SHORT INTEGUMENTS 2 Promotes Growth During Arabidopsis Reproductive Development

Corresponding author: Charles S. Gasser, Section of Molecular and Cellular Biology, University of California, 1 Shields Ave., Davis, CA 95616., csgasser{at}ucdavis.edu (E-mail)

Communicating editor: V. L. CHANDLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The short integuments 2 (sin2) mutation arrests cell division during integument development of the Arabidopsis ovule and also has subtle pleiotropic effects on both sepal and pistil morphology. Genetic interactions between sin2 and other ovule mutations show that cell division, directionality of growth, and cell expansion represent at least partially independent processes during integument development. Double-mutant analyses also reveal that SIN2 shares functional redundancy with HUELLENLOS in ovule primordium outgrowth and proximal-distal patterning and with TSO1 in promotion of normal morphological development of the four whorls of primary floral organs. All of these observations are consistent with SIN2 being a promoter of growth and cell division during reproductive development, with a primary role in these processes during integument development. On the basis of the floral pleiotropic effects observed in a majority of ovule mutants, including sin2, we postulate a relationship between ovule genes and the evolutionary origin of some processes regulating flower morphology.

PLANT morphogenesis is dependent on tight integration of cell division and cell expansion. Morphogenesis often involves coordinated growth among different cell lineages to form single structures, implying regulation through intercellular communication and non-cell-autonomous developmental signals. In recent years, genetic and molecular approaches have led to significant new insights into the regulation of some aspects of morphogenesis, including the control of floral organ identity and the maintenance of the shoot apex (for review see MEYEROWITZ 1997 ). Despite these advances, we have little information on the specifics of regulation of directional growth and cell division during formation of individual plant organs (MEYEROWITZ 1997 ; SCHNEITZ et al. 1998B ).

The bitegmic Arabidopsis ovule is being used as a morphogenetic model to help understand the regulation of growth and organogenesis, and a number of genes regulating ovule development have been identified through genetic mutant screens (reviewed in ANGENENT and COLOMBO 1996 ; GASSER et al. 1998 ; SCHNEITZ 1999 ). These genes can be separated in two different classes on the basis of their effects on growth. The first class encompasses genes that promote or suppress growth, mostly through regulation of cell division or cell expansion. huellenlos (hll) and aintegumenta (ant) mutations arrest integument growth prior to or during initiation (ELLIOTT et al. 1996 ; KLUCHER et al. 1996 ; BAKER et al. 1997 ; SCHNEITZ et al. 1998A ). ANT and HLL were also shown to be functionally redundant in promoting ovule primordia growth and patterning of the ovule (SCHNEITZ et al. 1998A ). SUPERMAN (SUP) and INNER NO OUTER (INO) act as growth suppressor and promoter of the outer integument, respectively, and both genes are needed for development of the asymmetric form of this structure (GAISER et al. 1995 ; SAKAI et al. 1995 ; VILLANUEVA et al. 1999 ). The short integuments 1 (sin1) mutations suppress growth of the integuments by impeding cellular expansion in these structures (ROBINSON-BEERS et al. 1992 ; RAY et al. 1996A ). The second group of ovule growth loci help determine the shape or identity of the growing tissue. Weak tso1 mutations affect directional control of cell growth and division, resulting in relatively disorganized integument tissues (HAUSER et al. 1998 ). In bell1 (bel1) mutants, an integument-like structure grows in place of the integuments, apparently due to loss of integument identity (ROBINSON-BEERS et al. 1992 ; MODRUSAN et al. 1994 ; RAY et al. 1994 ). The discovery of novel genes involved in ovule development shows the complexity and redundancy of growth regulation in a relatively simple structure and adds to our knowledge of regulation of plant morphogenesis.

Interestingly, mutations in a majority of the genes regulating ovule development also cause floral aberrations. For example, ap2 mutations affect ovule integuments and the identity of some floral organs (BOWMAN et al. 1989 ; KUNST et al. 1989 ; JOFUKU et al. 1994 ; MODRUSAN et al. 1994 ). SUP appears to control expression of the floral class B genes and the ovule gene INO through possibly similar non-cell-autonomous mechanisms (SAKAI et al. 1995 ; VILLANUEVA et al. 1999 ). These observations suggest a molecular relationship between floral and ovule morphogenic pathways.

