Results and Discussion

3.1. Floral mutants. The inflorescence of mutant T280 differs from that of the wild-type U390 line in that the basal bracts of the inflorescence contain in their axils, a reiteration of inflorescence initiation instead of individual flowers (Figure 2). Many of the floral primordia in these basal inflorescences do not show any obvious sepal, petal, stamen or carpel primordia at the same stage of development as wild type. Mature flowers develop from some of these aberrant primordia, but many abort before elongation of the floral organs. Other meristems produce normal-looking flowers. Nevertheless, very few seeds were obtained from selfing the coi mutants. F2 progeny segregated in a ratio that approximated 3:1.

We also obtained two lines (T149 and T148) that produce inflorescences, which were even more undeveloped than those of the coi mutant. The inflorescence was characterized by incompletely formed, secondary inflorescences composed of green, anomalous buds (data not shown). In contrast to the cauliflower-inflorescence mutants with unifoliate leaves previously described by Goplen (1967) and Micke (unpublished), this mutant has trifoliate leaves. The T149 and T148 mutants resembled the vegetable broccoli (brc) (Hirsch et al. 2000). Because the brc mutant was sterile, we were unable to do a genetic analysis.

Figure 3. U389 (left) has reddish stems whereas the Antho" mutant BT20 has green stems (right).

3.2. Flavonoid mutants. Fifteen independently isolated stable Antho" (anthocyanin-minus) mutants were generated in the U389 background using different mutagens. One Antho" mutant was found to be luteolin-minus (Hrazdina, LaRue, unpublished), but will not be described here. Three other Antho" mutants are described.

BT20 and BT65 are characterized by having green stems instead of the red stems that typify the U389 and U390 lines. BT19 has pale tan-colored stems. The green stems of BT20 are compared with the wild-type U389 line in Figure 3.

The Antho" mutants were crossed to U389 and Fi seeds were obtained. Analysis of the F2 population demonstrated a Mendelian ratio of 3 red to 1 green, indicating that the mutation segregated as a monogenic recessive. We have now intercrossed the various Antho" mutants for allelism tests.

Studies on nodulation and mycorrhizal establishment in the mutant roots are in progress. Preliminary results suggest that these three Antho" mutants are affected in their symbiotic interactions. Although BT19, BT20 and BT65 are nodulated by Rml021, the nodules show some unusual phenotypes. Moreover, their interaction with Glomus intraradices is impaired (Lum, Hirsch, unpublished).

Table 1. Effects of Sinorhizobium meliloti and Glomus intraradices on wild-type U389 and sym mutant sweetclover (Melilotus alba Desr.).

Table 1. Effects of Sinorhizobium meliloti and Glomus intraradices on wild-type U389 and sym mutant sweetclover (Melilotus alba Desr.).

Genotype/Mutant

Nodulation Phenotype*

Mycorrhizal Phenotype

Wildtype/U389

Hac+a, Infb, Nod+c

Pen+d, Ves+e, Arb+f

symllBT62, BT58, and BT35

Had+g, Inf; small, white nodules (12% of the plants) for BT62 but BT58 and BT35 are Nod"

Pen+, Ves+, Arb± (BT62), Pen", Ves", Arb" (BT35, BT58)

sym2i3T59

Hac+, Inf; similar to U389, white, ineffective nodules (25%), but 1.7% of the studied plants formed effective nodules

Pen+, Ves+, Arb+

sym3fST6\, BT69, and BT70

Has+h, Had", Hac", Inf, Nod-

Pen", Ves", Arb"

sym5iBTl\

Had^ Inf; occasional small, white nodules on 13% of the plants

Pen", Ves", Arb"

*Based on Utrup et al. (1993), Wu et al. (1996) and this report; aroot hair curling, i.e. shepherd's crook formation; infection thread formation; cnodule development; penetration of hyphae; evesicle formation; farbuscule formation; groot hair deformation; b root hair tip swelling.

3.3. Symbiotic mutants. The responses of the M. alba non-nodulating mutants to inoculation with Rml021 have already been extensively described (Utrup et al. 1993; Wu et al. 1996). We analyzed their responses to inoculation with G. intraradices and found that several of the non-nodulating mutants are Myc" as has been described for other legumes (Lum, Li, LaRue, Schwartz, Kapulnik, Hirsch, submitted). Interestingly, one of the non-nodulating sym mutants, sym2, appears to be colonized more extensively by mycorrhizal fungi than its wild-type parent (Table 1). This mutant is also responsive to S. melilotr, 25% of the plants were reported to develop white, ineffective nodules (Utrup et al. 1993).

The sym.3 mutant and its three alleles, BT61, BT69, and BT70, showed no penetration of the root by G. intraradices, even months after inoculation. Two syml mutants were also Myc", but a third one, BT62, exhibited some mycorrhizal formation. BT62 had previously been described as a weak allele by Utrup et al. (1993).

In conclusion, a number of single-gene recessive M. alba mutants are available for studying both symbiotic and developmental processes. In particular, white sweetclover has several distinct advantages as a legume model for studying the role of flavonoids in plant development as well as in symbiosis: (1) like Arabidopsis, there are a large number of available mutants, which are likely to be mutated in critical steps for flavonoid synthesis; and (2) sweetclover appears to have fewer copies of genes encoding enzymes for the phenylpropanoid pathway than either Medicago sativa (alfalfa) or M. truncatula (data not shown). It is well known that many of the flavonoids serve as important signaling molecules in plant-microbe interactions. Some recent data also indicates that flavonoids also impact plant growth and development (Woo et al. 1999). White sweetclover may also serve as a useful model for studying flower development because similar to Arabidopsis and Antirrhinum, the sweetclover inflorescence is determinate and consists of a simple raceme. Pea, in contrast, is indeterminate, and produces secondary inflorescences that generate one or more floral meristems (Ferrändiz et al. 1999; Singer et al. 1999). The study of legume floral mutants will help expand our knowledge of the genetic mechanisms of flowering by uncovering the key genes regulating inflorescence and floral development in plants beyond the model species.

4. References

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Hirsch AM et al. (2000) In Triplett EW (ed.) Prokaryotic Nitrogen Fixation: A Model System for the Analysis of a Biological Process, pp. 627-642, Horizon Scientific Press Kneen BE, LaRue TA (1988) Plant Sei. 58,177-182

Scheibe A, Micke A (1967) In Induzierte Mutationen und ihre Nutzung (Induced Mutations and their Utilization), Erwin-Baur Gedächtnisvorlesungen IV, Gatersleben 20-24 June 1966, pp. 231-236, Akademie-Verlag Berlin Singer S et al. (1999) Bot. Rev. 65, 385-410

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5. Acknowledgements

This research was supported in part by National Science Foundation grants 96-30842 and 97-23982, UC Mexus grant 017451, and a grant from the Council on Research from the UCLA Academic Senate to AMH. A USPHS National Research Service Award GM07185 and BioStar Grant S98-86 supported MRL.

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