Breeding Science 54 : 147-156 (2004) Developmental Course of and in Rice

Kyoko Ikeda, Hidehiko Sunohara and Yasuo Nagato*

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan

Seed production in rice (Oryza sativa L.) largely de- 2002, Kaplinsky and Freeling 2003). Also in rice, genetical pends on the number of flowers, which is in turn regu- studies on and flowers have recently been lated by the inflorescence architecture. The develop- carried out (Nagasawa et al. 1996, Takahashi et al. 1998, mental process of inflorescence and spikelet in grasses Komatsu et al. 2001, Komatsu et al. 2003, Nagasawa et al. including rice differs considerably from that in other 2003, Sunohara et al. 2003). However, the mechanisms of monocotyledonous and dicotyledonous species, and rice, most of the developmental processes are yet unknown, and an important crop plant, is often used as a model mono- further mutational studies would be indispensable for com- cot plant. Nevertheless, the developmental course of prehensive understanding. Description of mutant pheno- wild-type rice has not yet been well characterized. Thus, types and gene function would require exact understanding detailed description of rice inflorescence and spikelet of those in wild-type plants. In Arabidopsis, the develop- development would be valuable for characterizing mu- mental course of wild-type flower is elaborated into 12 tant phenotypes and also for comparing rice with other stages, using many landmark events (Smyth et al. 1990). grass species. In this study, we showed a number of This staging is now widely referred for describing mutant landmark events in the developmental courses of inflo- phenotypes. rescence and spikelet, and divided their development The structures of inflorescences, , and flowers into nine and eight stages, respectively. This staging sys- of grasses differ considerably from those of dicots and even tem would be useful as a reference for developmental other monocots. Their inflorescence is classified as a com- description. pound spike that is composed of a number of spikelets. Each spikelet contains several flowers (florets), thus the spikelet Key Words: Oryza sativa L., rice, inflorescence, rachis, itself is regarded as a small or secondary inflorescence. The spikelet, flower, developmental staging. architecture of grass inflorescence is usually described by the arrangement of spikelets rather than by the arrangement of flowers (Bell 1991). In many grass species, spikelets are not directly attached to the main axis, but are formed on the Introduction lateral branches. This type of branching is categorized as and observed in Oryza, Sorghum, Panicum, In dicotyledons, recent advances in molecular genetics Echinochloa, Setaria, Avena and so on. On the other hand, on plant development have been mainly achieved using a inflorescences of , wheat, Secale etc. are called spikes few model plants such as Arabidopsis thaliana. Develop- in which spikelets appear to be directly attached to the main mental molecular genetics has also been studied in mono- axis. Rice inflorescence is also categorized from the view- cots, especially in grasses. Differences in morphology and point of whole inflorescence shape as panicle, the conical related genes/gene functions between dicots and monocots inflorescence in which the degree of branching decreases indicate that monocots have their own developmental mech- acropetally. anisms and should be investigated independently. Recent In grasses, the main axis of the inflorescence is called studies including the genome project show that rice (Oryza the rachis (Bell 1991). Lateral branches directly attached to sativa L.), an important crop plant, is a useful model plant in the rachis are called primary (inflorescence) branches. In the monocots because of small genome size (ca. 400 Mbp), same manner, those ramified from the primary branch are abundant information of genome sequence, facility of trans- termed secondary branches. The term ‘branch’ is used only formation, accumulation of genetic information including a when it is sufficiently long to have potentially more than one large number of developmental mutants and so on (Hiei et spikelet. If the branch is short and comprises only one spike- al. 1994, Hong et al. 1995, Sasaki et al. 2002, Yu et al. 2002, let, it is called a peduncle. Goff et al. 2002). The spikelet/flower of grasses differ from those of Numerous mutants have been reported in that dicots and even other monocots. The spikelet meristem first show abnormalities in inflorescence and spikelets (Veit et al. differentiates a pair of called glumes in 1/2 alternate 1993, Chuck et al. 1998, Colombo et al. 1998, Chuck et al. phyllotaxy that are sterile and do not subtend flowers. Sub- sequent glumes subtend flowers at their axils. Grass ‘flow- Communicated by N. Kurata ers’ are also peculiar in that floral organs are enclosed by Received October 31, 2003. Accepted November 28, 2003. two glumes (lemma and palea) arranged in 1/2 alternate *Corresponding author (e-mail: [email protected]) phyllotaxy. The unit comprising lemma, palea and floral 148 Ikeda, Sunohara and Nagato organs is frequently called floret. Grass flowers lack organs labeled antisense probes were prepared from the coding corresponding to sepals. Organs