Genetic and developmental control of anthocyanin biosynthesis.

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GENETIC AND DEVELOPMENTAL CONTROL OF ANTHOCYANIN

Annu. Rev. Genet. 1991.25:173-199. Downloaded from www.annualreviews.org by Michigan State University Library on 03/12/13. For personal use only.

BIOSYNTHESIS Hugo K. Dooner and Timothy P. Robbins DNA Plant Technology Corporation, Oakland, California 94608

Richard A. Jorgensen Department of Environmental Horticulture, University of California, Davis, California 95616 KEY WORDS:

plants, flavanoids, regulatory genes,

trans interactions

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .

1 73

STRUCTURAL GENES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

174 1 74 1 75

The Anthocyanin Biosynthetic Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Isolation of Anthocyanin Structural Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . REGULATORY GENES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of Regulatory Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Isolation of Regulatory Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anthocyanin Regulation in Maize. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maize Regulatory Genes Encode Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo Studies of Regulatory Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural Gene Requirements for Trans-activation . . .... . . . .. . . . . . . . . . . . . . . . . .... Higher Levels of Anthocyanin Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ALLELIC AND ECTOPIC INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . .. . . Paramutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dominant CHS A lleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ectopic Transgene Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Speculations on trans Interaction Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 79 1 79 181 182 183 184 185 186 187 187 191 1 92 1 93

INTRODUCTION Most of the bright red and blue colors found in higher plants are anthocyanins. The conspicuous and dispensable nature of these pigments has made them favorite objects of study of plant geneticists and this, in tum, has resulted in 173

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DOONER, ROBBINS, & JORGENSEN

the accumulation of a wealth of knowledge concerning the structure, function, and interaction of genes involved in their synthesis . The advent of plant molecular biOlogy in the past decade has brought about renewed interest in the subject and a veritable explosion of molecular work on anthocyanin biosynthetic genes. Not surprisingly, the field of anthocyanin biosynthesis has been the subject of several comprehensive recent reviews (66, 102, 105). We approached the writing of this review with a twofold intention: to emphasize genetic, rather than purely physiological or biochemical, pheno menonology and to minimize, to the extent possible , overlap with the prev ious reviews. We did not want this review to be simply a directory of anthocyanin genes from many plant species or a compendium of anthocyanin biosynthesis in plants . We chose, therefore, to concentrate on genes that had been defined by mutations and for which there existed molecular information, criteria that led us to restrict our discussion largely to three plant species: maize, Antirrhinum, and petunia. We have divided our review into three sections, dealing with, respectively, the isolation of structural genes, i.e. those known to encode enzymes in the pathway, the isolation, and characterization of regulatory genes and their mode of action and, finally , the allelic and ectopic interactions evidenced .by anthocyanin biosynthetic genes. STRUCTURAL GENES

The Anthocyanin Biosynthetic Pathway Figure I shows the core linear pathway of anthocyanin synthesis. An thocyanins belong to the general class of phenolic compounds known as flavonoids. The first committed step in flavonoid biosynthesis is the con densation of three molecules of malonyl CoA and one of p-coumaroyl CoA by the enzyme chalcone synthase (CHS) to produce a yellow chalcone. The second step, the isomerization of the chalcone into a colorless flavanone, proceeds spontaneously at a low rate, but is accelerated by the enzyme chalcone-flavanone isomerase (CHI) . The flavanone so formed is hydroxy lated at the C3 position by the action of flavanone 3 -hydroxylase (F3H) to give an unpigmented dihydroflavonol that is reduced by dihydroflavonol 4reductase (DFR) to yield a still colorless leucoanthocyanidin. This compound is then converted into a colored anthocyanidin in either one or two steps, catalyzed by as yet undescribed enzymes (46). The last step shown, glycosyl ation of anthocyanidin to give an anthocyanin, is catalyzed by the enzyme UDPglucose flavonoid 3 -oxy-glucosyltransferase (UF3GT) . Table I lists the structural genes for each of the above enzymatic steps that have been defined mutationally and/or isolated molecularly in maize, Antirrhinum and petunia, the three best-characterized plants .

CONTROL OF ANTHOCYANIN BIOSYNTHESIS

175

CO-SCoA

6OH

CO-SCoA I

CH, I

coo·

p-Coumaroyl-CoA

3 Malonyl-CoA

Ic

HS


HO�OHO W OHO �

OH

l CHI

HO.-,,�o..n° -W OHO ...... HO

l

Chalcone

H

�OH OH

F3H

OH HO�on V--) OHOHo: DFR

Leucoanthocyanidin

�l*

HO

at

OH OH

l

Anthocyanldln

UF3GT

Q::x?°H OH

Figure 1

Dihydroflavonol

0

l

HO

Flavanone

Anthocyanin

Glucose

A simplified view of anthocyanin synthesis, showing the core linear pathway from the

CoA esters of malonic and p-coumaric acids to a glycosylated anthocyanin. See text for the full names of the various enzymes.

Molecular Isolation of Anthocyanin Structural Genes The isolation of anthocyanin structural genes has been accomplished by means of biochemical, genetic, and molecular strategies, often in combina tion. Biochemical strategies involve the use of antibodies prepared against purified gene products and are most useful for structural genes that encode enzymes that can be assayed in vitro and that are sufficiently stable during purification (56). Genetic strategies involve the use of transposable elements to induce mutations in an anthocyanin gene, which can then be isolated by the use of a physical probe for the transposon (29). Molecular strategies u tilize the fact that transcripts of anthocyanin structural genes are differentially

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JORGENSEN

Enzymes of the anthocyanin biosynthetic pathway for

which genes have been defined mutationally and/or isolated molecu larly in maize,

