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First published online March 26, 2004; 10.1104/pp.103.031971 Plant Physiology 134:1574-1585 (2004) © 2004 American Society of Plant Biologists
Regulation of Flower Development in Arabidopsis by SCF Complexes1Department of Biology and the Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802 (W.N., B.F., D.Z., J.A., H.M.); Institute of Molecular and Cell Biology, Singapore 11609 (D.X.); and Department of Biology, Adelphi University, Garden City, New York 11530 (L.H.)
SCF complexes are the largest and best studied family of E3 ubiquitin protein ligases that facilitate the ubiquitylation of proteins targeted for degradation. The SCF core components Skp1, Cul1, and Rbx1 serve in multiple SCF complexes involving different substrate-specific F-box proteins that are involved in diverse processes including cell cycle and development. In Arabidopsis, mutations in the F-box gene UNUSUAL FLORAL ORGANS (UFO) result in a number of defects in flower development. However, functions of the core components Cul1 and Rbx1 in flower development are poorly understood. In this study we analyzed floral phenotypes caused by altering function of Cul1 or Rbx1, as well as the effects of mutations in ASK1 and ASK2. Plants homozygous for a point mutation in the AtCUL1 gene showed reduced floral organ number and several defects in each of the four whorls. Similarly, plants with reduced AtRbx1 expression due to RNA interference also exhibited floral morphological defects. In addition, compared to the ask1 mutant, plants homozygous for ask1 and heterozygous for ask2 displayed enhanced reduction of B function, as well as other novel defects of flower development, including carpelloid sepals and an inhibition of petal development. Genetic analyses demonstrate that AGAMOUS (AG) is required for the novel phenotypes observed in the first and second whorls. Furthermore, the genetic interaction between UFO and AtCUL1 supports the idea that UFO regulates multiple aspects of flower development as a part of SCF complexes. These results suggest that SCF complexes regulate several aspects of floral development in Arabidopsis.
An Arabidopsis flower has four concentric whorls that contain four sepals, four petals, six stamens, and two carpels. After the transition from vegetative to reproductive development, the Arabidopsis apical meristem (inflorescence meristem) produces the floral meristem, which in turn undergoes a series of developmental stages to form a flower (Smyth et al., 1990  ). Genetic and molecular studies have uncovered a large number of genes that control different steps in flower development including flowering time, flower meristem identity, and flower organ identity (Zhao et al., 2001a). In particular, the ABC model has been proposed for the specification of floral organ identity (Coen and Meyerowitz, 1991; Ma, 1994; Weigel and Meyerowitz, 1994; Ma and dePamphilis, 2000). The combinatorial expression of ABC genes defines the organ type that differentiates in each whorl: A function alone specifies the sepal identity; A and B function together controls petal identity; B and C function together specifies stamen identity; and C function alone directs carpel identity.
The UNUSUAL FLORAL ORGANS (UFO) gene is involved in multiple aspects of floral development, including regulating floral meristem identity and floral organ development (Levin and Meyerowitz, 1995
The UFO gene encodes one of the approximately 700 F-box proteins that are believed to be components of the SCF-type E3 ubiquitin ligase (Gagne et al., 2002
Homologs of the core components of the SCF ubiquitin ligase have been found in Arabidopsis. At least five Cul homologs are expressed in the Arabidopsis genome. Among these, AtCUL1 and AtCUL2 are able to interact with ASK1 and F-box proteins in a yeast (Saccharomyces cerevisiae) two-hybrid assay (Risseeuw et al., 2003
Two Rbx1 homologs were uncovered in the Arabidopsis genome. The two Rbx1 proteins are highly similar to each other as well as to that of the human Rbx1 proteins. However, based on expression levels, AtRbx1a seems to be the dominant participant in SCF complexes (Gray et al., 2002
Among the 21 Arabidopsis Skp1 homologs (called ASK) in the Arabidopsis genome, at least 18 were found to be expressed under normal growth conditions, with a large subset of them detected in the inflorescence (Zhao et al., 2003b
ASK1 interacts genetically and in yeast two-hybrid assays with UFO (Samach et al., 1999
Floral Phenotypes of axr6-2, a Point Mutant in AtCUL1
A previous study showed that a null mutation in the AtCUL1 gene causes embryo lethality (Shen et al., 2002
Compared with the wild-type flower (Fig. 1A ), a typical axr6-2 mutant flower (Fig. 1B) has a slight reduction in the number of sepals, which are occasionally fused together (Table I). In the second whorl, the number of petals is reduced, and occasionally the size of petals is also reduced (Fig. 1B; Table I). In the third whorl, there are fewer than the normal number of stamens, and the stamen filaments are shorter than those of the wild type (data not shown). In addition, filamentous structures were also observed in the third whorl (Fig. 1B; Table I). Unlike ufo and ask1 flowers, no petal/stamen chimeric organs were observed in the axr6-2 flower (Table I), and whorls are clearly defined in the axr6-2 flower. In the fourth whorl, the number of carpels seems to be normal. However, about 25% of the gynoecia are curled (Fig. 1B), and occasionally carpels are not fused.
