Plant Physiol. (1998) 116: 935-946

An Embryo-Defective Mutant of Arabidopsis Disrupted in the Final Step of Biotin Synthesis

David A. Patton, Amy L. Schetter, Linda H. Franzmann, Karin Nelson, Eric R. Ward, and David W. Meinke^*

Novartis Crop Protection, P.O. Box 12257, Research Triangle Park, North Carolina 27709 (D.A.P., K.N., E.R.W.); and Department of Botany, Oklahoma State University, Stillwater, Oklahoma 74078 (A.L.S., L.H.F., D.W.M.)

	ABSTRACT

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Auxotrophic mutants have played an important role in the genetic dissection of biosynthetic pathways in microorganisms. Equivalent mutants have been more difficult to identify in plants. The bio1 auxotroph of Arabidopsis thaliana was shown previously to be defective in the synthesis of the biotin precursor 7,8-diaminopelargonic acid. A second biotin auxotroph of A. thaliana has now been identified. Arrested embryos from this bio2 mutant are defective in the final step of biotin synthesis, the conversion of dethiobiotin to biotin. This enzymatic reaction, catalyzed by the bioB product (biotin synthase) in Escherichia coli, has been studied extensively in plants and bacteria because it involves the unusual addition of sulfur to form a thiophene ring. Three lines of evidence indicate that bio2 is defective in biotin synthase production: mutant embryos are rescued by biotin but not dethiobiotin, the mutant allele maps to the same chromosomal location as the cloned biotin synthase gene, and gel-blot hybridizations and polymerase chain reaction amplifications revealed that homozygous mutant plants contain a deletion spanning the entire BIO2-coding region. Here we describe how the isolation and characterization of this null allele have provided valuable insights into biotin synthesis, auxotrophy, and gene redundancy in plants.

	INTRODUCTION

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Biotin is an essential vitamin that facilitates CO₂ transfer during carboxylation, decarboxylation, and transcarboxylation reactions (Dakshinamurti and Bhagavan, 1985; Knowles, 1989). Bacteria, plants, and some fungi are capable of synthesizing biotin directly from chemical intermediates. Other organisms must obtain biotin from their surrounding environment. The biosynthetic pathway for biotin was first elucidated in Escherichia coli and Bacillus subtilis through biochemical studies involving the analysis of auxotrophic mutants (Eisenberg, 1973; Pai, 1975). Biotin operons from several prokaryotes have now been sequenced and analyzed in detail (Otsuka et al., 1988; Cronan, 1989; Gloeckler et al., 1990; Bower et al., 1996). Enzymes required for biotin synthesis in E. coli and Bacillus sphaericus have been purified and their activities characterized in vitro (Izumi et al., 1981; Ploux and Marquet, 1992; Ploux et al., 1992; Alexeev et al., 1994; Huang et al., 1995). Related genes required for biotin synthesis have also been identified in a variety of other microorganisms (Zhang et al., 1994; Fleischmann et al., 1995; Bult et al., 1996). The entire biosynthetic pathway has therefore been the subject of extensive investigation.

The common pathway for biotin synthesis in plants and microorganisms is summarized in Figure 1. Although most steps in the pathway have been clearly documented in the literature, questions remain concerning the immediate precursor of pimeloyl CoA in different microorganisms, the role of specific proteins, cofactors, and intermediates associated with the final step of the pathway, and the nature of the sulfur donor required for biotin synthesis (Baxter et al., 1992; Marquet et al., 1993; Ifuku et al., 1994; Birch et al., 1995; Bower et al., 1996; Sanyal et al., 1996). The conversion of dethiobiotin into biotin, which is catalyzed in part by the enzyme biotin synthase, is perhaps the most fascinating and complex part of the pathway because it involves the addition of sulfur to form a thiophene ring. This reaction may also represent a rate-limiting step for biotin synthesis (Baldet et al., 1993b).

View larger version (12K): [in this window] [in a new window]   Figure 1. Biotin biosynthetic pathway in plants and microorganisms. Letters correspond to cloned bio genes of E. coli. Numbers correspond to mutant bio genes of Arabidopsis. The immediate precursor of pimeloyl CoA appears to be pimelic acid in many but not all organisms.

