| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
American Society of Plant Biologists The Identification of CVP1 Reveals a Role for Sterols in Vascular Patterning
a Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06511 1 To whom correspondence should be addressed. E-mail timothy.nelson{at}yale.edu; fax 203-432-5632
Vascular cell axialization refers to the uniform alignment of vascular strands. In the Arabidopsis cotyledon vascular pattern1 (cvp1) mutant, vascular cells are not arranged in parallel files and are misshapen, suggesting that CVP1 has a role in promoting vascular cell polarity and alignment. Characterization of an allelic series of cvp1 mutations revealed additional functions of CVP1 in organ expansion and elongation. We identified CVP1 and found that it encodes STEROL METHYLTRANSFERASE2 (SMT2), an enzyme in the sterol biosynthetic pathway. SMT2 and the functionally redundant SMT3 act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarization and axialization.
The plant vascular system is formed progressively during embryogenesis and postembryonic development. In organ primordia, provascular cells appear among ground cells in hierarchical patterns that give rise to the patterns of veins (Nelson and Dengler, 1997  ). Provascular cells proliferate and elongate in axialized files that appear to depend on the intercellular polar transport of auxin through the developing tissue. Screens for pattern-defective mutants have identified several genes that regulate or influence this process. The monopteros mutation causes a discontinuous venation pattern associated with improper vascular differentiation (Przemeck et al., 1996) and corresponds to an auxin response transcription factor (Hardtke and Berleth, 1998).
In gnom/emb30 mutants, which correspond to a brefeldin A–sensitive ADP-ribosylation factor guanine-nucleotide exchange factor (ARF-GEF), veins are unaxialized and patterned irregularly (Mayer et al., 1993
We recently described a number of additional mutants with various effects on cotyledon vascular pattern (Carland et al., 1999
Derivatives of the sterol pathway in plants and animals include both membrane sterols and signaling steroids. Recently, the membrane sterol cholesterol was shown to influence signaling and vectorial processes through its key role in the organization of lipid rafts (Simons and Ikonen, 1997
In contrast to animals and fungi, in which a single major sterol is accumulated, plants accumulate many sterols (Hartmann, 1998
The phenotypes of sterol biosynthetic mutants suggest that there are diverse sterol-dependent processes. The single SMT1 in yeast is defined by erg6 mutants, which were recovered in mutant screens for impaired Trp uptake, increased resistance to polyene antibiotics, hypersensitivity to various metals and ion salts, sensitivity to brefeldin A, suppression of vesicle trafficking, decreased mating efficiency, and modulation of steroid receptor signaling (Parks et al., 1999
Antisense SMT2 plants, which have lower sterol levels and higher campesterol levels, exhibit dwarfism accompanied by reduced apical dominance, floral organ elongation, and fertility (Schaeffer et al., 2001 We cloned the gene that corresponds to the cvp1 mutant described above and found that it encodes the Arabidopsis C-24 SMT2. As predicted from the role of SMT2 in the sterol biosynthetic pathway, sterol levels were altered significantly. Based on the phenotypes of an allelic series of cvp1 mutants, we propose that cvp1 mutants are defective in sterol signaling or an aspect of membrane organization that is essential for the polarization and axialization of vascular cells.
Additional Alleles of cvp1 Exhibit Defects Consistent with Aberrant Cell Expansion and Cell Elongation As reported previously, four alleles of cvp1 (cvp1-1, cvp1-2, cvp1-3, and cvp1-4) were identified from a screen of >34,000 ethyl methanesulfonate–mutagenized M2 seedlings derived from 10 M1 pools (Carland et al., 1999 ). cvp1-1 and cvp1-2 showed identical phenotypes and were found to have the same mutation (described below). Therefore, we compared in detail only the phenotypes of cvp1-1, cvp1-4, and cvp1-3, which correspond to weak, intermediate, and strong alleles, respectively. All cvp1 alleles exhibited a discontinuous cotyledon venation pattern that consisted of isolated patches (Figures 1A to 1D) of vascular tissue that lacked cellular axialization. There was a reduction of vein loops in cotyledons of cvp1-4 and cvp1-3, and the veins were thicker. The reduced cotyledon expansion of cvp1-4 and cvp1-3 in the proximal-distal axis resulted in a "soup spoon"–shaped cotyledon. cvp1-3 cotyledons were smaller than those of the wild type. No vascular aberrations were apparent in the rosette leaves of the cvp1 alleles (Figures 1E to 1H). Flowers in all cvp1 alleles were normal in gross morphology. However, petals appeared scalloped, with highly serrated margins (Figures 1I and 1J), and sepals were highly serrated, with ectopic protrusions extending from the apical margins (Figures 1K and 1L). These affected foliar organs often exhibited vascular alterations such as vein regions with nonuniform thickening.
