The Plant Cell, Vol. 2, 115-127, February 1990 O 1990 American Society of Plant Physiologists

Genetic Regulation and Photocontrol of Anthocyanin Accumulation in Maize Seedlings Loverine P. Taylor’ and Winslow R. Briggs2 Carnegie lnstitution of Washington, Department of Plant Biology, 290 Panama Street, Stanford, California 94305

The flavonoid pathway leading to anthocyanin biosynthesis in maize is controlled by multiple regulatory genes and induced by various developmental and environmental factors. We have investigated the effect of the regulatory loci R, B, and f / on anthocyanin accumulation and on the expression of four genes ( C2, A l , 621, and 822) in the biosynthetic pathway during an inductive light treatment. The results show that light-mediated anthocyanin biosynthesis is regulated solely by R; the contributions of B and f / are negligible in young seedlings. lnduction of the A1 and Bz2 genes by high fluence-rate white light requires the expression of a dominant R allele, whereas accumulation of C2 and B z l mRNA occurs with either a dominant or recessive allele at R. A1 and 822 mRNA accumulate only in response to high fluence-rate white light, but B z l is fully expressed in dim red light. Some C2 mRNA is induced by dim red light, but accumulation is far greater in high fluence-rate white light. Furthe’more, expression from both dominant and recessive alleles of the regulatory gene R is enhanced by high fluence-rate white light. Seedlings with a recessive allele at R produce functional chalcone synthase protein (the C2 gene product) but accumulate no anthocyanins, suggesting that, in contrast to the R-mediated coordinate regulation of C2 and B z l observed in the aleurone, C2 expression in seedlings is independent of R and appears to be regulated by a different light-sensitive pathway.

INTRODUCTION Genetic control of anthocyanin accumulation in maize is well documented, with more than 20 identified loci involved in the biosynthesis and tissue distribution of the pigments (Coe, Hoisington, and Neuffer, 1988). Severa1 genes encoding enzymatic activities have been identified and cloned via transposon tagging. These include the structural genes for dihydroquercetin reductase (A 7) (O’Reilly et al., 1985), chalcone synthase (C2) (Wienand et al., 1986), UDPgIucose-flavonol glucosyltransferase(bronze, Bz7 ) (Fedoroff, Furtek, and Nelson, 1984; Dooner et al., 1985), and Bronze-2(McLaughlinand Walbot, 1987; Theres, Scheele, and Starlinger, 1987). Although no gene product has as yet been identified for Bronze-2 ( B z ~ )precursor , feeding studies and accumulatedintermediatesindicate that it acts late in the pathway (Reddy and Coe, 1962; McCormick, 1978). Biochemical studies of mature kernels (Dooner and Nelson, 1977; Dooner, 1983) have suggested that R and C7 coordinately regulate enzymes that function at the beginning and end of the anthocyanin biosynthetic pathway. (See Figure 1 for relevant portions of the pathway.) Both chalcone synthase (CHS) and UDPglucose-flavonol gluco-

’ Current address: U.S. Department of Agriculture-A.R.S.,

Plant Gene Expression Center, 800 Buchanan St., Albany, CA 94710. To whom correspondence should be addressed.

syltransferase (UFGT) activily require the presence of at least one copy of a dominant allele at the R locus. No CHS (0% of wild type) and only very low levels of UFGT (2% to 3%)enzyme activity were detected in kernels homozygous for recessive alleles at R. Similarly, dihydroquercetin reductase activity, the product of the A7 gene, was measured in immature endosperm and aleurone tissue, and no enzymatic activity was found in material from plants homozygous for recessive alleles at R or C7 (Reddy et al., 1987). Although most maize tissues are competent to produce anthocyanin, the pattern of pigmentation in any plant is determined by the allelic combination at the regulatory loci. These genes show a broad range of variation with respect to developmental and tissue-specific expression (Coe et al., 1988). For example, regulation by C1 is restricted to the aleurone and scutellum of the kernel. On the other hand, although each of the scores of R alleles shows a characteristic expression pattern, as a group they function in almost all plant tissues. Elements of the R locus and the nearby Lc gene comprise a family of closely related sequences (Dellaporta et al., 1987; Ludwig et al., 1989), the number of which can vary from one to severa1 in different maize stocks. In many stocks R is a complex locus both structurally and functionally. Emerson (1921) showed that the R locus is responsible for anthocyanin pigmentation in both aleurone (outer

116

The Plant Cell

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anthocyanin (cyanidin 3-glucoside) Figure 1. A Portion of the Flavonoid Biosynthetic Pathway. The structural genes are indicated at their site of action. 822 is enclosed by dashed Unes to indicate that its enzymatic function has not been determined. C2 encodes chalcone synthase (EC 2.3.1.74); A l , dihydroquercetin reductase (EC number not assigned); and Bzl, UDPglucose-flavonol glucosyltransferase (EC

2.4.1.91).