We report here the characterization of short integuments 2 (sin2), a novel mutation arresting cell division in both integuments of Arabidopsis ovules. Genetic interactions between sin2 and other ovule mutations show that directional regulation, cell expansion, and cell division are partially independent processes governing integument development. SIN2 shares functional redundancies with at least two different genes regulating flower and ovule growth. Floral pleiotropic effects of sin2 and other ovule mutants lead us to postulate a connection between some genes regulating ovule development and the evolution of floral organs.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material:
sin2 was isolated from ethyl methanesulfonate-mutagenized Landsberg erecta (Ler) ecotype as described previously for other ovule mutants (ROBINSON-BEERS et al. 1992 ). Mutants were backcrossed at least three times to wild-type Ler plants prior to further analysis. Plants were grown as described previously (KRANZ and KIRCHHEIM 1987 ; ROBINSON-BEERS et al. 1992 ). Pistil measurements and ovule counts were performed under a Zeiss (Oberkochen, Germany) SV8 stereomicroscope. Epidermal cell counts were done from scanning electron micrographs. Because of the three-dimensional nature of the ovules, counts were compiled from multiple images and represent a best estimate of the true cell counts.

Genetic mapping of SIN2:
A mapping population (F₂ progeny) was generated by crossing sin2 and Co-3 (Columbia) wild-type plants. Using DNA samples (EDWARDS et al. 1991 ) from 34 sin2 plants in the mapping population, sin2 was mapped to chromosome II at a position 2.5 cM south of the cleaved amplified polymorphic sequence (CAPS) m429 marker (KONIECZNY and AUSUBEL 1993 ) and 3.5 cM north of the simple sequence length polymorphisms (SSLP) AthBIO2 (BELL and ECKER 1994 ). Further analysis was done by genotyping 918 plants from the mapping population for both m429 and AthBIO2 loci, which allowed for unambiguous determination of the genotype at the SIN2 locus.

Scanning electron microscopy:
Samples were prepared as described previously (HAUSER et al. 1998 ) and were examined with a Hitachi (Tokyo) S3500N scanning electron microscope at an accelerating voltage of 5 or 10 kV. Images were acquired digitally and were edited for publication in Photoshop 4.0 for Macintosh (Adobe Systems, Inc., San Jose).

Confocal laser scanning microscopy:
Arabidopsis inflorescences were fixed and stained with fluorescent periodic acid-Schiff reagent and were examined under a Zeiss LSM410 laser scanning confocal microscope as described previously (BAKER et al. 1997 ).

Double-mutant analyses:
Pollen from plants homozygous for a specific mutation was used to fertilize emasculated flowers of sin2 heterozygous plants. Seeds were collected from these crosses and sowed as described (KRANZ and KIRCHHEIM 1987 ; ROBINSON-BEERS et al. 1992 ). All F₁ plants were phenotypically wild type. F₂ families, showing segregation for both sin2 and the mutation under study, were further analyzed. When possible or in doubt, a putative double-mutant plant was backcrossed to a wild-type Ler plant to confirm the presence of both mutations by observation of their segregation in the backcross F₂' progeny.

