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First published online December 14, 2004; 10.1105/tpc.104.026666 © 2005 American Society of Plant Biologists
Activation of CRABS CLAW in the Nectaries and Carpels of Arabidopsis
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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). The main function of nectaries is to lure pollinators and protectors by providing sugary foods as rewards. Though there are reports of nectaries in ferns (Darwin, 1877) and in Gnetales (Porsch, 1910), nectaries are most widespread in angiosperms, particularly within flowers. A large proportion of angiosperm species require pollination by animals (Eriksson and Bremer, 1992), with pollinators in many cases being attracted to flowers to gather nectar as their food source. The fossil records of angiosperms and insects suggest that the timing of the radiation of angiosperms corresponds to the radiation of insects that consume nectar as a main food source (Meeuse, 1978; Crepet and Friis, 1987; Pellmyr, 1992). Therefore, it is likely that innovation or modification of genetic mechanisms to produce nectaries occurred during the evolution of flowering plants.
In species of the Brassicaceae, such as Arabidopsis thaliana, nectaries develop as a ring at the base of the stamens, with secretory glands associated with the stamens. In Arabidopsis crabs claw (crc) mutants, all traces of nectary development, both morphological and molecular, are lacking (Bowman and Smyth, 1999; Baum et al., 2001). CRC encodes a putative transcription factor containing a zinc finger and helix-loop-helix domain called the YABBY domain (Bowman and Smyth, 1999; Siegfried et al., 1999). CRC expression commences in two horseshoe-shaped domains thought to be the precursors of the nectary, and expression continues throughout the nectary beyond anthesis (Baum et al., 2001). Ectopic expression of CRC alone does not cause the development of ectopic nectaries, implying that other factors are required for nectary development. Some of these factors are meristem identity genes, gain- or loss-of-function alleles of which lead to ectopic nectaries at the base of the flower pedicel, suggesting that factors both intrinsic and extrinsic to the flower are required to localize CRC expression. Regardless of genetic background, CRC is always required for nectaries, indicating that it is one of the key genes directing nectary development in Arabidopsis (Baum et al., 2001).
The Arabidopsis genome contains six members of the YABBY gene family, and all are expressed abaxially in lateral organs (Bowman and Smyth, 1999; Sawa et al., 1999; Siegfried et al., 1999; Villanueva et al., 1999). Ectopic expression of two members, FILAMENTOUS FLOWER and YABBY3, in the adaxial regions of leaves is sufficient to promote abaxial cell fates, indicating a role for these genes in establishing the polarity of lateral organs (Sawa et al., 1999; Siegfried et al., 1999). Likewise, CRC is required for proper establishment of adaxial–abaxial polarity in the carpel. Whereas crc single mutants do not exhibit altered carpel polarity, crc kanadi1 carpels develop adaxial tissues in abaxial positions, and in addition, ectopic adaxial expression of CRC in lateral organs, such as leaves and petals, is sufficient to promote abaxial cell fates (Alvarez and Smyth, 1999; Eshed et al., 1999). CRC is expressed in the abaxial epidermis of the carpel, as well as in internal domains, in a complex and dynamic pattern, and this expression is critical for promotion of abaxial fates (Bowman and Smyth, 1999; Eshed et al., 1999). Whereas a role for CRC in carpels may be an ancestral function within angiosperms since the CRC ortholog in Oryza, DROOPING LEAF, promotes carpel identity, its role in nectaries could be a derived condition based on the proposed evolutionary origins of nectaries and the lack of evidence for any other YABBY gene family member expressing in the nectaries (Brown, 1938; Sawa et al., 1999; Siegfried et al., 1999; Villanueva et al., 1999; Yamaguchi et al., 2004).