corresponding to are regions of OSH1 and OsMADS45 of rice without poly(A) re- called lodicules but the number is two, in spite of the fact gion. In situ hybridization and immunological detection of that the basic number of floral organs in monocots is three. the signals were performed as reported by Kouchi and Hata Several investigations have described and categorized (1993). rice inflorescence and spikelet development (Matsushima and Manaka 1956, Takeoka et al. 1992). These anatomical Results and Discussion studies are valuable, but the results of recent genetic and mo- lecular studies should be incorporated into the developmen- Structure of mature inflorescence tal description. In this paper, we describe landmark events Rice inflorescence (panicle) consists of a rachis (main during inflorescence and spikelet development in rice, and axis), ten or more primary rachis branches and about 150 report a staging system established as a part of developmen- spikelets (Fig. 1A). Thus rice has two types of inflorescence tal ontology. meristems, rachis meristem and branch meristem. The fates of these two meristems differ as described below. The basal Materials and Methods end of the rachis is easily found by the presence of a trace of . In many cases, the lowermost primary branch is Cultivation of plants formed at its axil. In rice, the rachis meristem is not convert- Rice (Oryza sativa L.) cv. Taichung 65 was used as a ed to a spikelet meristem, but aborted and remains as a ves- reference cultivar, since a large number of mutants have the tige that is detected near the base of the uppermost primary genetic background of Taichung 65. Seeds were sown at the branch (Fig. 1B). The length of rachis is around 18 cm, end of April, and seedlings were transplanted to paddy field whereas the length of inflorescence (rachis length plus pri- at the end of May, and then grown under usual cultural con- mary branch length above rachis tip) is around 22 cm. The ditions. length of inflorescence/rachis and the number of primary branches vary widely among cultivars. Paraffin sectioning Primary inflorescence branches are formed in spiral Inflorescences at various stages were fixed in FAA phyllotaxy, completely different from that in vegetative (formalin : glacial acetic acid : 70 % ethanol = 1 : 1 : 18) and de- phase (1/2 alternate). The length of primary branch is the hydrated in graded ethanol series. Then, we replaced ethanol largest in the 4th or 5th one and then gradually declines to- with xylene, and embedded the samples in Paraplast Plus wards the apex (Fig. 1C). The primary branches bear second- (Oxford Labware, St. Louis, MO). Microtome sections (8 ary branches and spikelets (Fig. 1A). The number of second- µm thick) were stained with Delafield’s hematoxyline and ary branches is correlated with the primary branch length observed with a light microscope. (Fig. 1C). In contrast to the rachis meristem, primary and secondary branches have terminal flowers. Scanning electron microscopy (SEM) Samples were fixed in FAA, dehydrated in a graded Structure of spikelet ethanol series, and replaced with 3-methyl-butyl-acetate. A rice spikelet has only one floret (flower) (Fig. 1D). Samples were critical-point-dried, sputter-coated with Pt, On the peduncle (spikelet axis), two sterile glumes, which and observed under a scanning electron microscope (Hitachi are very small and called rudimentary glumes, are formed as S-4000, Tokyo) at an accelerating voltage of 10 kV. in the other grass species (Fig. 1D). Above the inner rudi- mentary glume, two more sterile glumes are produced. Clearing of young inflorescence These glumes are small but larger than the rudimentary Samples were hand-sectioned to about a 1-mm thick- glumes, and are called empty glumes (Fig. 1D and 1E). ness, fixed in FAA for about 16 h at 4°C and dehydrated in Then, two interlocking large glumes, lemma and palea, are a graded ethanol series. They were cleared in the benzyl- present (Fig. 1D and 1E). The lemma is larger than the palea, benzoate-four-and-a-half fluid devised by Herr (1982), and and has five vascular bundles whereas the palea has three then observed with a light microscope (Olympus IMT-2; (Fig. 1E). The arrangement of these six glumes is 1/2 alter- Olympus Optical Co., Ltd., Tokyo, Japan) equipped with nate, and is quite different from the subsequent floral organs. Nomarski differential interference-contrast optics. As described, sepals are lacking in grasses. Thus, inside the palea, two lodicules, corresponding to petals, are present in in situ hybridization the , but biased to the lemma side (Fig. 1E and 1F). Lod- Samples were fixed in 4 % paraformaldehyde and 0.25 icules are small and whitish, and considered to function in % glutaraldehyde in 0.1 M sodium phosphate buffer. Then the opening of the floret. Inside the lodicules, six stamens they were dehydrated in a graded ethanol series, replaced are positioned in a whorl, and one pistil in the center (Fig. 1E with xylene and embedded in Paraplast Plus. Microtome sec- and 1F). The floral organs are present in the whorl in con- tions (8 µm thick) were placed onto slide glasses coated with trast to 1/2 alternate arrangement of six glumes. Vectabond (Vector Lab. Burlingame, CA). Digoxygenin- Development of inflorescence and spikelet in rice 149