In situ hybridizations:
Sections of wild-type or sin2 inflorescences 8- to 10-µm thick were prepared and hybridized with digoxigenin-UTP-labeled probes as described previously (VIELLE-CALZADA et al. 1999 ). BEL1 probes were generated from pLR115 (gift from Linda Margossian and Robert L. Fischer, University of California, Berkeley, CA) as described (REISER et al. 1995 ). ANT probes were generated from pcDNA5 (gift from David Smyth, Monash University, Melbourne) as described (ELLIOTT et al. 1996 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Wild-type ovule development:
Detailed descriptions of ovule development in Arabidopsis have been presented previously (ROBINSON-BEERS et al. 1992 ; SCHNEITZ et al. 1995 ). A summary is presented here. The Arabidopsis ovule arises as a cylindrical primordium from the placental tissues found adjacent to both sides of the septum inside the pistil (Fig 1A). Initiation of both integuments occurs in the midzone of the ovule primordium and defines this zone as the chalaza (Fig 1C). The inner integument initiates as a ring of cells around the circumference of the ovule primordium and exhibits symmetrical cylindrical growth until it surrounds and encases the nucellus (Fig 1C and Fig E). After inner integument initiation, the outer integument initiates on the abaxial (toward the base of the carpel) side of the ovule primordium and has an asymmetric growth pattern (Fig 1C and Fig E). Parallel with the development of both integuments from the chalaza, the distal portion of the primordium differentiates to form the nucellus while the proximal portion will constitute the funiculus. Concomitant but opposite asymmetric growth of the funiculus and the outer integument give the ovule its final configuration (Fig 1G).

View larger version (141K): In this window In a new window Download PPT slide   Figure 1. Scanning electron micrographs of wild-type (A, C, E, and G) and sin2 (B, D, F, and H) ovules. Stages of ovule development according to SCHNEITZ et al. 1995 . (A) Stage 1-I, wild-type ovules and (B) stage 1-II, sin2 ovules arise as cylindrical primordia from placental tissue. (C and D) Stage 2-III, both integuments have initiated. (E) Stage 2-V, wild-type integuments grow toward the nucellus apex. (F) Stage 2-V, sin2 integument growth arrests before the nucelli are covered. (G) Stage 3-VI (anthesis), asymmetric growth of the outer integument and the funiculus results in amphitropous wild-type ovules. (H) Stage 3-VI (anthesis) sin2 integuments are short and do not encase the nucellus; f, funiculus; ii, inner integument; n, nucellus; oi, outer integument; p, primordia. Bars, 25 µm.

sin2 ovule phenotype and ontogeny:
sin2 is a single-locus recessive mutation that produced complete female sterility in homozygous plants. The frequency of mutant plants was lower than the expected 3:1 ratio in segregating populations [229 wt : 54 sin2 (ratio 4 : 1, ² = 0.15, P = 0.70 )]. This altered segregation ratio indicated either incomplete penetrance or reduced viability of sin2 plants. Using flanking markers, we determined the genotype at the SIN2 locus in 918 plants from a segregating mapping population (see MATERIALS AND METHODS). All 165 homozygous sin2 plants exhibited the mutant phenotype, demonstrating complete penetrance.

Ovules of sin2 plants developed as wild type up to the point when both integuments initiated (compare Fig 1A and Fig C, and Fig 1B and Fig D). The growth of both integuments arrested shortly after initiation, and at anthesis sin2 ovules had two short integuments comprising fewer cells than wild-type integuments (Fig 1F). The epidermis of wild-type outer integument is composed of ~200 cells arranged in 9–10 files whereas sin2 outer integument comprised only ~10–30 cells. Slight variations in integument length were observed even among ovules from a single sin2 pistil, but both integuments were always substantially shorter than wild type, leaving the nucelli fully exposed (Fig 1H). On the basis of these observations, SIN2 appears necessary for progression of integument growth following initiation of these structures during Arabidopsis ovule development. Confocal laser scanning microscopy observations of sin2 nucelli showed that megasporogenesis was arrested before formation of the megaspore mother cell (data not shown).

Sin2 floral phenotypes:
Besides having effects on ovule development, subtle morphological aberrations were also observed in the gynoecia (pistils) and sepals of sin2 flowers. At anthesis, a wild-type Arabidopsis pistil comprises an apical stigma, a short style, and two basal valves separated by a replum (Fig 2A). Most sin2 pistils had a cleft stigma and/or style (Fig 2C). This cleft was always in the axis of one of the valves and not in the plane of the replum. The valve on the cleft side of the stigma sometimes bore an outgrowth (Fig 2B and Fig C) and, less frequently, both valves of a pistil bore outgrowths (Table 1). The tissue forming the outgrowths had the appearance of valve tissue, except at the tip, where it did not resemble any floral tissue. These aberrant pistil phenotypes were not observed in all flowers of sin2 plants (Table 1) and the cleft and outgrowth varied in size (Fig 2B and Fig C). At anthesis, sin2 pistils were also shorter and bore fewer ovules than pistils from emasculated wild-type flowers (Table 1). Distribution of the ovules along the placenta was also affected in sin2 pistils with the distance between ovules being generally greater than in wild type (not shown).