Antirrhinum and petunia Maize

Enzyme

Antirrhinum

Locus

Clone

Locus

Clone

c2 whp

+

niv

+

+

inc pal canb

+

CHS

+

CHI F3H


DFR *

a

UF3GT 'See Figure

al a2 bzl I

and text

+ + +

+

b can is used here as the abbreviation for

+

Petunia

Locus

Clone

+ po an3 an6

+ + +

+ + candica

regulated by different alleles of regulatory genes. Thus, cDNA libraries prepared from tissues in which anthocyanin genes are expressed can be screened with RNA probes from the same tissues of genotypes that do not express these genes . Because several different genes are likely to be ident ified in this way, particular genes must be identified using additional tests (67). Once a gene has been isolated by any of these methods, it is often possible to use heterologous hybridization to isolate a homologous gene from another species , depending on the evolutionary distance between the two species and the degree of sequence conservation (73, 108). A powerful improvement over the heterologous hybridization method uses polymerase chain reaction (PCR) amplification to isolate genes from distantly related species . This approach requires sequence conservation over only a short length of DNA and so is potentially much more sensitive than is heterologous hybridization (40, 59). In order to determine unequivocally the identity of a clone isolated by heterologous hybridization or PCR amplification, it is necessary to perform appropriate biochemical and/or genetic tests . A simpler, though less certain, alternative is to compare the DNA sequence of a clone with a known gene. Sufficient similarity is often taken to indicate functional relatedness; however, because enzymes are known to sometimes evolve new functions, this approach provides only suggestive, though useful, information. CHALCONE SYNTHASE The first flavonoid gcnc to be physically isolated was a chalcone synthase (CHS) gene from parsley (Petroselinum hortense) (56). A cDNA library made from poly A+ mRNA from UV-irradiated suspension culture cells was screened first with radioactively labeled RNA from light-induced and dark-grown cells. Clones hybridizing specifically to the light-induced RNA probe were then screened with a CHS-specific anti-



177

serum in hybrid-arrested and hybrid-selected translation assays to identify a clone homologous to CHS mRNA. The maize c2 gene , which encodes a CHS enzyme expressed in the aleurone layer of the seed (23), was isolated by transposon tagging using the element SpmlEn ( 109) . Comparison of the DNA sequence of the maize c2 gene with that of the parsley gene was used to show that the genes are homologous. Subsequently , whp, a second CHS gene that is normally ex pressed in plant tissues other than the aleurone, was isolated by virtue of its homology to the c2 gene ( 107) . The nivea gene from Antirrhinum, encoding a floral CHS enzyme (88), was isolated by hybridization with the parsley CHS clone . Southern hybridization analysis of transposon-induced mutations and revertants of nivea was used to establish that this CHS-homologous clone corresponded to thc nivea locus (l08). Thc parsley CHS clone was also used as a probe to isolate CHS cDNA clones from petunia (82) . Because there are no known mutants of a CHS encoding gene in petunia, several pieces of evidence dependent on homology alone (hybrid-release translation, correlations between mRNA expression and CHS activity in flowers, and DNA sequence comparisons) were provided to argue that the clone corresponded to a CHS gene. Subsequently, it was shown that 12 CHS-homologous genes are present in the petunia genome, but that only two of these (CHS-A and CHS-J) are expressed at significant levels in flowers (54). CHS-A sequences were used to construct an antisense gene that was found to interfere with anthocyanin pigment production and transcription of both CHS-A and CHS-J, as well as CHS enzyme activity in flowers of transgenic petunias ( 100) . It is possible that both CHS-A and CHS-J encode a functional CHS , or that only one does. CHALCONE-FLA VANONE ISOMERASE The petunia po gene, which encodes chalcone-flavanone isomerase (30), was isolated from a cDNA expression library. Clones encoding CHI were detected with antiserum to purified CHI ( 104). Correspondence between these clones and the po gene was first indicated by RFLP mapping and then demonstrated unequivocally by com plementation of a po mutant with an introduced CHI gene ( 106) . An Anti rrhinum cDNA clone encoding a putative CHI gene was isolated by heterologous hybridization with the petunia clone, with which it was then found to be highly homologous (67). Recently, a maize CHI cDNA was isolated by PCR amplification using primers complementary to conseved sequences and shown to be regulated by the anthocyanin regulatory gene P (40).

A combination of differential cDNA clon ing and genetic screening of candidate clones was used to isolate a cDNA

FLAV ANONE 3-HYDROXYLASE


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clone corresponding to the incolorata (inc) locus in Antirrhinum, which is likely to encode flavanone 3-hydroxylase (F3H) (67). The differential cloning strategy utilized two alleles of the regulatory gene delila, which is known to control the expression of several other genes in the pathway. Candidate clones were screened against inc mutants and several were found to correspond to a message that was present in wild-type plants and absent in several inc lines. Restriction fragment length (RFLPs) were then used to show by linkage analysis that the clones originated from the inc locus. The petunia F3H , which appears to be encoded by the an3 locus (31), has been isolated by heterologous hybridization with thc Antirrhinum F3H gene (A. Prescott. T. Gerats, C. Martin, unpublished data). DIHYDROFLA VONOL 4-REDUCTASE Transposon tagging was used to isolate dihydroflavonol 4-reductase genes from maize and Antirrhinum. The maize DFR gene, aI, was isolated with the transposable element SpmlEn (75). The in vitro translated product of an Al cDNA was found to have dihydroflavonol 4-reductase activity (81). The Antirrhinum DFR gene, pallida, was isolated with the transposable element Tam3 (65 ). Comparison of the DNA sequences of the maize and Antirrhinum genes indicated that they were highly homologous and, therefore, likely to encode the same enzymatic function ( 1 9, 86). The Antirrhinum clone was used to isolate homologous clones from petunia, of which one was inferred to encode a DFR gene by comparison of its DNA sequence with that of the maize and Antirrhinum genes. This clone was postulated to correspond to the an6 gene, since the an6 mutant accumulates dihydroflavonols but has normal UF3GT activity (3).

A gene CONVERSION OF LEUCOANTHOCY ANIDIN TO ANTHOCYANIDIN involved in the conversion of leucoanthocyanidin to anthocyanidin has been identified and isolated in both maize and Antirrhinum. The maize gene, a2, was isolated by transposon tagging with SpmlEn (71), while the Antirrhinum gene, candica, was cloned by a combination of differential cloning and genetic analysis. DNA sequence comparisons strongly suggest that these genes encode the same enzyme. Because the enzyme shows homology to alpha-ketoglutarate-dependent dioxygenases, including F3H, it has been sug gested that a dioxygenase is involved in the conversion of leucoanthocyani dins to anthocyanidins (66). No gene has been cloned that corresponds to the hydratase that was proposed on biochemical grounds by Heller & Forkmann (46) to complete the conversion to anthocyanin. However, it has been sug gested that incolorata 1 (not allelic to incolorata) in Antirrhinum might encode this enzyme (66). The maize bzl gene encoding UF3GT (57) was isolated by transposon tagging with Ac

UDP-GLUCOSE FLAVONOID 3-0XY-GLUCOSYL TRANSFERASE


179


(28, 29). A putative UF3GT clone from Antirrhinum has been isolated by the method of heterologous hybridization using the maize UF3GT clone as a probe (67). DNA sequence comparisons of the Antirrhinum clone with the maize gene strongly suggest that this clone encodes UF3GT. OTHER STRUCTURAL GENES The maize bz2 gene, which acts late in the pathway but whose function remains unknown, was isolated independently by transposon tagging with Ds (97) and by a combination of transposon tag ging with Mul and a differential hybridization screen (69). In the latter effort, a genomic library from a line carrying a Mul insertion at bz2 was used to isolate clones homologous to the transposon. These clones were then screened by differential hybridization to identify a clone homologous to tran scripts under the control of anthocyanin regulatory genes. Final proof that a candidate clone corresponded to bz2 was obtained by Southern hybrid ization of insertion mutations and their revertants . No other genes that en code structural modifications to anthocyanins have been reported to be cloned.