Previous genetic analyses indicated that the floral homeotic genes that specify the identity of the other floral organs, commonly referred to as the ABC genes, influence nectary development (Baum et al., 2001). In both B (apetala3 [ap3] and pistillata [pi]) and C (agamous [ag]) class mutants, a reduction in nectary development is observed, and in BC double mutants, no nectaries develop. However, when A function is also compromised, nectary development is restored. CRC is active in the nectaries and carpels, regions of the flower in which B and C class genes are also active, raising the possibility that they may play a role as redundant activators in the nectary development pathway. To better understand the relationship between CRC and the floral homeotic genes and to elucidate how CRC is activated in nectaries and carpels, we undertook an analysis of its promoter by identifying evolutionarily conserved regulatory elements by comparison of CRC promoter sequences from related species, a process called phylogenetic footprinting (Duret and Bucher, 1997; Tautz, 2000; Colinas et al., 2002). Based on molecular and genetic analyses, we propose that a combination of floral homeotic gene activities act redundantly with each other and in combination with SEPALLATA (SEP) genes to activate CRC in the nectaries and carpels. These MADS box proteins may provide general floral factors that must work in conjunction with region-specific factors in the activation of CRC in the nectaries and carpels.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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). Carpel structure and morphology in Lepidium and Brassica are similar to that of Arabidopsis, suggesting that the CRC orthologs play similar functional roles in the three species (Polowick and Sawhney, 1986; Bowman and Smyth, 1998; Davis et al., 1998). That the Lepidium and Brassica genes described here are CRC orthologs is substantiated by expression patterns, functional studies, and phylogenetic analyses (J.-Y. Lee and J.L. Bowman, unpublished data). Five highly conserved regions were detected in the 5' upstream sequences of Arabidopsis, Lepidium, and Brassica CRC genes, and the regions used for analysis were denoted A, B, C, D, and E (Figure 1; see also supplemental data online for complete alignment). The regional analysis was primarily based on the conservation between Arabidopsis and Lepidium because the Lepidium CRC promoter driving ß-glucuronidase (GUS) expression in transgenic Arabidopsis exhibited a staining pattern indistinguishable from an Arabidopsis CRC:GUS transgene. In addition, an Arabidopsis CRC cDNA driven by the Lepidium CRC promoter complements the Arabidopsis crc-1 mutant phenotype in >90% of transformants (data not shown).
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). These were transformed into wild-type or crc-1 plants in which a GUS reporter gene was fused to the pOP promoter, which is activated by LhG4. Both wild-type and crc-1 plants were analyzed to ascertain whether autoregulation may play a role and to separately analyze regulation by CRC promoter regions in early versus late stages of nectary development. The GUS expression patterns of transgenic plants are summarized in Table 1 and Figure 2. Region A alone does not result in the transcription of GUS. However, chimeras between A and other regions result in GUS expression, partially reflecting the endogenous expression pattern of CRC. Regions C and E were responsible for most of the positive regulation of CRC. Both C and E drive GUS expression in the carpels, with E also responsible for nectary expression. By contrast, regions B and D did not result in any expression on their own in crc-1, but influenced expression patterns when combined with C and E (note the frequency of expression in valves and nectaries driven by E:D:C:B:A in Table 1), suggesting that the primary roles of regions B and D may be to negatively regulate CRC expression in the sepals and tissues of the gynoecium.