In addition, we also observed some small flower buds in the axr6-2 mutant which never opened (Fig. 1C). These flowers were found to contain aborted petals, stamens, and carpels inside relatively normal sepals.
Continuous silencing of AtRbx1 expression by double-strand RNA interference caused severe defects in plant growth and development (Lechner et al., 2002 All plants produce normal flowers without Dex treatment. Control plants transformed with an empty vector produced normal flowers after Dex treatment (Fig. 1D), except for occasional reductions in stamen number. In contrast, the flower of Dex-treated Rbx1 RNAi plants exhibited slightly reduced petal number and reduced stamen filament length (Fig. 1E). In addition, petal/stamen chimeras, filaments, and carpelloid organs were also observed in the middle whorls of the Dex-treated Rbx1 RNAi flowers (Fig. 1E; and data not shown), indicating a reduction of B function. Sometimes a curled gynoecium (Fig. 1E), or an axillary flower (Fig. 1F) was also observed in the AtRbx1 RNAi flowers.
The ask1-1 mutant contains a Ds insertion in the middle of the coding region (Fig. 2A
; Yang et al., 1999
The flower of ask2-1 (Fig. 1I; Table I) was indistinguishable from that of the wild type. In the F2 progeny of a cross between ask1 and ask2, we found that the ask1 ask2 double mutant is defective in embryo development and is seedling lethal (Liu et al., 2004 ). Further examination revealed that some F2 plants displayed a novel floral phenotype characterized by the near absence of petals. From 400 F2 plants, we observed 22 (close to 1/15) plants with the ask1-1-like flower and 46 (close to 2/15) plants with this novel phenotype. All other plants were similar to the wild type. PCR analysis demonstrated that all F2 plants with the ask1-like flower were ask1 single mutants (homozygous for the wild-type allele at the ASK2 locus), whereas all F2 plants with the novel petalless phenotype were of the ask1/ask1 ASK2/ask2 genotype. The ASK1/ask1 ask2/ask2 plants produced normal flowers. In addition, we performed PCR analysis on 192 F2 plants with no apparent phenotype, and none of them were ask1/ask1 ASK2/ask2. The RNA expression levels of the ASK2 gene were further tested using real-time PCR. Compared to the wild type, ASK2 gene expression is close to normal in the ask1 mutant, whereas its expression is reduced to about 48% in the ask1/ask1 ASK2/ask2 plant (Fig. 2C). Residual ASK2 expression was also detected in the ask2-1 mutant using primers C-terminal relative to the T-DNA insertion (data not shown).