REGULATORY GENES

Identification of Regulatory Genes Regulatory genes that act upon the structural genes of the anthocyanin biosynthetic pathway have bccn idcntificd in all plants in which anthocyanin genetics is well established. The best-characterized systems of regulatory genes are those found in Antirrhinum (64), petunia (110), and maize (17). More recently regulatory genes have been reported in Pisum sativum (42) and Lycopersicon esculentum (74), although regulation appears to be limited to the CHS step . We focus on regulatory genes that act in a more general fashion on two or more structural genes of the anthocyanin pathway. Evidence for such regulation can be obtained by either enzyme or mRNA assays for structural gene activity in plant material carrying different alleles at regulatory loci. Table 2 presents a summary of such information available for regulatory genes described in Antirrhinum, petunia, and maize. Three steps, in particular, have been intensively studied for evidence of regulation: CHS, DFR, and UF3GT. It is interesting to compare the type of regulation that occurs in the three species. In maize, the regulatory genes appear to act on all steps of the flavonoid pathway that are specific to anthocyanin biosynthesis (CHS, CHI, DFR, UFGT). One possible exception is the CHS step, which is under R gene regulation in the aleurone layer of the seed (23, 62) and in the scutellar node and mesocotyl of young seedlings (98), but apparently not in the leaf sheath of young seedlings (96). However, while the aleurone CHS is encoded by C2, the seedling CHS may or may not be encoded by the same gene .

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DOONER, ROBBINS, & JORGENSEN Table 2

Regulatory genes of the anthocyanin pathway described in selected species

and the structural genes upon which they act" Sample of structural genes assayed Species

Antirrhinum

Regulatory genes

Delila

CHS

DFR

NR (64, 67)b

R (I, 64,67)

R (I)


Eluta Rosea Petunia

Ani, 2 , 10,11

Maize

R(S)

UF3GT

E (64) R (67)

NR (67)

R (67)

R (67)

NR (66)

R (66)

R (66)

R (3)

E (3,36)

R (62)

E (26)

NR (96)

R (96)

R (96)

R (98)

R (98)

E (23) R (62)

myc

R(P) R(Sn) B

R (14)

E (33) R (14, 28)

Cl

E (23)

R (21)

E (26) R (21)

myb

PI P Vpl

E (33) R (39)

R (39)

E (23)

E (27) R (68)

• Trans-regulation of structural genes as demonstrated by enzyme assay (E) or steady state mRNA level (R). The prefix N indicates that no regulation was observed. The tissues assayed were, generally, corollas or subregions thereof for Antirrhinum and petunia, and aleurones,

seedlings or husk leaves for maize. The initial reports of quantitative CHS regulation by delila (I) have not been substantiated in more detailed experiments (67), It should be noted that tbe regulation of BzI by R(P) is based on the Northern rather than tbe slot blot data b References in parentheses

(96),

In Antirrhinum flowers, three regulatory genes , delila, eluta, and rosea, have been shown to act only on the latter section of the pathway ( 1, 66, 67) . While the early CHS and CHI steps show minimal regulation, four subsequent steps (F3H through UFGT) have an absolute requirement for the Delila gene product and show quantitative regulation by eluta and rosea, In petunia mutations at four loci, an} , an2, an}O and an} } , have similar regulatory effects on UF3GT enzyme and DFR mRNA levels (3, 36) . Since these mutants also accumulate dihydroflavonols (34), this suggests that CHS, CHI, and F3H are not regulated, indicating that petunia regulates the pathway from DFR onwards, one step further on than in Antirrhinum. It has been speculated that these species differences reflect selection for the synthesis of flavones and flavonols , products of the flavonoid pathway that contribute to copigmenta tion in Antirrhinum and petunia, but not in maize (67) . Alleles of certain regulatory genes alter the pattern rather than the amount of pigment produced. The best-characterized examples are the Rand B loci of maize that determine a wide range of pigmentation patterns over a diverse set



181

of plant tissues (95). In Antirrhinum, pigmentation of the corolla tube, but not the corolla lobe, requires the action of the delila gene that also affects pigmentation in the sepals, leaves, and stems (1). The Eluta allele restricts pigmentation to p roduce a distinct pattern in the corolla, whereas alleles at the rosea locus affect the distribution of pigment between inner and outer epidermis as well as in a variety of plant tissues (66). This diverse control of pigmentation patterns is reminiscent of the R and B loci in maize. In petunia, certain regulatory genes confer differences between tube and limb pigmenta tion of the corolla; this patterning is similar to that resulting from the action of Delila (110). In general, the tissues that are affected in all three species show an absolute requirement for a particular regulatory gene in dominant condition to cause pigmentation. Two exceptional negative regulators of pigmentation are the Eluta allele in Antirrhinum and the CI-I allele in maize .

Molecular Isolation of Regulatory Genes The technique of transposon tagging, well developed in both Antirrhinum and maize, has been extremely powerful in the isolation of anthocyanin regulatory genes where no prior knowledge existed concerning the nature of the gene product. Unstable alleles that arise from transposable element insertions were available for several regulatory loci in maize . The first to be isolated was the CIlocus using the SpmlEn transposable element (21, 79). Subsequently, the R and P loci were isolated using the Ac transposable clement (15, 22). Most recently, the VpI locus has been isolated using a Mutator induced allele (68). Following the isolation of these regulatory genes, it was possible to test whether additional regulatory loci could be isolated by virtue of DNA se quence homology. Using an RFLP approach, it was shown that a CI homologous sequence mapped to the PI locus (20) and subsequently that the R and B loci were also homologous (14). These homologies permitted the isolation of both the PI and B genes; they also define two families of maize regulatory genes, the R I The isolation of regulatory genes from both Antirrhinum and petunia has only recently been reported . In Antirrhinum, the delila gene has recently been isolated by transposon tagging using the Tam2 transposable element. A Delila cDNA has been isolated and appears to share homology with the R gene family of maize (W. 1. Goodrich, E. S. Coen, & R. C arpenter, personal communication). In petunia the AnI locus shows homology to the DFR structural gene (35). However, the AnI locus is not yet well characterized and it is conceivable that a DFR homologous sequence lies within a maize-type regulatory gene. It will be of interest to determine whether additional regula tory genes isolated from petunia and Antirrhinum have functional equivalents in maize and how this relates to the distinct levels of anthocyanin pathway regulation observed.