layer of the endosperm) and plant tissues. He designated four classes of pigmentation patterns: R-r (pigment in both aleurone and plant),R-g (pigmented aleurone, green plant), r-r (nonpigmented aleurone, pigment in plant), and r-g (nonpigmented aleurone, green plant). By convention, the seed component of the R locus is listed first. Stadler (1946, 1948) confirmed that the locus consists of two elements that can independently change from pigment-producing (dominant) to nonpigmented (recessive). S (R, dominant; r , recessive) is the seed component that functions in the aleurone and embryo, and P (-r, dominant; -g, recessive) is the plant component that determines color in seedlings and certain mature tissues, notably anthers. The genetic model of complexity was recently confirmed at the molecular leve1 by correlating the presence of distinctive crosshybridizing DNA restriction fragments with the genetic determination of plant (P) and seed (S) function in the R-cstandard allele (Dellaporta et al., 1987). Two other regulatory genes, B and PI, function in both the seed and plant but there is no evidence for complexity at either locus. Some B and R alleles can substitute for

each other in regulating kernel pigmentation. However, this relationship is not true for anthocyanin accumulation in plant tissues. Only plants with particular dominant alleles at B are intensely pigmented at maturity in tissues such as leaves, husks, and tassel glumes. When the PI gene is also present, the color is most intense and extends to inner husk and sheath regions not normally exposed to light. In contrast, in the presence of the recessive allele pl, B plants develop pigmentation only in those tissues directly exposed to light. Recently, these regulatory genes, which control expression of the unlinked structural genes, have been cloned. They include C7 (Cone, Burr, and Burr, 1986; Paz-Ares et al., 1986), R (Dellaporta et al., 1987; Ludwig et al., 1989), PI (K. Cone, personal communication), and B (Chandler et al., 1989). The genes fall into two classes by sequence similarity and function: R and 6 function as duplicate loci in controlling tissue-specific pigmentation; likewise, particular alleles at C7 and PI are functionally duplicate with respect to light-dependent pigmentation. This functional redundancy is reflected in sequence duplication:R and C7 gene sequences were used as hybridization probes to identify B and PI clones, respectively. Mechanistic features of regulatory gene action have been proposed based on sequence analysis of the C7 and R genes. The predicted protein product of the C7 locus has properties of known DNA binding and transcriptional activating proteins (PazAres et al., 1987). Sequence homology has been detected between an R gene (Lc) and a DNA binding portion of the proto-oncogene L-myc (Ludwig et al., 1989). Although the genetic constitution of a plant determines its potential to synthesize anthocyanin pigments, environmental factors, particularly light, influence the extent to which this potential is realized. An extensive literature exists on the fluence and wavelength requirements for pigment synthesis and deposition in many plant species (reviewed in Mancinelli, 1983; Beggs, Wellmann, and Grisebach, 1986). Unfortunately, these studies provide few clues as to the gene targets of the light-induced synthesis because most studies simply measured total pigment accumulation in genetically undefined material. An exception is the work of Hahlbrock and co-workers, who measured the rate of CHS transcription in parsley cell suspension cultures. In this species, UV light is the most efficient inducer of CHS mRNA, but red and blue light have a limited direct effect and can modulate the UV response (Hahlbrock and Scheel, 1989). The wealth of genetic information and material makes maize a logical choice in which to determine regulatory gene control of structural gene expression under controlled growth conditions. The experiments reported here are designed (1) to measure the response of anthocyanin regulatory and structural genes to light, (2) to identify regulated steps in the pathway, and (3) to determine which of the three known regulatory genes, R, B, and PI, control these steps. We utilized maize lines that differ by single

Genetic Regulationof Anthocyanin Accumulation

alleles (dominantor recessive) at these loci. Because these stocks have a common genetic background,we can compare the effect of changing a single regulatory allele on the steady-state mRNA levels of severa1 structural genes and on total pigment accumulation.

RESULTS Regulatory Gene Requirements and Light lnduction of Anthocyanin Accumulation The aim of these experiments was twofold: (1) to characterize light inductionof anthocyaninaccumulationin young maize seedlings in a defined genetic background and (2) to determine the combination of regulatory genes R, 8, and PI that are required to produce pigment. For each regulatory locus, we selected two alleles, dominant and recessive (see Methods). To determine the contribution of a particular regulatory allele, we measured anthocyanin accumulation in strains carrying all possible combinations of these six alleles (eight strains in all) in a common W23 inbred background (see Methods for details of strain construction). For this experiment, we are varying only the plant component of R ; the seed component remains constant in the recessive condition. Etiolated seedlings were used because they provide a developmentally uniform source of material that can be grown under controlled conditions. Anthocyanins were extracted from seedlings (1) grown in complete darkness for 3 days, (2) grown for an additional 48 hr in low fluence-rate red light (low RL), and (3) grown for a further 12 hr to 48 hr in high fluencerate white light (high WL). Figure 2 shows that only those seedlings with the dominant form of the plant component of the R locus (-r) accumulate pigment. Although neither B nor PI alone or together can support pigment production at this developmentalstage, they may modify the response in seedlings with an -r allele: plants with the dominant alleles €3 and PI produce somewhat more pigment than those with the recessive alleles b and pl. Plants with an -r allele accumulate pigment only in response to high WL. No anthocyanin is detected during the 72 hr of growth in the dark or during the 48 hr of low RL exposure (-48 hr and O hr timepoints) nor when plants are exposed continuously (5 days) to low RL or kept in total darkness for the entire period (data not shown). Following an 8-hr to 12-hr lag period after the start of high WL, pigment levels rise and continue to do so throughout the period of illumination.

Effect of the Seed-Pigmenting Component on Anthocyanin Accumulation in Seedlings Genetic analysis indicates that the seed and plant components of R function independently to regulate anthocy-

117

anin deposition in various tissues (Coe et al., 1988). To confirm that the seed component does not control anthocyanin accumulationin the plant body, pigment was measured in sheath tissues of young seedlings with all four allelic combinations at the R locus. In this experiment, regulatory loci B and PI are present in the recessive state, b and pl. As is apparent in Figure 3, only those plants with a dominant plant component allele (-r) accumulate pigment in response to high intensity white light. Plants with a dominant allele at the seed component 2nd a recessive allele at the plant component (R-g) produce no measurable anthocyanin in the sheath under these light conditions. The independent effects of R in the kernel and the seedling are also reflected in the mRNA levels of a structural gene in the anthocyanin pathway. Both r-r and R-r leaf sheaths produce A 7-homologous transcripts, but R-g seedlings grown from fully colored kernels produce no

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Figure 2. Anthocyanin Accumulation in Seedling Leaf Sheaths Requires High lntensity Light and a Dominant Allele at the Plant Component of R.