The identity of the sulfur donor in biotin synthesis has long been questioned. Candidate molecules have included Cys, Met, and S-adenosylmethionine. Recent studies have provided contradictory evidence. Birch et al. (1995) reported that Cys may be the sulfur donor based on their ability to label biotin with [³⁵S]Cys in a cell-free extract of an E. coli strain designed to overexpress the cloned biotin synthase gene. Sanyal et al. (1996) could not detect a similar incorporation of label into biotin using a highly purified preparation of biotin synthase. The purified E. coli enzyme appears to be a dimer that contains two iron atoms and two acid-labile sulfur atoms per monomer (Sanyal et al., 1994). Several additional proteins and cofactors appear to be required for efficient biotin synthesis in cell-free extracts. These include flavodoxin, flavodoxin reductase, NADPH, and perhaps several compounds of low molecular weight present in crude extracts (Ifuku et al., 1994; Birch et al., 1995; Sanyal et al., 1996). A potential intermediate in this final step of the pathway (9-mercaptodethiobiotin) has also been identified, synthesized in the laboratory, and examined for its role in biotin synthesis (Baxter et al., 1992; Baldet et al., 1993b; Marquet et al., 1993).

Several different strategies have been used to study biotin synthesis and utilization in plants. One approach has been to analyze biotinylated proteins (Nikolau et al., 1985). Four biotin-dependent carboxylases have been examined to date (Harwood, 1988; Wurtele and Nikolau, 1990, 1992; Alban et al., 1993). Another biotinylated protein identified in pea seeds may play a role in sequestering biotin late in embryogenesis for subsequent use during germination (Duval et al., 1993, 1994). A holocarboxylase synthetase responsible for biotin attachment to plant proteins has also been identified (Tissot et al., 1996, 1997). Other studies have dealt with biotin content and the relative amounts of free and protein-bound biotin in plant cells (Baldet et al., 1993a). Biotin synthesis has been examined in part by noting the fate of radioactive precursors and identifying known chemical intermediates in plant tissues (Baldet et al., 1993b). These biochemical studies have for the most part confirmed the similarity of the prokaryotic and eukaryotic pathways for biotin synthesis, although questions remain concerning details of the initial and final steps.

The intracellular location of biotin synthesis also remains to be defined. Some studies suggest that biotin synthesis occurs primarily in the chloroplast, whereas other results are more consistent with a cytosolic or mitochondrial localization (Shellhammer, 1991; Baldet et al., 1993a; Patton et al., 1996a; Weaver et al., 1996). These studies have been complicated by the extremely small amounts of biotin produced by most plant cells. Molecular characterization of the biosynthetic pathway has dealt primarily with the biotin synthase gene. Three laboratories have recently cloned the cDNA corresponding to this gene from Arabidopsis (Baldet and Ruffet, 1996; Patton et al., 1996a; Weaver et al., 1996).

An alternative approach used to study biotin synthesis in plants has been the isolation and characterization of auxotrophic mutants. All biotin auxotrophs identified to date have been obtained from collections of embryo-defective mutants of Arabidopsis (Meinke, 1994). The bio1 mutant was the first plant auxotroph shown to result in embryo lethality (Schneider et al., 1989). Mutant bio1 embryos remain pale throughout development, typically arrest between the heart and cotyledon stages of embryogenesis, contain severely reduced levels of biotin, and can be rescued by biotin, dethiobiotin, or 7,8-diaminopelargonic acid (Shellhammer and Meinke, 1990). The initial model that this mutant was defective in the conversion of 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid (Shellhammer, 1991) has recently been confirmed by demonstrating that mutant plants can be rescued by introduction of a functional copy of the bioA gene of E. coli through Agrobacterium-mediated transformation (Patton et al., 1996b).