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Anthocyanin Regulation in Maize Maize represents a paradigm for the study of anthocyanin regulation since several genetically defined regulatory genes have been isolated. Of the 20 or so genes that affect anthocyanin production in maize (17), six have es tablished regulatory functions as defined by the criteria in Table 2; R(includes Lc and Snl), B, CI, PI, P, and VpI. With the exception of P and VpI, this regulation is highly specific to 3 -hydroxy anthocyanin production . The P locus regulates the 3 -deoxy anthocyanin biosynthetic pathway, which shares certain intermediates , and VpI has pleiotropic effects on seed dormancy, suggesting a more general role. We consider the first five factors in some detail before returning to the role of the latter in anthocyanin regulation during development. The alleles at R show more phenotypic variation than at any other locus in higher plants, but typically condition pigmentation of the aleurone, in addi tion to other tissues of the embryo, seedling, and mature plant (89, 90). The structurally related, R ( uniquely pigment the leaf blade, scutellar node, and mesocotyl (25, 3 2) . The R locus i tself may comprise duplicate genes that are organized in a tandem array, each gene conferring a distinct pattern of pigmentation (91). In contrast to R, color but frequently condition extensive color in mature plant tissues, notably the leaf sheath and husk leaves. Despite these differences , certain R and B alleles pigment the same tissue (e . g. R(S) and in addition show duplicate factor inheritance . This led to the notion that the Rand B loci are duplicate genes (95) , a prediction that was later confirmed at the molecular level (14). An R or B allele is , however, not sufficient to produce pigmentation in a particular tissue without the appropriate allele of either CI or Pl. Although the related CI and PI factors display tissue specificity in the regulation of anthocyanin biosynthesis, there is evidence, as well, that these genes play a role in the regulation of the pathway by light. The C I gene is required for the synthesis of anthocyanin in the aleurone in combination with a suitable R or B allele. The recessive alleles fail to accumulate pigment in the kernel, but one allele, clop, displays a dependence on high light intensity for pigmentation during germination (52) . An interesting dominant allele, CI-I, inhibits the synthesis of pigment in a C l-lICI heterozygote. The Pl gene displays a similar control over the light dependence of pigment accumulation in a variety l

In this review we use the following nomenclature for the members of the R gene family: R(P).

the plant-pigmenting component of the R-r complex; R(S). the seed-pigmenting component of

R-r; R(Lc), the leaf-pigmenting component of the Lc displaced repeat; R(Sn), the scutellar node-pigmenting component of the Sn displaced repeat.



183

of mature plant tissues. The recessive allele pi results in a requirement of direct sunlight for pigmentation in a variety of mature plant tissues, depending on the RIB constitution. The dominant allele PI renders this pigmentation less light-dependent and has a considerable intensifying effect. Thus, it is possible for inner husk leaves, which would be green in a B pi genotype, to achieve intense coloration in a B PI line. The P locus shows a high degree of tissue and pathway specificity. The P locus controls the biosynthesis of anthocyanin-derived pigments called phlo baphenes in the ovary wall (pericarp) and a variety of other floral parts. Since the products of the P locus are 3-deoxy anthocyanins, distinct from the 3-hydroxy anthocyanins conditioned by R, B, Cl, and PI, it has been pro posed that P regulates a parallel biosynthetic pathway that shares certain intermediates (94). Interestingly, this is not a metabolic limitation of pericarp tissue since certain R alleles (known as the R-cherry alleles) can condition 3-hydroxy anthocyanin biosynthesis in pericarp. Since P alone is sufficient for peri carp color, it is not clear whether P acts as a single factor or requires a cofactor similar to RIB and ClIPI.

Maize Regulatory Genes Encode Transcription Factors The C 1 locus appears to encode at least two C l-specific transcripts of 1.4 and 1.6 kb in aleurone tissue (21, 79) . A putative full-length cDNA was found to encode a 273-amino acid protein (78) . At the C terminus the protein contains a short acidic region that may adopt an alpha helical configuration characteris tic of many transcription activation domains (77). Consistent with such a role in transcription activation, the N-terminal region contains a 114-amino acid basic region with 40% homology to the myb proto-oncogenes defined in mammalian systems. These genes are known to encode nuclear proteins with DNA-binding capacity that act as transcriptional activators. The sequence of the PI gene shows 90% homology with Cl, although the transcription unit has still to be defined (20). Three R alleles have been characterized at the transcriptional level, R(P), R(S), and R(Lc) (62). All three produce a single 2 . 5-kb transcript in tissues pigmented by the respective alleles. A cDNA that encodes a 610-amino acid protein was isolated for R(Lc), the R gene that exhibited the highest level of expression (62). The putative protein has an extensive acidic N-terminal region (220 amino acids) characteristic of a transciptional activator. A shorter basic region at the C terminus (93 amino acids) shares homology with the helix-loop-helix (HLH) motif characteristic of the myc family of proto oncogenes (63). Additional R sequences have been reported following the isolation of cDNAs from tissues that are pigmented by the R(S) and R(Sn) alleles (80, 98). The R(S) gene shows 95% homology to the original R(Lc) cDNA at the DNA level, with most differences being either conservative or in


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the untranslated leader. The sequences of two B alleles, B-Peru and B-1, are also highly homologous to the other members of the R gene family (38). The P locus has a complex pattern of transcription. Several P-specific transcripts are present in w ild-type and absent in an insertion mutation (58). The sequence of two cDNAs has been reported that appear to arise from alternative splicing (39). The longer of the two transcripts encodes a protein w ith a 1I8-amino acid N-terminal sequence that shows 40% homology w ith members of the myb family of proto-oncogenes . S ignificantly, the homology w ith the CI protein sequence was even greater (70%). Taken in conjunction w ith a conserved exon/intron structure between CI and P, this suggests thatP may be a distantly related member of the C l IPI factors.