Time course of anthocyanin accumulation in seedling leaf sheaths in complete darkness (-48 hr), after 48 hr of low-intensity red light (O hr), and high-intensity white light (12 hr to 48 hr) (see Methods for fluence rates and light quality). The eight lines tested are homozygous dominant for the structural genes required for anthocyanin pigmentation in a W23 background. They vary by the presence of dominant or recessive alleles at three regulatory loci: R,B,and PI. The regulatory gene combinations in each line are noted at the right. For 6 and PI, the dominant allele is indicated by upper case symbols and recessive by lower case. The two elements at the R locus are ordered with the seed component first (in this case only the recessive r allele is present) followed by the plant component (-r is the dominant allele, q is recessive). Total acid-extractable pigment was determined spectrophotometrically at 530 nm. Individual optical density readings were normalized to 1 g dry weight tissue. For example, a relative OD of 100 is derived from a spectrophotometric reading of 0.39 extracted from 0.078 g dry weight of tissue. The symbol / / indicates 48 hr elapsed time.

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Figure 3. Time Course of Anthocyanin Accumulation in Seedling Leaf Sheaths with Combinations of Dominant and Recessive Alleles at the Seed and Plant Components of the R Locus.

Seedlings were harvested after growth in the dark (-48 hr), lowintensity red light (O hr), and high-intensity white light (12 hr to 72 hr), and total acid-extractable pigment was measured spectrophotometrically at 530 nm.

detectable A7 transcript (data not shown). Thus, the two elements at the R locus function independently in regulating anthocyanin accumulation in different tissues and organs.

R Gene Expression in Seedlings The results above indicate that R gene function is required for light-stimulated pigment production in seedling leaf sheaths. If the regulatory gene itself is part of the transducing mechanism of the light signal, then its expression may be light-modulated. To determine whether a light stimulus identical to that required for pigment production can affect R gene expression, we measured R-homologous transcript accumulation under various light regimens. As before, seedlings of genotypes r-r b pl and r-g b pl were grown in complete darkness for 72 hr and then moved to low RL for 48 hr. At this point, the group was split, half remaining in low RL and half moved to high WL for 12 hr. Seedlings were then returned to darkness for a further 48 hr. A control group was kept in complete darkness throughout the entire experimental period. Total RNA was isolated from pooled seedling leaf sheaths at each timepoint, and the relative amount of R-homologous transcript present was determined by slot-blot analysis. Figure 4A shows the accumulation pattern of R transcripts in darkness (solid line), low RL (dotted line), and high WL (dashed line). A low level of R-homologous transcript accumulates during the 48 hr following 72 hr of dark growth, regardless

of whether the seedlings remain in the dark or are exposed to low RL. In the absence of high WL, the rate of transcript accumulation eventually levels off. In both genotypes, accumulation of R-homologous transcripts is stimulated by high WL, and high transcript levels are maintained even after the plants are returned to darkness for 48 hr. We have shown that an -r allele and high WL are necessary for anthocyanin production in seedlings and that r-r b pl lines produce R-homologous transcripts in response to high WL. It is intriguing that r-g b pl seedlings, which produce no anthocyanin in response to light, produce R-homologous transcripts with an accumulation pattern similar to f-r b pl lines. To characterize this RNA, poly(A') RNA was isolated from seedlings after 12 hr of high WL and examined by gel blot analysis. Comparison of the r-r b pl and r-g b pl seedling mRNAs hybridized with an R gene cDNA probe (Figure 48) shows no obvious differences that can account for the functional disparity displayed by the two alleles. The righthand lane of Figure 48 contains poly(A') RNA from pigmented R-f kernel tissue, here used as a control source of R-homologous mRNA. This line is dominant for Lc, a member of the R gene family that is highly expressed in the pericarp (Ludwig et al., 1989). Because transcripts from Lc and R are identical by RNA blot analysis (Ludwig et al., 1989), it is not clear whether the 2.5-kb transcript in the right lane (R-r)of Figure4 8 is from the seed component of R, Lc, or both. The cytosolic glyceraldehyde phosphate dehydrogenase (GAPDH) gene (Brinkmann et al., 1987) was used to estimate the relative amount of RNA per lane (Figure 4C). Comparison of the hybridization intensities indicates that r-r b pl lines produce slightly more R-homologous transcript than r-g b pl lines and that kernel tissues accumulate very high levels relative to seedling tissue. Quantitative estimates should be treated with caution because all aleurone and pericarp cells can produce anthocyanin, whereas, in the sheath tissue, pigment accumulates primarily in the epidermal layer of the outer sheath. A comparison of CHS enzymatic activity in the outer versus inner sheaths of r-r b pl seedlings shows greater than a 100-fold difference (L. Taylor, unpublished data).