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) and continues until after anthesis (Figures 2J to 2K and 2O). In a crc-1 background, expression in the nectary anlagen, the region where nectaries arise in the wild-type flower, indicates that region E is responsible for initial CRC expression in nectaries before the cell divisions that mark the initiation of the nectary glands (Figures 2J to 2K). In addition to driving transcription in nectaries, region E also drives expression in carpels, initially visible at stage 6 (Figures 2L and 2N) but conspicuous by stage 7 (Figures 2L and 2M). GUS reporter gene expression appears to be throughout the entire carpels and ovules. After stage 13, expression in ovules disappears (Figure 2I), but expression in carpels is still present even after stage 15, although by this stage it is only present in the upper region of carpels, including the style and stigma (Figures 2F and 2O). Thus, the expression pattern driven by region E is in tissues that endogenously express CRC and, in addition, some tissues where CRC is not normally expressed. It is likely that the additional expression observed when the reporter gene is driven by region E is suppressed by other regions, presumably region B and/or D because reporter gene expression driven by a chimeric promoter combining all the conserved regions mimics the pattern driven by the intact 3.8-kb promoter (Figures 2G, 2H, 2Q, and 2R). GUS expression driven by the intact 3.8-kb promoter and the composite conserved region promoter (E:D:C:B:A) commences in carpels at stage 6 and continues until stage 14. In contrast with E:A>>GUS, GUS is not expressed in the septum or ovules at stage 12 (Figure 2P). In nectaries, E:D:C:B:A>>GUS expression commences by stage 9 (Figure 2Q), about the same stage when the nectary expression is initiated by region E. These results suggest that the sequences conserved in three closely related species are sufficient for regulating CRC.
Although region E contains qualitative elements for nectary expression of CRC, for proper quantitative expression of CRC in nectaries, region A was also required. As a comparison, when region E fused with a CaMV-TATA:GUS was introduced into Arabidopsis, GUS activity in nectaries was reduced in frequency and intensity (data not shown), suggesting that region A might act as a core promoter for transcriptional initiation (Smale, 2001).
Complementation of crc Mutants by Promoter Regions
Because regions C and E are largely responsible for CRC expression in carpels and nectaries, we examined the extent to which these regions are able to complement crc mutants (Figure 3). Transgenic lines with E:A:LhG4, C:A:LhG4, and E:D:C:B:A:LhG4 were generated in a line with OP:CRC to determine the extent of complementation of the phenotypic defects in carpels and nectaries in a crc mutant (Figure 3). Driving CRC expression with the composite promoter, E:D:C:B:A, is sufficient to rescue carpel defects, as exemplified by normal fruit development in these transgenic lines (Figure 3D). By contrast, CRC expression driven by E:A was able to rescue the style defects associated with crc mutations, but was unable to fully complement fruit growth, suggesting that rescue of ovary wall defects was not complete (Figure 3C). Surprisingly, expression of CRC by C:A was unable to rescue most carpel defects despite C:A driving expression early in carpel development (Figure 3B).
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Binding Sites for LEAFY and MADS Box Proteins and the Regulation of CRC
Previous genetic studies implicated several known transcription factors in the regulation of CRC. MADS box genes are candidate regulators because pi ag flowers lack nectaries (Baum et al., 2001). In addition to the regulation by MADS box genes, two genes, LEAFY (LFY) and UFO, thought to regulate both the homeotic genes and the formation of the third whorl, also affect nectary development (Baum et al., 2001). In the case of LFY, it appears to be involved both in inducing nectary development within the flower and suppressing nectary development outside the flower. Mutations in another MADS box gene, AP1, also result in ectopic nectary development (Baum et al., 2001). Thus, we investigated whether any of these genes may directly regulate CRC in nectaries.
To begin to address this question, we searched the 5' upstream sequences of CRC in all three species for potential LFY and MADS box protein binding sites (Dolan and Fields, 1991; Treisman, 1992; Busch et al., 1999). In the region spanning 3.8 kb upstream of the Arabidopsis CRC coding region, the region found to be necessary and sufficient for proper CRC expression, four putative LFY binding sites (CCANTG) and two potential binding sites for MADS box proteins, known as CArG boxes [CC(A/T)6GG], were identified (Figure 1; see also supplemental data online). In L. africanum, five LFY binding domains and four CArG boxes were identified within 5 kb upstream of CRC, whereas in B. oleracea, two LFY binding domains and four CArG boxes were identified within 4.5 kb upstream of CRC. Some of these binding sites are found not only in the regions conserved among the three species but also in nonconserved regions (Figure 1). The CArG boxes located in Arabidopsis region E were identical to those of Lepidium; however, some base changes in Brassica resulted in slight deviations from the consensus CArG box sequence (see supplemental data online). LFY binding domains in regions E and C were also 100% identical between Arabidopsis and Lepidium but base changes were found in Brassica. We focused on the binding sites located in conserved regions because these sites might be functionally significant for regulating CRC in the context of the other cis-regulatory elements resident in conserved regions.