Fig. 1. Mature inflorescence and spikelet of rice. (A) Mature inflorescence just after heading having 13 primary inflorescence branches. pib: primary inflorescence branch, sib: secondary inflorescence branch and sp: spikelet. (B) Close up of the region surrounded by square in (A), showing vestige of rachis meristem (arrowhead). (C) Positional change of primary branch length and the number of spikelets on each primary branch. Vertical bar represents standard error. (D) External appearance of spikelet. (E) Cross section of mature spikelet. (F) Mature flower. Glumes are removed. Spikelet comprises a pair of rudimentary glumes (rg), a pair of empty glumes (eg), two lodicules (lo), six stamens (st) and one pistil (pi). Bar = 5 cm for A, 1 mm for D, E and F.

Development of inflorescence In the vegetative shoot apex, plastochron 2 (P2) leaf primor- We undertook anatomical and histological observation dium is higher than the shoot meristem and covers more than of inflorescence development by SEM, clearing method, and half of it, when P1 primordium is just formed (Fig. 2B), in situ hybridization to clarify the way in which inflores- whereas the tip of the flag leaf primordium is comparable in cence architecture is established. height with that of the rachis meristem when bract 1 primor- The transition from vegetative to reproductive phase dium is just formed (Fig. 2D). occurs in response to an environmental change such as short Then bract 2 and first primary branch primordia are day length and high temperature. After producing the last formed almost simultaneously. Subsequently, ten or more foliage leaf, the flag leaf, the shoot apical meristem (SAM) is bracts and primary rachis branches are rapidly produced in converted to an inflorescence meristem (in this case, rachis spiral arrangement. The rachis meristem reaches maximum meristem). The rachis meristem before producing the first size approximately when bract 3 primordium is formed (Fig. bract (bract 1) differs from the vegetative meristem in that 2E), 1.2 fold in diameter and 1.5 fold in height of vegetative the rachis meristem is taller and slightly wider than the veg- meristem. Subsequently, primary branch primordia are etative meristem (Fig. 2A and 2C). The rachis meristem formed in spiral arrangement (Fig. 2F–2H). The proximal when producing bract 1 primordium can also be identified primary branch primordia, although formed earlier, do not by the relative position of meristem and bract primordium. elongate before most of branch primordia are produced (Fig. 2G). 150 Ikeda, Sunohara and Nagato