View larger version (178K): In this window In a new window Download PPT slide   Figure 2. sin2 floral effects. Stages of floral development according to BOWMAN 1994 . (A) At anthesis (stage 13), a wild-type Arabidopsis pistil is composed of a stigma, a short style, and two valves joined by the replum. (B) Stage 13 sin2 pistil bearing an outgrowth on one valve (arrowhead). (C) Stage 16 sin2 pistil showing postpollination characteristics of wild-type pistils (elongated valve cells and interspersed stomata; BOWMAN 1994 ) but with the style/stigma split in the axis of one of the valves (arrow) and an outgrowth (arrowhead) present on the valve in the axis of the cleft stigma. (D) Stage 12 wild-type floral bud. Sepal tips have a smooth edge made up of small uniform cells. (E) Stage 11 sin2 floral bud. Sepals have jagged tips made up of cells of variable size and shape. p, petal; r, replum; se, sepal; sg, stigma; st, style, v, valve. Bars, 250 µm.

Sepals were also affected in sin2 flowers. Wild-type sepal margins are smooth and consist of very small rounded cells (Fig 2D; BOWMAN 1994 ). The sepals of all sin2 flowers had fewer such cells on their margins, especially toward the tips. The absence of margin tissue made the tips of sin2 sepals jagged (Fig 2E). While sin2 plants reached the same final size as wild-type plants, they exhibited a slightly slower growth rate. No other vegetative effects of sin2 were observed.

Genetic interactions:
Double mutants were generated to investigate the interactions between SIN2 and other genes regulating ovule growth and development. The observed segregation ratios were as expected for each genetic interaction examined (Table 2) and no partial dominance was observed for any of the segregating mutations. Except as noted otherwise, flowers of double-mutant plants had phenotypes that were consistent with simple addition of the floral effects of the two single mutations (data not shown).

sin2 ant-5: ANT encodes a putative transcription factor containing two AP2 domains (ELLIOTT et al. 1996 ; KLUCHER et al. 1996 ) and has recently been shown to promote growth of all Arabidopsis lateral organs (MIZUKAMI and FISCHER 2000 ). During floral morphogenesis, ant mutations affect the expansion and the number of primary floral organs, but have more severe effects on integument development (ELLIOTT et al. 1996 ; KLUCHER et al. 1996 ; BAKER et al. 1997 ; KRIZEK 1999 ). Ovules of putative null ant-5 mutants (Gln227 to stop codon, eliminating the C-terminal half of the ANT protein including both AP2 domains; B. A. KRIZEK, personal communication) fail to develop integuments, forming at most a small integumentary ridge from the chalazal region (Fig 3A; BAKER et al. 1997 ). ANT is thought to be necessary for promotion of integument primordia initiation and growth (ELLIOTT et al. 1996 ; KLUCHER et al. 1996 ; BAKER et al. 1997 ; SCHNEITZ et al. 1998A ). sin2 ant-5 double-mutant ovules were not different in appearance from those of ant-5 single mutants (Fig 3B), indicating that ant is epistatic to sin2 with respect to ovule development.