To determine the roles of these putative binding sites, site-directed mutagenesis was performed to alter the sequence of three LFY binding sites of regions C and E and two CArG boxes of region E (Figure 1; Tilly et al., 1998; Busch et al., 1999). The expression of GUS controlled by regions C and E or the fusion of all five conserved domains containing mutagenized sites was analyzed and is summarized in Tables 2 (LFY) and 3 (CArG boxes). Site-directed mutagenesis of the LFY binding site (CCANTG 
AAANTG) in region E led to slightly decreased levels of expression but did not affect the pattern of expression. A similar result was obtained for the LFY binding sites in region C. Mutagenesis of all three LFY binding sites in the context of the fusion of the five conserved domains does not change the expression pattern or the frequency and level of expression (Table 2).
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AA(A/T)6GG] alters the expression pattern driven by E:A dramatically (Table 3). No expression was observed in the nectaries in any of the transgenic lines, with only a few lines exhibiting a low level of expression in the stigma. The mutagenesis of the CArG boxes also affected GUS expression driven by the fusion of the five conserved domains. Only six out of 25 transgenic lines exhibited expression in nectary anlagen in crc-1, with expression in nectaries commencing later than with the wild-type fusion promoter. This suggests that additional elements responsible for CRC nectary expression exist, but for consistent early nectary expression binding of MADS box protein(s) is essential. The expression pattern in the carpels was also affected significantly in the promoters in which the CArG boxes were mutated. Only 10 out of 25 transgenic lines had expression in the valves, similar to transgenic lines with region C alone (seven out of 23 lines), suggesting that MADS box proteins also regulate carpel expression in a redundant manner.
LEAFY Regulation of CRC
Though the site-directed mutagenesis of the LFY binding sites did not significantly affect the expression pattern in vivo, other studies indicate the involvement of LFY in the regulation of CRC. In plants carrying both CRC:GUS and LFY:LFY:VP16 (Parcy et al., 1998) transgenes, the level of GUS expression is greatly enhanced, whereas the domain of expression is not altered (data not shown). In a lfy-6 background, CRC:GUS is expressed at the abaxial base of pedicels, where small outgrowths resembling nectaries arise (Baum et al., 2001). However, whereas LFY influences the extent and pattern of CRC expression, a 35S:LFY:VP16 transgene is not sufficient to activate CRC in seedlings (data not shown), in contrast with the activation of AG by 35S:LFY:VP16 (Parcy et al., 1998).
Expression of CRC in Plants with Altered MADS Box Gene Activity
Previous genetic analyses of nectary development in floral homeotic mutants suggests that although nectary development depends on the presence of the third whorl in flowers of Arabidopsis (Figures 4A and 4E), it is not generally affected by homeotic changes of floral organs (Baum et al., 2001). The single exception is that nectaries fail to develop in pi ag flowers, in which both B and C class gene activities are missing (Bowman et al., 1991), and this phenotype was attributed to the lack of third whorl development in this genotype (Baum et al., 2001). Mutations in the SEP genes, which are required for B and C gene activity (Pelaz et al., 2000, 2001; Honma and Goto, 2001), also result in a failure in nectary development. The flowers of sep1 sep2 sep3 triple mutants consist entirely of sepals (Figure 4B), similar to pi ag double mutants, but in contrast with pi ag flowers, a third whorl appears to be present in sep1 sep2 sep3 flowers (Pelaz et al., 2000). Despite possessing a third whorl, nectaries are lacking in sep1 sep2 sep3 flowers (Figure 4H), suggesting that the SEP genes are required for CRC activation in the third whorl, consistent with the loss of CArG boxes resulting in a loss of CRC expression. That ap3 and pi flowers lack a third whorl, whereas sep1 sep2 sep3 flowers appear to have a third whorl, suggests a SEP independent role for the B class genes in patterning the floral ground plan.