As mentioned above, the most dramatic phenotype in the ask1/ask1 ASK2/ask2 plants is the absence of second whorl organs in most flowers (Fig. 1, J–N; Table I), with an average number of 0.14 petals per flower. In addition, carpelloid stamens and filaments were observed in the third whorl of the ask1/ask1 ASK2/ask2 flowers (Fig. 1, J and K; Table I). Other defects that were also observed in whorls 2 and 3 include a variable number of stamens, both fewer and more than six, and chimeras of petal/stamen and sepal/carpel (Fig. 1K). Sometimes organs in the same whorl, or between different whorls, were fused together (Fig. 1L). The first and fourth whorls of the ask1/ask1 ASK2/ask2 flower were mostly normal, with a slightly reduced number and variable size of sepals, and a slightly increased number of carpels (Table I; and data not shown). Occasionally, carpelloid sepals (Fig. 1M; Table I), fused sepals (Fig. 1N), curled carpels or unfused carpels (Fig. 1, K and N) were also observed; these defects were more severe in late flowers (data not shown). The carpelloid sepals and absence of organs in whorl 2 in the ask1/ask1 ASK2/ask2 flower are similar to the phenotype of ap2 mutants, in which the C function gene AGAMOUS (AG) expands to the first and second whorls (Kunst et al., 1989
We have also generated ASK1 RNAi transgenic plants (Zhao et al., 2003b To further verify that the enhanced and novel phenotypes in ask1/ask1 ASK2/ask2 flowers were caused by the combination of ask1 and ask2 mutations, we carried out a functional complementation of the ask1/ask1 ASK2/ask2 mutant with either a genomic ASK1 or a fusion of the ASK2 cDNA with the 35S promoter of the cauliflower mosaic virus (CaMV). The flowers of ask1/ask1 ASK2/ask2 mutant with a transgenic genomic ASK1 were restored to normal (Fig. 1P; and data not shown). In addition, the flowers of ask1/ask1 ASK2/ask2 mutant with the 35S-ASK2 construct exhibited phenotypes similar to those of the ask1 single mutant, including less reduction in petal number compared to the ask1/ask1 ASK2/ask2 flowers and correct organ identities for sepal, stamen, and carpel (Fig. 1Q). However, filaments were still occasionally observed in these plants, and the petal number was less than that in the ask1 mutant. This may be due to a possible difference in the expression pattern of the 35S promoter from that of the endogenous ASK2 regulatory elements. In conclusion, our results demonstrated that reduced expression of ASK1 and ASK2 is responsible for the ap2-like phenotype in the outer two whorls, the enhanced reduction of B function in the third whorl, and the defects in carpel identity in the fourth whorl of the ask1/ask1 ASK2/ask2 flower. We have also made a cross between axr6-2 and ask1-1. Similar to the ask1/ask1 ASK2/ask2 flower, carpelloid stamens (data not shown), carpelloid first whorl sepals, and unfused fourth whorl carpels (Fig. 1R) were also observed in the axr6-2 ask1 flower.
Previous studies in our laboratory have shown that UFO interacts with ASK1 genetically to positively regulate B function gene expression (Zhao et al., 2001b The strong ufo-2 mutant flower exhibited obvious defects in organ identity in the middle whorls, with sepal-like petals and filament-like structures occupying most of the organ positions in the second and third whorl, respectively (Fig. 1S). The first whorl sepals are largely normal. Occasionally two sepals are fused together. Enlarged or increased number of carpels was observed in the fourth whorls. In the weak ufo-6 mutant, sepals and carpels are generally normal, whereas the number and identity of petals and stamens are somewhat altered (Fig. 1T; Table I). Filaments and occasionally sepal-like petals were observed (Table I), suggesting a slightly reduced B function. In addition, petal/stamen chimeric organs were frequently observed. The inflorescence of the ufo-6 mutant is normal. In ufo-6 axr6-2 double mutant flowers, fused sepals were more frequently observed compared to the axr6-2 single mutant. Occasionally carpel-like sepals were observed in the first whorl (Fig. 1U; Table I). Compared to the axr6-2 and two ufo single mutants, the number of second whorl organs in the ufo-6 axr6-2 double mutant was greatly reduced, similar to those found in ask1/ask1 ASK2/ask2 flowers (0.09 per flower; Table I). Most flowers in the double mutant had no petals (Fig. 1, U and V). Occasionally a petal, or sepal-like petal, was observed in the second whorl (Table I). In the third whorl, the number of stamens was further reduced compared to the ufo-6 and axr6-2 single mutants, whereas carpel-like structures and filaments were increased in the double mutant (Fig. 1V; Table I). In the fourth whorl, the phenotypes of double mutant carpels were similar to those of axr6-2 single mutant. The increased carpelloid organs and filaments in the third whorl of ufo-6 axr6-2 double mutant flower support the hypothesis that these two genes act together to promote B function; there also seems to be a unique aspect of the phenotypes that may not result from B function, suggesting additional roles for AtCUL1, particularly in organ formation and carpel development. Moreover, we observed inflorescence defects in the ufo-6 axr6-2 double mutant, including a filament-like structure instead of a normal flower (Fig. 1W), and termination of an inflorescence by a pistil-like structure (Fig. 1X), after the production of only four to five flowers. These defects were not observed in ufo-6 or axr6-2 single mutant.