cvp1-1 and cvp1-4 were similar to wild-type plants in general morphology and growth rate (Figure 1N), whereas cvp1-3 plants had retarded growth and were smaller at both the seedling and rosette stages (Figures 1M and 1N). The compact cvp1-3 rosette was composed of small vegetative leaves that failed to expand to wild-type size. This strong cvp1-3 allele also exhibited delayed flowering time and senescence and reduced plant height and apical dominance.
cvp1 siliques were shorter (wild type, 13.1 ± 1.1 mm; cvp1, 10.1 ± 0.5 mm [n = 61 siliques from 12 plants]) and wider than wild-type siliques (Figure 1O). However, a reduction in fertility occurred only in the strong cvp1-3 allele. As described previously, cvp1-1 had a decrease in internodal stem elongation in the apex of primary and secondary inflorescences in
CVP1 Encodes SMT2
Individual TAC clones were introduced into cvp1-1 mutants and scored for complementation of the cvp1 mutant phenotype. A complementing TAC contained six unique genes relative to overlapping noncomplementing TACs, based on gene annotation that had become available by that date. These six genes were sequenced in the cvp1-1 background, and a mutation was identified in a previously identified gene encoding SMT2 (Bouvier-Nave et al., 1997 ). The additional cvp1 alleles contained mutations in this gene. cvp1 was complemented by transformation with the SMT2 genomic region, confirming the identity of CVP1 (Figure 2B). Although cvp1-1 and cvp1-2 were identified from different M1 pools and therefore should be independent alleles, they contain the same mutation in an invariant residue within the first S-adenosyl Met binding site. cvp1-4 was found to have a nonsense mutation that is predicted to produce a truncated protein. The cvp1-3 allele contained a missense mutation in the initiation codon (Figure 2C) that should result in a null allele, consistent with its more severe phenotype. The next in-frame Met residue follows the sterol binding site.
Features of the SMT2 Protein
SMT2 Is Expressed in Several Developing Organs We examined the effects of the cvp1 molecular lesions on SMT2 transcript levels by RNA gel blot analysis. RNA isolated from 6-day-old seedlings revealed decreases in SMT2 transcript levels in all alleles (Figure 4A) in proportion to their phenotypic severity, even though the mutations were predicted to alter translational products rather than transcription. There were undetectable levels of SMT2 transcripts in the severe cvp1-3 allele, confirming the designation of cvp1-3 as a null allele.
The cvp1 mutations in SMT2 also affected the accumulation of transcripts for SMT1 and SMT3. The transcript levels of the highly homologous SMT3 and of SMT1, determined with gene-specific probes, were increased in cvp1-4 but were unaffected in cvp1-1. Remarkably, neither transcript was detected in cvp1-3, suggesting that the null allele is deficient in all SMT activity. We reproduced these measurements several times, because the results were unexpected. Some effects on SMT2 and SMT3 transcript levels were reported previously for smt1 mutants, but these were not as great as the effects we observed in cvp1 alleles (Diener et al., 2000 ). The increased SMT1 and SMT3 transcript levels in cvp1-4 and the decreases in cvp1-3 may reflect a feedback mechanism between sterol production and activation of the SMT genes. Alleles that differ in severity may disrupt domains in SMT2 involved in distinct aspects of this regulation.
The SMT2 gene was expressed in most developing tissues in a pattern that differed slightly from that of SMT3 and SMT1. RNA gel blot analysis showed that SMT2 mRNA was present in all developing tissues surveyed and revealed low-level expression in roots and siliques, moderate levels in seedling and rosette leaves, and abundant levels in stems and inflorescences (Figure 4A). SMT3 showed a similar expression profile, except that mRNA was at undetectable levels in siliques (Figure 4B). Likewise, the pattern of SMT1 mRNA (data not shown) resembled that of SMT2, in agreement with a previous report (Diener et al., 2000 The expression of SMT2 was influenced by several hormones. We measured SMT2 and SMT3 mRNA after treatment with ethylene, cytokinin, indoleacetic acid, epibrassinolide, or gibberellic acid, as described in Methods. Hormone induction studies indicated that SMT2 was induced strongly and moderately by ethylene and cytokinin, respectively. SMT3 was induced strongly by cytokinin. SMT2 and SMT3 were induced weakly and moderately, respectively, by indoleacetic acid. The BR epibrassinolide and gibberellic acid did not appear to have any effect on SMT transcript levels. This finding suggests that SMT2 and SMT3 are induced differentially by at least three hormones that promote cell division and/or elongation.