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). Because B and C gene products interact with SEP gene products (Egea-Cortines et al., 1999; Honma and Goto, 2001), other MADS box encoding genes are attractive candidates for the redundant factors. SHATTERPROOF1 (SHP1) and SHP2 encode proteins similar to AG, are negatively regulated by AP2, and are ectopically expressed in ap2 ag flowers (Savidge et al., 1995; Flanagan et al., 1996; Pinyopich et al., 2003). Flowers of ap2 pi ag shp1 shp2 plants consists entirely of leaf-like organs produced from an indeterminate flower meristem (Figure 4C), and these flowers lack nectaries (Figure 4I), suggesting that SHP proteins may interact with SEP proteins to activate CRC in the third whorl of the flower. Whereas the SHP genes are expressed in wild-type nectaries (Baum et al., 2001), loss of SHP activity in shp1 shp2 flowers does not result in morphological defects in nectary development (Figure 4F).
If SHP1 and SHP2 are acting with SEP to activate CRC in an ap2 pi ag background, nectary development in a sep1 sep2 sep3 ap2 background is predicted to be absent. Thus, we examined nectaries in this background. Because of the instability of the sep2 allele (Pelaz et al., 2000), the interpretation of the carpelloid organs produced in such flowers (Figure 4D) is ambiguous because all quadruple mutant plants displayed some reversion of the sep2 mutant allele to the wild type at the molecular level as assayed by PCR. However, in all flowers examined, no trace of nectary development was observed (Figure 4G), suggesting that the primary pathway negatively regulated by AP2 is one that acts in conjunction with SEP proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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) differently from nonfunctional regions, and this conservation of modules is recognizable in sequences from species within the same family, Brassicaceae in this case (Wray et al., 2003). Each of five modules conserved between Arabidopsis and Lepidium has a discrete regulatory function, and only in combination do they reflect the complete regulatory pattern of the endogenous CRC promoter. Module E primarily directs CRC expression in nectaries and carpels, and C acts redundantly with E to direct CRC expression in valves of developing gynoecia. However, additional modules (B and D) are needed to repress expression in specific tissues of the carpels and sepals. Thus, the regulation of CRC expression is a combination of positive and negative regulation acting through different modules of the promoter. Five regions of the 5' upstream sequences of CRC are conserved between Arabidopsis and Brassica, but each conserved region does not precisely overlap with the regions conserved between Arabidopsis and Lepidium. The nonoverlapping conservation is likely because of differential selection pressure acting upon the conserved regions after the separation from the last common ancestor of the three species. Whether the regions conserved in a nonoverlapping manner reflects functional redundancy or is the result of selection is not clear until those regions are functionally tested.
Two factors that could contribute to the relatively large size of the conserved regions are the evolutionary distances between species compared (e.g., linkage disequilibrium) and the complexity of gene regulation. We compared sequences from closely related species that have diverged less than 
10 to 14 million years ago (Arabidopsis and Lepidium) and 20 million years ago (Arabidopsis and Brassica) based on the fossil record of Rorippa, and consistent with their phylogenetic relationships, within the conserved regions, Arabidopsis CRC promoter sequences are more similar to those of Lepidium than to those of Brassica (Mai, 1995; Koch et al., 2001a). Other studies comparing orthologous regulatory sequences from Brassicaceae species facilitated identification of conserved regulatory elements (Hill et al., 1998; Koch et al., 2001b; Hong et al., 2003). Comparisons of the AP3 and CHALCONE SYNTHASE promoters, consisting of 500 bases 5' to the transcription start site, from 22 species identified conserved elements, some of which have been experimentally tested for functionality, amidst sequences that are difficult or impossible to align (Hill et al., 1998; Tilly et al., 1998; Koch et al., 2001b). By contrast, sequences of the second intron of AG were unambiguously aligned over their entire length of 3000 bases (Hong et al., 2003). Comparisons of the regulatory sequences of CRC were similar to those of AP3 and CHALCONE SYNTHESIS in that conserved sequences are flanked by sequences that cannot be aligned because of extensive divergence.
