they exist in the same cells. Thus, once PAP2 acquired specialized machinery allowing it to be transcribed in the SAM upon reproductive transition, its interaction with SAM-expressed AP1/FUL MADS box genes, such as MADS14 and MADS15, is expected. This enables PAP2 to be involved in the regulation of IM identity. This is a clear example of gene duplication leading to diversification in the location and timing of expression, resulting in the creation of a novel function. (Kobayashi et al., 2010). PAP2 also functions as a suppressor of the extra elongation of glumes (Gao et al., 2010; Kobayashi et al., 2010). In general, leaf growth is extensively suppressed during reproductive growth. Assuming that suppression of glumes is likely to be essential to secure the SM identity in grass inflorescence development, an attractive interpretation of PAP2 function is that it ensures transition to the SM in two ways, namely, induction of SM identity and suppression of glumes. The new role of PAP2 as a positive regulator of IM identity revealed in this study further expands the importance of PAP2 in the regulation of rice inflorescence development. In all three roles, PAP2 positively regulates the progression of the steps in inflorescence development. Generation of the PAP2 gene in grasses may be a molecular innovation in inflorescence development. Although the functions of PAP2 orthologs in other grass species remain to be analyzed, the maize (Zea mays) and wheat orthologs MADS4 and AGLG, respectively, exhibit expression patterns similar to that of PAP2, suggesting that their function may be conserved in grass species (Yan et al., 2003; Danilevskaya et al., 2008). METHODS Sample Collection for LM The Role of PAP2 in Rice Inflorescence Development The timing of the meristem phase change is crucial for plant development, and subtle modifications of this timing readily result in the emergence of novel morphologies (Prusinkiewicz et al., 2007). During rice inflorescence development, newly formed meristems acquire BM or SM identity depending upon the timing and position of their occurrence. We showed previously that PAP2 functions as a positive regulator of SM identity Rice (Oryza sativa ssp japonica cv Nipponbare) plants grown in a paddy field in Tsukuba, Japan (;36°N) under field conditions during the 2008 cultivation season were used. Shoot apices were collected at different growth stages. The samples were fixed in 75% ethanol and 25% acetate and embedded in paraffin using a microwave processor (Energy Beam Sciences) according to Takahashi et al. (2010). The paraffin-embedded samples were cut into 10-µm-thick sections using a microtome (Leica RM2255). LM was performed using the Veritas Laser Microdissection System LCC1704 (Arcturus). 10 of 12 The Plant Cell Microarray Analysis Total RNA was extracted from the collected cells with a Pico-Pure RNA isolation kit (Arcturus) according to the manufacturer’s protocol. The quantity and quality of RNAs were checked using the Agilent 2100 Bioanalyzer (Agilent Technologies). One-color spike mix was added to the total RNA prior to the labeling reaction, and labeling was performed using a Quick Amp Labeling Kit, One-Color (Agilent Technologies). The cyanine-3 (Cy3)-labeled cRNA was purified using the RNeasy mini kit (Qiagen), and the quantity was checked using a NanoDrop ND-1000 UV-VIS spectrophotomer (NanoDrop Technologies). The Cy3-labeled cRNA sample was fragmented and hybridized with 4 3 44k microarray of rice (Rice Annotation Project-Data Base) gene expression microarray slides (Agilent; G2519F#15241) at 65°C for 17 h. The hybridization and washing were performed according to the manufacturer’s instructions. The slides were scanned on an Agilent G2505B DNA microarray scanner, and background correction of the Cy3 raw signals was performed using Agilent’s Feature Extraction software (version 9.5.3.1). We detected 36,100 independent signals corresponding to 27,201 annotated loci and 340 microRNA precursors annotated in the Rice Annotation Project-Data Base. We selected 19,881 probes that each had a raw signal intensity above 50 in at least one of the 24 microarray data. The numbers of microarrays examined are three for the V1, V2, V/R, R3, and R4 stages, four for the R1 stage, and five for the R2 