We describe in this report the isolation of a second biotin auxotroph of Arabidopsis disrupted at a different step in the biosynthetic pathway, the conversion of dethiobiotin to biotin. Once again, the vitamin requirement of this bio2 mutant was identified by comparing the growth of mutant embryos on minimal and enriched media. We demonstrate here that the bio2 mutation corresponds to a deletion of the entire genomic coding region for biotin synthase in Arabidopsis. This observation has allowed us to establish the cellular and developmental consequences of a complete loss of biotin synthesis in plants. The isolation of a second biotin auxotroph and the failure to identify other types of nutritional mutants among embryonic lethals also raises fundamental questions concerning the scarcity of plant auxotrophs. We conclude that although alternative methods may be devised to identify additional auxotrophs in the future, the number of mutants with defined nutritional requirements that can be identified in plants will continue to be limited by widespread redundancy of essential genes and biochemical pathways.

	MATERIALS AND METHODS

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Mutant Isolation and Plant Maintenance

Genetic Analysis of Mutant Plants

Screen for Auxotrophic Mutants

Vitamin plates were supplemented with 5 µm each of p-amino-benzoic acid, nicotinamide, calcium pantothenate, pyridoxine hydrochloride, biotin, and riboflavin-5-P, and 25 µm choline chloride. Enriched plates were supplemented with the vitamins noted above, 50 µm each of 20 l-amino acids, and 50 µm each of five nucleosides. Organic supplements were sterilized with a 0.2-µm syringe filter (Gelman, Ann Arbor, MI) and then added to autoclaved media. Chemicals used in media preparation were obtained from Sigma and Fisher Scientific. For initial screens, 20 to 40 seeds and embryos from each mutant line were tested on each type of medium. Growth responses were noted after 2 to 4 weeks in culture. The biotin requirement of bio2 embryos was confirmed using media supplemented with 1 to 10 µm biotin or dethiobiotin. Several rescued plantlets with roots were transferred to soil and watered daily with 1 mm biotin as described by Schneider et al. (1989). Mature seeds from rescued heterozygotes were germinated on media without phytohormones. Rescued seedlings from biotin plates were transplanted to soil approximately 2 weeks after germination.

Mapping the Cloned BIO2 Gene

PCR Analysis of the bio2 Allele

DNA and RNA Gel-Blot Analysis

Co-Segregation of the bio2 Deletion and Nonbolting Phenotype

	RESULTS

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The bio2 Auxotroph Is an Embryo-Defective Mutant

Embryo-defective mutants have traditionally been grouped into phenotypic classes based on the size and shape of mutant embryos at maturity (Muller, 1963; Meinke, 1994). The bio2 auxotroph was originally classified as a transition mutant. Arrested embryos remain white or pale yellow-green throughout development, often reach a globular or heart shape prior to desiccation, and consistently fail to germinate at maturity. Arrested bio2 embryos are typically smaller and blocked earlier in development than arrested bio1 embryos. Terminal phenotypes of these two mutants are compared in Figure 2. When aborted seeds from selfed bio1 and bio2 heterozygotes grown under identical conditions were compared, most of the bio2 embryos examined arrested before the torpedo stage of development, whereas every bio1 embryo examined reached a more advanced cotyledon stage. The bio2 mutation therefore appears to disrupt embryogenesis to a greater extent than the bio1 mutation.

View larger version (32K): [in this window] [in a new window]   Figure 2. Terminal phenotypes of bio1 and bio2 embryos obtained from heterozygous siliques. A, Examples of phenotypic classes observed. Embryos ranged from the globular (A) to cotyledon (E-G) stages. B, Distribution of phenotypic classes (A-G) in random samples of 125 arrested embryos from each mutant. Class X embryos were too small to be visible under a dissecting microscope. Note the different distribution of embryo phenotypes found in bio1 and bio2 mutant seeds.

Examination of cleared mutant seeds with Nomarski optics revealed a variety of early defects in cell division patterns and a characteristic elongation of the mutant embryo proper. Examples of these developmental abnormalities are shown in Figure 3. The earliest defect observed in bio2 seeds occurred when the embryo proper was composed of a single cell (Fig. 3A). Related but less-severe abnormalities were found in bio1 mutant seeds. These defects were not caused by another linked mutation because they disappeared in heterozygous plants watered with biotin. The unusual pattern of morphogenesis observed in some bio2 embryos (Fig. 3C) demonstrates that disrupting a general metabolic function such as biotin synthesis can have developmental consequences that may resemble defects in pattern formation. This supports the conclusion that interesting defects in cell division patterns are not necessarily the result of mutations in genes that play a direct role in the regulation of morphogenesis (Meinke, 1996).