Steady-State RNA Accumulation Patterns of Four Anthocyanin Structural Genes To ascertain whether the control of anthocyanin accumulation by -r is at the level of mRNA abundance, we measured the steady-state RNA levels of four structural genes that act at the beginning (C2),middle ( A l ) , or end (Bzl, Bz2) of the biosynthetic pathway (Figure 1). Total RNA isolated from seedlings with the eight combinations of R, B, and Pl regulatory genes was analyzed by slot blot hybridizationto DNA probes for the four structural genes and for the control GAPDH gene. Samples were harvested at five timepoints from each line: after dark (-48 hr), low

Genetic Regulation of Anthocyanin Accumulation

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Time (hours) Figure 4. Time Course and RNA Blot Analysis of ft-Homologous Transcript Accumulation. (A) Seedlings of r-r b pi and r-g b pi genotypes were grown in the dark (——), low-intensity red light (• • •), and high-intensity white light (- - -) and samples harvested at the indicated times. The transfer of seedlings from one light source to another is denoted by a change in line style. The relative abundance of transcripts homologous to pR236, a 900-bp cDNA clone of an R gene, was determined by slot blot hybridization of total RNA isolated from pooled seedling leaf sheaths. A correction for loading variation was determined by hybridizing the same filters with pZm9, a cDNA probe to maize cytosolic GAPDH after the fl signal was removed. (B) Poly(A+) RNA from seedling leaf sheaths after 12 hr of high-intensity white light treatment was electrophoresed in a 1.4% agarose gel with 2.2 M formaldehyde, transferred to nylon membrane, and hybridized with pR236, the R gene probe. The genetic constitution at the R locus is indicated above each lane and at the right are the positions of RNA size standards (Bethesda Research Laboratories). Lane R-r contains poly(A*) RNA isolated from 20-day post-pollination kernels with the genetic constitution R-r Lc, where Lc is a duplication of the R locus that allows pigment to be produced in the pericarp (Coe et al., 1988). The harvested tissue was a mixture of aleurone and pericarp cells. (C) The R gene signal was removed and the filter hybridized to the GAPDH probe as described above.

RL (0 hr), and high WL treatment (12 hr, 24 hr, 48 hr). Figure 5 shows a representative slot blot experiment, with total RNA from the 0-hr and 24-hr specimens of all eight genotypes hybridized to the structural gene probes. Different patterns of mRNA accumulation are apparent: all lines produce C2 and Bz1 RNA, whereas only lines with the -r allele produce significant levels of A1 and Bz2 RNA. Production of Bz1 RNA appears to be independent of high WL induction, but the other structural genes are expressed to high levels only in the 24-hr (high WL) samples. The presence of low levels of C2-homologous transcripts are evident in the low RL-treated material (0 hr). As seen in Figure 2, anthocyanin production is only weakly dependent

on the B and PI genotypes; hence, only data from r-r b pi, r-r B PI, r-g b pi, and r-g B PI lines are shown in subsequent figures. When the results of several independent slot blot experiments are pooled, three patterns of RNA accumulation are apparent, as shown in Figure 6. First, A1 and Bz2 RNA accumulation parallels what is observed for pigment production in Figure 2: significant levels of transcripts from these genes are detected only in the presence of a dominant allele at the plant component of R (-r) and only in response to high WL. This rapid accumulation of transcripts precedes the detection of pigment by approximately 12 hr. Second, significant amounts of C2 mRNA

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The Plant Cell

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610 nm, the cutoff point of the red filter used in these experiments. An RNA gel blot of poly(A+) RNA isolated from plants with either a recessive or dominant allele at the plant component of R was used to confirm that the slot blot signals came from the expected mRNAs. RNA was isolated from seedlings after 24 hr of high WL and hybridized with a cDNA probe to the C2 gene. Figure 7A shows that all -g or -r lines, whether in combination with B PI or b pi, contain a single species of C2-homologous mRNA of the expected size (Wienand et al., 1986). When the same filter is hybridized with the A1 probe (Figure 7B), only -r lines contain a 1.4-kb transcript homologous to that probe. Both -/- and -g lines also hybridize to the Bz1 probe and contain the expected 1.8-kb mRNA (Figure 7C). This result confirms the slot blot pattern of structural gene transcript accumulation and indicates that -g lines produce a C2homologous transcript that is indistinguishable by RNA gel blot analysis from the transcript produced by -r lines. C2homologous transcripts from the aleurone layer of the kernel (lane 6) likewise appear identical with those from seedlings of both genotypes. These results raise the possibility that the anthocyanin-less -g seedlings can produce chalcone synthase protein.

24

Chalcone Synthase Protein and Enzymatic Activity Figure 5. A Representative Slot Blot Experiment. The four anthocyanin gene probes are described in Methods. From the usual five specimens collected per experiment, two of each genotype are displayed in the duplicate filters. The slot designated 0 contains RNA isolated from seedlings grown in low fluence-rate RL, and those designated 24 contain RNA from seedlings harvested after an additional 24 hr of high fluence-rate WL. The right lane is a reprobing of the A1 filter with the insert from pZm9, a clone of the maize cytosolic GAPDH gene after the A1 hybridization signal had been removed. The Bz2 signal in the 0 hr specimen of r-g B pi is the result of RNA overloading in this

slot.

are produced by plants with a recessive allele (-g) at R, at levels only slightly less than those produced by seedlings with a dominant allele (-r). In addition, this transcript is responsive to light, showing similar kinetics of accumulation in -g and -r plants. C2-homologous RNA also shows a modest but consistent induction in low RL in both -r and -g material. Transcripts homologous to the Bz1 gene show a third pattern of regulation. Dark-grown seedlings (-48 hr) of both -r and -g lines have a measurable level of Bz7 transcript, and this can be increased by low RL treatment with virtually identical kinetics of accumulation. There is no further increase in steady-state levels of Bzl RNA upon exposure to high WL. This result indicates that the system is saturated by low fluence rates of wavelengths above