View larger version (143K): In this window In a new window Download PPT slide   Figure 3. Scanning electron micrographs of single- and double-mutant ovules at anthesis (stage 3-VI; SCHNEITZ et al. 1995 ). (A) ant-5 ovules have integumentary ridges in place of integuments. (B) sin2 ant-5 ovules are similar to ant-5 ovules. (C) ino-1 ovules fail to initiate outer integuments but development of the inner integuments is not impaired. (D) sin2 ino-1 ovules lack outer integuments and bear short inner integuments. (E) bel1-6 ovules have ILS in place of integuments. Protuberances (arrowheads) formed from the ILS. The funiculi consist of more cells than those of wild type. (F) sin2 bel1-6 ovules had smaller ILS than bel-6 ovules and protuberances were present. The funiculi are shorter than bel1-6 ovules and display an increase in diameter relative to sin2 ovules. (G) sin1-1 integuments are made up of the same numbers of cells as wild type, but are short due to impeded cell expansion. (H) sin2 sin1-1 integument cell numbers are as in sin2 ovules but with reduced cell expansion. (I) hll-1 ovules have short funiculi and nucelli, and only limited integument initiation from the chalaza is sometimes observed. Occurrence of collapsed cells in the distal portion can be observed. (J) sin2 hll-1 pistils bear only abortive ovule primordia where cell collapse is observed. (K) sup-5 outer integuments are more radially symmetrical than wild type. (L) sin2 sup-5 ovules are similar to sin2 ovules. f, funiculus; ii, inner integument; ils, integument-like structure; n, nucellus; oi, outer integument. Bars, 50 µm.

sin2 ino-1: ino mutations are specific to ovules and affect only outer integument development. In the strong ino-1 allele, ovules fail to initiate outer integuments but development of the inner integuments is not impaired and is similar to wild type (Fig 3C; BAKER et al. 1997 ). sin2 ino double mutants displayed additivity as their ovules lacked outer integuments and bore short inner integuments at anthesis (Fig 3D).

sin2 bel1-6: Mature bel1-6 ovules do not have integuments but bear an integument-like structure (ILS) in the chalazal region (Fig 3E; ROBINSON-BEERS et al. 1992 ; MODRUSAN et al. 1994 ; RAY et al. 1994 ). At anthesis, protuberances are often observed on the distal surface of the ILS that may take on nucellar or carpel identity later during its development (ROBINSON-BEERS et al. 1992 ; MODRUSAN et al. 1994 ; RAY et al. 1994 ; HERR 1995 ; GASSER et al. 1998 ). bel1 funiculi contain more cells and can be longer than those of wild-type ovules (Fig 3F; ROBINSON-BEERS et al. 1992 ; SCHNEITZ et al. 1997 ). The BEL1 homeodomain protein, a putative transcription factor (REISER et al. 1995 ), appears to be necessary for establishment of integument identity and for cessation of cell division in the funiculus (ROBINSON-BEERS et al. 1992 ; MODRUSAN et al. 1994 ; RAY et al. 1994 ). At anthesis, the ovules of sin2 bel1-6 plants bore smaller ILS than bel-6 ovules. The funiculi did not show the abnormal elongation observed in bel1 single mutants, but did show a marked increase in diameter relative to sin2 single mutants (Fig 3F). Protuberances were present on the ILS of the sin2 bel1-6 ovules at anthesis (Fig 3F) and some of the ovules became carpelloid (not shown).

sin2 sin1-2: In the Ler ecotype, integuments of sin1-2 ovules do not fully elongate, and the ovules superficially resemble those of sin2. But in contrast to sin2 mutants, integuments of sin1-2 ovules have the same number of cells as wild type and their short length results from a reduction in cell elongation (Fig 3G; ROBINSON-BEERS et al. 1992 ; LANG et al. 1994 ; RAY et al. 1996A , RAY et al. 1996B ). The integuments of sin2 sin1-2 ovules were more reduced in size than in either of the single mutants (Fig 3H). The number of cells in both integuments appeared similar to those of sin2 single mutants, but most of these cells were smaller than in those of sin2 single mutants (Fig 3H). These results show that some expansion must occur in the integument cells of sin2 ovules.

sin2 hll-1: Strong hll alleles (e.g., hll-1) arrest ovule development at an early stage. At anthesis, ovules of hll-1 plants have short funiculi and nucelli, and only limited integument initiation from the chalaza is sometimes observed (Fig 3I). A striking phenotype of the hll-1 allele is the occurrence of collapsed cells in the distal portion of the ovules (Fig 3I; SCHNEITZ et al. 1998A ). HLL appears to have a role in the early steps of ovule development and in regulation of cell death in these structures (SCHNEITZ et al. 1998A ). The combination of sin2 and hll-1 had an even greater affect on ovule development than either single mutant. At anthesis, sin2 hll-1 pistils bore only rudimentary ovule primordia that appeared to have arrested following only very limited growth, and cell death was observed in the entire abortive primordia (Fig 3J). This synergistic phenotype indicates a role for SIN2 in promotion of the earliest stages of ovule primordia growth (Fig 3J).