View larger version (169K): [in this window] [in a new window]   Figure 3. Early defects in development of bio2 seeds revealed after treatment with Hoyer's solution and examination with Nomarski optics. A and C, Immature mutant seeds. Note unusual elongation of apical cell (A) and embryo proper (C) in mutant seeds. B and D, Wild-type seeds at the proembryo (B) and late-globular (D) stages. Scale bars = 20 µm.

Homozygous Mutant Embryos Are Rescued by Biotin

A more significant difference between bio1 and bio2 mutants was found in the response of arrested embryos to dethiobiotin, the immediate precursor of biotin in plants and microorganisms. These results are summarized in Table I and Figure 4A. Arrested bio2 embryos were not rescued by dethiobiotin, whereas bio1 embryos included as positive controls on the same plate were rescued. The failure of bio2 embryos to grow on dethiobiotin was therefore not caused by culture conditions but, rather, by the inability of mutant embryos to convert this intermediate into biotin. Even high concentrations of dethiobiotin (10 µm) were unable to promote sustained growth of bio2 embryos in culture. These results are consistent with the conclusion that bio2 is defective in the final step of biotin synthesis in plants, the conversion of dethiobiotin into biotin.

View larger version (95K): [in this window] [in a new window]   Figure 4. Response of immature mutant embryos (A) and mature rescued seeds (B) in culture. A, Arrested embryos were removed from immature siliques of heterozygous plants, plated on basal and enriched media, and observed after several weeks in culture. The top row of plates contained bio2 embryos on the left half of each plate and bio1 embryos on the right half. The left plate is a basal medium; the right plate contains 1 µm dethiobiotin. The bottom row of plates contains plants derived from bio2 embryos rescued on 1 µm biotin. Note that bio2 embryos were rescued only in the presence of biotin. B, Mature seeds from heterozygous (bio2/BIO2) plants grown in pots watered with biotin were plated on basal medium (right) and 1 µm biotin (left). Each plate received seeds from a single silique. Segregating mutant seedlings appeared pale in the absence of biotin (right) but normal in the presence of biotin (left).

Most bio2 plantlets rescued on biotin appeared normal but were difficult to transplant to soil because roots were not well established. An alternate method was therefore devised to generate large numbers of homozygous mutant plants for analysis. Pots containing a mixture of bio2/BIO2 and BIO2/BIO2 seedlings were watered daily with 1 mm biotin. This treatment rescued many of the mutant seeds produced by heterozygous (bio2/BIO2) plants later in development. Several plants in these pots were identified as heterozygotes based on the presence of a few pale seeds. Mature siliques from these rescued heterozygotes were then harvested and the resulting seeds were germinated on plates in the presence and absence of biotin. Examples of such plates are shown in Figure 4B. Approximately 25% of the seedlings grown on a basal medium became pale shortly after germination and failed to produce a viable rosette. In contrast, all of the seedlings grown in the presence of biotin were phenotypically normal. These results indicate that many homozygous mutant seeds produced by heterozygous plants watered with biotin were indeed rescued, survived desiccation, and germinated to produce normal seedlings.