Chalcone synthase activity encoded by C2 is crucial to the biosynthesis of all classes of flavonoids, which include aurones, flavones, flavonols, and proanthocyanidins in addition to anthocyanins. Although maize stocks with a recessive R allele at the plant component (-g) produce no measurable anthocyanin pigments, nor A1 or Bz2 mRNA, they may require CHS activity to produce these other classes of flavonoids. To ascertain whether the C2-homologous mRNA produced in -g lines is translated, proteins isolated from r-r b pi and r-g b pi plants were separated by denaturing polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with a rabbit antiserum raised against the chalcone synthase of parsley. As shown in Figure 8, this antibody recognizes a single protein species of approximately 42 kD in both -g and -r lines. This matches the size of chalcone synthase proteins isolated from a number of sources (Mol et al., 1983; Rail and Hemleben, 1984; Hrazdina, Zobel, and Hoch, 1987; Schmelzer, Jahnen, and Hahlbrock, 1988). The maximum signal is obtained from the soluble protein fraction of -r and -g plants (lanes 3 and 4), although low levels can be detected in the microsomal fractions (lanes 1 and 2). The detection of a monocotyledonous chalcone synthase protein by an antibody raised against a dicot enzyme is not unexpected because chalcone synthase genes from a variety of sources, including parsley and maize, show >80% conservation at the amino acid level (Niesbach-Klbsgen et al., 1987).

Genetic Regulation of Anthocyanin Accumulation

121

Time (hours) Figure 6. Light-lnduced Expression of Anthocyanin Structural Genes in Plants with Different Regulatory Gene Combinations. Time course of RNA accumulation in seedling leaf sheaths grown under the light conditions described in Figure 2 and Methods. The relative amount of RNA within each experiment was determined by slot blot analysis of total RNA isolated from seedlings whose regulatory gene constitution is indicated at the right. The blots were hybridized with the DNA probes for transcribed regions of the A7, 822, C2, and Bzl loci (exact clones described in Methods). The standard error of the mean ranged from a high value of 12.9 relative units for the O-hr value of the r-g B PI line with the Bz7 probe to 0.8 relative units for the 12-hr value of the r-r b pl line with the A7 probe. Most standard errors of ttie mean ranged between 2 and 8 relative units. The curves were calculated from data averaged from 11 independent experiments with five sets of independentiy generated plant material for C2, 10 experiments with five sets for A7, seven experiments with four sets for B Z 7 , and five experiments with four sets for €322.

To determine whether the chalcone synthase protein is enzymatically active, assays were performed on extracts from r-r b pl and r-g b pl seedlings after 48 hr of low RL and after an additional24 hr of high WL illumination. Table 1 shows that protein extracts from high WL-grown seedlings with the -g allele convert ''C-malonyl COA into naringenin chalcone at about 50% of the rate catalyzed by -r seedling extracts. This analysis also shows that the highintensity light induction of mRNA (Figure 6) is followed by

a concomitant increase in active enzyme levels in both -r and -g seedlings.

D,SCUSS,ON We measured the effect of different alleles at three regulatory loci, R , B,and Pl, on anthocyanin production and on

122

The Plant Cell

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Figure 7. RNA Gel Blot Analysis of C2,A1, and Bz1 Transcripts in Seedlings with Different Regulatory Gene Combinations. (A) Poly(A+) RNA was isolated from seedling leaf sheaths after 24 hr of high white light treatment, electrophoresed in a 1.4% agarose gel with 2.2 M formaldehyde, blotted, fixed to nylon membrane, and hybridized with the C2 probe. For each genotype, a dilution series of RNA was tested, with fivefold less RNA in the second lane of each pair (the exception is right lane 3, which has about 20-fold less RNA relative to left lane 3). The number following the regulatory genotype is the relative amount of poly(A+) RNA in the first lane of each pair determined by probing the blot with pZm9, a cDNA probe of the constitutively expressed cytosolic GAPDH gene. Lane 1, r-r b pi, (2); lane 2, R-r b pi, (1); lane 3, Rr B PI, (2.5); lane 4, r-g b pi, (2); lane 5, R-g B PI, (0.8); lane 6 contains RNA from R-r B PI (0.1) 18 day post-pollination aleurone and pericarp tissue. RNA size standards in kilobases are indicated at the right. (B) The same blot probed with a 600-bp Pstl fragment of the transcribed portion of the A1 gene. (C) The Bz1 hybridization signal from the same blot.

the level of anthocyanin accumulation (Figure 2) and transcript abundance (Figure 6). Preliminary data indicate that, after 96 hr of high WL, lines with the genetic combination of r-g B begin to produce significant amounts of pigment and A1 mRNA (L. Taylor, unpublished data). Hence, B does play a direct role in regulating anthocyanin accumulation in older plants. This result agrees with Gerats et al. (1984), who found that, at 52 days after planting (anthesis occurred at day 55), only plants with a dominant allele at B express significant levels of pigment or UFGT activity. The contribution of the -r allele to overall anthocyanin or UFGT activity was less than 1% of the levels found in plants with a B allele. The steady-state mRNA levels of four structural genes involved in pigment biosynthesis show a complex pattern off? gene regulation. In young seedlings, A1 and Bz2 gene expression is absolutely dependent on a dominant plantpigmenting allele, -r. Expression of the C2 and Bz1 genes is independent of the R locus because transcripts are produced in both -r and -g lines. However, R may modulate the absolute amounts of C2 mRNA produced because seedlings with the recessive allele (-g) have lower levels of