SMT2 appeared to be expressed predominantly in regions with active cell division. We visualized SMT2 mRNA accumulation by in situ RNA hybridization (Figure 5). As reported previously (Diener et al., 2000
 -Glucuronidase (GUS) reporter analysis revealed some additional features of the SMT2 and SMT3 expression patterns. Both were expressed specifically in the elongation zone of the roots (Figures 6A and 6B). In rosettes, GUS histochemical staining was apparent in cotyledons, expanding leaves, and the apical tips of young leaves (Figures 6C and 6D). These are regions in the leaf that are undergoing cell expansion and reflect the basipetal maturation of leaves. In SMT2::GUS and SMT3::GUS flowers, staining was localized to the apical region of the sepal, particularly in the vascular bundles (Figures 6E and 6F). In SMT2::GUS siliques, GUS staining also was detected in ovules (Figure 6E). These data, in conjunction with the RNA gel blot analysis of organs and hormone application, indicate that SMT2 and SMT3 have similar but not identical expression patterns.
SMT2 and SMT3 Are Functionally Redundant SMT2 and SMT3 are highly homologous, act on the same substrate, and exhibit similar but distinguishable expression patterns (Figures 4 and 6), suggesting that the proteins are functionally redundant. Because SMT3 expression is restricted to the outer cell layers of the developing embryo, and thus is excluded from the majority of procambial cells of developing cotyledons (Diener et al., 2000 ), it appears that the function is provided solely by SMT2 in these locations. This probably explains the aberrant venation of cvp1 mutants and provided an experimental system to test for functional redundancy in vivo. We expressed either SMT3 or SMT2 in the cvp1-1 background, under the control of the 35S promoter of Cauliflower mosaic virus, and observed full restoration of the vascular patterning, petal and sepal margin, and silique elongation defects in cvp1-1 (Figures 7A to 7F and data not shown). However, the stem elongation defect was not rescued by SMT3 (Figure 7G). This finding appears to reveal a role for SMT2 in the stem that is not shared by SMT3, indicating that the functional overlap is extensive but not complete.
SMT2 and SMT3 have overlapping functions but are not completely functionally redundant. The overexpression of SMT2 and SMT3 yielded high transcript levels in the cvp1-1 background but did not produce any phenotypic abnormalities (Figures 7H and 7I). In addition, SMT2 overexpression did not have a detectable effect on SMT3 transcript levels, and likewise, SMT3 overexpression did not have an effect on SMT2 transcript levels, indicating that the cvp1 complementation is attributable specifically to SMT3 ectopic expression. The overexpression of SMT3 in the sense and antisense orientations in the wild-type background in  60 transgenic plants also did not produce a phenotype (data not shown).
Based on the SMT3 complementation of cvp1 and the observation that SMT3 is not expressed independently from SMT2, we propose that the lack of a phenotype in SMT3 antisense plants is caused by the functional redundancy of SMT2 and SMT3. This redundancy also may provide an explanation for the wild-type gross morphology of cvp1-4. cvp1-4 has only 35% of SMT2 wild-type RNA levels and is predicted to have a truncated SMT2 protein. However, increased levels of SMT3 mRNA accumulate in this allele. We propose that the wild-type morphology of cvp1-4 is maintained by the increase in SMT3 activity, which compensates for the decrease in SMT2 activity. The overlap in SMT2 and SMT3 expression patterns does not include the majority of cotyledon procambial cells, so a vascular patterning defect remains (Diener et al., 2000
cvp1 Mutants Alter the Balance between Sterols and BRs
These precursors to the BRs were increased in cvp1 relative to the wild type. For example, the null allele cvp1-3 showed more than a 25-fold increase in 24-methylenecholesterol. Cholesterol lacks methyl additions and is formed by bypassing the methyl addition reactions. As predicted, cholesterol was higher in cvp1 mutant alleles. Although there were gross changes in individual sterol levels, the total sterol content of cvp1 was not markedly different from that of the wild type. Therefore, sterol levels were altered significantly in cvp1, most notably in the null allele cvp1-3, by shifting the balance of intermediates in favor of the BR pathway at the expense of the sterol pathway.