The growth of bracts is restricted. Among them, bract 1 is lose its activity after producing a cultivar-specific number of the largest and encloses the rachis meristem but its margins primary branches. do not overlap unlike foliage leaves (Fig. 2H and 2I). The When all primary inflorescence branch primordia have growth of subsequent bracts is severely suppressed, and distal formed, they commence elongation almost simultaneously cells of third and subsequent bracts become hairs (Fig. 2G (Fig. 3A). Subsequent development is faster in distal branches and 2H). than in proximal ones (Fig. 3A). The apex of each primary The conspicuous feature in the early reproductive phase branch retains meristematic activity and produces lateral or- is the drastic change to spiral phyllotaxy from 1/2 alternate gans: bracts, secondary inflorescence branches and spikelets in vegetative phase. This change does not occur abruptly but (Fig. 3B). As in the primary branches, bracts of secondary proceeds rather gradually. The divergence angles of the first branches become hairs (Fig. 3B). Secondary branches and two bracts are slightly smaller than 180° (Fig. 2I), about spikelets do not show 1/2 alternate phyllotaxy, but show in- 160°–170°; they gradually converge to 144° (Fig. 2J). The stead a biased distichous phyllotaxy (superposition in two direction of spiral is either clockwise or counter-clockwise, rows with divergence angle of about 110° (Figs. 3C–3E). depending on which margin of flag leaf primordium be- The secondary branch and spikelets are mutually indis- comes inside. tinguishable at their primordial stages. The secondary In the mature inflorescence, we can find a small vesti- branch meristem formed in the distal region of primary gial protrusion at the base of the highest primary branch branch is immediately transformed to a spikelet meristem (Fig. 1B), but can not find a terminal flower of the rachis. without producing lateral organs (Fig. 3D). In this case, sec- Thus in rice, rachis meristem is assumed to abort at an early ondary branch axis is regarded as peduncle. The fate of stage after producing ten or more primary branch primordia. primary branch meristems and secondary branch meristems To confirm this, we examined the expression of the OSH1 differs from that of a rachis meristem. They are invariably gene, which marks indeterminate cells in the meristem and is converted to a spikelet meristem to form terminal spikelets not expressed in determinate or aborted cells (Sato et al. (Fig. 3D). The terminal spikelet is discriminated from lateral 1996). The OSH1 mRNA was detected constantly in rachis spikelets by the direction of its insertion and the position of meristem during the production of primary branch primordia glumes (Fig. 3D and 3E). When inflorescence becomes (Fig. 2K), but soon after the last (12th in this case) primary about 40 mm long and all floral organ primordia are formed, branch primordium protruded, the OSH1 expression disap- rachis and branches start rapid elongation. During this stage, peared from the rachis meristem (Fig. 2L). These results in- enlargement of organs and differentiation of ovule and pol- dicate that the wild-type rachis meristem is programmed to lens take place.

Fig. 2. Early stages of inflorescence development in rice. (A)–(G) Cleared shoot and inflorescence apices. (A) and (B): Mature vegetative shoot apices. In (A), tips of P1 leaf primordium and shoot meristem are almost same in height. When P1 leaf primordium has just protruded, the tip of P2 leaf is higher than that of the shoot meristem (B). (C): Initial stage of rachis apex before first bract formation. Rachis meristem is larger than the vegetative shoot apex in (A). (D): First bract primordium formation. Tip of the flag leaf primordium is not higher than that of rachis meristem (E): Formation of two primary branch primordiia. Rachis meristem is the largest at this stage (F): Rachis meristem forming several primary branch primor- dia. (G): Final stage of primary branch formation. In this rachis, about ten primary branches are present and bracts are differentiating hairs (white arrowhead). (H): SEM image of young inflorescence. Tip of the bract become hairs (white arrowhead). (I) Rachis apex showing first three branch primordia (1–3). Divergence angles of the first and second branch primordia are close to 180°. (J) Rachis apex showing 2nd to 6th branch primordia. Divergence angles of 4th to 6th primordia are approximately 144°. In (A) and (B), primary branch primordia are numbered from the oldest one. (K) and (L) Expression of OSH1 in rachis apex. OSH1 is expressed in the rachis meristem producing branch primordia (K), but not expressed in the rachis meristem after producing final primary branch primordia (L). Meristems are indicated by black arrowheads. : primary branch meristem, fl: flag leaf, b1: first bract, and b2: second bract. Bar = 150 µm for (A) to (G), 100 µm for (H) to (L). Development of inflorescence and spikelet in rice 151

Fig. 3. Late stages of inflorescence development. (A) Elongation of primary branches (). (B) Differentiation of secondary branch primordia (). Distal cells of bracts of secondary branches become hairs (arrows). (C) Top view of (B) show- ing biased distichous phyllotaxy of secondary branches (). (D) Spikelet formation in primary branch apex. Lateral spikelets (ls) are arranged with the divergence angle of about 110°. (E) Schematic presentation of terminal spikelet (ts) and lateral spikelet (ls) disposition in the pri- mary branch apex. Terminal spikelet is distinguished from lateral spikelets by its direction of insertion and the position of glumes (arrowheads indicate lemma side). Bar = 100 µm.