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Figure 8. Chalcone Synthase Protein Is Detected in Seedlings with a Recessive Allele at the Plant Component of R. the expression of flavonoid biosynthetic genes after light treatment in seedlings. Our findings show that a dominant plant component allele (-r) at the R locus is both necessary and sufficient for pigment production in the leaf sheath of young seedling tissue. No pain/vise combination of B and PI is sufficient to condition pigmentation at this stage of development, but they may provide a modulating effect on

Total proteins were isolated from seedling leaf sheaths after 24 hr of high WL exposure, separated by SDS-polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose, and incubated with rabbit antisera raised against parsley chalcone synthase protein. Lanes 1 and 3 contain 50 ^9 of protein from microsomal and soluble fractions, respectively, isolated from r-g b pi lines. Lanes 2 and 4 are the similar fractions from r-r b pi material.

Genetic Regulation of Anthocyanin Accumulation

Table 1. lncorporation of Mal0nyl-2-['~C] COA into Naringenin Chalcone by Protein Extracts from Seedlings with a Dominant (-r) or Recessive (-9) Allele at the Plant Component of the R

Locus 14Clncorporation

Genotype

O hr

24 hr

1515

6039

607

12003

Protein extracts isolated from seedling leaf sheaths grown for 48 hr in low RL (O hr) and then an additional24 hr in high WL (24 hr) were assayed for CHS activity as described in Methods. Values were normalized to wet weight of leaf sheath and are reported in counts per minute.

transcripts and CHS activity than do those with the dominant allele (-r). We propose that C2 and Bz7 expression in seedlings is not a rate-limitingfactor for anthocyanin production. Klein and Nelson (1983) showed that, in the aleurone, as little as 0.5% of the wild-type levels of UFGT enzyme are sufficient to produce wild-type pigment levels. We did not measure UFGT activity in seedlings, but we assume that the abundant Bz7 transcript produced in g lines is fully functional. Likewise, plants with a -g allele produce active CHS enzyme and abundant C2-homologous transcripts. Styles and Ceska (1981) measured the abundance of pollen flavonols and showed that -g plants have nearly the same levels as do -r plants (89% versus 100%). An examination of the pathway (Figure 1) shows that the branch point for flavonol production is just prior to the action of the A7 gene, and that CHS activity is necessary for production of flavonol precursors. Consequently, we favor the model that the lack of pigment in -g seedlings is caused by the tight regulation of A 7 (and 822) by the R locus and that C2 regulation is mediated by factors independent of R. One complication in interpreting the presence of C2homologous transcripts is the existence of a duplicate locus, Whp (white pollen), which presumably also encodes a chalcone synthase (Coe, McCormick, and Modena, 1981). Whp contributes a minor percentage of chalcone synthase in tissues other than the tapetum; kernels recessive for C2 (c2) and dominant for Whp have no aleurone COlOr (Coe et al., 1981, 1988). The W23 lines we employed are homozygous for the dominant Whp allele; c2 Whp seedlings in this inbred background produce very low levels of anthocyanin even after 48 hr of high WL (L. Taylor, unpublished results). Mature plants eventually accumulate some pigment, primarily in the husk and sheath regions (Coe et al., 1988). To estimate the contribution of Whp to the overall chalcone synthase transcript level, we measured C2-homologous mRNA by slot blot analysis in plants with the C2/dfa7 gene, which is allelic to, and acts

123

as a dominant inhibitor of, C2 function (Greenblatt, 1975). C2-Homologous transcripts were detected only in 24-hr high WL-treated seedlings, at levels about 2% of that present in C2 whp and C2 Whp material (L. P. Taylor and W. R. Briggs, manuscript in preparation). Thus, we hypothesize that the high levels of chalcone synthase mRNA detected in r-g seedlings are likely to arise mainly from the C2 locus. We are currently testing this hypothesis. Table 2 shows that light is absolutely required for anthocyanin production in maize seedlings and that steady-state mRNA levels of both structural and regulatory genes in the pathway increase after light treatment. A7 and 822 are not transcribed in dark-grown seedlings of genotype r-r b pl, despite the substantial amount of R-homologous transcript present. Following high WL treatment, there is increased production of R-homologous mRNA, A 7 and 822 genes are expressed, and pigment is produced. Whether the light induction of A7 and 822 expression is mediated through the regulatory gene R (-r) or whether all three genes respond independently to the light signal is not clear. The major portion of C2-homologous mRNA is induced by high WL, the rest by low RL, possibly mediated by phytochrome. High WL, in the absence of a functional R allele, can induce C2-homologoustranscripts to high levels (Figure 6) and produce active CHS protein (Table 1). If C2 mRNA is produced in the absence of a functional R allele, then how is C2 expression regulated in the seedling? We propose that C2 expression in seedlings (and possibly other tissues) is regulated principally by light through a pathway other than that mediated by the anthocyanin regulatory gene R. Supporting this view is the observation that -g seedlings have high levels of C2-homologous transcripts yet never produce pigment in response to light. Chalcone synthase genes from a variety of plants show light induction (reviewed in Dangl, Hahlbrock, and Schell, 1989). Using promoter deletions and in vivo genomic footprinting techniques, Schulze-Lefert et al. (1989) have defined two light-responsive cis-acting units in the parsley chalcone synthase gene promoter. In addition, they found an upstream region that enhances the light responsiveness of the chalcone synthase promoter twofold to fivefold.