sin2 sup-5: Arabidopsis sup flowers have supernumerary stamens, reduced carpels, and aberrant ovules (SCHULTZ et al. 1991 ; BOWMAN et al. 1992 ; GAISER et al. 1995 ; SAKAI et al. 1995 ). Compared to wild type, sup ovules have greater growth of the outer integument on their adaxial side, leading to a more symmetrical outer integument (Fig 3K; GAISER et al. 1995 ; SAKAI et al. 1995 ). SUP has been proposed to regulate cell division on the adaxial side of the outer integument during ovule development (GAISER et al. 1995 ; SAKAI et al. 1995 ), possibly through negative regulation of INO (VILLANUEVA et al. 1999 ). sin2 sup-5 ovules were indistinguishable from those of sin2 single mutants, indicating that sin2 is epistatic to sup during ovule morphogenesis (Fig 3L).

sin2 tso1-3: While strong tso1 mutants develop highly reduced, aberrant organs in the three inner floral whorls (LIU et al. 1997 ), the weaker tso1-3 allele produces relatively normal flowers with aberrant ovule integuments (Fig 4A and Fig C; HAUSER et al. 1998 ). In tso1-3 ovules, cells of both integuments are misshapen and are not organized in files due to an apparent loss of directional regulation of cell elongation and division (Fig 4A; HAUSER et al. 1998 ). Plants homozygous for both sin2 and tso1-3 had flowers exhibiting morphological defects in all the primary floral organs. In the first-formed sin2 tso1-3 flowers, the margins of organs in the first three whorls were jagged and the pistils had split styles/stigmas. Later flowers had reduced pistils and more severely affected floral organs (Fig 4D). This suggests that SIN2 plays a larger role in floral organ development than indicated by the single-mutant phenotype and that SIN2 and TSO1 are genetically partially redundant during flower development. Because of the strong acropetal effect, only the first sin2 tso1-3 flowers developed pistils bearing ovules. These ovules had short integuments with slight evidence of cell disorganization (Fig 4B). This result suggests that sin2 is additive with tso1-3 during ovule development.

View larger version (173K): In this window In a new window Download PPT slide   Figure 4. Scanning electron micrographs of interactions between tso1-3 and sin2. (A) tso1-3 ovules have disorganized integument tissues. (B) sin2 tso1-3 ovules have short integuments with evidence of tissue disorganization. (C) tso1-3 flowers have jagged sepal tips and may display slight pistil aberrations. (D) sin2 tso1-3 flowers have reduced pistils and severe floral organ aberrations. f, funiculus; ii, inner integument; oi, outer integument; n, nucellus; p, petal; se, sepal; sg, stigma; sm, stamen; v, valve. Bars, 50 µm (A and B) and 250 µm (C and D).

Expression of BEL1 and ANT in sin2 ovules:
To learn more about the basis of the phenotypic effects of the sin2 mutation, we investigated the patterns of expression of ANT and BEL1 during sin2 ovule development through in situ hybridization. In wild-type ovules, both ANT and BEL1 have been shown to be initially expressed throughout the ovule primordia, but in later stages of development expression is restricted to the chalazal region and the emerging integuments (REISER et al. 1995 ; ELLIOTT et al. 1996 ; KLUCHER et al. 1996 ). Comparisons between wild-type and sin2 ovules were performed on emerging ovule primordia (Fig 5A, Fig B, Fig E, and Fig F) and on ovules where both integuments had initiated (corresponding to the final sin2 ovule phenotype; Fig 5C, Fig D, Fig G, and Fig H). For both stages of development, no differences were observed for the patterns of ANT or BEL1 mRNA accumulation (Fig 5) between wild-type and sin2 ovules.