Development of spikelet lemma and palea, are arranged in 1/2 alternate phyllotaxy in The primary and secondary branch meristems produce contrast with the arrangement of the subsequent floral or- spikelet primordia. Their tips also function as meristems gans. (spikelet meristems) and differentiate lateral organs consti- Three kinds of floral organs are formed following the tuting a spikelet. The spikelet meristem first differentiates a palea. Thus the meristem at this stage may be called floral pair of sterile glumes in 1/2 alternate arrangement. These meristem. As stated earlier, organs homologous to sepal are glumes subtend no buds at their axils and are rudimentary in lacking in grasses. The first floral organ consists of two lod- shape (Fig. 1D and 4A). They are called rudimentary glumes icules (petals), positioned on the lemma side (Fig. 4E). Then in rice or merely ‘glumes’ in other grasses. In the grass spe- six stamen primordia are formed in the whorl. At around cies such as barley and wheat, several florets are produced carpel primordium formation, stamens differentiate into fil- after the pair of (rudimentary) glumes. Thus the glumes pro- aments and anthers (Fig. 4F). At last, the carpel primordium duced after the two sterile glumes are fertile and the first is differentiated from the lemma side of the floral meristem glume of each floret is called lemma. The meristem produc- (Fig. 4G), and grows to enclose floral meristem. Two stig- ing a floret is called the floret meristem. The subsequent de- mas protrude after the carpel encloses the meristem. Then velopmental course in rice, however, differs from that in the the meristem loses the indeterminacy and is transformed to other grasses in two aspects. First, each spikelet comprises ovule primordium in which megagametogenesis occurs to only one floret. Second, the two glumes following the two form an embryo sac (Fig. 4H and 4I). rudimentary glumes are also sterile and are called empty Recent molecular studies have revealed several genes glumes (Fig. 4B). The empty glumes are much larger than expressed during flower development. The OsMADS45 gene rudimentary glumes, but are much smaller than the lemma. (Greco et al. 1997) is expressed in floral organs but not in The pair of empty glumes is followed by two large glumes, glumes (Fig. 4J). Expression of rice homeotic gene lemma and palea (Fig. 4C and 4D). It is marked that these six SUPERWOMAN1 (SPW1)/OsMADS16 is specifically ob- glumes, i.e., two rudimentary glumes, two empty glumes, served in lodicules and stamens (Nagasawa et al. 2003). 152 Ikeda, Sunohara and Nagato

Fig. 4. Developmental course of spikelet. (A) Rudimentary glume primordium (arrowhead) formation. (B) Empty glume primordia (arrowheads) formation. (C) Lemma primordi- um (arrowhead) formation. (D) Palea primordium (arrowhead) formation. (E) Lodicule primordia (arrowhead) formation. (F) Stamen primordia () formation. (G) Carpel primordium (arrowhead) formation. (H) Down-regulation of OSH1 expression in floral meristem/ ovule primordium (arrowhead). (I) Ovule formation. Inner and outer integuments (arrowheads) are growing from the base of ovule. (J) Expression of OsMADS45 in all floral organs including carpel (). Bar = 50 µm.

Rice homologs of Arabidopsis homeotic genes also show difficult to identify the rachis meristem before bract forma- specific expression in flowers: RAP1A (Arabidopsis AP1 ho- tion, careful observation reveals the enlargement of the meri- mologue) is expressed in spikelet meristem, lemma, palea stem immediately after the conversion of SAM to rachis and lodicules, RAG (AG homologue) is found in stamens and meristem. During this stage the rachis meristem would es- pistil, OsMADS2 (PI homologue) is found in lodicules and tablish its identity through the induction of inflorescence- stamens (Kyozuka et al. 2000), and OsMADS13 is expressed specific gene expression. The −3 and −4 stem internodes have in ovule (Lopez-Dee et al. 1999). already started their elongation and the −2 internode starts elongation at this stage. Staging of inflorescence development stage In 2 Formation of primary rachis branches I (inflo- As described above, the developmental course of inflo- rescence length 0.1–0.2 mm) rescence has a variety of stage-specific landmark events. The stage In 2 starts when the first primary branch pri- Thus it would be convenient to categorize the developmental mordium protrudes from rachis meristem, and ends when the course into several stages as a reference of description of size of rachis meristem reaches maximum (at around the mutant phenotypes (Table 1). For staging, data thus far accu- formation of second primary branch primordium and bract mulated, especially those on genes and mutants, are also in- 3 primordium) (Fig. 2E and 2F). At this stage, most of the corporated. Since stem length is an important agronomical positions of bracts would be determined. trait and is determined during inflorescence development, In pla1 mutant, primary branches are converted to veg- information on stem internode elongation is also added. In etative shoots, and bracts grow vigorously like foliage leaves rice, it is convenient to number the stem internodes from the (Itoh et al. 1998). top, because only the upper four or five internodes show stage In 3 Formation of primary rachis branches II substantial elongation irrespective of the number of inter- (inflorescence length 0.2–0.4 mm) nodes formed. Thus, we designate the internode just below This stage is the period of primary branch formation af- the uppermost one (the second internode from the top) as the ter the stage In 2 (Fig. 2G and 2H). The number of primary −1 internode. In the same manner, the third, fourth and fifth branches would be closely associated with the time when the internodes counted from the top were designated as −2, −3 rachis meristem loses meristematic activity, because the and −4 internodes, respectively. OSH1 expression in the meristem is down regulated just stage In 1 Establishment of rachis meristem (inflores- after the last primary branch primordium is formed. The cence length 0.05–0.1 mm) duration of In 1 to In 3 is estimated to be 3 to 4 days. The The stage In 1 represents the period from the conver- elongation of −1 stem internode starts at this stage. sion of the SAM to rachis meristem (flag leaf protrusion) In the lax mutant, development of primary branches is until the bract 1 formation (Fig. 2C and 2D). Although it is suppressed (Komatsu et al. 2001), and the number of primary Development of inflorescence and spikelet in rice 153

Table 1. Staging of inflorescence development in rice

Stage Inflorescence Cloned Related Meristem identity Events a) Symbol Name length (mm) gene mutants In 1 Establishment of Rachis meristem Convertion of vegetative meristem to rachis 0.05–0.1 rachis meristem meristem Enlargement of rachis meristem Formation bract 1 primordium Onset of −2 stem internode elongation (Fig. 2C, D) In 2 Formation of pri- Rachis meristem Rachis meristem reach maximum size 0.1–0.2 pla11), lax2) mary branches I Branch meristem Formation of first two primary branch primor- dia and bract 2 and 3 primordia (Fig. 2E,) In 3 Formation of pri- Rachis meristem Formation of primasy branch primordia in spi- 0.2–0.4 lax2), pap13) mary branches II Branch meristem ral arrangement Abortion of rachis meristem at the end of this period Onset of −1 stem internode elongation (Fig. 2F, G, H, I, J, K) In 4 Elongation of pri- Branch meristem Simultaneous elongation of primary branches 0.4–0.6 sp4) mary branches (Fig. 3A) In 5 Formation of Branch meristem Formation of secondary and tertially branches 0.6–0.9 lax2), Dn15), higher-order Onset of uppermost stem elongation Dn26), Dn37) branches (Fig. 3B, C) In 6 Differentiation of Spikelet meristem Differentiation of two rudimentary glumes, two 0.9–1.5 (See Table 2) (See Table 2) glumes Floret meristem empty glumes, lemma and palea in 1/2 alter- nate arrangement (Fig. 3D) In 7 Differentiation of Floral meristem Differentiation of two lodicules, six stamens 1.5–40 (See Table 2) (See Table 2) floral organs and a carpel in whorl (Fig. 3E) In 8 Rapid elongation Loss of meristem Rachis and branches elongate exponentially. 40–220 (See Table 2) (See Table 2) of rachis and Completion of anther and ovule development branches In 9 Heading and Inflorescence protrudes from the sheath of flag ca. 220 flowering leaf a) Only major mutants are cited. For other mutants, see http://www.shigen.nig.ac.jp/rice/oryzabase/genes/geneClasses.jsp 1) Itoh et al. (1998), 2) Komatsu et al. (2001), 3) Takahashi et al. (1998), 4) Iwata and Omura (1971b), 5) Nagao and Takahashi (1963), 6) Jones (1952), 7) Futsuhara et al. (1979) branches is increased in pap1 mutant (Takahashi et al. 1998). florescence 0.6–0.9 mm) Although Matsushima and Manaka (1956) subdivided We define the stage In 5 as the period when the primary the period of primary branch formation into three substages, rachis branch meristem produces lateral branches, but spike- the distinction of the three substages is unclear. Thus, we let formation has not yet occurred (Fig. 3B). Basal secondary divided the period into two stages by the time when rachis branches frequently produce tertiary branches. An estimated meristem reaches the maximum size. 7–9 days elapses from the conversion of meristem until the stage In 4 Elongation of primary branches (inflorescence end of this stage. The uppermost stem inrternode starts length 0.4–0.6 mm) elongation at this stage. By this time, −3 and −4 internode Proximal primary branch primordia, in spite of earlier elongation is completed. formation, seem not to elongate until the last primordium is The lax mutation also affects the development of formed (Fig. 3A). All the primordia almost simultaneously secondary branches and spikelets on primary branches start to elongate. Thus In 4 is the stage when primary (Komatsu et al. 2001). The Dn mutants affect the number of branches elongate without differentiating lateral organs. secondary branches and spikelets (Jones 1952, Nagao and Elongation of hairs derived from epidermal cells of bracts is Takahashi 1963, Futsuhara et al. 1979). remarkable at this stage. stage In 6 Differentiation of glumes (inflorescence length Several proximal primary branches fail to develop in sp 0.9–1.5 mm) mutant (Iwata and Omura 1971a). Primary and secondary branch meristems are converted stage In 5 Differentiation of higher-order branches (in- into terminal spikelet merisitems and form rudimentary 154 Ikeda, Sunohara and Nagato glumes are formed (Fig. 3D). Subsequently lateral meri- 2003). stems become lateral spikelet meristems. Lateral spikelets stage Sp 2 Formation of a pair of empty glume primordia are arranged in biased distichous phyllotaxy with divergence Following two rudimentary glumes, a pair of empty angle of 110°. After two rudimentary glumes, two empty glumes is formed in 1/2 alternate arrangement (Fig. 4B). The glumes, lemma and palea are formed. FZP gene (Komatsu et two rudimentary glumes are vestigial, and their growth ceases al. 2003), which is expressed just above the rudimentary at this stage. glume primordia, would be a useful marker representing the stage Sp 3 Formation of lemma primordium early phase of this stage. Lemma primordium is formed at a position 180° apart stage In 7 Differentiation of floral organs (inflorescence from the second empty glume (Fig. 4C). The two empty length 1.5–40 mm) glumes continue growing. When the panicle becomes longer than ca. 1.5 mm in The LHS gene regulates the identity of lemma and length, each floret starts floral organ differentiation (Fig. 4). palea (Jeon et al. 2000), and RAP1A is expressed in lemma At this stage, although the floral organs differentiate rapidly, (Kyozuka et al. 2000). The eg mutant shows a duplication of the inflorescence (rachis) elongates slowly. Carpel is ob- lemma (Iwata and Omura 1971a). served in all flowers when the panicle is 10 mm long. Two stage Sp 4 Formation of palea primordium organ identity genes are known, SPW1 for lodicule and sta- Palea primordium is formed at a position 180° apart men identities and DL for carpel identity (Nagasawa et al. from lemma (Fig. 4D). The two empty glumes and lemma 2003). The −2 stem internode elongates rapidly. are growing. stage In 8 Rapid elongation of rachis and maturation of RAP1A is expressed in palea (Kyozuka et al. 2000). reproductive organs (inflorescence length 40–230 mm) Many mutants affecting palea development are known, for When the inflorescence reaches ca. 40 mm in length, it example, dp1 and dp2 mutants show underdevelopment of begins rapid elongation. Each organ increases its size. Dur- palea (Iwata and Omura 1971a, 1971b). ing this stage, maturation of reproductive organs, formation stage Sp 5 Formation of lodicule primordia of pollen grains and embryo sac, takes place. The Two lodicule primordia are formed, biased to lemma OsMADS13 gene is estimated to function in establishing sides (Fig. 4E). The two empty glumes, lemma and palea are ovule identity (Lopez-Dee et al. 1999), and the MAP1 gene growing. is closely associated with male and female sporogenesis RAP1A is also expressed in lodicules (Kyozuka et al. (Nonomura et al. 2003). At this stage, the uppermost and −1 2000). The expression of, OsMADS45, OsMADS2 and stem internodes rapidly elongate. SPW1 genes is detected in lodicule primordia from this stage stage In 9 Heading and flowering (Fig. 4J, Kyozuka et al. 2000, Nagasawa et al. 2003). In A few days after completion of flowers, the inflores- spw1, lodicules are transformed to glumes (Nagasawa et al. cence protrudes from the sheath of flag leaf, and flowering 2003). The number of lodicules is increased in fon1 and fon2 occurs. mutants (Nagasawa et al. 1996). stage Sp 6 Formation of stamen primordia Staging of spikelet development Six stamen primodia are formed in the whorl (Fig. 4F). The spikelet development is not synchronized among The expressions of OsMADS45, SPW1 and RAG are de- primary branches and even within a primary branch. Gener- tected in stamen primordia (Fig. 4J, Nagasawa et al. 2003, ally, spikelets formed on the upper branches develop faster Kyozuka et al. 2000). The stamens are transformed to car- than those formed on the lower branches. In the primary pels in spw1 mutant (Nagasawa et al. 2003). The number of branch, except for the terminal spikelet that develops first, stamens is increased in fon1 and fon2 mutants (Nagasawa the lower spikelets develop earlier than the upper ones. et al. 1996). Thus, in an inflorescence, spikelets with different develop- stage Sp 7 Formation of carpel primordium mental stages coexist, and it is difficult to incorporate the Carpel primordium is formed from the lemma side of staging of spikelet development into that of inflorescence. floral meristem (Fig. 4G). In the stamens, differentiation into At present, spikelet development is categorized into eight anther and filament is apparent. stages (Sp1~Sp8) based on the identity of lateral organs The expression of OsMADS45 and RAG is detected in (Table 2), independently of inflorescence staging. In Table 2, carpel primordiium (Fig. 4J, Kyozuka et al. 2000). In dl cloned genes and mutants related to each stage are included. mutant, carpel is transformed to stamens (Nagasawa et al. stage Sp 1 Formation of a pair of rudimentary glume pri- 2003). The fon1 and fon2 increase the number of pistils mordia (Nagasawa et al. 1996). A pair of rudimentary glumes is formed in 1/2 alternate stage Sp 8 Formation of ovule and pollens arrangement, the outer one being positioned on the adaxial Floral meristem loses the indeterminacy and is convert- side of the spikelet meristem (Fig. 4A). The inner rudimenta- ed to ovule primordium as visualized by the down regulation ry glume is formed in a 1/2 alternate arrangement. of OSH1 gene (Fig. 4H). Carpel primordium extends to en- FZP gene is expressed at this stage and is considered to close the ovule at the early this stage (Fig. 4I). The expres- be required for floret meristem formation (Komatsu et al. sion of OsMADS45 gene is detected in ovule primordium Development of inflorescence and spikelet in rice 155

Table 2. Staging of spikelet development in rice Stage Events Cloned gene Related mutantsa) Symbol Name Sp1 Formation of a pair of rudimentary Formation of two rudimentary glumes in 1/2 FZP1) fzp1) glume primordia alternate phyllotaxy (Fig. 4A) Sp2 Formation of a pair of empty Formation of two empty glumes in 1/2 alternate glume primordia phyllotaxy (Fig. 4B) Sp3 Formation of lemma primordium Formation of lemma in 1/2 alternate phyllotaxy LHS2), RAP13) lhs2),, eg8) Elongation of empty glumes (Fig. 4C) Sp4 Formation of palea primordium Formation of palea in d1/2 alternate phyllotaxy LHS2), RAP13) lhs2), dp18), dp29) Elongation of empty glumes and lemma (Fig. 4D) Sp5 Formation of lodicule primordia Formation of lodicules in whorl RAP13), SPW14), spw14), fon110), Elongation of empty glumes lemma and palea OsMADS23), fon210) (Fig. 4E, I) OsMADS455) Sp6 Formation of stamen primordia Formation of six stamen primordia in whorl SPW14), spw14), fon110), (Fig. 4F, I) OsMADS23), RAG3), fon210) OsMADS455) Sp7 Formation of carpel primordium Formation of carpel RAG3), dl4), fon110), fon210) Differentiation of stamen into filament and anther OsMADS455) (Fig. 4G, J) Sp8 Formation of ovule and pollens Differentiation of integuments and embryo sac. MSP16), msp16) Pollen differentiation OsMADS137), (Fig. 4H) OsMADS455) a) Only major mutants are cited. For other mutants, see http://www.shigen.nig.ac.jp/rice/oryzabase/genes/geneClasses.jsp 1) Komatsu et al. (2003), 2) Jeon et al. (2000), 3) Kyozuka et al. (2000), 4) Nagasawa et al. (2003), 5) Greco et al. (1997), 6) Nonomura et al. (2003), 7) Lopez-Dee et al. (1999), 8) Iwata and Omura (1971b), 9) Iwata and Omura (1971a), 10) Nagasawa et al. (1996)

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