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First published online January 21, 2005; 10.1104/pp.104.055368 Plant Physiology 137:651-662 (2005) © 2005 American Society of Plant Biologists Multiple Protein Regions Contribute to Differential Activities of YABBY Proteins inReproductive Development1,[w]Section of Molecular and Cellular Biology (R.J.M., C.S.G.) and Section of Plant Biology (H.O., J.L.B.), University of California, Davis, California 95616
Members of the YABBY family of putative transcription factors participate in abaxial-adaxial identity determination in lateral organs in Arabidopsis (Arabidopsis thaliana). Two YABBY genes specifically expressed in reproductive structures, CRABS CLAW (CRC) and INNER NO OUTER (INO), have additional activities, with CRC promoting nectary development and carpel fusion, and INO responding to spatial regulation by SUPERMAN during ovule development. All YABBY coding regions, except YABBY5, were able to restore outer integument growth in ino-1 mutants when expressed from the INO promoter (PROINO). However, INO was the only YABBY family member that responded correctly to SUPERMAN to maintain the wild-type gynoapical-gynobasal asymmetry of the outer integument. By contrast, INO, FILAMENTOUS FLOWER, and YABBY3 failed to complement crc-1 when expressed from PROCRC. Roles of individual regions of CRC and INO in these effects were assessed using chimeric proteins with PROINO and PROCRC and the relatively constitutive cauliflower mosaic virus PRO35S. Regions of CRC were found to contribute additively to CRC-specific functions in nectary and carpel formation, with a nearly direct relationship between the amount of CRC included and the degree of complementation of crc-1. When combined with INO sequences, the central and carboxyl-terminal regions of CRC were individually sufficient to overcome inhibitory effects of SUPERMAN within the outer integument. Reproductive phenotypes resulting from constitutive expression were dependent on the nature of the central region with some contributions from the amino terminus. Thus, the YABBY family members have both unique and common functional capacities, and residues involved in differential activities are distributed throughout the protein sequences.
Functional characterization of plant transcription factors has been facilitated by genetic and transgenic analysis, and the activities of many such proteins have been clearly linked to diverse regulatory processes (Zhang, 2003  ). Transcription factors have been classified into families, such as the MADS and MYB families, based on sequence conservation of protein regions, most commonly DNA-binding domains (Riechmann, 2002). In many instances, members of the different families have become functionally diversified, resulting either from critical changes in protein coding sequence or from spatial and temporal changes in expression patterns. The distinct roles of the MADS-domain transcription factors AGAMOUS and APETELA3 in floral development represent an example of the former process (Riechmann et al., 1996; Riechmann and Meyerowitz, 1997), whereas the apparent interchangeability of the protein coding sequences of the MYB-domain proteins WEREWOLF and GLABARA1 exemplifies the latter process (Lee and Schiefelbein, 2001). Experimental interchange of protein coding sequences and regulatory regions has facilitated the understanding of both in vivo roles and mechanisms of functional divergence of such gene family members.
The YABBY family of putative transcription factors was identified in Arabidopsis (Arabidopsis thaliana; Bowman and Smyth, 1999
Asymmetric growth of the outer integument of ovules, mediated by interactions between INO and SUPERMAN (SUP; Meister et al., 2002 With diverged expression patterns and in vivo roles, we exploited the YABBY gene family as a model for mechanisms of functional divergence among plant transcription factors. Using both tissue-specific and constitutive promoters, we characterized the effects of expression of native or chimeric YABBY coding sequences on plant morphology. We demonstrate that FIL, YAB2, YAB3, and CRC could restore integument growth in ino-1 mutants but appear to be insensitive to the inhibitory effects of SUP. By contrast, INO, FIL, and YAB3 were unable to compensate for the loss of CRC in directing the proper development of both the nectaries and the gynoecium. Analysis of chimeric coding sequences suggested that each of three putative protein regions contributes to the observed functional differences in at least one of three expression domains examined and that the zinc finger motif may be dependent on an adjacent conserved sequence region for functionality in some expression domains. In addition to differences in expression domains, we speculate that differences in protein-protein interactions, due to changes in the coding regions, may make a significant contribution to functional divergence of the YABBY protein family.