In vivo Studies of Regulatory Genes One approach to support the inferred role of the R, B, CI, PI, and P gene products as transcription factors is to test for DNA-binding activity that is specific to the promoters of the structural genes that are regulated. This has been attempted for the CI product that, when synthesized in vitro , was unexpectedly shown to bind to 3 I untranslated sequences in addition to promoter regions of the regulated Al (DFR) gene (107) . An alternative approach, with more direct relevance to in vivo gene regulation , is to use the maize transient expression system . Microprojectile bombardment of develop ing aleurones makes it possible to complement structural gene mutations, resulting in pigment accumulation (53). Using structural gene promoter fu sions to a luciferase reporter gene , it is possible to study single gene, as distinct from whole pathway, regulation. Using this system, the Al (DFR) and BzI (UFGT) structural genes were shown to require both CI and R for e xpression in aleurones . Thus cJ r, Cl r or cJ R genotypes failed to induce transcription of Al or Bzl promoters, consistent with previous expression studies (Table 2) and the unpigmented phenotypes conferred. The first demonstration of complementation using a regulatory gene in volved the R(Lc) coding sequence under the control of the CaMV 35S promoter (61). This "constitutive" promoter allowed the expression of pigmentation in tissues other than those normally pigmented by R(Lc). This extended pattern of R gene expression was still restricted by the action of the PI gene or light, suggesting that constitutive expression of an RI is not sufficient for pigmentation. Goff et al (38) have used similar constructs based on two B alleles (B-Peru and B-1) to investigate the requirements for transactivation of both Al and BzI promoters linked to a luciferase reporter. They showed that either the B-Peru or B-1 gene products were able to transactivate these structural gene promoters in aleurones of r b CI genotype. They extended the analysis to normally unpigmented embryogenic callus



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and demonstrated a requirement for both B and C 1 gene products in r b cl material. This system has made possible the in vitro manipulation of both the RIB and Cl proteins to study the nature of their in vivo interaction with the Bzl promoter. Substantial progress has been made in the functional dissection of the Cl gene product (37) . Both Cl genomic sequences and CaMV 35S promoter fusions to Cl coding sequences can provide a Cl function and induce pigmentation in c1 R aleurones. Using the B z system, it was shown that frameshift mutations in either the basic or acidic domains could abolish the Cl trans-activation function. The Cl acidic do main can act as a nonspecific transcriptional activation domain when fused to th e DNA -binding domain of the yeast GAL4 transcription factor. When this fusion was cobombarded with a GAL4-induced promoter fused to a luciferase gene, trans-activation of luciferase was observed in the maize aleurones. However, in the converse experiment, the GAL4 activation domain was only partially successful in substituting for the Cl C-terminal region. Surprisingly, GAL4 acidic domain substitutions were nevertheless capable of activating the entire pathway, resulting in weak aleurone pigmentation (37) . The transient expression system has also been valuable in examining the basis of dominant inhibition by the C 1-1 allele. A sequence comparison of C 1 and Cl-l revealed several minor changes in the coding region (77). Most significantly, the sequence predicts a premature termination that results in the loss of most of the acidic activator domain. Since the basic DNA binding region is only slightly altered, this led to a model for Cl-/ inhibition involving a competition for binding sites in structural gene promoters (77) . In terestingly, a frameshift mutation introduced in vitro into the acidic domain of Cl was able to inhibit trans-activation by the normal Cl allele in the transient expression system (37) . However, C-terminal substitution of Cl-/ for Cl sequences was not as effective for inhibition as N-terminal substitutions including the basic domain of Cl-/ (37) . This indicates that the minor alterations in the DNA-binding domain are also relevant to the inhibitory phenotype. -

Structural Gene Requirements for trans-activation The maize transient assay system has also been used to study the requirements of the structural gene promoter for trans-activation by a regulatory gene. The Bzl promoter has been dissected by a series of deletion and linker scanning constructs and cis-regulatory sequences have been defined (53, 84). This analysis is similar to stable transformation approaches often used to study cis-regulatory sequences (102). However, the maize system offers the advan tage of studying genetically defined trans-activators such as Cl and R. This approach has revealed that a 31bp promoter sequence is essential for both


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Rand Cl regulation (84) . This region includes sequences similar to those recognized by both myc and myb type proteins. In vitro mutagenesis of the myc type binding site of Bzl resulted in a 30-fold reduction in BICl trans activation, whereas the myb type binding site appears to play a minimal role (38) . In Antirrhinum, structural gene promoters have been analyzed using com binations of promoter mutations induced by transposable elements and muta· tions in regulatory genes. An allelic series has been described for the pallida (pal) gene (DFR) based on the imprecise excision of Tam3 from a position 70bp upstream of the start of transcription (19) . Certain pal alleles carry deletions, yet low levels of pigmentation persist in both the corolla l obe and tube . Almeida et al (1) studied the transcription of these promoter mutants in delila (del) mutant backgrounds. Promoter deletions of up to 20bp had no effect on the del regulation of pal transcription. However, a deletion extend· ing almost IOObp 5 ' and an extensive inversion were found to abolish transcriptional regulation of pal by del. This indicates that a region between ·170 to ·70 of the pal promoter is essential for trans-activation by the Del gene product. The recent observation that the Del gene product is homologous to the RIB gene family of maize (W . J. Goodrich & E. S. Coen, personal communication) should allow a comparison of trans-activator binding sites in the two systems. It will also be interesting to test whether the Del and RIB gene products are interchangeable, for example, will Del function as RIB does in the maize transient expression system?

Higher Levels of Anthocyanin Regulation It is anticipated that the anthocyanin regulatory genes are responsive to a variety of developmental and environmental cues that lead to the initiation of anthocyanin biosynthesis. One example of a gene that is implicated in such a response is the viviparous-l (Vpl) gene in maize. Whereas the cl and r mutations only affect anthocyanin biosynthesis, the anthocyaninless pheno· type of the vpJ mutant is associated with a general failure of the maturation process, resulting in viviparous development of the embryo and pleiotropic enzyme deficiences in the aleurone (24). There is evidence that the vpJ mutant has reduced sensitivity to ABA, an important regulator of seed maturation (83) . The VpJ gene encodes a 2. 5·kb transcript that accumulates i n both embryo and endosperm prior to the expression of CJ (68) . In vpJ mutant tissues, no inducti on of CJ is observed. This may explain the anthocyaninless aspect of the phenotype , although R expression may also be affected. The Vpl gene appears to encode a transcriptional activator that can function to trans-activate the CJ gene in a transient expression system (44) . The dissection of the Cl promoter has revealed a 27bp region responsible for both VpJ and ABA



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regulation but it is not clear whether the VpJ gene product acts directly on the C I gene or via one or more intermediate steps in a regulatory hierarchy. Despite the isolation of several regulatory genes in maize, the mechanism by which these regulatory genes determine tissue-specific patterns of pigmentation is not known. Although the activity of regulatory genes can be detected in pigmented tissues, it is not yet apparent what factors are respons ible for activation in specific tissues. This is illustrated by the Rand B genes, which are expressed principally in different tissues, but which encode similar gene products as judged by transient assays (38, 61) . This result is not surprising in light of the observation that R al1e1es that give different patterns of pigmentation share a conserved region that can be exchanged by recombination, without altering tissue specificity (50) . The interesting ques tion will be the dissection of the different R or B promoter regions that confer tissue specificity of regulation. This may now be approached by means of the transient expression systems, although analysis of R promoter insertion mutants may also be informative (51) .

