Plant Physiol. (1999) 120: 615-622

white anther: A Petunia Mutant That Abolishes Pollen Flavonol Accumulation, Induces Male Sterility, and Is Complemented by a Chalcone Synthase Transgene¹

Carolyn A. Napoli, Deirdre Fahy, Huai-Yu Wang², and Loverine P. Taylor^*

Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721 (C.A.N., H.-Y.W.); and Genetics and Cell Biology Department, Washington State University, Pullman, Washington 99164-4234 (D.F., L.P.T.)

	ABSTRACT

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A mutation in an inbred line of petunia (Petunia hybrida) produces a reduction in the deep-purple corolla pigmentation and changes the anther color from yellow to white. In addition, the mutant, designated white anther (wha), is functionally male sterile. The inability of pollen from wha plants to germinate in vitro provides a physiological basis for the lack of seed set observed in self-crosses of the mutant. Biochemical complementation with nanomolar amounts of kaempferol, a flavonol aglycone, confirms that the inability of the wha pollen to germinate is due to a lack of this essential compound. Transgenic complementation with a functional ChsA (Chalcone synthase A) cDNA suggests that the genetic lesion responsible for the wha phenotype is in Chs, the gene for the first enzyme in the flavonol biosynthesis pathway. The genetic background of the parental line, as well as the pollen phenotype, allowed us to deduce that the wha mutation is in ChsA. To our knowledge, wha is the first induced, nontransgenic Chs mutant described in petunia, and analysis of the mutation confirms earlier molecular and genetic observations that only two Chs genes (A and J) are expressed in reproductive tissues and that they are differentially regulated in corolla and anther.

	INTRODUCTION

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Flavonoids are plant-specific compounds that accumulate in virtually all tissues of plants, ranging from mosses to angiosperms (Koes et al., 1994). They are classified according to the oxidation state of the central pyran ring. A variety of functions have been attributed to the different classes of flavonoids, e. g. the red and blue anthocyanin pigments in combination with UV-absorbing flavonol copigments act as attractants for insect pollinators (Harborne, 1976; Stafford, 1990; Koes et al., 1994). More recently, flavonols, particularly kaempferol and quercetin (Vogt et al., 1995), have been shown to be essential for pollen germination and tube growth in petunia (Petunia hybrida) and maize (Mo et al., 1992; Taylor and Jorgensen, 1992; Pollak et al., 1995).

The flavonoid pathway in petunia, as in many other species, has been well characterized, and many structural and regulatory genes have been identified (Dooner et al., 1991; Martin, 1993; Forkmann, 1994). Sequences encoding Chs (Chalcone synthase), the gene encoding the first enzyme in flavonoid synthesis, have been isolated and characterized from a variety of plant sources (Dooner et al., 1991). In petunia 8 to 10 copies of Chs sequences have been identified in the genome of the inbred line V30 (Koes et al., 1989b). RNase protection analyses with gene-specific probes have shown that only two genes, ChsA and ChsJ, are expressed in petunia anthers and corollas (Koes et al., 1989a) and that ChsJ transcripts are from 5% to 20% of the ChsA levels. In addition, several regulatory loci have been identified, which transcriptionally control expression of the flavonoid structural genes in a temporal or tissue-specific manner. One of these genes, An4, controls flavonoid production in the anthers by regulating ChsJ but not ChsA (Quattrocchio et al., 1993). Results of RNase protection assays suggested that plants that are homozygous recessive at an4 express both ChsA and ChsJ transcripts in corollas, but anther tissue accumulates ChsA mRNA exclusively (Quattrocchio et al., 1993).