Table 2. mRNA Accumulation

mRNA Homologous to: R Allele (Plant)

R C2 A1 Bz2 Bzl _ _ _ _ _ _ _ _ _ _ _ ~ L" D L D L D L D L D

-r(pigment)

+++ ++ +++ - +++ - +++ - +++ + +++ + ++ - - - - - + + +

-g(nopigment)

The degree of response can only be compared within a gene, not between different genes. * L, light; D, dark.

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The Plant Cell

Although they observed no in vivo footprint in this region, it is possible that the mechanism of increased light responsiveness is mediated by protein-DNA interaction at this site. We see enhancement of light-mediatedC2 expression in the presence of a functional R gene and speculate that this may be a result of direct DNA binding of the R gene product (Ludwig et al., 1989) to upstream C2 sequences. In the aleurone, structural gene expression is coordinately regulated by R, including the initial step, which is catalyzed by chalcone synthase (Dooner and Nelson, 1977; Dooner, 1983). In young seedlings, the structural genes C2, A l , B z l , and Bz2 are not coordinately expressed nor is chalcone synthase expression regulated by the R locus (-r). R gene regulation is first detected at the Al-catalyzed step, which is well into the anthocyanin pathway. Why is C2 expression regulated so differently in the seedling as compared with the aleurone? Perhaps the dominant alleles of the plant- and seed-pigmenting components of the R locus are not equivalent. They appear indistinguishableby RNA blot analysis (Figure 4B; Ludwig et al., 1989), but small sequence differences or posttranslational modification may yield functionally distinct proteins. Our finding of a similar-sized transcript in -g lines offers an opportunity to identify the sequences required for a functional -r gene. Although the -g allele does not provide R function in regulating anthocyanin, it should be noted that it does retain light inducibility with accumulation kinetics similar to the functional allele. Another possibility to account for tissue differences in R-mediated regulation of C2 is that, in young tissue (developing seedlings), there is a heavy demand for the products of CHS activity. In situ localization of CHS protein with antibody showed that the highest levels of protein were in newly unfolding leaves of parsley seedlings, and, as the tissue aged, the protein dropped to near undetectable levels (Jahnen and Hahlbrock, 1988). The requirement to produce flavonoid compounds in response to developmental or environmental signals may not exist in the kernel. In terms of light-induced stress under field conditions, the developing kernels on the ear see very low levels of light (Taylor, 1988) in contrast to the high fluence rates of ultraviolet and visible light encountered by emerging seedlings. A third explanation for the functional difference between seed and plant R regulation is that tissue-specific factors may be required to interact with the regulatory gene, structural genes, or both. Coe (1985) described how functional differences are determined not only by genotype but by the interaction of genes with “effectors” such as position, time, place, stage, conditions, and history. A large number of regulatory and structural genes that are differentially responsive to developmental signals and environmental stress can result in sensitive modulation of the flavonoid biosynthetic pathway. Therefore, the complexity of anthocyanin regulation is not unexpected, and it is not surprising to find that anthocyanin regulation is not identical in all tissues at all times.

METHODS

Genetic Material

The seed stocks for these studies were provided by E.H. Coe, Jr. and D. Hoisington, Department of Agronomy, University of Missouri, Columbia, MO (stock numbers available on request). All lines were homozygousdominant for the structural genes required for anthocyanin biosynthesis in the plant. For comparisons between regulatory alleles, sister strains were made homozygous dominant or recessive at the three regulatory loci listed in Table 3. The sister strains were established by a series of intercrosses between lines previously converged into a W23 backgroundby a minimum of three back-crosses (excludingthe initial cross). When all loci were established in the homozygous state, the maximum probabilityof a modifying factor co-segregatingin one of the lines is 0.0625 (assuminga single unlinked factor present in one or the other original parent).However, in many comparisons, the degree of isogenicity was greater than this. The source of Pl, Al, A2, Bzl, 822, C2, and f r alleles may be assumed to be W23, and the W23 parent is the specific source of the b and pl alleles. The 8-1 and R-r genes plus derivatives are from Coe stocks. The source of the Lc gene used in the RNA blot in Figure 4 is the Sn-s allele (a gift of G. Gavazzi, Universita di Milano). Growth Conditions and Light Treatment

Seeds of the appropriate genotype were imbibed (20 min), sterilized with 5% hypochlorite (10 min), rinsed, and planted in small flats of vermiculite in room light. After growth for 3 days in total darkness at 29’C, a “dark” specimen (-48 hr in the figures) was harvested consisting of the coleoptile and enclosed tissue (average size = 2.5 cm). The flats were irradiatedwith low fluence-rate RL for 48 hr, during which time the seedlings reached the twoleaf stage and were photosynthetically competent. They were then transferred to high fluence-rate WL, and specimens were harvested after 12 hr, 24 hr, 48 hr, or more hours of illumination. The time of transfer is designated the O-hr timepoint. Continuous red illumination (2 pnol m+ sec-’) was provided by four 25-W incandescent bulbs filtered through red acrylic (Shinkolite 102, Argo Plastic Co., Los Angeles, CA) and included wavelengths between 610 nm and 800 nm. The high-intensitywhite light was generated by 1000-W metal halide high-intensityflood lamps (GE R1000, Multi Vapor),which generate a broad band spectrum from 290 nm to 800 nm. The young seedlings received 1200 wmol m-’ sec-’ of white light. This fluence rate was determined with a Li-

Table 3. Regulatory Loci Used in This Study

Locus

Dominant Allele (Pigmenting)

Recessive Allele (Nonpigmenting)

R -r 8-1 PI

r

R (complex)

Seed component Plant component B PI

-9 b

P’