Complexity of gene regulation may also be responsible for maintaining large regions of sequence conservation, especially within region E, which is almost 500-bp long and is responsible for most of the nectary and effective carpel expression. Region E is a combination of three highly conserved regions separated by very short nonconserved sequences. When region E was dissected further using the three conserved subregions individually fused with a CaMV-TATA:GUS, we observed nectary expression only when all three subregions are combined, suggesting possible interactions between the subregions (J.-Y. Lee, unpublished data). However, because expression levels conferred by region E are significantly decreased without region A, analysis of the subregions of E combined with region A is required to verify these results. Sequencing of CRC regulatory sequences from additional Brassicaceae species could help discriminate between linkage disequilibrium and regulatory complexity as causes for the large size of conserved regions observed in this study. Analyses of potential binding sites of MADS box proteins demonstrate their importance for activating CRC in nectaries and carpels. However, conservation in the sequence immediately surrounding the CArG boxes is not as high as in the remainder of region E. A similar pattern is observed in LFY binding sites in the AG promoter (Hong et al., 2003). The lack of sequence conservation may be because of these transcription factors relying primarily on their specific binding sites and not on surrounding sequence context.
Complementation of crc Mutant Phenotypes
The ability of both the intact 3.8-kb CRC promoter and the E:D:C:B:A composite promoter to complement the crc mutant phenotype suggests that all necessary promoter elements are found within conserved DNA sequences among species. Although at a lower frequency, CRC regulated by region E alone could partially complement both the carpel and nectary phenotypes. Neither E:D:C:B:A nor E:A fully complemented the crc nectary phenotype because medial nectaries were lacking and stomata were reduced in number on the lateral nectaries. One possible explanation is that additional sequences, perhaps those immediately flanking the highly conserved regions, may be required for fine-tuning quantitative or qualitative CRC expression. Surprisingly, despite region C driving high levels of expression in the carpel, it was unable to complement the crc carpel phenotype, in contrast with E:A>>CRC, which partially complements the crc carpel phenotype. Thus, a combination of sequences in C and E is required for proper spatial and temporal regulation within the carpel, and these promoter elements are at least partially redundant because both C and E are sufficient to drive expression independently.
Regulation of CRC by Floral Genes in Arabidopsis
Genetic analysis of nectary development in floral homeotic mutants demonstrated that nectaries can develop in the absence of the activity of the ABC genes (Baum et al., 2001). Although nectaries are normally associated with stamens in wild-type flowers, when the identity of the third whorl organs is altered to carpels (as in ap3 and pi flowers) or petals (as in ag flowers), nectaries still develop at the abaxial base of the third whorl organs. Conversely, in 35S:PI 35S:AP3 ap2 flowers, in which stamens occupy all floral whorls, nectaries are only found associated with the third whorl stamens. In superman mutants and in 35S:UFO flowers, multiple whorls of nectaries are associated with the supernumerary whorls of stamens produced interior to the third whorl. One interpretation is that nectary development depends on the formation of the third whorl and is independent of the development of the other floral organs (Baum et al., 2001). However, the sizes and positions of nectaries are affected by mutations in the ABC genes (Baum et al., 2001). For example, in both B and C class mutants, the extent of nectary gland development is reduced, and in both lfy and ufo single mutants, nectaries were rarely found. Finally, in BC double mutants (ag pi or ag ap3) no sign of nectary development is observed, but nectary development is restored when AP2 activity is also compromised. Thus, whereas the ABC genes are not absolutely required, these genes influence the extent of nectary development.