INO Has a Unique Ability to Maintain Asymmetric Growth of the Outer Integument
Ovule development has been characterized previously through morphological, genetic, and molecular analyses (Gasser et al., 1998
INO expression is limited to the site of outer integument initiation and the outermost (abaxial) cell layer of the developing outer integument (Villanueva et al., 1999 ; Balasubramanian and Schneitz, 2000). The identification of a genomic fragment (PROINO) sufficient to duplicate the endogenous expression of INO was described previously (Villanueva et al., 1999; Meister et al., 2002, 2004). Using PROINO, the ability of each YABBY member to complement the ino-1 mutant phenotype was assessed. Transgenic plants in this study exhibited a range of ovule morphologies. Ovules of transgenic plants were classified as wild type (complementing) if normal ovule development was fully restored (Fig. 1F) or "ino-like" if the ino-1 effects were not mitigated (Fig. 1I). Ovules resembling those of sup-5 were also identified and termed "sup-like" (Fig. 1D). In addition, two intermediate phenotypes were observed and referred to as "weak-sup" and "weak-ino." In weak-sup ovules, integument growth from the gynoapical side of the ovule primordium was often less than observed in sup-5, but still greater than in wild type (Fig. 1E). Outer integument growth in weak-ino ovules resembled that of the ino-4 allele (Villanueva et al., 1999), where this structure partially covers but never fully envelops the inner integument yet still exhibits the characteristic asymmetric growth (Fig. 1, G and H).
Based on their phenotypic effects on ino-1 ovules, enumerated in Table I, the YABBY proteins partitioned into three statistically separable classes. As described previously (Villanueva et al., 1999
In contrast with INO, the majority of other Arabidopsis YABBY coding regions produced results similar to those observed for the PROINO:CRC construct. For CRC, YAB3, FIL, and YAB2, integument growth was at least partially restored, but the majority, or a significant fraction, of the transgenic lines also exhibited ectopic outer integument growth on the gynoapical side of the ovule primordia (Table I). Within this group, there was variation both in the ability to promote outer integument growth and for evidence of spatial regulation of this growth. PROINO:YAB3 and PROINO:CRC transgenics appeared to be most efficient at growth promotion and also had a higher frequency of ectopic integument growth from the gynoapical side of the ovule primordium relative to PROINO:FIL or PROINO:YAB2 transformants. Among these transgenes, only PROINO:FIL produced a significant fraction of plants (22%) with apparently wild-type ovules. PROINO:YAB5 was unable to support outer integument growth in any transformant analyzed and, thus, by itself represents a third class of transgene.
Chimeric cDNAs, combining regions of INO and CRC coding sequences, were produced to identify protein regions responsible for the different effects of expression of these two coding regions on integument development. The chimeras represented permutations of three regions: the amino-terminal region (including the conserved zinc finger motif), the central variable region, and the carboxyl-terminal region (beginning at the start of the conserved YABBY region). Boundaries of the different regions are illustrated in Figure 2 and were placed at the junctions of the previously described conserved regions and the adjacent residues (Bowman and Smyth, 1999
Chimeric coding regions were fused to PROINO and assessed in an ino-1 mutant background. As with PROINO-directed expression of the endogenous YABBY members, observed ovule phenotypes could be grouped into five classes: sup-like, weak-sup, wild type, weak-ino, and ino-like (Fig. 1). In general, we found that all chimeras were able to support some growth of the outer integument in at least a subset of transformants. Effects of the amino-terminal region exchange appeared to be sensitive to the conserved residues adjacent to the zinc finger motif. As evidenced by the CiII chimera (and the later described CiIC chimera), the inclusion of the INO sequence in this region in conjunction with the CRC amino terminus resulted in a severe reduction of the ability to promote integument growth. By contrast, the CcII coding region was able to support outer integument growth in most transformants. Since more than one-half of the CcII transformants had weak-ino ovules, this chimera was less effective than INO at promoting integument growth, but no transformants had weak-sup or sup-like integument growth, indicating a normal response to SUP effects. The INO amino-terminal region appeared to be less dependent on the nature of the adjacent sequence, as both the IcCC and IiCC chimeras were found to promote integument growth in a manner that was not statistically different from the effects of CRC (Table I). Replacement of either the central variable or carboxyl-terminal regions of INO with CRC sequences produced transgenes that could promote growth of the outer integument from the gynoapical side of the ovule primordium. The IcCI chimera was the most effective; all PROINO:IcCI transgenics with substantial outer integument growth exhibited visible outer integument growth on the gynoapical side of the ovule. Both the IiCI and IiIC chimeras produced plants with either weak-sup or wild-type ovules. Uniquely, PROINO:IiIC transformants producing weak-ino or ino-like ovules were not identified. Of these three chimeras, both IcCI and IiIC produced effects that were statistically distinct from effects of INO. Although exchange of the variable or carboxyl-terminal regions of CRC resulted in the identification of plants containing wild-type ovules, which was not observed with the PROINO:CRC transgene, neither exchange eliminated the ability of the chimera to elicit growth of the outer integument from the gynoapical side of the ovule primordium in some transformants. However, due to the large percentage of wild-type ovules in transgenic plants, the CcIC chimera was statistically distinct from CRC, and CcCI was marginally distinct from CRC. Consistent with the apparent deleterious effects of the CiII amino-terminal exchange, the CiIC chimera appeared to be compromised in its ability to promote integument growth, producing mostly weak-ino or ino-like transformants.