Because of the visible and nonessential nature of anthocyanins, mutations in flavonoid pathway genes are easily recognized and maintained. In contrast to maize and Arabidopsis, in which spontaneous and induced Chs mutations have been described (Dooner et al., 1991), no such Chs mutations have been identified in petunia. However, there are several examples of homology-dependent suppression of Chs transcription in petunia (Napoli et al., 1990; van der Krol et al., 1990). Transgenic plants suppressed for Chs in anthers were instrumental in identifying an essential role for flavonols in pollen function (Mo et al., 1992; Taylor and Jorgensen, 1992; Ylstra et al., 1994). The lack of CHS protein in both petunia and maize anthers results in white pollen that is devoid of all flavonols and is unable to germinate or produce a functional pollen tube in self-pollinations (Mo et al., 1992; Pollak et al., 1993). However, when this flavonol-deficient pollen was crossed to wild-type stigmas, the pollen germinated and seeds were produced, thereby demonstrating that the reproductive defect is conditional. Plants expressing this phenotype were designated CMF by Taylor and Jorgensen (1992). The bioactive compound from wild-type petunia stigmas was identified as kaempferol, a flavonol aglycone. Although the mechanism of flavonol induction of pollen germination is unknown, biochemical complementation of CMF pollen (pollen rescue) established that the response to exogenously added flavonols was sensitive (maximum germination at 0.4 µM) and specific for flavonol aglycones (Vogt et al., 1995).

Here we describe a novel male-sterile mutant that was found in an M₂ population derived from EMS-mutagenized V26 petunia seeds. This mutant displays both a reduction in the purple color of the corolla and produces white, rather than wild-type yellow, pollen. Pollen from the mutant was nonfunctional in self-crosses. The similarity of this EMS-induced phenotype to the transgene-induced CMF phenotype suggested a mutation at a Chs gene. In this report we describe the genetic and biochemical characterization of the reproductive tissues of the wha (white anther) mutant and show that the mutation is complemented by a functional ChsA cDNA.

	MATERIALS AND METHODS

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Plant Materials and Plant Culture

Plant Transformation

Agrobacterium tumefaciens-mediated plant transformation was carried out as described by Jorgensen et al. (1996), except for the following changes: Explants were grown on one-half-strength Murashige and Skoog salts and 2% Suc; acetosyringone was omitted from the cocultivation medium; after removal of the plates from dark incubation, the explants were transferred to fresh selection/regeneration medium; the concentration of carbenicillin was decreased to 200 µg/mL; and adventitious shoots were transferred to magenta boxes, not plates, for rooting. The kanamycin assay to confirm the identity of transformed shoots was performed on 0.5 µM IAA and 0.5 µM benzoaminopurine.

In Vitro Pollen Germination and Rescue

HPLC of Anther and Corolla Extracts

HPLC analysis was performed using a Millennium 2.1 chromatographic workstation (Waters) equipped with a dual-pump system (model 600, Waters) and photodiode array detector (model 996, Waters). Chromatographic separations were accomplished with a 0- to 15-min linear gradient of 100% solvent A (10% [v/v] acetonitrile in water and 0.04% [v/v] trifluoroacetic acid) to 100% solvent B (100% [v/v] acetonitrile and 0.04% [v/v] trifluoroacetic acid). The flow rate was 1.5 mL min¹ on a C₁₈ reversed-phase matrix (150- × 3.9-mm i.d., particle size 4 mm, Nova-pak, Waters). Flavonols were detected at 365 nm. Kaempferol and quercetin were identified by retention time and spectral comparisons with authentic standards.

Anthocyanin Quantitation

Immunoblotting

1

RNA-Blot Analysis

	RESULTS

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Identification of the Mutant Phenotype

Rescue of Pollen Germination

The lack of pollen tube growth in the absence of exogenous flavonols was confirmed in planta. In more than 75 self-crosses of plants homozygous for the wha mutation, no seed was set. However, when the mutants were self-crossed in the presence of added kaempferol, 14% of the crosses of class 1 plants produced seed capsules, and class 2 plants, which showed a higher frequency of early tube outgrowth, produced 21% successful self-crosses. More than 50% of the crosses of wild-type pollen to wha stigmas from both class 1 and class 2 were successful, confirming that the lack of seed set in the nonsupplemented self-crosses was due to nonfunctional pollen.