Because SMT2 regulates the biosynthetic flux that controls the amount of sterol and campesterol, the alteration in the flux often is expressed as a ratio of campesterol to sitosterol. In the wild type, this ratio was equal to 0.2, whereas in cvp1, the ratio was 1.9 or greater. In general, the decrease in sterol levels of the three alleles was proportional to the severity of their phenotypes. Although the cvp1-3 null allele exhibited a reduction in transcript levels in all SMTs, it was able to produce alkylated sterols, albeit at reduced levels. The presence of sterols was reported in a null smt1 allele (Diener et al., 2000
The BR-Specific Inhibitor Brassinazole Does Not Rescue the cvp1 Mutant Phenotype As shown in Figure 8A, Brz application did not rescue this aspect of the mutant phenotype. Subsequent to treatment, seeds were isolated and the cotyledon vascular patterning defect was assessed in seedlings. Brz application during embryogenesis did not rescue the cvp1 vascular patterning defect, nor did it affect the wild-type venation pattern (Figures 8B and 8C), suggesting that the phenotype is not the result of altered BR levels. The cvp1 alleles exhibited a significant reduction in sitosterol and stigmasterol levels. We were unsuccessful in altering cvp1 by supplementation with these compounds, although supplementation is made difficult by the limited solubility of the compounds.
The first recognizable event associated with the differentiation of provascular tissue is cell polarization and the formation of axialized procambial files. Our analysis of mutations that disrupt the SMT2 gene demonstrates that this process is dependent on a functional sterol pathway. Arabidopsis has two very similar proteins (SMT2 and SMT3) acting at the same step in sterol biosynthesis, but only SMT2 is expressed in cotyledon vascular cells. This was important for two reasons. First, it allowed the critical observation of aberrant venation in the cvp1 mutants, establishing a vascular role for the pathway. Second, it provided an experimental opportunity to introduce ectopic SMT3, demonstrating functional redundancy. SMT2 acts at the bifurcation of the sterol and BR pathways, at which the substrate 24-methylenelophenol is methylated to enter the sterol pathway or is not further methylated and enters the BR pathway. Thus, SMT2 regulates the ratio of campesterol, precursor of the BRs, to sitosterol, the major sterol in plants. In cvp1 mutants, we showed that this ratio, which normally favors the sterol pathway, was inverted to favor the BR pathway, possibly most dramatically in cotyledons. The phenotypic consequences could be the result of the enrichment of one or more BRs or of the depletion of one or more sterols, or a combination of both effects.
We favor the second of these possibilities. We believe that the potential increase of BR levels is unlikely to be the cause of the cvp1 phenotype, for several reasons. First, we were unable to phenocopy the cotyledon vascular defect by supplementation of wild-type plants with brassinolide. Second, we were unable to cure the cvp1 mutant phenotype by the application of Brz, an inhibitor that acts at a point in the BR pathway downstream from campesterol. Third, the BR pathway appears to be highly regulated, such that increases of early intermediates do not necessarily result in increased levels of the downstream active BR, brassinolide (Clouse and Sasse, 1998 Sterols as membrane-ordering components are likely to have roles in organizing the many asymmetric structures and processes that are essential for the development of axialized files of polarized cells, such as the provascular cells and procambial strands that are affected in cvp1 mutants. In addition, the sterol branch of the pathway may produce compounds distinct from BRs, with roles in intercellular signaling for the alignment of cell files and elongation axes or in conferring cell identity distinct from neighbor cells. We discuss each of these processes below in relation to the cvp1 phenotype.