The Arabidopsis gynoecium initiates development as a continuous ring of tissue that is believed to represent two congenitally fused carpel primordia (Smyth et al., 1990
To assess the extent to which native YABBY proteins were able to complement crc-1 mutant plants, we used light microscopy to examine carpel defects and an enhancer trap line (ET668; Baum et al., 2001 ) that directs expression of the  -glucuronidase (GUS) coding sequence during nectary differentiation to assess nectary development. Using these assays, a PROCRC:CRC transgene was able to complement the crc-1 carpel and nectary defects as well as activate expression of the GUS reporter gene within the nectaries in almost all transformants examined (Fig. 3, J and K), as enumerated in Table II. In contrast with CRC, the PROCRC:INO transgene was unable to complement either mutant defect of crc-1 (Fig. 3, I and M; Table II). In the PROCRC:INO transformants, the carpels did not fuse in the apical region and GUS activity was not detected at the predicted site of nectary formation. In similar studies, the production of FIL or YAB3 regulated by PROCRC also failed to complement crc-1 mutant defects (Y. Eshed and J. Bowman, unpublished data).
Chimeric Coding Sequences Assess ProteinDomain-Dependent Transgene Effects within the Gynoecium and Nectaries The inability of INO to substitute for CRC within the nectaries and gynoecium contrasted with the ability of CRC to partially substitute for INO in outer integument development. To determine whether a specific protein region was responsible for this inactivity, INO-CRC chimeras were expressed under control of PROCRC. As with the PROCRC:INO transgene, the ability of the chimeras to complement the nectary and gynoecium defects of crc-1 was assayed in such mutants carrying the nectary-specific enhancer trap (Fig. 3; Table II). In some transformants, the transgene appeared to partially complement the nectary and/or carpel defects. For nectary development, this was evidenced by detectable GUS activity even when the nectaries were reduced in size or number of glands as compared to wild type. In regard to gynoecium development, partial complementation resulted in an incomplete fusion of the gynoecium apex and an overall length of the gynoecium intermediate between crc-1 and wild type (Fig. 3). The numbers of transgenic lines in each category (noncomplementation, partial complementation, or complete complementation of nectary of fusion defects) are enumerated in Table II. Proteins including any two regions of CRC and only one region of INO (the IcCC, CiIC, or CcCI chimeras) appeared to have slightly less ability to complement the nectary defects of the crc-1 mutant relative to intact CRC (Table II), with CiIC most closely approximating the full CRC effect. Effects of these chimeras on closure of the gynoecium could not be statistically separated from effects of intact CRC (Table II; Supplemental Table III), but wild-type silique growth was not restored by any of the chimeras (Fig. 3). The relatively efficient complementation with the CiIC chimera shows that, in contrast to the observations for complementation of ino defects, the association between the amino-terminal region and adjacent conserved residues was not critical for complementation of crc defects. All three proteins containing only a single CRC region were significantly less active in complementation of crc-1 effects than were those with two CRC regions. A combination of the CRC carboxyl-terminal region with the other regions of INO was similar to INO in its inability to complement the nectary or gynoecium growth defects of crc-1. By contrast, replacement of either the amino-terminal region or the central diverged region of INO with the corresponding region of CRC led to a higher frequency of partial complementation of the crc-1 gynoecium defects than observed for INO, but still apparently less complementation than was observed for any of the chimeras containing two CRC regions. Overall, the results indicate that all three regions of CRC contribute to its unique ability to function in nectary and gynoecium development. The amino-terminal and central diverged regions play the most significant roles, with a lesser role for the carboxyl-terminal region. The positive contribution of the carboxyl-terminal region to CRC function was most apparent when examined in combination with one of the other two regions (compare results of CiII with CiIC and results of IcCI with IcCC in Table II).