Complementation of the wha Mutation

Confirmation that the wha phenotype resulted from a Chs mutation was obtained by transgenic complementation of the mutant using a ChsA cDNA expressed from a CaMV 35S promoter (Que et al., 1997). A total of 63 transgenic plants homozygous recessive for wha were produced from two independent transformation experiments. Corolla pigmentation was evaluated visually for the entire population. Three classes of phenotypes were observed and the data are reported in Table III. Three-quarters (47 plants) of the 63 transgenotes had corolla pigmentation complementation, as evidenced by either a phenotype indistinguishable from the wild type or one intermediate between the mutant and the wild type. Complementation in the anthers did not occur as frequently as corolla complementation; only 34 of the 47 transgenotes had a restoration of yellow anther pigmentation. A second class of plants comprised 8 transgenotes with white flowers and anthers, resulting most likely from transgene silencing that is common to introduced copies of Chs. The third class included 8 transgenotes that retained the wha plant phenotype, showing no complementation in either the corolla or anther pigmentation. The latter two classes were not analyzed.

Quantitative Flavonoid Analyses

Flavonol levels in the anthers of two of the complemented transgenotes (Chs8705.5 and Chs8705.6) were virtually the same as wild-type levels. Accompanying this accumulation of flavonols was a restoration of pollen germination frequency to wild-type levels (Table IV). Flavonol and anthocyanin levels in the corollas of all three transgenotes increased but showed more variation than the increases in pollen flavonols. Although anthocyanin levels reached only 65% to 82% of wild-type levels, the complemented corollas were visually indistinguishable from the deep-purple wild-type corollas. This indicates that visual estimates of pigmentation are not reliable at high anthocyanin concentrations.

It is interesting that the third transgenote, Chs8705.3, exhibited complementation in the corolla but not in the anther. Phenotypically, anther color reflected the restoration of flavonol production; transgenotes Chs8705.5 and Chs8705.6 had yellow anthers and Chs8705.3 retained the mutant white anthers. Of these three transgenotes, Chs8705.6 produced one capsule in three self-pollinations, Chs8705.5 set seed in two of three self-pollinations, and Chs8705.3 produced no capsules from three self-pollination attempts.

mRNA Accumulation of Flavonoid Biosynthetic Genes in Anthers and Corollas

View larger version (62K): [in this window] [in a new window]   Figure 1. Expression of flavonoid transcripts and CHS protein in anthers and corollas of wha and transgenic complemented wha plants. A, Total RNA was extracted from stage 4-6 anthers or corolla tissue, electrophoresed in formaldehyde agarose gels, transferred to nylon filters, and hybridized to 32P-labeled inserts of Chs, F3h, and Fls cDNA clones from petunia. The correction for loading differences was calculated following hybridization with a GAP cDNA probe:     Probe Anther Corolla V26 wha6.1 wha7.10 CMF V26 wha6.1 CMF Chs 85 0 0 0 91 3 7 F3h 72 7 7 33 112 99 137 Fls 29 28 34 22 49 36 35 B, Ten micrograms of anther total RNA from two complemented transgenotes and the parental wha line was hybridized to a Chs probe. The rDNA hybridization pattern was used to detect loading differences. C, Western analysis of CHS protein in corollas and anthers. A recombinant maize CHS protein (lane C) was included as a positive control (Pollak et al., 1993).

The expression of two other flavonoid biosynthesis genes was also determined. F3h acts downstream of Chs to convert flavanones to dihydroflavonols, which are precursors of both flavonols and anthocyanins. Fls converts dihydroflavonols to flavonols and is not involved in anthocyanin production. F3h expression in the corolla was not affected by the wha mutation but was decreased in anthers from both the wha and CMF mutant plants. The level of the F3h transcript in wha anthers was 10% that of the wild type, and the cosuppressed CMF mutant had a 50% reduction (relative to the wild type) of F3h mRNA levels. On the other hand, Fls expression was not affected in the corolla or anthers of either mutant.