The bio2 Mutant Contains a Deletion Spanning the Biotin Synthase Gene

View larger version (48K): [in this window] [in a new window]   Figure 5. Molecular characterization of the bio2 locus. A, Structure of the wild-type (BIO2) gene. Thin lines represent introns; thick bars correspond to exons. Horizontal arrows note the locations of primers used to confirm the presence of a deletion in mutant plants. The vertical arrow denotes a region within the first intron that contains a "CT" repeat detectable as an SSLP between Landsberg erecta and Columbia ecotypes. B, Analysis of PCR products generated using template DNA from wild-type (BIO2/BIO2) and mutant (bio2/bio2) plants. "BIO2" lanes contain PCR products generated using primers spanning the BIO2 locus. Locations of these primer pairs are designated with arrows in A. "Control" lanes contain products generated using primers specific for seven different single-copy genes in Arabidopsis. Lanes with identical numbers used the same primer pairs. Reference lanes contain a 1-kb ladder. Note that DNA isolated from mutant plants yielded PCR products in all cases tested except with BIO2 primers. C, Gel blot of genomic DNA isolated from wild-type (BIO2) and mutant (bio2) plants and digested with EcoRI. The same blot was probed sequentially with the full-length BIO2 cDNA (1); a random single-copy gene used as a positive control (2); and a  clone containing the BIO2 gene located within a 22-kb insert (3). There was no hybridization between the BIO2 cDNA probe and DNA isolated from mutant plants. Three bands marked with arrows (13 kb total) are missing from the blot of bio2 DNA probed with the  BIO2 genomic clone.

PCR primers covering the BIO2 locus were then used to compare DNA isolated from wild-type and rescued bio2 plants. As shown in Figure 5B, the expected BIO2 products were recovered from wild-type plants but not from mutant plants. PCR products generated using primers within other single-copy (control) genes were recovered equally from both mutant and wild-type plants. These results suggested that bio2 plants contained a deletion that spanned the entire coding region of the biotin synthase gene. Results of Southern blots of genomic DNA isolated from wild-type and bio2 mutant plants and probed with full-length BIO2 cDNA and genomic clones were consistent with this interpretation. A typical DNA gel blot is shown in Figure 5C. These results suggested that BIO2 is a single-copy gene and that mutant plants are missing at least 13 kb of DNA surrounding the BIO2 locus. In addition, RNA gel blots revealed the presence of a transcript complementary to the BIO2 cDNA probe in wild-type plants but not in rescued mutant plants (data not shown). Taken together, these results indicate that the bio2 mutation is associated with a deletion that spans the entire coding region of the biotin synthase gene and probably extends into adjacent regions of the genome. Although large deletions are not common following EMS mutagenesis, they have been reported to occur in other organisms when high doses of mutagen were involved (Ashburner, 1989).

Mapping the distribution of aborted seeds in siliques of selfed heterozygotes has been used previously to identify mutations that interfere with both embryogenesis and pollen-tube growth (Meinke, 1982, 1985). In cases where gametophytic expression of the target EMB gene is required for normal pollen-tube growth, aborted seeds are rarely found at the base of heterozygous siliques because mutant pollen tubes are at a competitive growth disadvantage. Results of segregation tests involving bio2 and bio1 heterozygotes are summarized in Table II. The deletion associated with the bio2 mutation does not appear to interfere with pollen development or pollen-tube growth because mutant seeds were distributed randomly along the length of heterozygous siliques and segregation ratios were not significantly different from those expected for a single recessive mutation. Pollen grains obtained from selfed bio2 heterozygotes also appeared normal. Thus, even the complete loss of biotin synthesis in mutant pollen grains does not appear to interfere with normal pollen-tube growth. Furthermore, the deletion appears to cover a small segment of the genome because heterozygotes do not exhibit the pollen abortion characteristic of large deletions and chromosome aberrations.

Mutant Plants Do Not Flower in the Presence of Biotin

All rescued bio2 homozygotes identified by PCR failed to bolt after extended growth under either long days or continuous light. Conversely, all plants that flowered contained at least one copy of the BIO2 gene. Mutant rosettes produced dark green leaves that were slightly reduced in size but otherwise appeared normal. The low ratio of mutant plants identified in these populations (12.7 instead of 25.0%) probably resulted from experimental selection against defective seeds and weak seedlings that corresponded to mutant seeds incompletely rescued by the biotin provided to parent plants. The failure of mutant plants to bolt was likely not caused by inadequate transport of biotin to the shoot apex because rescued bio1 homozygotes flower without difficulty in the presence of biotin. Rescued bio2 heterozygotes watered with biotin also produce nearly 100% phenotypically normal seeds. A more likely explanation is that a second gene covered by the deletion is required for bolting. Since none of the known late-flowering mutants has been mapped to this precise region of the genome, despite extensive screens in several different laboratories, the loss of more than one gene adjacent to the BIO2 locus may be required to produce the nonbolting phenotype.