Genetic Regulationof Anthocyanin Accumulation

1800 spectroradiometer (Licor, Lincoln, NE). Material harvested consistedof the sheath and underlyingtissue dissectedjust above the coleoptilar node to the auricle of the first leaf (except for the “dark” specimen, mentioned above). In most cases, each timepoint consisted of a pool of 12 to 15 seedlings (minimumof 6). Anthocyanin and RNA lsolation

Pooled seedling tissue from each timepoint was ground to a fine powder in a mortar and pestle in liquid nitrogen. An aliquot was placed in 1% HCI, and the total anthocyanins were extracted accordingto the procedureof Pettipher(1986).The optical density at 530 nm was normalized to 1 g of dry weight tissue. Total RNA for slot blots was extracted from the remainingsample according to Cone et al. (1986), followed by two lithium chloride precipitations. Poly(A+) RNA for gel blot analysis was isolated using oligo(dT)-cellulose as described by Maniatis, Fritsch, and Sambrook (1982).

Slot and RNA Gel Blot Analysis

RNA gel analysis, radiolabeling, hybridization, and subsequent signal removal conditions are described in Taylor and Walbot (1987). lsolated insert fragments from each plasmid were radiolabeledand added to the hybridizationmixture at 2 x 1O6 counts per milliliter of solution. For RNA slot blot analysis,a dilution series of total RNA (usually 5 pg, 1O pg, and 20 pg) was denatured by heating to 70°C in a 6% formaldehyde solution in 20 mM sodium phosphate, adjusted to 1 M ammonium acetate, and applied to Genatran nylon membrane (Plasco) using a Manifold II slot blot apparatus (Schleicher & Schuell). RNA was fixed to the filters by UV cross-linking. Each filter was sequentially probed with the various anthocyanin gene probes using the same hybridization conditions used for the RNA gel blot analysis. After each hybridization and exposure, the signal was removed and the blot monitored by autoradiography to confirm that no residual radioactivity remained. Lastly, each filter was probed with the insert from pZm9, a cDNA clone containing the cytosolic glyceraldehyde-3phosphate dehydrogenase (GAPDH) sequences from maize (Brinkmann et al., 1987). All autoradiographs were scanned densitometrically and each value was normalized against the GAPDH hybridization signal to correct for loading variation. Each slot blot experiment was repeated at least three times with different material. The following anthocyanin gene probes were used in this study and kindly providedby the indicated authors: C2, pC2-c46, a cDNA probe of the C2 locus (Wienandet al., 1986);A 7 , a 600bp Pstl fragment of pALC-2 encompassing a transcribed portion of the gene (Schwartz-Sommer et al., 1987); 6z7,pD3MS9, a 620-bp probe of the transcribed region (Schiefelbeinet al., 1985); 622, pP300, a 300-bp Pstl segment that hybridizes to 622 RNA (McLaughlinand Walbot, 1987);and pR236, a 900-bpcDNA clone of Lc. a member of the R gene family (Ludwiget al., 1989). Protein Analysis and Enzyme Assays

CHS enzyme assays were performed as described in Feinbaum and Ausubel(1988),with the followingmodification.The extraction

125

procedure was modifiedby the addition of insoluble polyvinylpyrrolidone and quartz sand during grinding in a buffer consisting of 0.1 M phosphate, pH 7.2,20 mM ascorbate, 10 mM dithiothreitol, and 7.5 mg/mL BSA (adaptedfrom Britsch and Grisebach,1985). Each assay used 25 pL of plant extract, and 1O0 pL of the ethyl acetate phase was spotted on a glass-fiber disc and quantitated by scintillationcounting. Thin-layer chromatography showed that greater than 98% of the i4C in this fraction co-migrated with naringenin. For protein gels, maize leaf sheaths of the appropriate genotypes were homogenizedin the buffer described above, omitting the BSA. After removal of cellular debris by low-speedcentrifugation, the total soluble proteinswere precipitatedwith 10% TCA and resuspended in 50 mM sodium carbonate, and 50 pg of protein was subjectedto SDS-PAGE in gels consisting of a 10% to 18% gradient of polyacrylamide.The separated polypeptides were electrotransferredto nitrocellulose and incubatedwith rabbit antiserum raised against parsley chalcone synthase protein (Schroder et al., 1979). Bound IgG was visualized with a commercial avidin biotinylated horseradish peroxidase conjugate following manufacturer’s directions (Vector Laboratories, Burlingame, CA). Total protein was determined by the Bradfordmethod (1976) using BSA as the standard. The CHS antiserum was the kind gift of K. Hahlbrock, Max-Planck-lnstitut,Koln, Federal Republic of Germany.

ACKNOWLEDGMENTS

We thank E.H. Coe, Jr. and David Hoisington, University of Missouri, for the generous gifts of plant material and insightful discussions; Rijdiger Cerff of the Universite de Grenoble for the GAPDH clone; Rhonda Feinbaum for advice and the gift of p coumaroyl-COA;Timothy Short for advice and help with protein gel blots; and Neil Hoffman, Bruce Baker, Virginia Walbot, Rosemary Redfield, and Gary Peter for critical reading of the manuscript. We also thank Vicki Chandler and Susan Wessler for prepublication results. L.P.T. was supported throughout the period of this research by National lnstitute of General Medical Sciences fellowship award 1F32 GM 10893. This is Carnegie lnstitute of Washington Department of Plant Biology publication no. 1049.

ReceivedJune 26,1989; revised November 20,1989.

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Genetic Regulation of Anthocyanin Accumulation

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