Because both LFY and MADS box proteins are implicated in the regulation of CRC, we searched for their respective binding sites in the CRC promoter. Two CArG boxes and four LFY binding sites were found in the Arabidopsis CRC promoter. CArG boxes were found in region E, and site-directed mutagenesis of the two boxes dramatically disrupted transcriptional regulation by region E, suggesting that a MADS box protein(s) is critical for the transcriptional regulation of CRC. Based on the observation that B and C class gene products interact with SEP proteins in the specification of floral organ identity (Egea-Cortines et al., 1999; Honma and Goto, 2001; Pelaz et al., 2001) and the lack of nectary development in sep1 sep2 sep3 flowers, a compelling scenario is that a complex including SEP proteins and C and/or B MADS box proteins activates CRC through binding of the CArG boxes in region E. The lack of nectary formation in BC double mutants, but their presence in B and C single mutants, would reflect redundancy of these proteins in the complex with the SEP proteins.
The restoration of nectary development in ap2 pi ag triple mutants suggests that AP2 represses another redundant pathway or factor(s) that can act with the SEP proteins to activate CRC. SHP1 and SHP2 are obvious candidates representing such a redundant pathway because they are negatively regulated by AP2, encode proteins similar to AG, and are ectopically expressed in an ap2 ag background (Savidge et al., 1995; Flanagan et al., 1996; Pinyopich et al., 2003). In this scenario, in an ap2 pi ag background a complex of SHP and SEP proteins activates CRC in the nectaries. Consistent with this hypothesis, ap2 pi ag shp1 shp2 flowers lack nectaries. That sep123 ap2 flowers also lack nectaries indicates that the primary pathway negatively regulated by AP2 is through repression of genes encoding MADS box proteins. Thus, in wild-type flowers, a complex of SEPs + B and/or C would activate CRC, whereas in an ABC triple mutant SEPs + SHPs would activate CRC. The control of CRC expression by complexes of MADS box genes represents a mechanism by which CRC is activated specifically in the flowers. However, because the combination of SEPs + B and/or C would have the potential to activate CRC throughout the inner three whorls of the flower, these complexes must act in conjunction with other, presently unidentified, spatial regulators such that activation is restricted to the nectary anlagen and specific regions of the carpels. The spatial regulators must also act through promoter sequences contained within regions C and E (Figure 5).
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). Thus, although LFY activity is critical for proper regulation of CRC, its action is unlikely to be direct, perhaps acting through LFY-mediated regulation of MADS box genes (Parcy et al., 1998; Schmid et al., 2003). As with other genes whose regulation is complex both in terms of spatial and temporal patterns, several modules act positively and negatively in a combinatorial fashion to control CRC expression, as summarized in Figure 5. The SEP + B/C MADS box genes activate CRC in the flower, but other whorl-specific factors are required to restrict CRC expression to the nectaries and specific tissues of the gynoecium. Finally, that the E module is not easily dissected suggests that the whorl-specific factors may be required to interact directly with the more general flower activation factors to modulate CRC expression.
The regulation of CRC has implications concerning nectary evolution in flowering plants. Nectaries in basal angiosperms are usually associated with the perianth, whose structure is not as distinct or elaborate as in core eudicots and monocots. By contrast, in core eudicots, nectaries are usually located near or on the reproductive organs (Brown, 1938; Fahn, 1953; Endress, 2001). Expression analysis of CRC in developing nectaries of several species of eudicots suggests that CRC might be a general regulator for nectary development in core eudicot lineages (J.-Y. Lee, unpublished data). The functional significance of CArG boxes for the regulation of CRC suggests that the establishment of the regulatory pathway between MADS box proteins and CRC may have facilitated restricting nectaries to the reproductive whorls of flowers.