	DISCUSSION

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Embryo-defective mutants of Arabidopsis represent a valuable source of genes with essential functions during plant growth and development (Meinke, 1995). More than 500 mutants disrupted in embryogenesis have been identified following chemical and insertional mutagenesis (Jur-gens et al., 1994; Meinke, 1994; Yadegari et al., 1994; Devic et al., 1996; Lindsey et al., 1996). Several mutants defective in known cellular functions have been examined in detail (Shevell et al., 1994; Springer et al., 1995; Traas et al., 1995; Lukowitz et al., 1996; Tsugeki et al., 1996). The molecular basis of abnormal development in many other mutants remains to be determined. The present study was designed to expand the search for auxotrophs among existing collections of emb mutants. The assumption as outlined by Langridge (1958) and confirmed by Schneider et al. (1989) was that some auxotrophs escape detection at the seedling stage because they arrest early in development. The discovery reported here of another emb mutant altered in biotin synthesis, when combined with previous work on two different bio1 mutant alleles, raises a fundamental question concerning the nature of plant auxotrophs in relation to plant embryogenesis. Although screens for auxotrophic emb mutants have not yet approached saturation, the prevalence of biotin auxotrophs and the absence of other auxotrophs identified among more than 60 emb mutants examined to date is intriguing. What is peculiar about the biotin pathway in Arabidopsis that has allowed these auxotrophs to be identified at a moderate frequency among embryo-defective mutants, while auxotrophs disrupted in other biosynthetic pathways continue to escape detection?

Isolation of the bio1 auxotroph initially demonstrated that biotin serves an essential function during plant growth and development (Schneider et al., 1989). Analysis of this mutant allowed the disrupted step to be identified and suggested that the biotin pathway might be conserved between plants and bacteria (Shellhammer and Meinke, 1990; Patton et al., 1996b). Further interpretation of the mutant phenotype was limited by the availability of a single mutant allele of unknown strength. This made it difficult to determine whether mutant embryos survived to the cotyledon stage because they obtained biotin from surrounding maternal tissues, retained some activity of the altered protein, or expressed a duplicate gene or biosynthetic pathway. This has been a common problem when trying to predict which auxotrophs should arrest during embryogenesis and which might survive to the seedling stage. Analysis of the bio2 deletion mutant has finally resolved this question with respect to biotin by establishing the null phenotype and demonstrating the consequence of a complete loss of biotin synthesis during embryo development. Mutant bio2 embryos typically arrest at the globular stage, apparently because maternal tissues cannot supply enough biotin to support the rapid cell division and increased lipid biosynthesis associated with later stages of development. Further growth of bio1-1 embryos probably results from residual activity of a hypomorphic allele. The identification of a second bio1 allele that arrests slightly earlier in embryogenesis (R. Fischer and N. Ohad, University of California, Berkley, personal communication) is consistent with this model. The somewhat variable phenotype of bio2 embryos most likely reflects minor differences in maternal biotin available from heterozygous siliques produced under different conditions.

We propose that biotin auxotrophs have been easy to identify among embryo-defective mutants because the nutrient deficiency cannot be corrected by duplicate genes or maternal sources of biotin and because mutant embryos arrest at a stage of development when they can be effectively rescued in culture. This model predicts that auxotrophs defective in other steps of the biotin pathway should also result in embryonic lethality, provided the corresponding genes are not duplicated, and that mutant embryos should arrest between the globular and cotyledon stages. It should therefore be possible to complete a genetic dissection of the entire biotin pathway in plants by analyzing collections of embryo-defective mutants. The bio2 mutant should be particularly useful in future attempts to identify additional cofactors required for biotin synthase activity (Birch et al., 1995; Sanyal et al., 1996), resolve the significance of the putative intermediate 9-mercaptodethiobiotin in biotin synthesis (Baxter et al., 1992; Baldet et al., 1993b), and determine whether ecotypes of Arabidopsis reported to have temperature-sensitive growth defects that can be rescued with supplemental biotin (Langridge and Griffing, 1959; Langridge, 1965) contain deleterious mutations within the BIO2 gene.