View larger version (112K): In this window In a new window Download PPT slide   Figure 5. Comparison of ANT and BEL mRNA accumulation in wild-type (A, C, E, and G) and sin2 (B, D, F, and H) ovules by in situ hybridization. In young flowers of both wild type (A) and sin2 (B), ANT is expressed in the emerging ovule primordia. In stage 2-III wild-type (C) and sin2 (D) ovules, ANT is expressed at highest levels in the chalaza and emerging integument primordia. BEL mRNA was found throughout stage 1-I wild-type (E) and sin2 (F) ovule primordia. In stage 2-III wild-type (G) and sin2 (H) ovules, BEL1 mRNA was at highest levels in the developing integuments and throughout the chalaza. f, funiculus; ii, inner integument; n, nucellus; oi, outer integument; ovp, ovule primordia. Bars, 50 µm (A and B), 25 µm (C, D, G, and H), and 10 µm (E and F).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

SIN2 regulates growth throughout reproductive development:
All of the mutant phenotypes described herein support a role for SIN2 as a primary promoter of integument growth and as a secondary growth promoter during other aspects of reproductive morphogenesis. In all combinations with mutations producing at least one integumentary structure, sin2 led to reduction in the number of cells in such structures. This resulted in additive interactions with sin1, bel1, ino, and tso1. Such interactions imply that SIN2 regulates early stages of integument growth and suggest that elongation (SIN1), directionality of growth (TSO1), and cell division (SIN2) are at least partially parallel processes that must interact closely to generate appropriate morphogenesis of both integuments. The epistasis of sin2 over sup (Fig 3L) was consistent with sup affecting only later stages of integument growth that never occur in sin2 mutants.

The strong synergism between sin2 and hll, which nearly eliminates ovule development (Fig 3J), could not have been predicted from either single-mutant phenotype. A similar phenotype was described for ant hll double mutants (SCHNEITZ et al. 1998A ), suggesting that SIN2, ANT, and HLL have redundant functions in ovule primordia growth and the proximal-distal patterning of ovules. A role for SIN2 in funiculus growth is further indicated by the shorter length of the funiculi of sin2 bel1 mutants relative to bel1 single mutants. Thus, at least three genes appear to promote ovule primordium outgrowth and funiculus elongation. HLL must be a key regulator of these processes as reduced growth is observed in hll single mutants (Fig 3I). Because ant, sin2, and ant sin2 ovules had wild-type funiculi, both ANT and SIN2 must have secondary roles in these processes that become apparent only in the hll background. The similar synergistic effects of either ant or sin2 with hll may be an indication that ANT and SIN2 act in a common secondary pathway. ANT could act first in this secondary pathway and be a positive regulator of SIN2 activity. This is supported by the observation that transcriptional regulation of ANT is not affected in sin2 ovules (Fig 5B and Fig D). We also hypothesize that a low level of SIN2 activity is still present in ant mutants to explain the slightly stronger effects on ovule development observed in sin2 hll compared to ant hll plants (SCHNEITZ et al. 1998A ) and the fact that no floral effects were reported in ant tso1-3 double-mutant plants (HAUSER et al. 1998 ; see below).

An obvious explanation for the sterility of sin2 ovules is the arrest in megaspore mother cell development. While it is possible that SIN2 plays a direct role in the regulation of megagasporogenesis, it was observed previously that mutations leaving exposed nucelli also display arrested megagametogenesis as a secondary effect of the absence of integuments (BAKER et al. 1997 ; GASSER et al. 1998 ). A similar hypothesis can be made for the sin2 ovules.

The plasticity of integument growth observed among sin2 ovules might result from variation in SIN2 activity, suggesting that our only sin2 isolate might not represent a null allele. sin2 was isolated through a genetic screen based on female sterility and searches for additional sin2 alleles have not yet been fruitful. Since SIN2 could be expressed throughout the flower, putative null alleles might generate a stronger floral phenotype and would not have been identified as being putative alleles of this mutation. Ongoing efforts to find other alleles in populations arising from mutagenized heterozygous SIN2/sin2 seeds are underway. On the basis of the latest classical genetic map of chromosome 2 (http://www.arabidopsis.org/chromosomes/), other characterized mutations can be found in the vicinity of the SIN2 locus (65 cM), including some that cause embryo lethality. Because the correlation between physical and genetic maps is imprecise, the potential allelism between sin2 and these other mutations must be tested on an individual basis.