Common and distinct effects of ectopic expression of INO and CRC have been described previously (Eshed et al., 1999 The occurrence of vegetative and floral phenotypic effects in plants ectopically expressing the INO-CRC chimeras are illustrated and enumerated in Figure 4 and Table III, respectively. Among the proteins that included two regions of CRC, those containing the amino-terminal or carboxyl-terminal regions of INO (IiCC, IcCC, and CcCI) most closely approximated the activity of intact CRC and could not be statistically separated from this activity. The CcIC and CiIC proteins produced more divergent phenotypes, including the INO-like production of supernumerary floral organs (CcIC), but were still on the border of statistical separation from effects of CRC (Table III; Supplemental Table V). Thus, the central diverged region appears to have the largest role in effects of ectopic CRC expression on flower development. This contrasts with CRC complementation of crc-1 defects, where replacement of the central region with that of INO led to the smallest decrease in complementation.
None of the chimeras were able to duplicate the frequency of production of supernumerary floral organs observed for ectopic production of INO (Table III). Chimeras that did result in this relatively INO-specific effect included IiIC, IiCI, and CcIC. Statistical analysis (Supplemental Table V) indicated that the IiIC and IiCI chimeras were the closest to producing INO activity, indicating a likely importance of the amino-terminal region in this activity, with clear additive contributions from the other regions. The detrimental effects on function of combining the amino-terminal region of CRC with the adjacent conserved region of INO seen in the PROINO experiments were again observed, with the CiII chimera failing to elicit effects on vegetative or floral development in most transformants. By contrast, the CiIC chimera was able to elicit a phenotypic response. Thus, consistent with the positive effect seen in PROINO expression, the presence of the CRC carboxyl-terminal region appeared to mitigate the apparent detrimental effect of the CRC amino-terminal region in conjunction with the adjacent conserved region of INO. In summary, our results suggest that the central variable region, with contributions from either the amino-terminal region (in some instances) or the region adjacent to the zinc finger motif, is the most significant determinant of the INO- or CRC-specific phenotypic responses observed from ectopic expression.
Members of the YABBY gene family are hypothesized to function in the promotion of abaxial identity (Bowman, 2000 ; Bowman et al., 2002). Abaxial identity has been proposed as one component required for both proximal-distal and laminar extension of lateral organs (Waites and Hudson, 1995; McConnell and Barton, 1998). We observed that several YABBY members were sufficient to compensate for the loss of INO in promoting extension of the outer ovule integument when expressed using PROINO. As seen for other YABBY genes (Siegfried et al., 1999), INO and CRC were also able to promote abaxial epidermal identity in adaxial epidermal cells in vegetative tissues. These observations support a common functional capacity of the YABBY members in promotion of abaxial identity and the associated lateral organ growth. This also implies a common DNA-binding capacity for YABBY proteins in their functions in ovule and leaf development. However, this work indicates that YABBY family members involved in reproductive development have apparently evolved additional specialized functions.
INO is involved in an antagonistic relationship with SUP with respect to the regulation of PROINO-driven expression (Meister et al., 2002
Chimeric proteins have been used to evaluate regions responsible for differential function of members of other families of plant transcription factors. Serikawa and Zambryski (1997) Our studies on chimeric INO-CRC proteins showed that there was a quantitative effect of the amount of CRC included in the chimeric protein on the protein's ability to complement crc-1. This additive effect was independent of which of the regions derived from CRC. When two CRC regions were included in the chimeric proteins, they commonly produced a degree of complementation close to that observed for the complete CRC protein, but still produced some transgenic plants that were deficient in nectary formation and carpel fusion. When a single CRC region was included in the chimeras, either the amino-terminal or central variable region in conjunction with the remaining INO protein was sufficient to partially complement crc-1, correcting the carpel phenotype more efficiently than the nectary defects. For complementation of ino-1, most chimeric proteins could compensate for loss of outer integument growth, but only some responded correctly to the inhibitory effects of SUP. In contrast with the crc complementation analysis, replacement of either the variable or carboxyl-terminal regions with those of CRC resulted in relative insensitivity to SUP. Only the amino-terminal exchange (CcII) retained any ability to both promote integument growth and respond correctly to SUP, albeit at a reduced frequency. Consistently, replacement of any two regions of INO resulted in even more pronounced sup-like integument growth. In ectopic expression analysis, we demonstrated that exchange of either the CRC or INO variable region or the INO amino-terminal region was individually sufficient to alter the floral phenotype produced by the chimeric protein. Taken together, our results imply that all three regions have roles in CRC- or INO-specific functional activity, but the relative contribution of each protein region was specific to the expression domain and function being complemented. This implies that all three regions of the protein participate in function-specific activities. In complementation of ino-1, the majority of INO-CRC chimeric proteins (and even other YABBY proteins) supported integument growth, indicating a common capacity to bind at least the majority of downstream targets of INO. However, since replacing any single region of INO with the corresponding region of CRC led to a decrease in the response to SUP, all three regions of INO must have some participation in this process, which likely depends on protein-protein interactions.