stage. The selected raw signal intensities were subjected to quantile normalization for interarray comparison and then transformed to a log2 scale using the R program (http://www.r-project.org/) and the limma package (Smyth, 2005). For redundant probes with the same locus ID, we calculated the average normalized values and thereby obtained the expression profile for 15,193 genes. The median expression values across the seven stages were subtracted for each gene, and the gene-normalized values were assigned as relative expression values. subsequently transferred to SD conditions (12 h light at 28°C, 12 h dark at 24°C). Meristem samples were treated with acetone on ice for 15 min. After washing with 100 mM NaPO4 buffer, pH7.0, the samples were incubated in GUS staining solution (100 mM NaPO4 buffer, pH 7.0, 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% Triton X-100, and 0.5 mg/mL 5-bromo-4-chrolo-3-indolyl-b-Dglucuronide) at 37°C in the dark. Scanning Electron Microscopy Samples were fixed in 2.5% glutaraldehyde overnight at 4°C and dehydrated in a series of ethanol solutions, and then the final ethanol solution was substituted with 3-methylbutyl acetate. The samples were then dried at critical point, sputter-coated with platinum, and observed under a scanning electron microscope (S-4000; Hitachi) at an accelerating voltage of 10 kV. Real-Time RT-PCR Analysis Total RNA was extracted using the Plant RNA Isolation Mini Kit (Agilent Technologies). After DNase I treatment, first-strand cDNA was synthesized by SuperScript III reverse transcriptase (Invitrogen). RT-PCR was performed using the primer sets shown in Supplemental Table 1 online. The relative abundance of mRNAs were measured with a Light Cycler 480 system (Roche Applied Science) and normalized against the expression of ubiquitin. For real-time PCR, three replicates were used for each sample. In Situ Hybridization Analysis In situ hybridization of the APO1 and PAP2 genes was performed as previously described (Ikeda-Kawakatsu et al., 2009; Kobayashi et al., 2010). Vector Construction A 520-bp fragment from MADS14 containing significantly high homology with MADS15 and MADS18 was used to generate the RNAi trigger. This fragment was amplified using the primers OM14i-110XbaF and OM14i630XbaR (see Supplemental Table 1 online) and then cloned into the pZH2Bi KXB SpeBam vector (Kuroda et al., 2010) in a tail-to-tail orientation flanking the intron from a rice aspartic protease gene, resulting in the vector pOsMADS14;15;18i. Similarly, 67-, 77-, and 64-bp fragments were used as RNAi triggers to generate single knockdown lines of MADS14, MADS15, and MADS18, respectively. These fragments were amplified using the primer sets OM14iXba-F/OM14iXba-R (MADS14), OM15iXba-F/OM15iXba-R (MADS15), and OM18iXba-F/OM18iXba-R (MADS18) (see Supplemental Table 1 online) and then cloned into pZH2Bi KXB SpeBam in a tail-to-tail orientation flanking the intron from a rice aspartic protease gene, resulting in the vectors pMADS14i, pMADS15i, and pMADS18i, respectively. To construct pPAP2:GUS, a 2.2-kb PAP2 promoter region was amplified using the primers shown in Supplemental Table 1 online. This fragment was cloned into the pENTR/D-TOPO vector (Invitrogen). The resulting vector was inserted pGWB3 by the LR recombination reaction (Invitrogen) (Nakagawa et al., 2007). Rice Transformation Rice transformation was performed as described by Hiei et al. (1994). Wild-type (cv Nipponbare) and pap2-1 (Kobayashi et al., 2010) seeds were used to induce callus for transformation. Hygromycin-selected T1 generation transgenic plants were grown in a growth chamber. GUS Staining Transgenic plants containing the PAP2:GUS gene were grown under LD conditions (14 h light at 28°C, 10 h dark at 24°C) in a chamber for 7 d and Yeast Two-Hybrid Analysis The coding regions of PAP2, MADS14, MADS15, and MADS18 were amplified using the primer sets shown in Supplemental Table 1 online, cloned into pTA2 (TOYOBO) by TA cloning, and then transferred to the yeast two-hybrid vector pGBKT7 containing the GAL4 DNA binding domain and the TRP1 gene. PAP2 and MADS15 were cloned into pGADT7 containing the GAL4 activation domain and LEU2. Plasmids were cotransformed into the AH109 strain of Saccharomyces cerevisiae with the Matchmaker