Role of SMT2 in Signaling
Although we did not measure brassinolide, the active signal in Arabidopsis, directly, it is unlikely that the cvp1 defect is caused by excessive BR levels, for the reasons described above. Instead, another sterol-derived signal may be affected in cvp1 and fackel mutants. There is indirect evidence for such signaling sterols in the spatial organization of various organs. Sterol/lipid binding domains (START) have been identified in a subtype of homeodomain/Leu zipper–containing proteins, which includes Athb-8, Athb-9, Athb-14, and REVOLUTA/INTERFASCICULAR FIBERLESS (Sessa et al., 1998
SMT2 and the Synthesis of Cellulose Microfibrils
Because sitosterol is reduced greatly in cvp1, both sitosterol-
Lipid Rafts and the Compartmentalization of Proteins Rafts are assembled initially with endoplasmic reticulum–derived sterols in the Golgi, the site of lipid synthesis, and then are transported to the plasma membrane carrying proteins destined for polarized delivery. After protein delivery, rafts are endocytosed from the cell surface and then recycled either directly back to the plasma membrane or indirectly by endosomes. In plants, there are low sterol levels in the endoplasmic reticulum, where sterols are synthesized as corroborated by the presence of signal anchors on SMT2 and SMT3, and high levels in the plasma membrane, providing support for this mode of transport.
Although lipid rafts have not been observed in plants, their small size may have precluded observation by conventional microscopy. In other systems, raft components have been resolved through advanced microscopy techniques coupled with biochemical methods. Recently, a GPI-anchored protein was found to be the product of the Arabidopsis COBRA gene, whose mutant phenotype includes a loss of polarized, longitudinal expansion (Schindelman et al., 2001
Other candidates for lipid raft localization in plants are the PIN family of auxin efflux carriers (Galweiler et al., 1998
It is tempting to speculate that PIN asymmetric delivery acts in association with lipid rafts. Alternatively, PIN may reside in plasma membrane microdomains rich in sterols and lipids. Although pin mutants have not been reported to exhibit any cotyledon vascular patterning discontinuities, emb30/gnom mutants exhibit a loss of vascular cell axialization similar to that observed in cvp1 mutants, albeit much more severely (Mayer et al., 1993 Based on the known role of sterols in mammalian and yeast cells, it is likely that sterols act in more than one process. We propose that the cvp1 mutant phenotype is not caused by a disruption in membrane permeability but rather reflects either (1) the mistargeting of proteins involved in polarizing and aligning vascular cells or (2) the synthesis of novel sterol signaling molecules. These two possibilities are not mutually exclusive. Sterol signaling may involve a receptor that subsequently initiates a signaling cascade that resides in a plasma membrane microdomain. The further analysis of cvp1 and other sterol-defective mutants should reveal the roles of sterols in vascular pattern formation and other developmental processes.
Plant Growth Conditions All cvp1 alleles were recovered from an ethyl methanesulfonate–mutagenized population of Arabidopsis thaliana ecotype Columbia in the screen described previously (Carland et al., 1999 ) and backcrossed at least three times. Seeds were surface-sterilized, plated on Murashige and Skoog (1962) (MS) medium (Sigma) with (for transgenic plants) or without antibiotics, and grown under continuous light. Approximately 1-week-old seedlings were either collected for RNA gel blot analysis, -glucuronidase (GUS) staining, or sterol analysis or transplanted to soil and grown to maturity for additional manipulations. Wild-type and mutant plants were at the same developmental stage for comparative morphological studies. Plants in soil (Metromix; Scotts, Hope, AR) were grown at 22°C with a 16-h day and a light intensity of 200 µmol·m-2·s-1. The phenotype of cvp1-3 plants grown at a higher light intensity (290 µmol·m-2·s-1) was not as severe.
Histology and GUS Histochemical Staining
Plant organs were placed in 5-bromo-4-chloro-3-indolyl-
Positional Cloning of CVP1
CVP1 was localized further by searching for polymorphic markers. To obtain new polymorphic markers, as BAC sequences became available in this region, 0.8-kb regions of DNA were amplified by PCR from Landsberg erecta, sequenced, and compared with the Columbia sequence. We designed several novel markers with this method and used them to further define the position of CVP1 to an Although eight TAC clones were transformed into cvp1-1, seeds were obtained from only five TAC clones because of low transformation frequency. Restriction analysis and gene annotation indicated the presence of six unique genes in a complementing TAC relative to an overlapping noncomplementing TAC. Upon sequencing cvp1-1 alleles of these genes, a mutation was identified only in the SMT2 gene. The remaining cvp1 alleles were amplified by PCR, cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA), and sequenced to identify base-pair changes within SMT2.