Our phenotypic analysis of Dex-treated AtRbx1 RNAi and ask1/ask1 ASK2/ask2 plants suggests that B function gene expression may be reduced in these plants. To evaluate this possibility, we performed RNA in situ hybridization with an AP3 probe. In the inflorescence of Dex-treated pTA2 control plants, AP3 RNA was detected at a high level in petal and stamen primordia of a young floral bud (Fig. 3A
). In the young buds of Dex-treated AtRbx1 RNAi plants, the expression of AP3 was detected at a lower level (Fig. 3B). In addition, compared to the wild type (Fig. 3C), the ask1-1 flower showed slightly reduced AP3 signals (Fig. 3D; Zhao et al., 2001b
To further confirm a reduction of AP3 RNA expression in the ask1 mutant and an enhanced reduction of AP3 expression in the ask1ask1/ ASK2/ask2 plant, we performed real-time PCR using the RNA isolated from young floral buds up to stage 8 (Smyth et al., 1990 ) from the wild-type, ask1, and ask1/ask1 ASK2/ask2 plants. Compared to the wild type, AP3 expression was reduced to approximately 45% in the ask1 mutant, and further reduced to about 33% in the ask1/ask1 ASK2/ask2 plant (Fig. 4
).
In addition, the ap2-1 like phenotype of ask1/ask1 ASK2/ask2 flowers suggests an expansion of C function to the first and second whorl. To test whether C function is altered at the transcription level in the ask1/ask1 ASK2/ask2 flower, we performed RNA in situ hybridization in the wild-type, ask1, and ask1/ask1 ASK2/ask2 flowers with an AG probe. AG RNA was detected in the central dome of wild-type flowers starting at early stage 3, and thereafter was restricted to stamen and carpel primordia (Fig. 3F). Nonspecific signal was detected in the upper part of sepals in late stage flowers (starting from stage 7), as observed previously (Drews et al., 1991 ; data not shown). The ask1 flower showed a normal spatial and temporal pattern of AG expression, and the expression level was also close to normal (Fig. 3G). The ask1/ask1 ASK2/ask2 flower exhibits a slightly reduced AG expression level in the center (Fig. 3H), whereas the expression pattern was close to normal. No significant AG signal was detected in early sepal primordia (Fig. 3H). Compared to the wild-type flower, slightly earlier and stronger signals seemed to be detected at the upper part of sepals in the mutant flower (data not shown). However it is not certain whether these signals reflect altered AG expression pattern or are only nonspecific signals.
To further test if AG is epistatic to ASK1 and ASK2 in terms of the ap2-like phenotypes in the ask1/ask1 ASK2/ask2 flower, we crossed the ask1/ask1 ASK2/ask2 into ag-1, a strong ag allele in which stamens are converted to petals, and whorl 4 is replaced by another flower (Bowman et al., 1989
Ubiquitin-mediated protein degradation has been recognized as a very important mechanism for regulating many cellular events. In particular, the SCF ubiquitin-protein ligases are known to control cell cycle regulation, signal transduction, transcription, and other biological events (Bai et al., 1996 ; Hershko and Ciechanover, 1998; Schulman et al., 2000; DeSalle and Pagano, 2001; Conaway et al., 2002; Zheng et al., 2002). In Arabidopsis, several SCF complexes that are involved in hormone signaling or cell division have been characterized (Gray et al., 1999; del Pozo et al., 2002a; Xu et al., 2002). The existence of a large number of F-box genes in the Arabidopsis genome suggests that plants make extensive use of SCF complexes to regulate multiple biological processes (Gagne et al., 2002; Risseeuw et al., 2003).
Expression analysis indicates that many ASK genes are expressed in the inflorescence, suggesting that SCF complexes may play multiple roles in flower development (Zhao et al., 2003b
Previous studies have suggested that AtCUL1 and AtRbx1a encode core components of many SCF complexes in Arabidopsis and that they play critical roles throughout development (Gray et al., 2002 Although several similar floral phenotypes were observed in all of the ask1/ask1 ASK2/ask2, axr6-2, and AtRbx1 RNAi plants, distinctive floral phenotypes were also observed in plants of each of these genotypes. One of the explanations for the distinctive phenotypes is the partial functional redundancy within different members of ASK genes and cullin homologs. It is also possible that the AtCUL1 point mutation might affect its interaction with some F-box proteins more so than that with others. In addition, although all of the ASK1, ASK2, AtCUL1, and AtRbx1 genes encode core components of SCF complexes, we cannot rule out the possibility that these proteins can also function as a subunit of non-SCF complexes so that each gene may have its distinctive function.