Two transgenotes, Chs8705.3 and Chs8705.6, were examined by northern analysis to determine whether an increase in Chs transcripts correlated with complementation of pigmentation and germination of the pollen and pigmentation in the corolla. The wha mutant parent was included as a control (Fig. 1B). Based on the biochemical analysis and germination results, transgenote Chs8705.6 was complemented in both the anther and the corolla, which was confirmed by the detection of Chs-homologous transcripts in both organs. Transgenote Chs8705.3 showed differential complementation; it accumulated flavonols and anthocyanins in the corolla but the pollen remained devoid of flavonols and was nonfunctional (Table IV). As expected, no Chs-homologous transcripts were detected in anthers but high steady-state levels were present in the corolla.

CHS Protein Accumulation in wha Reproductive Tissues

	DISCUSSION

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A combination of molecular and biochemical data supports the hypothesis that the wha mutation is in ChsA. Furthermore, our results provide genetic confirmation that, in the absence of a functional An4 allele, ChsJ is not expressed in anthers. Koes et al. (1989a) suggested that the two Chs genes expressed in petunia anthers were differentially regulated: Expression of ChsJ, but not ChsA, was controlled by An4. Thus, our use of the V26 inbred as the progenitor line for the EMS mutagenesis was a fortuitous choice. In a recessive an4 genetic background such as V26, a chsA mutation should result in flavonoid-deficient anthers containing viable pollen that is unable to germinate unless supplemented with kaempferol, a flavonol aglycone. Furthermore, because ChsJ is not regulated by An4 in corollas, it would be expressed at its normal level (3% of that of ChsA), resulting in corollas with reduced anthocyanin pigmentation. Thus, the phenotype of the wha mutant reflects a mutation of ChsA and the differential expression of ChsJ in the relevant tissues of the wild type.

The mutation could have been mapped using restriction fragment-length polymorphism markers, but given that mapping indicates only linkage and petunia shows extensive regions of recombination suppression (Robbins et al., 1995), we believed a more straightforward approach would be to use transgenic complementation. Clearly, our results show that introduction and expression of a ChsA cDNA completely restores flavonoid production and pollen germination to the wha mutant. Additional evidence that wha plants require a functional Chs gene for restoration of wild-type pigmentation comes from Que et al. (1997), who engineered a frame-shift mutation in the ChsA cDNA with the intent to prematurely terminate transcription. When the wha mutant was transformed with the engineered frame shift, the altered cDNA failed to complement the mutation. The molecular and biochemical basis for differential complementation seen in 28% of the transgenotes with wild-type corolla pigmentation (e.g. Chs8705.3) bears investigation. It suggests that the CaMV 35S promoter may not be as active in anthers as corollas, a suggestion that is supported by the lack of anther transformation observed in numerous antisense experiments using the CaMV 35S promoter.

The characterization of the wha mutant confirmed previous molecular data that suggested that only two of the eight Chs genes of petunia were expressed in reproductive tissue. Using RNase protection assays to distinguish transcripts, Koes et al. (1989a) estimated that ChsJ contributed about 10% to the total Chs expression in anthers, tube, and corolla with ChsA providing the bulk. Analyzing wha plants, which express only ChsJ, we detected Chs transcripts in the corolla at levels approximately 3% of that found in wild-type corollas, which express both ChsA and ChsJ. This level of expression is limiting for both flavonol and anthocyanin production but is more limiting for the former class, as evidenced by the different effects on flavonol and anthocyanin accumulation shown in Table IV. This may indicate that the dihydroflavonol reductase enzyme involved in anthocyanin production is more efficient than flavonol synthase at competing for the common substrate dihydroflavonol. The wha mutant also provides an opportunity to determine the effect on pollen germination of reducing flavonol content in pollen. This is accomplished by introgressing a functional An4 allele into the wha homozygous recessive background and selecting for the appropriate corolla and anther pigmentation. This genotype would express ChsJ in anthers but at 3% of the levels of mRNA as the wild type. Whether the expected lower levels of pollen flavonols would be sufficient for the pollen to function awaits analysis of these plants.