We observed complete penetrance of sin2, showing that the unexpectedly low segregation ratio of this mutation (closer to 4:1 than the expected 3:1 for a recessive trait) was due to a reduced frequency of homozygous sin2 plants in the mature segregating population. The deficiency in homozygous mutants could result from reduced production, viability, or vigor of either sin2 embryo sacs or pollen. It could also be due to reduced germination efficiency or increased seedling mortality of sin2 plants. Experiments to differentiate among these possibilities have thus far been inconclusive, but are still in progress.

SIN2 roles in primary floral organ formation:
The synergistic effects on floral development observed in sin2 tso1-3 double mutants (Fig 4D) suggest that SIN2 might be expressed throughout flowers, consistent with the partially aberrant sepals and pistils observed in sin2 single-mutant flowers. The double-mutant phenotype also suggests that directional cell expansion and cell division are at least partially compensatory processes in floral organ formation. A compensatory mechanism has been proposed to be responsible for the relatively normal shape of maize leaves in the tangled-1 mutant, where cell division planes are highly aberrant (SMITH et al. 1996 ). Small perturbations in either cell division, as in sin2, or directional expansion, as in tso1-3 (HAUSER et al. 1998 ), lead to slight organ deformities, but a combination of these two defects appears to prevent compensation, resulting in malformed organs. An alternative explanation of our results implicates TSO1 as a negative regulator of SIN2 expression in the flower. The tso1 mutation would lead to a higher ectopic expression of altered SIN2 protein activity throughout the flower, leading to the observed tso1 sin2 floral phenotype.

Pleiotropic floral effects are common among ovule mutants:
A majority of ovule mutants described to date also have pleiotropic effects on other aspects of flower development. These mutants include sup (SCHULTZ et al. 1991 ; BOWMAN et al. 1992 ; GAISER et al. 1995 ; SAKAI et al. 1995 ), tso1 (LIU et al. 1997 ; HAUSER et al. 1998 ), leunig (LIU and MEYEROWITZ 1995 ), apetala2 (BOWMAN et al. 1989 ; KUNST et al. 1989 ; JOFUKU et al. 1994 ; MODRUSAN et al. 1994 ), and sin2 (this study). The pleiotropic roles of these genes may be an indication of the evolutionary origin of some of the genes regulating flower development. Ovules preceded flowers in the evolution of seed plants. The lateral floral organs represent structures that must have been added to, or modified within, the reproductive axis below the "ancestral" ovule (DOYLE 1994 ). Pathways for regulation of formation of the lateral floral organs would have to derive from genes in preexisting developmental pathways. Genes regulating ancestral ovule morphogenesis may have taken on additional roles in regulation of aspects of floral development, while maintaining their roles in ovule development, and would thus exhibit pleiotropic mutant phenotypes like those described above. A comparison of whole-genome expression analyses between each floral organ and ovules might help to identify and differentiate genes with dual roles and those that are exclusive to either floral or ovule development. Genes involved in both developmental processes might have their origin in the ovule morphogenetic pathway while genes specific for lateral floral organs would have been recruited from other pathways. Such analyses would help clarify the sources of genes that contributed to the evolution of floral structures.

	FOOTNOTES

¹ Present address: Illumina, Inc., Suite 200, 9390 Towne Centre Dr., San Diego, CA 92121.

	ACKNOWLEDGMENTS

We thank David Smyth, and Linda Margossian and Robert L. Fischer for the ANT and BEL1 cDNA clones, respectively. We also thank Beth A. Krizek for sharing unpublished data and we thank reviewers for constructive comments on the manuscript. We are grateful to the members of the Bowman lab for exchanging ideas and help with the in situ hybridization experiments and Rick Harris for help with the scanning electron microscopy. Thanks to past and present members of the Gasser lab for discussions. This work was supported by a grant from the National Science Foundation (IBN98-08395).

Manuscript received November 30, 1999; Accepted for publication March 2, 2000.

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