Plasmid Construction and Plant Transformation
For overexpression of SMT2 or SMT3, because there are no introns, a genomic region representing the cDNA of SMT2 (MT1 [5'-GGTCTTCCTCACTCTTAACG-3'] and MT9) or SMT3 (SMT3-6 [5'-CAGAGTCGTGAACTTAACG-3'] and SMT3-7 [5'-CCAATAGAATTTCCCGGC-3']) was amplified by PCR and eventually cloned into pZP35 (Hajdukiewicz et al., 1994
Plant transformations were conducted (Bechtold and Pelletier, 1998
Hormone Induction Studies and RNA Gel Blot Analysis
For RNA gel blot analysis, RNA was isolated using the Trizol method (Gibco BRL). Unless noted otherwise, 10 µg of RNA was loaded onto formaldehyde gels, electrophoresed under denaturing conditions, and transferred to a nylon membrane (Zetaprobe; Bio-Rad). To ensure equal loading of RNA, filters were stained in 0.02% methylene blue and 0.3 M NaOAc, pH 5.5, according to a published method (Herrin, 1988
RNA in Situ Hybridization
Measurement of Sterol Levels Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
Accession Number
We are grateful to Brian Keith (University of Pennsylvania) for providing the cvp1 alleles and to Asami Tadao (Institute of Physical and Chemical Research) for providing Brz. We thank Lien Lai (Ohio State University) for advice on positional cloning. We gratefully acknowledge Neil McHale (Connecticut Agricultural Experiment Station) for a critical review of the manuscript. We acknowledge the ABRC for providing the TAC filter and BAC and TAC clones used in the cloning of CVP1. This research was supported by Grants IBN-9808295 and IBN-0110730 from the National Science Foundation.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003939. Received April 18, 2002; accepted May 27, 2002.
Asami, T., and Yoshida, S. (1999). Brassinosteroid biosynthesis inhibitors. Trends Plant Sci. 4, 348–353.[CrossRef][ISI][Medline] Bechtold, N., and Pelletier, G. (1998). In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259–266.[Medline] Benveniste, P. (1986). Sterol biosynthesis. Annu. Rev. Plant Physiol. 37, 275–308. Bouvier-Nave, P., Husselstein, T., Desprez, T., and Benveniste, P. (1997). Identification of cDNAs encoding sterol methyl-transferases involved in the second methylation step of plant sterol biosynthesis. Eur. J. Biochem. 246, 518–529.[Medline] Busch, M., Mayer, U., and Jurgens, G. (1996). Molecular analysis of the Arabidopsis pattern formation of gene GNOM: Gene structure and intragenic complementation. Mol. Gen. Genet. 250, 681–691.[ISI][Medline]
Carland, F.M., Berg, B.L., FitzGerald, J.N., Jinamornphongs, S., Nelson, T., and Keith, B. (1999). Genetic regulation of vascular tissue patterning in Arabidopsis. Plant Cell 11, 2123–2137. Carland, F.M., and McHale, N.A. (1996). LOP1: A gene involved in auxin transport and vascular patterning in Arabidopsis. Development 122, 1811–1819.[Abstract]
Choe, S., Dilkes, B.P., Gregory, B.D., Ross, A.S., Yuan, H., Noguchi, T., Fujioka, S., Takatsuto, S., Tanaka, A., Yoshida, S., Tax, F.E., and Feldmann, K.A. (1999a). The Arabidopsis dwarf1 mutant is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis. Plant Physiol. 119, 897–907.
Choe, S., Noguchi, T., Fujioka, S., Takatsuto, S., Tissier, C.P., Gregory, B.D., Ross, A.S., Tanaka, A., Yoshida, S., Tax, F.E., and Feldmann, K.A. (1999b). The Arabidopsis dwf7/ste1 mutant is defective in the delta7 sterol C-5 desaturation step leading to brassinosteroid biosynthesis. Plant Cell 11, 207–221. Choe, S., Tanaka, A., Noguchi, T., Fujioka, S., Takatsuto, S., Ross, A.S., Tax, F.E., Yoshida, S., and Feldmann, K.A. (2000). Lesions in the sterol delta reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis. Plant J. 21, 431–443.[CrossRef][ISI][Medline] Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.[CrossRef][ISI][Medline] Clouse, S.D., and Sasse, J.M. (1998). Brassinosteroids: Essential regulators of plant growth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 427–451.[CrossRef][ISI] Cnops, G., Wang, X., Linstead, P., Van Montagu, M., Van Lijsebettens, M., and Dolan, L. (2000). Tornado1 and tornado2 are required for the specification of radial and circumferential pattern in the Arabidopsis root. Development 127, 3385–3394.[Abstract] Deyholos, M.K., Cordner, G., Beebe, D., and Sieburth, L.E. (2000). The SCARFACE gene is required for cotyledon and leaf vein patterning. Development 127, 3205–3213.[Abstract]
Diener, A.C., Li, H., Zhou, W., Whoriskey, W.J., Nes, W.D., and Fink, G.R. (2000). Sterol methyltransferase 1 controls the level of cholesterol in plants. Plant Cell 12, 853–870.
Galweiler, L., Guan, C., Muller, A., Wisman, E., Mendgen, K., Yephremov, A., and Palme, K. (1998). Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 282, 2226–2230. Geldner, N., Friml, J., Stierhof, Y.D., Jurgens, G., and Palme, K. (2001). Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413, 425–428.[CrossRef][Medline] Gil, P., Liu, Y., Orbovic, V., Verkamp, E., Poff, K.L., and Green, P.J. (1994). Characterization of the auxin-inducible SAUR-AC1 gene for use as a molecular genetic tool in Arabidopsis. Plant Physiol. 104, 777–784.[Abstract]
Grebe, M., Gadea, J., Steinmann, T., Kientz, M., Rahfeld, J.U., Salchert, K., Koncz, C., and Jurgens, G. (2000). A conserved domain of the Arabidopsis GNOM protein mediates subunit interaction and cyclophilin 5 binding. Plant Cell 12, 343–356. Hajdukiewicz, P., Svab, Z., and Maliga, P. (1994). The small, versatile pZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994.[CrossRef][ISI][Medline] Hardtke, C.S., and Berleth, T. (1998). The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO J. 17, 1405–1411.[CrossRef][ISI][Medline] Hartmann, M.-A. (1998). Plant sterols and the membrane environment. Trends Plant Sci. 3, 170–175.[CrossRef][ISI] Herrin, D.L. (1988). Rapid, reversible staining of northern blots prior to hybridization. Biotechniques 6, 196–198.[ISI][Medline] Husselstein, T., Gachotte, D., Desprez, T., Bard, M., and Benveniste, P. (1996). Transformation of Saccharomyces cerevisiae with a cDNA encoding a sterol C-methyltransferase from Arabidopsis thaliana results in the synthesis of 24-ethyl sterols. FEBS Lett. 381, 87–92.[Medline]
Jang, J.C., Fujioka, S., Tasaka, M., Seto, H., Takatsuto, S., Ishii, A., Aida, M., Yoshida, S., and Sheen, J. (2000). A critical role of sterols in embryonic patterning and meristem programming revealed by the fackel mutants of Arabidopsis thaliana. Genes Dev. 14, 1485–1497.
Klahre, U., Noguchi, T., Fujioka, S., Takatsuto, S., Yokota, T., Nomura, T., Yoshida, S., and Chua, N.H. (1998). The Arabidopsis DIMINUTO/DWARF1 gene encodes a protein involved in steroid synthesis. Plant Cell 10, 1677–1690. Koizumi, K., Sugiyama, M., and Fukuda, H. (2000). A series of novel mutants of Arabidopsis thaliana that are defective in the formation of continuous vascular network: Calling the auxin signal flow canalization hypothesis into question. Development 127, 3197–3204.[Abstract] Li, J., and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90, 929–938.[CrossRef][ISI][Medline]
Liu, Y.G., Shirano, Y., Fukaki, H., Yanai, Y., Tasaka, M., Tabata, S., and Shibata, D. (1999). Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning. Proc. Natl. Acad. Sci. USA 96, 6535–6540. Long, J.A., Moan, E.I., Medford, J.I., and Barton, M.K. (1996). A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379, 66–69.[CrossRef][Medline] Malamy, J.E., and Benfey, P.N. (1997). Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124, 33–44.[Abstract] Mayer, U., Büttner, G., and Jurgens, G. (1993). Apical-basal pattern formation in the Arabidopsis embryo: Studies on the role of the gnom gene. Development 117, 149–162.[Abstract] McConnell, J.R., Emery, J., Eshed, Y., Bao, N., Bowman, J., and Barton, M.K. (2001). Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411, 709–713.[CrossRef][Medline] Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497.[CrossRef] Nelson, T., and Dengler, N. (1997). Leaf vascular pattern formation. Plant Cell 9, 1121–1135.[CrossRef][ISI] |
TOP
Abstract
INTRODUCTION