The floral phenotypes of axr6-2, axr6-2 ask1, and Dex-induced AtRbx1 RNAi plants suggest a reduction of B function in these flowers. In situ results further suggested a reduction of AP3 gene expression in the Dex-induced strong AtRbx1 RNAi flower. Furthermore, the ufo-6 axr6-2 double mutant showed an increased number of carpelloid organs and filaments in the third whorl, suggesting a further reduction in B function compared to either single mutant. Previous studies in our laboratory indicated that UFO interacts with ASK1 and LEAFY genetically to regulate B function gene expression (Zhao et al., 1999
Consistent with previous results, we observed a slight reduction of B function in the ask1-1 mutant flower (Zhao et al., 1999
The carpelloid sepals in the first whorl and near absence of petal in both the ufo-6 axr6-2 and ask1/ask1 ASK2/ask2 flowers were similar to those observed in ap2 mutants, in which expression of the C function gene AG expands to the first and second whorls (Bowman et al., 1991
Unlike the ufo-14 mutant, flowers in the ufo-6 axr6-2 double mutant exhibited carpelloid sepals in the first whorl. Carpelloid sepals were also observed in strong ufo alleles in ecotype Landsberg erecta of Arabidopsis (Ler) background (Wilkinson and Haughn, 1995
Our in situ results suggest that AG RNA did not expand to the sepal primordia in the ask1/ask1 ASK2/ask2 flower. Similarly, AG RNA was not detected in the second whorl of the newly isolated petalless ufo alleles (Durfee et al., 2003
SCFUFO and other SCF complexes can repress AG protein function in the first and second whorls either through a direct repression of AG function, or through an indirect repression by activating a repressor of AG function. Because ectopic expression of UFO throughout the flower does not lead to an ag phenotype (Lee et al., 1997
The curled or unfused carpel in the fourth whorl of the ask1/ask1 ASK2/ask2 flowers suggests a reduced C function, as supported by the in situ hybridization results. Unfused carpels were also found in the ufo-1 mutant when grown under short day (SD) condition (Wilkinson and Haughn, 1995
The inflorescence of the ufo-6 axr6-2 double mutant exhibited a filament-like structure instead of a normal flower and terminal inflorescence meristem, similar to those found in strong ufo mutants (Levin and Meyerowitz, 1995
An axillary flower similar to those found in ap1 mutants (Bowman et al., 1993
The axr6-2 flower exhibited a reduction of floral organ number in all four whorls, indicating that SCF complexes also regulate floral organ number, probably through regulating cell division in the floral meristem. In addition, fused sepals, petals, and stamens were observed in ask1/ask1 ASK2/ask2 and strong ASK1 RNAi plants, which indicate that SCF complexes are also required for organ separation, probably through the regulation of cell division in each individual whorl. Furthermore, increased number or size of carpel was found in the ask1/ask1 ASK2/ask2 flower. Similar phenotypes were also observed in strong ufo mutants. UFO seemed to be required for the restriction of cell division in the center of a stage 2 flower (Samach et al., 1999 In conclusion, our results indicate that SCF complexes regulate several aspects of floral development in Arabidopsis. Further functional studies on additional F-box proteins in flower development, as well as target proteins regulated by these SCF complexes, will provide insights into the network of flower development regulation by SCF complexes in Arabidopsis.
Plant Materials and Growth Conditions
The axr6-2 mutant is in the Columbia background (Hobbie et al., 2000 The axr6-2 mutant was backcrossed to the wild type eight times before phenotypic analysis. Among the rootless homozygous axr6-2 seedlings cultured on Murashige and Skoog medium for 10 d, the few seedlings that developed roots were transferred to soil for further characterization. For floral phenotypic analysis of AtRbx1 RNAi plants, a strong line (dsRNA-2), a weak line (dsRNA-64), and a control line transformed with an empty vector were treated with Dex for 5 to 7 d at 2 to 3 weeks of age, then transferred to Dex-free conditions for further culture and analysis. Dex was dissolved in ethanol and kept at a concentration of 30 mM, and was directly added at a concentration of 1 µM to the medium or dripped onto plants in a solution at 10 µM (with 0.01% Tween 20). For RNA in situ hybridization, 3-week-old dsRNAi-2 plants and control plants were treated with Dex for 5 d, then fixed in formaldehyde-acetic acid fixative. Plants were cultured on Murashige and Skoog medium, or grown on Metro-Mix 360 (Scotts-Sierra Horticultural Products, Marysville, OH) at 22°C (16 h light, 8 h dark).