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METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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). They are indicated with black bars on the top of the plots in Figure 1, and they were used for expression analysis. Including Brassica sequences, three sequences were aligned with AVID, a global alignment program (Bray et al., 2003), and conserved regions shared by three species were detected using mVISTA with the parameter of 75% of identity and a 50 base sliding window (Dubchak et al., 2000; Mayor et al., 2000), which fits well with the local alignment result between Arabidopsis and Lepidium (Figure 1). Three-way species comparison used in mVISTA identifies conserved domains by choosing regions conserved in all three species using intersection/union analysis (Dubchak et al., 2000). The conserved regions of 300 to 550 bp were conspicuous relative to surrounding sequence because in most cases the adjacent sequences were impossible to align with any degree of confidence (Figure 1).
Transgene Construction for Promoter Analysis
The five domains (A, B, C, D, and E) conserved between the 5' upstream sequences of Arabidopsis thaliana CRC and L. africanum CRC were isolated by PCR and subcloned such that they were 5' to the chimeric transcription factor, LhG4 (Moore et al., 1998), with a 3' octopine synthase terminator after the LhG4 coding sequences. Six constructs were generated: A:LhG4, B:A:LhG4, C:A:LhG4, D:A:LhG4, E:A:LhG4, and E:D:C:B:A:LhG4. These constructs were subcloned into the binary vector pMLBART and transformed into Agrobacterium tumefaciens strain ASE by electroporation. Each construct was transformed into wild-type Ler or crc-1 mutants containing the transgene 2OP:GUS by floral dipping (Weigel and Glazebrook, 2002), and the transgenic plants were selected with BASTA.
Site-directed mutagenesis was accomplished using a PCR-based method. Primers having mutant sequences were used to amplify (Pfu polymerase) the CRC promoter region of interest. The template plasmid was removed by treating the PCR mixture with the restriction enzyme DpnI, then the mixture was transformed into Escherichia coli. The mutagenesis was confirmed by sequencing.
GUS Staining
Inflorescences were fixed in 90% ice-cold acetone for 20 min and rinsed twice with a GUS working solution [25 mM phosphate buffer, pH 7.0, 1.25 mM K3Fe(CN)6, 1.25 mM K4Fe(CN)6, 0.25% Triton X-100, and 0.25 µM EDTA] for 20 min each time. After rinsing, tissue was incubated with 5-bromo-4-chloro-3-indoyl ß-D-glucuronide cyclohexylamine salt, added to GUS working solution to the final concentration of 1.25 mg/mL, at 37°C overnight. The reaction was terminated and tissue was cleared in 70% ethanol added fresh once a day for a week.
Microscopy
Inflorescences incubated with X-Gluc were fixed in formaldehyde-acetic acid (3.7% formaldehyde, 5% acetic acid, and 50% ethanol) for 2 h and dehydrated through an ethanol series and embedded in paraffin. Inflorescences were sectioned at 8 µm of thickness. Sectioned tissue was viewed using dark-field optics. For scanning electron microscopy, tissue was fixed overnight with 3% glutaraldehyde, phosphate buffered to pH 7, followed by a second overnight fixation in 0.5% osmium tetraoxide. Tissue was dehydrated in ethanol and critical point dried. After sputter coating with gold/palladium, tissue was observed on a Hitachi S-3500N scanning electron microscope (Tokyo, Japan).
Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY703987 for 6090 bases of L. africanum CRC 5' upstream and partial coding regions and AY703986 for 5462 bases of B. oleracea CRC 5' upstream and partial coding regions.
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Acknowledgments |
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Footnotes |
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2 Current address: United States Patent and Trademark Office, 400 Dulany, Alexandria, VA 22312.
3 Current address: Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100 Israel.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: John L. Bowman (jlbowman{at}ucdavis.edu).
Online version contains Web-only data.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.026666.
Received August 5, 2004; accepted October 20, 2004.
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