No other YABBY coding region could complement crc-1, and replacement of any two regions of CRC with the corresponding INO regions decreased the ability of the protein to carry out any function of CRC. This indicates that all three regions may participate in CRC-specific interactions with other proteins or with DNA, or a combination of these processes. These results indicate that all three regions of YABBY proteins participate in both DNA-binding and hypothesized protein-protein interactions necessary for YABBY function. Our results are consistent with the recent publication by Sieber et al. (2004)
We observed that the attachment of the CRC amino-terminal region with the immediately adjacent INO sequence (CiII) had a negative influence on protein activity when expressed from the PROINO or PRO35S regulatory regions. This effect was partially suppressed by replacing the INO region immediately adjacent to the putative zinc finger motif with that from CRC (CcII), even though the remainder of the central diverged region still derived from INO. The effect was also partially suppressed by exchanging the noncontiguous carboxyl-terminal region for that of CRC (CiIC). The combination of these two changes (CcIC) led to even greater activity that approached that of intact CRC in at least the PROINO expression domain. These results support two hypotheses. First, an independently functional zinc finger domain may extend outside the previously described conserved region, reminiscent of experiments demonstrating that residues adjacent to other zinc finger motifs were critical for functionality (Takatsuji, 1998
The YABBY family members are hypothesized to function as DNA-binding transcriptional regulators. Although the YABBYs are thought to specify abaxial identity, differences in ectopic expression phenotypes and ability to complement ino or crc mutant defects in this and prior studies (Eshed et al., 1999
A second component of the functional differences of the YABBY members may be a differential ability to interact with distinct protein partners. Thus, DNA-binding activity may be similar for all of the chimeric proteins (as suggested by Kanaya et al. [2002]
Construct Assembly
Chimeric Coding Sequences
PROINO Constructs
PROCRC Constructs
Constitutive Expression Constructs
Lines containing the enhancer trap ET668 were stained for GUS activity as described by McConnell and Barton (1998)
Transgenes for plant transformation were shuttled as NotI fragments into pMLBART (a gift from Kim Richardson, HortResearch, Auckland, New Zealand) and transferred into the Agrobacterium strain ASE by triparental matings (Figurski and Helinski, 1979
Sequences were aligned using ClustalX version 1.8 for Macintosh (Thompson et al., 1997
We thank members of the Gasser and Bowman labs for helpful research discussions and Mitchell Watnik for statistical analysis. We appreciate the technical support of Christian Nelson, Louren Kotow, Peter Luu, Erin Goodson, and Chris Roxas. Received October 21, 2004; returned for revision December 3, 2004; accepted December 6, 2004.
1 This work was supported by a U.S. Department of Agriculture National Research Initiative Competitive Grant (2001–35304–09989 to C.S.G.); a grant from the Department of Energy, Division of Biosciences (DE–FG03–97ER20272 to J.L.B); a grant from the National Science Foundation (IBN 0077984 to J.L.B.); a National Science Foundation Plant Cell Biology Training Grant (to R.M.); and a University of California, Davis, Jastro-Shields Fellowship (to R.M.). 
2 Present address: Department of Trait and Technology Development, Pioneer Hi-Bred International, Inc., 7250 NW 62nd Avenue, Johnston, IA 50131.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.055368. * Corresponding author; e-mail csgasser{at}ucdavis.edu; fax 530–752–3085.
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TOP
ABSTRACT
RESULTS
-value that would represent the likelihood that two transgenes had similar phenotypic effects was determined. The Bonferroni adjustment (0.05/number of comparisons performed; 