Several complementary approaches have been used in the past to identify mutants defective in amino acid, vitamin, and nucleoside biosynthesis in higher plants (Schneider et al., 1989). The initial approach was to screen populations of M₂ seedlings for growth defects that could be reversed with supplemental nutrients. In Arabidopsis, this strategy led almost exclusively to the isolation of thiamine auxotrophs (Li and Redei, 1969; Redei and Acedo, 1976), several of which have subsequently been examined in detail (Komeda et al., 1988; Ribeiro et al., 1996). Attention then shifted to the isolation of auxotrophic cell lines in culture, using strategies employed successfully with microorganisms (Blonstein, 1986). These efforts resulted in the isolation of a number of auxotrophs, particularly among members of the Solanaceae (Negrutiu et al., 1992), but plant regeneration and transmission of the phenotype through successive generations often proved to be difficult. A third approach has been to apply microbial selection strategies to M₂ seedlings of Arabidopsis. This approach has been particularly useful for studying Trp biosynthesis (Rose and Last, 1994). A common assumption when auxotrophs have not been recovered at the seedling stage has been that the desired mutants arrested early in development. Results presented here challenge this assumption because they raise the possibility that biotin mutants may be the only auxotrophs that can be readily identified among embryo defectives of Arabidopsis.

Several alternative models for this apparent scarcity of plant auxotrophs must be considered. One model postulates that additional auxotrophs are present among embryo-defective mutants, but maternal supplies of the missing nutrient are quickly depleted and the embryo arrests very early in development. Such auxotrophs could have escaped detection in previous screens because the small mutant seeds are difficult to manipulate in culture. Arrested embryos lacking a nutrient with many essential functions in growth and development might also be more difficult to rescue and identify in culture than arrested embryos from biotin auxotrophs, which should be disrupted only in a small number of biotin-dependent enzymes. An alternative model, which includes the missing auxotrophs among gametophytic lethals, cannot be eliminated based on available evidence but may be difficult to test without exposing large numbers of flowers to supplemental nutrients. In some cases, supplements containing the required nutrient may still fail to rescue the corresponding mutant because transport and utilization of the essential component are disrupted. The most probable explanation for the scarcity of plant auxotrophs appears to be the widespread existence of multigene families. Even in plants with small genomes, many proteins with essential cellular and developmental functions are encoded by duplicated genes (McGrath et al., 1993; Pickett and Meeks-Wagner, 1995). Results of mapping large numbers of EMB genes in Arabidopsis suggest that the number of target genes that can mutate to give an embryo-defective phenotype is far less than the total number of genes expressed at this stage of the life cycle (Franzmann et al., 1995). Molecular data supporting the concept of gene redundancy have also been obtained for a number of biosynthetic pathways (Rose and Last, 1994; Tada et al., 1994; Bogdanova et al., 1995). Additional examples of gene duplications in amino acid and vitamin biosynthesis are likely to be uncovered with continued advances in the Arabidopsis genome project. Molecular genetic dissection of biosynthetic pathways in plants may therefore need to rely increasingly on the use of reverse genetics (McKinney et al., 1995), gene trapping with reporter fusions (Sundaresan et al., 1995), and homology searches of genome databases (Fleischmann et al., 1995) to identify desired target genes, analyze potential chemical intermediates, and characterize the phenotypic consequences of loss-of-function mutations.

	FOOTNOTES

   Received July 18, 1997; accepted November 25, 1997.

	ABBREVIATIONS

Abbreviations: cM, centiMorgan(s). EMS, ethyl methanesulfonate. RI, recombinant inbred. SSLP, simple sequence-length polymorphism.

	ACKNOWLEDGMENTS

We thank Jeni Strednak for technical assistance with embryo culture of transition mutants, Randy Dinkins for providing the initial seed source for the bio2 mutant, Jennifer Goldman and Forrest Bath for assistance with plant maintenance, and Scott Uknes and Mary-Dell Chilton for helpful comments concerning the manuscript.

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