twohybrid system (Clontech). Two-hybrid interactions were assayed on selective synthetic defined media, lacking Leu and TRP (-LT), Leu, Trp, and His (-LTH), or Leu, TRP, His, and adenine (-LTHA). Transient Expression in Nicotiana benthamiana and Coimmunoprecipitation Assays To express the tag-fused proteins, the coding regions of PAP2, MADS14, and MADS15 were amplified using the primer sets shown in Supplemental Table 1 online and cloned into pENTR/D-TOPO (Invitrogen), then cloned into pGWB11 (35S promoter, C-FLAG) or pGWB17 (35S promoter, C-4xMyc) using LR clonase mix (Invitrogen) (Nakagawa et al., 2007). The expression constructs were introduced into Agrobacterium tumefaciens strain EHA105. For transient expression, Agrobacterium strains carrying each construct were infiltrated into leaves of 3- to 4-week-old N. benthamiana as previously described (Kinoshita et al., 2010). Protein was extracted from N. benthamiana leaves 3 d after infiltration. Total protein was extracted according to Gregis et al. (2009). Both total and immunoprecipitated protein were analyzed by SDS-PAGE and immunoblotting using anti-FLAG (anti-ECS [DDDDK]) (Bethyl; A190-101A) and anti c-Myc (Bethyl; A190-104A) antibodies. Goat IgG horseradish peroxidase–conjugated secondary antibody was used (R and D systems; HAF017). Inflorescence Meristem Identity of Rice Accession Numbers Sequence data from this article can be found in the GenBank/EMBL databases under the following accession numbers: Os-MADS34, Q6Q9H6; Os-MADS18, Q0D4T4; Os-MADS20, Q2QQA3; Os-MADS14, P0C5B1; and Os-MADS15, Q6Q912. Supplemental Data The following materials are available in the online version of this article. Supplemental Figure 1. Phylogenetic Analysis of the FUL/AP1, AGL6-Like, and SEP Subfamilies of MADS Box Proteins in Grasses. Supplemental Figure 2. In Situ Hybridization Analysis of APO1 Expression in Meristems at Transition (V/R) Stage. Supplemental Figure 3. The Overall Profile of Gene Expression during Meristem Phase Transition. Supplemental Figure 4. Phenotype of MADS14, MADS15, or MADS18 Single Knockdown Plants. Supplemental Figure 5. Relative Expression Levels of MADS14, MADS15, MADS18, MADS20, and PAP2 in Transgenic Plants Containing an OsMADS14;15;18iRNAi Construct. Supplemental Figure 6. Correlation between the Reduction of Expression Levels and the Severity of the Phenotype. Supplemental Figure 7. Inflorescence Development Is Not Affected in the Triple Knockdown Plants. Supplemental Table 1. Primers Used in This Study. Supplemental Data Set 1. Text File of the Alignment Used for the Phylogenetic Analysis Shown in Supplemental Figure 1. ACKNOWLEDGMENTS We thank Ko Shimamoto for prolC-Hd3a:GFP, Yasufumi Daimon for phylogenetic analysis, and Rebecca Harcourt for critical reading of the article. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (22119008, 22247004, and 21027012 to J.K. and 23580012 to H.Y.), by the Ministry of Agriculture, Forestry and Fisheries (Genomics for Agricultural Innovation; IPG0001 to J.K. and N.Y.), by Genomics for Agricultural Innovation (RTR0002 to Y.S. and Y.N.), by Genomics for Agricultural Innovation (GAM-207 to H.Y.), and by the Program for Promotion of Basic Research Activities for Innovation Bioscience (PROBRAIN; to J.K.). AUTHOR CONTRIBUTIONS K.K., N.Y., Y.S., M.Y., R.Y., M.K., H.Y., Y.N., and J.K. designed and performed the research. K.K., Y.S., and J.K. analyzed data. J.K. wrote the article. Received February 15, 2012; revised April 10, 2012; accepted April 21, 2012; published May 8, 2012. REFERENCES Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y., Yamaguchi, A., Ikeda, Y., Ichinoki, H., Notaguchi, M., Goto, K., and Araki, T. (2005). 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Inflorescence Meristem Identity in Rice Is Specified by Overlapping Functions of Three AP1/FUL -Like MADS Box Genes and PAP2, a SEPALLATA MADS Box Gene Kaoru Kobayashi, Naoko Yasuno, Yutaka Sato, Masahiro Yoda, Ryo Yamazaki, Mayumi Kimizu, Hitoshi Yoshida, Yoshiaki Nagamura and Junko Kyozuka Plant Cell; originally published online May 8, 2012; DOI 10.1105/tpc.112.097105 This information is current as of June 16, 2017 Supplemental Data /content/suppl/2012/05/02/tpc.112.097105.DC1.html Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY