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Home , Cellular differentiation, Regional differentiation

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8117-8122, July 1996 Plant

A rice , OSHJ, is expressed before organ differentiation in a specific region during early embryogenesis (/in situ hybridization/organless mutant) YUTAKA SATO*, SOON-KWAN HONGt, AKEMI TAGIRIt, HIDEMI KITANO§, NAOKI YAMAMOTOt, YASUO NAGATOt, AND MAKOTO MATSUOKA*¶ *Nagoya University, BioScience Center, Chikusa, Nagoya 464-01, Japan; tFaculty of Agriculture, University of Tokyo, Tokyo 113, Japan; tNational Institute of Agrobiological Resources, Tsukuba, Ibaraki 305, Japan; and §Department of Biology, Aichi University of Education, Kariya 448, Japan Communicated by Takayoshi Higuchi, Nihon University, Tokyo, Japan, March 25, 1996 (received for review October 20, 1995)

ABSTRACT Homeobox encode a large family of One of the powerful approaches for understanding the homeodomain proteins that play a key role in the pattern molecular mechanisms involved in plant embryogenesis is to formation of . By analogy, homeobox genes in identify molecular rnarkers that can be used both to monitor plants are thought to mediate important processes in their specification events during early embryogenesis and to embryogenesis, but there is very little evidence to support this gain an entry into regulatory networks that are activated in notion. Here we described the temporal and spatial expression different embryonic regions after fertilization (10). With this patterns of a rice homeobox gene, OSHI, during rice embry- approach, it has been demonstrated that some genes are ogenesis. In situ hybridization analysis revealed that in the expressed in specific cell types, regions, and organs of embryo wild-type embryo, OSHI was first expressed at the globular (11, 12). The information gained from these studies helps the stage, much earlier than started, in a ventral way to elucidate the functional roles of these genes. region where shoot apical and would later In , the principles of the genetic control during develop. This localized expression of OSHI indicates that the embryogenesis have been unraveled through the combined has already occurred at this stage. At genetic and molecular approaches (for review, see ref. 13). later stages after organogenesis had initiated, OSHI expres- These approaches demonstrate that the homeobox genes, sion was observed in shoot apical meristem [except in the Li which encode evolutionarily conserved 61 amino acid domains (tunica) layer], epiblast, radicle, and their intervening tissues called homeodomains, play important roles in cellular or in descending strength of expression level with embryonic of Drosophila embryo. In plants, ho- maturation. We also performed in situ hybridization analysis meobox genes have been isolated from several such as with a rice organless embryo mutant, orll, that develops no maize (14, 15), rice (16, 17),Arabidopsis (18-21), and soybean embryonic organs. In the orl) embryo, the expression pattern (22). Plant homeobox genes, by analogy of the functional roles of OSHI was the same as that in the wild-type embryo in spite of animal homeobox genes, have been expected to encode of the lack of embryonic organs. This shows that OSHI is not transcriptional regulators that mediate important develop- directly associated with organ differentiation, but may be mental processes during embryogenesis (19). It has not been related to a regulatory process before or independent of the demonstrated, however, that the plant homeobox genes are organ determination. The results described here strongly directly involved in embryogenesis, although ectopic expres- suggest that, like animal homeobox genes, OSHI plays an sions of the homeobox genes have caused the abnormal leaf important role in regionalization of cell identity during early development in the vegetative phase of the transgenic plants embryogenesis. (16, 21, 23-25). Only very recently, the expression of maize homeobox genes was observed at the localized region of The fundamental body plan in higher plants is established developing maize embryo (26, 27). during the embryogenesis, although most morphogenetic To elucidate the possibility that plant homeobox genes are events occur in the postembryonic phase of the life cycle. In the involved in embryo development, we examined the temporal early stage of embryogenesis, several body axes that cause and spatial expressions of a rice homeobox gene, OSH1, during apical-, dorsal-ventral, and radial patterns are estab- rice embryogenesis in wild-type and in organless embryo lished, and two apical (shoot and root) are formed. mutant, orll, that develops no embryonic organs (2). The The apical meristems successively produce various tissues and results described here suggest that OSH1 is not directly asso- organs throughout the plant life cycle while maintaining ciated with shoot development, but may function to specify cell themselves as stem-like cells. In Poaceae species, which have identity and provide regional information for the formation of highly developed embryos, mature embryos contain almost all shoot and its adjacent tissues. organs seen in the vegetative phase such as shoot apex, leaves, vascular systems, and root (1, 2). To elucidate the genetic control of the and/or the organ differenti- MATERIALS AND METHODS ation during plant embryogenesis, many embryonic mutants Plant Materials. To observe developmental course of wild- have been isolated in Arabidopsis (3-5), maize (6, 7), and rice type embryos-and expression pattern of OSH1, embryos from (2, 8). The study of available embryo mutants of rice indicate the rice (Oryza sativa L.) cultivar Taichung 65 were used. We the existence of several major developmental processes taking also used embryos from orll mutant, a monogenic, recessive place during early embryogenesis before morphogenetic mutant of rice derived from chemical mutagenesis with N- events start (9). Despite the intensive efforts by researchers, methyl-N-nitroso-urea (2). little is known about the molecular mechanisms regulating Preparation of Rice Embryo Sections. Developmental these developmental processes. course of wild-type rice embryos was examined by standard paraffin method. For paraffin sectioning, seeds at various The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: DAP, days after pollination. accordance with 18 U.S.C. §1734 solely to indicate this fact. 1To whom reprint requests should be addressed.

8117 Downloaded by guest on September 29, 2021 8118 Plant Biology: Sato et al. Proc. Natl. Acad. Sci. USA 93 (1996) developmental stages were fixed in FAA (formalin/glacial formed successively at 5, 6, and 8 DAP, respectively, in an acetic acid/70% ethanol, 5:5:90), dehydrated in graded etha- alternate phyllotaxis (Fig. 1 E-G). nol series, and embedded in paraffin. Samples were sectioned Based on the developmental course of rice embryo de- at 10 ,tm and stained with hematoxylin. scribed above, we performed in situ hybridization analysis on In Situ Hybridization. In situ hybridization with digoxigenin- embryos at 2-7 DAP to determine the expression pattern of labeled RNA produced from the OSH1 coding region without OSH1 during rice embryogenesis. When the sense-strand poly(A) region was conducted as described (28). Tissues were probe was used, no specific hybridization signal was observed fixed with 4% (wt/vol) paraformaldehyde and 0.25% glutar- in any experiments (data not shown). Therefore, signals with aldehyde in 0.1 M sodium phosphate buffer and embedded in the antisense probe were caused by the specific hybridization Paraplast Plus (Oxford Labware, St. Louis). Microtome sec- between the OSH1 transcripts and the probe. tions (7-10 gm thick) were applied to slide glass treated with We first examined the OSH1 expression in globular em- Vectabond (Vector Laboratories). Hybridization and immu- bryos. The hybridization signals were first detected in globular nological detection of the hybridized probe were performed by embryos comprising around 100 cells and were about 50-55 method of and Hata (28). gm long at late 2 DAP or early 3 DAP (Fig. 2A). At this stage, the Kouchi embryos show no sign of organ differentiation. It is clear that the expression is restricted to a small region just below the RESULTS center of the ventral region of the embryo, where the shoot apex arises later (Fig. 2A). In the late globular through Expression of a Rice Homeobox Gene, OSHJ, During Em- coleoptilar stages of embryos, OSH1 was expressed in an bryogenesis in Wild-Type Plants. Rice embryos develop much enlarged ventral region of embryo (Fig. 2B and C). The signals more quickly than those of other cereal plants such as maize were detected in the outermost several cell layers just below and barley. They complete most of the morphogenetic events the coleoptilar protrusion extending to the basal (Fig. 2B) and within 1 week under normal conditions. Globular stage lasts inner part of the embryo corresponding to the expected nearly 3 days after pollination (DAP) (Fig. 1 A and B). epiblast and radicle, respectively (Fig. 2C). Although organogenetic events are not observed before 3-4 At the shoot apex differentiation stage when embryos are DAP, a dorsiventral polarity is evident from the gradient of 250-300 ,um long (4 DAP), OSHI was expressed in the shoot cell size in the late globular embryos at 2-3 DAP (Fig. 1B). The apex, epiblast, radicle primordia, and in their intervening first morphological differentiation is recognized as a ventral tissues (Fig. 2D), but not in coleoptile, scutellum, or a region protrusion of coleoptile at the late 3 DAP or early 4 DAP when where the first leafwould differentiate. The expression pattern the embryo reaches 100-110 ,um long and comprises 800-900 in the radicle is different from that in the shoot. Serial sections cells (Fig. 1C). Shoot and radicle apices are first observed at of the embryo revealed that the OSH1 expression in the radicle 4 DAP (Fig. 1D). The first through third foliage leaves are was observed in the cells surrounding the root apical meristem A B C D f w7-'p ..

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a; < \ 8 ; E *a" \;ii .. ti.;8 KS<5Ft.* .;; 'tSS ,, *wiSS X,h.. Zpl* 11iSYE! Eb E t: s ':' J- F o'*H''s; ;,B' { ,... ..: S..- '. , ... .i so- - f.r FIG. 1. Developmental course of wild-type rice embryo. Micrographs shown are the median longitudinal sections. (A) Early globular stage (2 DAP). No morphological differentiation is observed. (B) Late globular stage (3 DAP). While a dorsiventral polarity is evident from the gradient of cell size, no organogenetic events are observed. (C) Coleoptilar stage (early 4 DAP). The first morphological differentiation is recognized as a ventral protrusion of coleoptile at this stage. (D) Apical meristem differentiation stage (4 DAP). Shoot and radicle apices are observed. (E) First leaf differentiation stage (5 DAP). The shoot apical meristem is flat at this stage. (F) Second leaf primordium differentiation stage (6 DAP). The shoot apical meristem become dome-like at this stage. (G) Nearly mature embryo at 9 DAP. By this time, the first to third foliage leaves are formed successively in an alternate phyllotaxis. Arrow, shoot apex; arrowhead, radicle apex; cp, coleoptile; sc, scutellum; ep, epiblast; lpl, first leaf primordium; lp2, second leaf primordium. (Bar = 100 ,um.) Downloaded by guest on September 29, 2021 Plant Biology: Sato et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8119

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FIG. 2. Detection of the OSHJ transcript in wild-type rice embryos by in situ hybridization. Median longitudinal sections of embryos are shown except in C and E, which are sectioned adjacent to the median longitudinal plane. Positive hybridization signals are indicated by blue to violet color. (A) Globular embryo at 3 DAP. Signals are localized in the ventral region of embryo, where shoot apex will differentiate later. (B) Coleoptilar stage embryo at early 4 DAP. Signals are visible in enlarged ventral region just below the coleoptilar protrusion. (C) The section of coleoptilar stage embryo at early 4 DAP adjacent to the median longitudinal plane. The hybridization pattern is different from that on the median section, and signals are observed in the inner part of the embryo corresponding to the region where radicle will arise. The section on the opposite side against the median plane shows the identical hybridization pattern. (D) Embryo at late 4 DAP in which shoot apex is being formed. Pattern of OSHI expression is similar to that in B. (E) The section of the embryo at late 4 DAP that is swerved from the median longitudinal plane. Signals are observed at the region surrounding the root apical meristem like a donut shape. The swerved section of the opposite side against the median plane showed the hybridization pattern identical to this fig. (F) First leaf differentiating embryo at 5 DAP. Signals are visible in shoot apex, epiblast, and the region at the ventral side of vascular . (G) Enlarged view of the shoot apex region in F. Signals are absent in both Li (tunica) layer and first leaf primordia. (H) Second leaf differentiating embryo at 7 DAP. Similar pattern to that in F is observed but the intensity of signals is much lower than that at 5 DAP. Arrow, shoot apex; arrowhead, radicle apex; cp, coleoptile; sc, scutellum; ep, epiblast; Li, Li (tunica) layer; vt; ; lpl, first leaf primordium; lp2, second leaf primordium. (Bar = 100 ,um.) in a donut shape but not in the meristem (Fig. 2E). At a more stage (Fig. 2F). An enlarged view of the shoot apex shows that advanced stage when the first leaf primordium was evident and no signal was detected in the Li (tunica) layer of the meristem embryos were 400-500 ,um in length (5 DAP), the expression and the first leaf primordium (Fig. 2G). This spatial pattern of pattern of OSH1 was basically the same as that in the previous OSH1 expression is maintained in the following stage when the Downloaded by guest on September 29, 2021 8120 Plant Biology: Sato et al. Proc. Natl. Acad. Sci. USA 93 (1996) second leaf primordium is formed, although the intensity of hybridization signals becomes much weaker (Fig. 2H), indi- W. B cating that the OSH1 expression is reduced and/or the OSH1 transcripts become unstable at the late embryonic stage. .4 It is noteworthy that the onset of the OSHI expression is 6 much earlier than that of organ differentiation and the ex- !lji,; pression is restricted to a specific region on the ventral side. Although the hybridization signals are detected in the shoot, epiblast, radicle (except the meristem), and the intervening

tissues, clearly there is no relation in cell lineage among them. a J This suggests that the OSHI expression does not reflect cell . 'ef t. ar lineage, but may be related to regionalization in globular to

onic organs including shoot and radicle, although the embryos - z'<.Re_4- %< i, develop relatively large (longer than 1000 ,um) (2). At the D globular stage (1-3 DAP), they do not show any abnormality or .s and are indistinguishable from the wild-type embryo. At 4 DAP, a small coleoptile-like protrusion frequently appears on .@+ sF't,. the ventral side of the embryo but the embryo continues t .a _ .s - %. growing without further organogenetic events (Fig. 3B). Mi- w. .. e } s croscopically, we cannot detect any sign of shoot and radicle =t ;t.. .. t differentiation in the orll embryo. At 4 DAP and later stage, .l orll embryos are morphologically distinguishable from the l, .i: wild-type embryos by the absence of shoot and radicle. 8;^. fl k i t :'

We also examined the OSH1 expression in the orll embryos isS, i. *S. r

by in situ hybridization to elucidate the epistatic relationship w-. \ b --'. \, X ', is between the OSHI expression and organ differentiation. In - S this experiment, we used embryos set on heterozygous plants $ 7-~ a for orll, because the homozygous orll embryos became lethal. Consequently, we can expect the frequency of homozygous orll embryos to be 25% in the panicles of the heterozygous FIG. 3. Expression of OSHI in rice orll mutant embryo showing no plants. At the 3 DAP, all globular embryos examined showed organ differentiation. All images shown are median longitudinal the same pattern of hybridization signals as with the wild-type sections of orll embryos. (A) Embryo at 4 DAP. The expression of embryos, indicating that both temporally and spatially the OSH1 is observed in the ventral side of the embryo. This pattern of OSH1 expression is normally regulated in the orll embryos. At expression is essentially the same as that in the wild-type embryo at 3 DAP (Fig. 2A1). (B) Embryo with coleoptile-like protrusion._ .at @..y4 DAP. 4 DAP, orl embryos, retaining globular shape (Fig. 3A) or In orll mutant embryos, a small coleoptile-like protrusion frequently showing small coleoptile-like protrusions (Fig. 3B), maintain a appears on the ventral side of the embryo. The embryo continues its normal pattern of the OSH1 expression, although neither growth without any further organogenetic events. The expression shoot nor radicle is differentiated (Fig. 3B). Hybridization pattern of OSHI is similar to that observed in A. (C) Embryo with signals were detected in the ventral region corresponding to a coleoptile-like protrusion at 6 DAP. Compared with the wild-type place where shoot, epiblast, and radicle would differentiate. At embryo at 6 DAP (Fig. IF), the sever defect in organ differentiation 6 DAP, the same expression pattern was observed as at 4 DAP, is observed. The expression pattern of OSHJ observed is similar to that but the expression level was reduced (Fig. 3C). In the large orll in B, but the expression level is reduced. (D) Embryo at 7 DAP. The embryos at 7 DAP, no signal was detected (Fig. 3D). Thus, the embryo grows up with showing no organ differentiation. No OSHI OSHJ transcript is decreasing in orll embryo with the embry- expression is observed. (Bar = 100 ,um.) onic maturation as in the wild-type embryo. opment, we need to monitor their expression going back to the These results indicate that both temporal and spatial pat- In this have examined the terns of the OSH] expression are exactly maintained even in embryonic phase. paper, we spatial the orll embryos lacking shoot and radicle. Consequently, the and temporal expression patterns of rice homeobox gene, OSHI expression is not directly associated with organ differ- OSH1, during rice embryogenesis. OSHI expression was first entiation. Taken together with the observation made in the detected in a specific ventral region of globular embryo. In wild-type and mutant embryo, it is clear that OSHI is first embryos at this stage, however, no morphological differenti- expressed much earlier than the onset of morphological dif- ation is observed, although regional differentiation such as ferentiation, and is considered to function in a regulatory dorsiventral pattern is implied by the gradual change of cell process before or independent of organ determination. size along the dorsiventral axis. This indicates that the cellular differentiation at the level has already oc- curred at this stage. Although we do not know anything about DISCUSSION the initial signal for such cellular differentiation in embryo, it Do plant homeobox genes act to determine the regional and is plausible that OSH1 may play an important role in the cellular identities during embryogenesis like their animal cellular differentiation preceding organ formation. Consider- homologs? Expression of plant homeobox genes in shoot ing the possibility that the putative in vivo function of OSHI is meristems strongly suggested their involvement in organ for- a trans-acting factor, OSHI may function as a regulator mation from shoot meristems (29-31), but it has not been switching on and off the developmental program of embryonic reported so far that plant homeobox genes also mediate some cells located in a specific region. With the advancement of organ formation processes in embryogenesis as in animal embryonic maturation, the expression level of OSHJ is re- embryos. For evaluating their functional significance in devel- duced (Fig. 2H). The down-regulation of OSHJ expression at Downloaded by guest on September 29, 2021 Plant Biology: Sato et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8121

I Pattern formation |Detailed regionalization IDeterminationi Organ differentiation *1 OSH1 expression

high level expression low level expression start

FIG. 4. OSHI expression and schematic representation of hypothetical regulatory processes during rice embryogenesis. The OSH] expression is first observed in a specific region of the ventral side of the globular stage embryo that has no visible organ differentiation. The high level expression of OSH1 keeps during the late globular stage to the first leaf primordia differentiation stage. Then the expression of OSHJ is reduced as the embryo maturation proceeds. From the observation in the orll mutant, the localized expression of OSH] is not affected by the organ differentiation. These indicate that OSHI functions in the early stages of the embryogenesis and may play a role in the specification of cell identity or in the detailed regionalization after embryonic pattern formation is roughly established.

later stages suggests that its primary function resides in the ever, OSH1 is expressed from the middle globular stage (ca. early embryogenesis. This result also suggests a possibility that 100-cell stage), so it would not be directly involved in the the main function of OSH1 in embryo is to establish cellular pattern formation, but rather may function in specifying cell identity in the ventral region at the globular stage. Once the identity or in the detailed regionalization after embryonic ventral identity is established and then the organogenesis is pattern is roughly established. Otherwise there is another started, OSH1 may finish its function in early embryogenesis possibility that the rice embryonic pattern could be established and decrease its expression level. later than Arabidopsis. OSH1 has been considered as a rice counterpart gene of To date, a loss of function mutation of OSHJ has not been maize KN1 gene because the primary structures of the two isolated. Consequently, we cannot yet specify the exact func- genes are quite similar and the overexpression of the genes tion of OSH1 during embryogenesis, although the temporal cause similar alterations in morphology in transgenic tobacco and spatial patterns of OSHI expression imply an important plants (16). KN1 is expressed in the vegetative shoot apex, role of OSH1 in early embryogenesis. An Arabidopsis muta- except in the Li layer and leaf primordia (30). From the tion, shoot meristemlessl (stml), caused the loss of shoot apical expression pattern of KNI in the wild-type and dominant Knl meristem in embryo (34). Recently, STMJ gene has been mutant plants, Hake and colleagues (29) proposed a hypoth- characterized as a KNJ -type homeobox gene (35). Considering esis that KNI functions to promote indeterminate growth and the fact that homeobox gene functions are often conserved its down-regulation is important for the entry of leaf founder among as different as mice and Drosophila, the cells into a determinate developmental pathway. Recently, function of OSH1 may also be involved in the formation of the Hake et al. examined the expression of KNI during maize shoot apex in embryo by providing regional information for embryogenesis and showed that the KNI expression is closely shoot differentiation. In fact, among the rice embryo mutants associated with the shoot apex differentiation, because KN1 that show abnormal OSH1 expression pattern, a rice shoot-less expression is first detected when and where shoot apex pri- mutant is included (unpublished data). mordium is anatomically visible (26). This observation with the The present data also show that OSHJ can be used as a KN1 expression in maize embryo is different from our obser- temporal or region-specific marker of embryogenesis. Normal vation that the onset of OSHI expression occurs much earlier expression of OSHI in orll embryo implies that orll is a defect than the formation of shoot meristem, although the spatial of a gene operating downstream of OSH1. That is, in orl] expression pattern of KN1 in embryo coincides with that of embryo, pattern formation is normally accomplished, but the OSHI at the shoot apex-differentiation stage. This indicates after organ differentiation would be impaired. Our prelimi- that OSH1 may function at much earlier stage during embry- nary data suggests that the expression pattern of OSH1 differs mutants similar ogenesis than KN1 does, and therefore, there may be func- among embryo exhibiting phenotypes (unpub- lished data). Thus, by examining the expression pattern of tional differences between OSHI and KN1 . Normal expression of OSH] in the organless orll embryos OSH1, we can characterize embryo mutants more precisely in (Fig. 3) strongly suggests that OSH1 is not directly associated relation to pattern formation, which, in turn, enriches our understandings on the functions of plant homeobox genes. with shoot apex differentiation, but is related to a regulatory process operating prior to or independently of the shoot apex This work was supported by a Grant-in-Aid for Scientific Research differentiation. many rice mutants, By analyzing embryo on Priority Areas (The Molecular Bases of Flexible Organ Plans in Kitano et al. (9) presented a scheme for regulatory processes Plants) from the Ministry of Education, Science and Culture (Japan) active during rice embryogenesis (Fig. 4). In this scheme, they and by the special coordination funds for COE from the Science and have proposed several regulatory processes before morpho- Technology Agency. genetic events take place: pattern formation (apical-basal, dorsal-ventral), determination of organ differentiation, posi- 1. Randolph, L. F. (1936) J. Agric. Res. 53, 881-916. tional regulation, and size regulation. Embryonic pattern is 2. Hong, S. K., Aoki, T., Kitano, H., Satoh, H. & Nagato, Y. (1995) estimated to be determined quite early in embryogenesis, Dev. Genet. 16, 298-310. 3. Castle, L. A. & Meinke, D. (1993) Semin. Dev. Biol. 4, 31-39. because the genes of such as pattern-determining Arabidopsis, 4. Errampalli, D., Patton, D., Castle, L., Hansen, K., Schnall, J., GNOM and MONOPTEROS, function at the very early stage Feldmann, K. & Meinke, D. (1991) Plant Cell 3, 149-157. of globular embryo (32 33). Our data on the OSHI expression 5. Jurgens, G., Mayer, U., Ruiz, R. A. 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