An interesting observation was the decreased expression in anthers, but not corollas, of a flavonoid biosynthesis gene, F3h, in the absence of Chs activity. In maize Chs mutations can also have an effect on other genes in the flavonoid pathway; transcripts from the A1 (DFR) and Bz1 (UFGT) genes are altered in C2-Idf plants (U. Weinand, personal communication). F3h acts downstream of Chs to oxidize flavanones to dihydroflavonols, a class characterized by a hydroxyl group at the C3 position of the C-ring. This 3-hydroxyl group is the major site for glycosylation of pollen flavonols, converting them from potentially cytotoxic compounds to a water-soluble form, which can be sequestered (Vogt et al., 1994; Xu et al., 1997). F3h expression in anthers may be controlled by substrate availability. In maize anthers F3h has been shown to control flavonol production in a stage-specific manner (Deboo et al., 1995). All other flavonol biosynthesis genes are constitutively expressed or are transcriptionally activated following the appearance of the F3h transcript at the uninuclear stage.

Rescue of pollen germination is extremely sensitive to small amounts of flavonol; tube outgrowth can be detected after adding as little as 0.4 µM kaempferol (Mo et al., 1992; V. Guyon, unpublished data). The differences in the percentage of germination and rescue (Table II) suggest that there is variation in the expression of the wha phenotype in a population of the mutants. This may reflect differences in the penetrance of the mutation, since the EMS-induced change is most likely a point mutation that might be affected by environmental influences. However, the absolute amount of flavonols in any one plant is likely to be quite small because no flavonols were detected by HPLC (detection limit 5 pmol). In any event, the few pollen tubes that formed were nonfunctional, since no seed was ever produced from mutant self-crosses.

Although 70% to 80% of the pollen grains from wha mutants could be successfully rescued in vitro (Tables II and IV), seed set was often lower than expected in both kaempferol-supplemented self-crosses and reciprocal crosses to wild-type stigmas (Tables I and II). The basis of this reduced fecundity is not known, but it should be noted that the highly inbred parental line V26 exhibits variable fertility, which may respond to environmental fluctuations. Vogt et al. (1994) were able to increase seed set in self-crosses of V26 by more than 100% by wounding the flower 24 h before pollinating. Wounding was accompanied by a dramatic increase in stigmatic kaempferol levels.

CHS is the first enzyme in flavonoid biosynthesis, and an alteration in CHS activity would be expected to affect accumulation of all classes of these compounds. Although both spontaneous and chemically induced mutations in Chs have been characterized in maize and Arabidopsis, to our knowledge, this is the first nontransgenically induced Chs mutant described in petunia. Although cosuppressed plants (CMF) were useful in establishing the pollen germination requirement for flavonols, these plants are phenotypically unstable and revert to coexpression (restoration of pigmentation) in certain branches on older plants (Jorgensen et al., 1996). Thus, a nontransgenic Chs mutant in petunia is extremely useful as the progenitor of transgenic plants to test various gene functions during pollen development and germination in the absence of flavonols. They will be especially useful for testing whether flavonoid regulatory genes respond to product accumulation or for determining the role of flavonoid-modifying activities in cellular processes (Vogt and Taylor, 1995).

	FOOTNOTES

   Received December 14, 1998; accepted March 8, 1999.

	ABBREVIATIONS

Abbreviations: CaMV, cauliflower mosaic virus. CMF, conditionally male fertile. EMS, ethane methanesulfonate. GAP, glyceraldehyde phosphate dehydrogenase.

	ACKNOWLEDGMENTS

L.P.T. is thankful for the valuable contribution of Michaela Witcher for the experiments presented in Table II as part of a Howard Hughes undergraduate research project at Washington State University, Pullman.

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