ALLELIC AND ECTOPIC INTERACTIONS Genes concerned with anthocyanin biosynthesis have figured prominently in the detection, description and analysis of several unusual genetic phenomena involving interactions between alleles and/or between homologous unlinked loci. At present, understanding of these phenomena is limited. This subject is discussed here because we believe that a full understanding of the mech anisms underlying these phenomena is likely to be significant in deciphering general mechanisms of gene expression and control in higher plants.

Paramutation A basic tenet of Mendelian genetics is that the passage of two alleles through the same nucleus for one heterozygous generation will not affect the expres sion of either allele. The phenomenon of paramutation constitutes an apparent exception to that rule. As defined by Brink (8), paramutation is "an interac tion between al1eles that leads to a directed, heritable change at the locus with high frequency and sometimes invariably within the time span of a genera tion". R LOCUS IN MAIZE Paramutation was first de scribed at the R locus in maize (5). A vast body of genetic literature docu ments paramutation at R (see 8 and 11 for reviews), but for this review, we need to concern ourselves with only three al1eles, R-r, R-st, and r-g. R-r conditions anthocyanin pigmentation in both the aleurone (the outer cell layer of the endosperm) and in the plant; R-st gives a spotted or stippled pattern in PARAMUTATION AT THE

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the aleurone, and

r-g is a null allele, producing no pigmentation in either

aleurone or plant. It should be added at this point that the residual genetic background was maintained constant through all the studies of

R locus

paramutation.

R-r is the prototype of R alleles whose level of expression in the aleurone R allele with which they coexisted in the previous generation. The level of expression of R-r is assayed by the amount of aleurone pigment formed in kernels obtained from crosses of r-g r-g females to males carrying R-r. (The aleurone in maize is a triploid tissue, receiving two genomes from the female parent and one from the male parent.) If the R-r allele is extracted from an R-rIR-r homozygote, it will produce a mottled aleurone; if from an R-r Ir-g heterozygote, a darkly mottled to fully colored aleurone, and if from an R-r IR-st heterozygote, a very weakly pigmented aleurone. The R-r allele that is sensitive to the allelic interaction is said to be paramutable (or paramutant, and designated R -r' after exposure to R -Sf) and the R-st allele that suppresses the level of pigmentation of R-r is said to be paramutagenic. Since the r-g effect is mimicked by an r deficiency, r-g is considered to be paragenetically amorphic (92, 93). It follows from the above


depends on the particular

,

observations that the capacity for a heritable change in action is a built-in character of

R-r. The "paramutagenic" R-st allele simply amplifies a kind of R-r is capable of undergoing by itself. The paramutational changes at R -r take place progressively during develop ment of the plant. That paramutation at R is a somatic phenomenon was shown by sampling pollen grains from different tassel branches of R-rIR-st heterozygotes and demonstrating that they carried R -r alleles with varying aleurone-pigmenting potentials (85). The changes are also progressive through successive plant generations. The pigmenting potential of an R-r genetic change that

allele continues to decrease or increase, respectively, if maintained over

R-st or r-g (70, 92). R paramutation is that changes in the structure of the chromosome where the R gene is located have profound effects on the paramutability of R-r. The R locus lies on the long arm of chromosome 10 (10L). Reciprocal translocations (T) involving a break in 10L either proximal or distal to R reduce the sensitivity of R-r to paramutation in T R-rIR-st heterozygotes (6, 9, 10, 12). Similarly, a large, subterminal, heterochromatic knob, known as K1O, renders R-r less sensitive to paramuta tion in both R-r K1OIR-st (13) and R-rIR-st KlO (7) heterozygotes. Alkylating agents also interfere with paramutation. Treatment of R-rIR-st seeds with diethyl sulfate enhances the pigment-producing potential of R-r compared with untreated R-rIR-st controls (2). The isolation of the R gene (22) has allowed an investigation of the molecular basis of paramutation at the R locus. Alleman & Kermicle (perseveral generations of heterozygosity with either An intriguing observation about



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sonal communication) have found that the DNA of the paramutant R is hypermethylated relative to that of its progenitor, based on gel blot analysis using restriction enzyme isoschizomers . This hypermethylation occurs in all the components of the R-r complex. The same pattern of methylation in the paramutant R-r ' root, and seedling. However, the methylation pattern observed in endosperm DNA differed somewhat from that of other tissues. Just as paramutational changes in R-r occur progressively through successive plant generations, the DNA of R crossing to R-st and less methylated after one generation of heterozygos ity with r-g. Similarly, treatment of R -r I sults in an enhancement of the R-r allele's pigment-producing potential and in demethylation of its DNA. In general, then, paramutational changes at R appear to be correlated with changes in the degree of methyla tion of the R-r allele. B LOCUS IN MAIZE A similar type of directed genetic change has been observed in maize at the B locus on chromosome 2 (16) . Interestingly, R and B are duplicate genes (14, 95), although most B alleles affect pigmentation in the plant, rather than in the aleurone. Normally, the B-1 allele condition strong pigmentation in the husks, sheaths, and culm. When passed through a heterozygote with a weakly pigmenting, paramutagenic B I allele, the B a B I type, i.e. it becomes weakly pigmenting and paramutagenic itself. Changes of B B-II There are differences, however, between B and R paramutation with re spect to the origin of the paramutagenic allele, the stability of the paramutant condition, the effect of chromosomal aberrations and, possibly, the de velopmental timing of the paramutational event. (a) Whereas strongly paramutagenic B ' apparently in one step ( 16), the strongly paramutagenic R-st and the paramut able R-r alleles trace back to two different and largely nonoverlapping geo graphic areas (103) . The R-r allele becomes weakly paramutagenic after exposure to R generations. (b) A paramutant R-r ' to R stable ( 1 6) . (c) Paramutation at R , chromosome structure, such as translocations and knobs. (d) Changes of R-r to R-r' occur somatically during development of a R-r IR of B to B ' do not occur somatically, at least not during the first five days of development of a B I

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the sporophyte (16). Coe ( 1 6) has proposed that the change of B to B ' occurs at or near the time of meiosis. Recent molecular data suggest additiollal differences between B and R paramutation. Initial studies revealed that B plants accumulated 1 0-20 times more B transcript than B ' plants (76). Subsequently , Patterson & Chandler (personal communication) analyzed 40 restriction sites throughout the coding regions of B and B ' and several kb on either side using methylation-sensitive restriction endonucleases . They found no differences in the pattern of methylation of the two DNAs, except for two sites 1 kb upstream of the start of transcription that were more methylated in the B allele. Though the meaning of the last observation is unclear, it does not appear that methylation differences are correlated with the differential expression of B and B ' alleles.


�

NIVEA LOCUS IN ANTIRRHINUM An interaction that results in a predictable alteration in the expression of a particular allele has also been described at nivea, one of the loci that affects anthocyanin pigmentation in Antirrhinum . The mUltiple allelic series at this locus includes Niv + , niv, and nivrec types of alleles, which specify fully red, white, and variegated flowers, respectively. Many nivrec/niv heterozygotes have var iegated flowers . However, heterozygotes resulting from crosses between female parents carrying the niv allele niv-44 and male parents carrying the nivrec allele niv-53 are mostly nonvariegated and have, instead , a granular flush over the corolla that fades as the flower ages. Fz progenies produced by selfing nonvariegated F 1 plants continue to produce flowers without variega tion . Since the change in expression of niv-53 appears to be directed and heritable, it has been referred to as paramutation (43). In this interpretation of the phenomenon, niv-53 is considered to be the paramutable allele and niv-44, the paramutagenic allele , although cosegregation of niv-44 with paramutage nicity has not been demonstrated. The molecular isolation of the nivea locus (1 08) has allowed a molecular characterization of niv alleles . Both niv-53 and niv-44 contain insertions in the niv gene. The niv-53 allele has a 1 7-kb transposable element, called Tam } , in the niv promoter (4) and the niv-44 allele, a 5-kb insert, called Tam2, in the first exon-intron junction of the niv gene (99) . Tam} and Tam2 share inverted terminal repeats and are present in mUltiple copies in both the niv-53 and the niv-44 lines. The variegation or flaking in niv-53 is due to frequent excision of Tam } that restores wild-type expression of the niv gene (4) . The disappearance of variegation in niv-53/niv-44 heterozygotes and their progeny would be due presumably to suppression of Tam} transposition by either the niv-44 allele or some other factor in the genetic background of the niv-44 line. Since variega tion is not observed in subsequent generations, the change in niv-53 must PARAMUTATION AT THE



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persist i n the absence o f niv-44. Southern blot analysis has revealed n o major rearrangement in the DNA of niv-53 in either the F I or Fz generation (55) . An allele resulting from excision of Tam2 from niv-44 has been isolated. Heterozygous plants carrying the new allele and niv-53 produce flowers that are indistinguishable from niv-44Iniv-53 , so Tam2 does not have to be present at the niv locus for changes in the varicgated expression of niv-53 to occur (55 ) . Models have been advanced that explain the suppression o f variegation seen in niv-53Iniv-44 heterozygotes in light of the more recent molecular data (48 , 55) . It is proposed that factors repressing the excision of Tam2 and TamI are present in line 1144 , which contains the niv-44 allele. These factors could either positively regulate production of a repressor that binds to the common inverted repeats of Tam} and Tam2 . resulting in its progressive accumulation in the F I heterozygote and in subsequent generations, or heritably modify (methylate) Tam } and Tam2 , thereby inactivating them (48). According to this view , the directed, heritable change in niv-53 expression that occurs in niv-53Iniv-44 heterozygotes would be due to the residual genetic background and not to an interaction between alleles , and if so, should no longer be considered a case of paramutation. There is, however, an unexpected twist to the interactions that take place in Antirrhinum plants that contain hybrid 53/44 backgrounds . Three stable white derivatives from the variegating niv-53 have been described: niv-46 and niv-49 arose by identical 5-bp terminal deletions in TamI (45) and niv-99, by a complex rearrangement involving the TamI element at the niv locus (48). Surprisingly, heterozygotes between any of these and the white-flowered, "paramutagenic" niv-44 allele can be variegated, as a consequence of Tam2 excision from niv-44. Since Tam2 excision is not observed in niv-53Iniv-44 heterozygotes , it may be that excision of Tam2 from the niv-44 allele de pends , in addition to other genetic factors , on the organization of the TamI carrying allele in the homologue . This is consistent with the postulate that Tam2 is a defective element that can be complemented by an active TamI element (45 , 48) .

Dominant CHS Alleles An interesting series of dominant and semidominant alleles of the nivea (CHS) locus in Antirrhinum illustrates another type of rare allelic interaction ( 1 8 ; E. H . Coen, unpublished data; C. Martin, unpublished data). Each of these trans-acting alleles was derived from a Tam3 insertion in the promoter of the nivea locus, niv-98 . Each trans-acting allele is the result of a Tam3associated rearrangement that generated an inverted duplication of part of the nivea locus centered on the point of Tam3 insertion in the promoter. In several cases Tam3 is still present at the locus but outside the duplication . In at least

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one case it has been shown that the presence of Tam3 is not necessary for the mutant allele to be dominant to a normally functional Nivea allele. Models invoking either titration of transcription factors or antisense RNA are consid ered unlikely explanations for the transdominant effect. Instead, a model requiring direct physical association between alleles, as has been proposed for transvection in Drosophila, is preferred ( 1 8) .


Ectopic Transgene Interactions Allelic interactions that lead to alterations in the expression of one or both alleles are not common. However, the recent advent of plant transformation has made it possible to examine interactions between copies of the same sequence placed at nonallelic (ectopic) locations. Perhaps surprisingly, it has been observed that ectopic transgenes frequently interact with homologous genes in ways that lead to changes in gene expression at either or both locations. These observations have been summarized in a recent review (49) . Among the most informative of these observations are those involving an thocyanin genes, namely, CHS and DFR. Introduction of a chimeric trans gene to petunia was found to result in suppression of the expression of the homologous, endogenous CHS or DFR gene (72, 101). Both alleles of the endogenous gene were affected, and the effect was found to be specific to genes homologous to the introduced gene, i . e . no gene other than the one homologous to the transgene was affected. Importantly, it was found that expression of the introduced transgene was also suppressed in plants in which the endogenous gene was suppressed, suggesting the term cosuppression to refer to this effect. Another interesting trans-interaction effect involving anthocyanin trans genes was also observed in petunias . A chimeric maize DFR gene that was introduced into a petunia line that normally accumulates dihydroflavanols in the flowers enabled the plants to produce anthocyanins. It was found that the color phenotype was generally unstable in plants that carried more than one copy of the transgene, and generally stable in the presence of a single copy of the transgene (60) . This suggests that the maize DFR gene can interact in trans with additional maize DFR genes but not with the petunia DFR gene. It is perhaps important that the sequence of the maize DFR gene is only approximately 70% homologous to the petunia gene, possibly precluding interactions between the transgene and the endogenous DFR gene. It may also be significant, however, that no petunia DFR mRNA accumulates in the parental petunia line. A correlation was found between methylation of the promoter of the transgene and the instability in floral coloration. These interactions involving transgenes in plants might be compared to the phenomenon of premeiotic inactivation in filamentous fungi, a process where by gene duplication results in the inactivation of both the introduced and the l�ndogenQUS copies of the gene, accompanied by hypermethylation of the


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duplicated sequences and, in Neurospora, by a high frequency of mutation within the homologous pair of sequences, a process known as RIP (repeat induced point mutation) (87).


Speculations on trans Interaction Phenomena The trans interaction phenomena described above strongly suggest the possibility of direct interactions between homologous genes in somatic cells. Various features of these phenomena suggest further that these interactions are somehow dependent on differing sequence contexts between the interact ing alleles or loci and that the exchange of information between homologous genes probably involves a homogenization or directional alteration of chroma tin configuration, e.g. via exchange of chromatin-associated proteins and possibly DNA methylation patterns, but not DNA sequence . We have consid ered two types of hypotheses that attempt to describe the possible nature of such interactions. The first hypothesis assumes the existence of a mechanism that normally prevents homologous sequences from interacting and explains trans interac tion phenomena as rare failures of this normal mechanism. This hypothesis is supported by evidence that in plants homologous chromosomes are spatially separated from each other in somatic nuclei (47). This spatial separation might reflect the operation of a mechanism that keeps alleles from pairing somatically under normal conditions. However, in certain instances , unusual alleles or genes could pair with homologous genes and cause an alteration in gene expression. A gene introduced by transformation would be able to evade the system for prevention of pairing purely by virtue of its random ectopic localization. In R paramutation, a paramutable allele would possess sequences that are more likely to be altered in interactions with certain other alleles, while a paramutagenic allele possesses sequences that promote somatic homologous interactions. In the absence of a paramutagenic allele , the para mutant allele would revert to the original state. The fact that reversion is gradual can be understood in terms whereby methylation , which was induced by the alIelic interaction, is removed progressively. In trans-dominant nivea alleles and ectopic transgenes, reversion is immediate and complete in the absence of the causative allele or gene. This is not in conflict with the often progressive loss of paramutations , but requires that the condition leading to the altered state of expression be reversible through sexual reproduction. The second hypothesis makes the assumption that homologous sequences normally interact, possibly to prevent alleles from adopting chromatin con figurations that could lead to alterations in gene expression. This hypothesis proposes that trans interaction phenomena are observed where alleles or homologous genes differ substantially in sequence context, permitting one gene to impose a change in chromatin configuration on the other. Thus, an ectopic gene might adopt a chromatin configuration that is functionally


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different from that of the endogenous gene and that would be imposed on the normal gene during the proposed normal interaction of homologous sequ ences. In the absence of the ectopic gene, the normal gene would be able to revert to its original state. According to this hypothesis, a paramutable R allele would possess sequences that are susceptible to change during interac tion with a sufficiently different allele, while a paramutagenic allele possesses sequences that permit it to adopt a functionally different chromatin configura tion, perhaps in metastable fashion. The latter hypothesis would require a highly efficient homology-searching process in somatic cells, resulting in the exchange of information residing in chromatin . Although there is no evidence for high efficiency somatic genetic exchange between homologous sequences in plants, we would point out that the mechanism proposed does not require any physical exchange of DNA sequence information. That highly efficient homology-searching is physically possible is illustrated by the demonstration in yeast that ectopic gene conver sion can be as efficient as allelic gene conversion (4 1 ) . The "homology sensing" machinery in filamentous fungi that results in premeiotic inactivation may be analogous to the one in Saccharomyces, except that the outcomes of the homology search would differ in the two fungal species (4 1 ) . Similarly, plants may possess a specialization of a homology-searching process that results in an exchange of functional chromatin-associated information without any exchange of DNA sequences. We anticipate that the continuous use of anthocyanin biosynthetic genes as "reporters" in the study of trans in teractions in plants will lead to the elucidation of new mechanisms of gene regulation. ACKNOWLEDGMENTS

We are very grateful to our many colleagues who communicated to us unpublished observations or sent us preprints of manuscripts . We also thank Ed Ralston and Neal Gutterson for helpful comments, Joyce Hayashi for Figure 1 , and Lily Kruger for typing assistance. This is paper No. 5-22 from DNA Plant Technology . Literature Cited 1 . Almeida, J . , Carpenter,R . , Robbins, T. P . , Martin, C . , Coen,E. S. 1 989. Ge

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noid synthesis in Petunia hybrida: par tial characterization of dihydroflavonol4-reductase genes. Plant Mol . Bioi.

1 3 : 49 1 -502 4. Bonas, U . , Sommer, H . , Harrison, B. J . , Saedler, H. 1 984. The transposable element Tam 1 of Antirrhinum majus is 17 kb long. Mol. Gen. Genet. 1 94: 1 3843

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origin, and mechanism of conversion type inheritance at the B locus in maize. Genetics 53 : 1 035-63 Coe, E. H . , Neuffcr, M. G . , Hoising ton, D. A. 1 988. The genetics of com. In Corn and Corn Improvement, ed. G. F. Sprague, J. W . Dudley, pp. 8 1-258. Madison, WI: ASA Coen, E . S . , Carpenter, R. 1 988. A semi-dominant allele, niv-525, acts in trans to inhibit expression of its wild type homologue in Antirrhinum majus. EMBO J. 7:877-83 Coen, E. S . , Carpenter, R . , Martin, C. 1 986. Transposable elements generate novel patterns of gene expression in Antirrhinum majus. Cell 47: 285-96 Cone, K. c . , Burr, B . 198 8 . Molecular and genetic analyses of the light require ment for anthocyanin synthesis in maize.

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