The single mutants used for phenotypic analysis and comparison were from self-pollination of either homozygous (ufo-2, ufo-6, ask2-1) or heterozygous (axr6-2, ask1-1, ag-1) plants. To construct a ufo-6 axr6-2 double mutant, a heterozygous axr6-2 plant was used as the male parent in a cross with a homozygous ufo-6 plant. The genotype of the double mutant was determined by sequencing PCR products. For determining the homozygous ufo-6, genomic DNA was amplified with primers oMC 834 (5'-CTTTGCCACGGCTTTGTAGCTTG-3') and oMC 835 (5'-GACCCACAGCCAGCTTTTTCTCA-3'). For determining the homozygous axr6-2, DNA was amplified with primers oMC 836 (5'-TGTGGTTAGGTTTTGCCTGCGTT-3') and oMC 837 (5'-AGCAGGGCCCTATCAATCTGCTC-3').
To construct an ask1-1 ask2-1 double mutant, pollens from a homozygous ask2-1 plant were used to pollinate homozygous ask1-1 pistils. The genotypes of F2 plants were determined by PCR. The ask1-1 mutant harbors a Ds transposon in ASK1 at the position 237 bp downstream of the ATG start codon (Yang et al., 1999
For the complementation of ask1/ask1 ASK2/ask2 floral phenotypes with ASK1, we crossed the ask2-1 mutant with an ask1-1 mutant harboring an ASK1 transgene (tASK1), which contains a 5,578 bp HindIII/EcoRV genomic fragment including the ASK1 gene and 4,183 bp upstream of the ATG (Zhao et al., 2003a
For the complementation of ask1/ask1 ASK2/ask2 with ASK2, we performed a cross between the ask2-1 mutant and an ask1-1 mutant harboring an ASK2 transgene (tASK2), which contains an ASK2 cDNA fused with the 35S promoter (Zhao et al., 2003a To construct an ask1-1 axr6-2 double mutant, pollens from a heterozygous axr6-2 plant were used to pollinate homozygous ask1-1 pistils. To construct an ask1/ask1 ASK2/ask2 ag-1 mutant, pollens from a heterozygous ag-1 plant were used to pollinate ASK1/ask1 ask2/ask2 pistils. The genotypes of F2 plants were determined by PCR as described before.
Inflorescences from wild-type and mutant plants were harvested from 3- to 4-week-old plants and immediately fixed in a formaldehyde-acetic acid fixative. RNA in situ hybridizations with radioactive probes were performed as previously described (Drews et al., 1991
RNA was isolated from the young inflorescence including stage 0 to 8 young floral buds (Smyth et al., 1990 The cDNA was amplified using the SYBR Green PCR Master Mix (Stratagene, La Jolla, CA) on the ABI PRISM 7700 thermocycler (ABI). The PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 1 min. The cycle threshold values were used to calculate differences in fold changes. At the end of PCR cycles, the data were analyzed with the ABI Sequence Detection Systems (SDS) version 1.7 (ABI). To check the specificity of annealing of the PCR products, a dissociation kinetics was performed by the machine at the end of the experiment. In addition, PCR products were verified by sequencing directly. Negative control using the same amount of RNA did not produce any PCR product. In one experiment, at least three replications were performed for each sample. The experiments were repeated at least twice independently.
We thank P. Genschik for kindly providing the AtRbx1 RNAi lines, E. Risseeuw and W.L. Crosby for providing the tASK1 and 35S:ASK2 constructs, and N. Wei for the 35S:UFO-myc seeds. In addition, we thank Y. Hu for technical assistance, and A. Omeis and J. Wang for plant care. We are grateful for helpful comments from C.L. Hendrix, W. Hu, and L.M. Zahn. We thank two anonymous reviewers for their helpful comments. Received August 19, 2003; returned for revision November 3, 2003; accepted January 12, 2004.
1 This work was supported by the National Science Foundation (grant nos. MCB–9896340 and MCB–0092075 to H.M. and IBN–998926 to L.H.), and by funds from the Department of Biology and the Huck Institutes of Life Sciences at the Pennsylvania State University.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.031971. * Corresponding author; e-mail hxm16{at}psu.edu; fax 814–863–1357.
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