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The Journal of Heredity 2002:93(6)
© 2002 The American Genetic Association 93:421-428

The Identification and Characterization of Two Dominant r1 Haplotype-Specific Inhibitors of Aleurone Color in Zea mays

P. S. Stinard, and M. M. Sachs

From the U.S. Department of Agriculture/Agricultural Research Service, Soybean/Maize Germplasm, Pathology and Genetics Research Unit, Urbana, Illinois (Stinard and Sachs), and Department of Crop Sciences, University of Illinois, Urbana, Illinois (Sachs).

Address correspondence to Martin M. Sachs, Maize Genetics Cooperation Stock Center, S-123 Turner Hall, 1102 S. Goodwin Ave., Urbana, IL 61801, or e-mail: msachs{at}uiuc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We report the identification and characterization of two novel dominant inhibitors of aleurone color in Zea mays that interact with specific haplotypes of the r1 locus. One inhibitor locus, inr1 (inhibitor of r1 aleurone color 1), maps to the long arm of chromosome 10, distal to the TB-10L19 breakpoint and tightly linked to dull1, and the second inhibitor locus, inr2 (inhibitor of r1 aleurone color 2), maps to the long arm of chromosome 9. Dominant inhibitory alleles of inr1 and inr2 act by suppressing aleurone color conditioned by certain r1 haplotypes. Two haplotypes, R1-ch:Stadler and R1-Randolph, exhibit nearly complete suppression of aleurone color in the presence of inhibitory alleles of inr1 or inr2. Two members of the R1-d class of haplotypes, R1-d:Catspaw and R1-d:Arapaho, show partial suppression. Other haplotypes tested were not visibly affected. The response of r1 haplotypes to inhibitory inr1 and inr2 alleles provides another means of analyzing the complex behavior of the seed color components of r1 haplotypes. Possible mechanisms of action of inr1 and inr2 are discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Aleurone color in maize is determined by a group of regulatory and structural genes in the anthocyanin biosynthetic pathway (Dooner et al. 1991). The regulatory genes that have been most intensively studied are the b1/r1 gene family, which codes for myc-like transcriptional activators (Ludwig et al. 1989; Radicella et al. 1991), and the c1/pl1 gene family, which codes for myb-like transcriptional activators (Cone et al. 1993; Paz-Ares et al. 1987). These genes appear to be capable of up-regulating all of the structural genes in the anthocyanin biosynthetic pathway (Bruce et al. 2000). Structural genes in the anthocyanin pathway that have been identified in maize include a1, a2, c2, bz1, and bz2 (Bruce et al. 2000). Most typically, b1 and pl1 control anthocyanin pigmentation of plant parts, and r1 and c1 control anthocyanin pigmentation in the aleurone and other seed parts. However, there are exceptions, and a certain amount of overlap in the tissue specificity of certain alleles (Coe et al. 1988).

Dominant inhibitory mutants of aleurone color have been identified at the c1 locus (C1-I), the c2 locus (C2-Idf), and the in1 locus (In1-D) (Coe et al. 1988). Molecular analysis of the c1 locus indicates that functional alleles code for a myb-related transcriptional activator that has a DNA binding domain and a transcription activating domain (Paz-Ares et al. 1987, 1990). The lesions in the C1-I allele appear to affect both the DNA binding domain and the transcription activating domain of the C1-I protein product (Goff et al. 1991). The exact mechanism of inhibition is not known (suggested mechanisms include competition by the defective C1-I protein for activation binding sites at the structural gene loci, or formation of inactive mixed heterodimers with normal C1 product), but it appears that lesions in both domains are necessary for the greatest amount of inhibition (Goff et al. 1991).

The c2 locus in maize is one of two duplicate loci that code for chalcone synthase, one of the structural genes in the anthocyanin biosynthetic pathway. The C2-Idf allele carries multiple copies of rearrangements or partial deletions of functional C2 sequences (Wienand et al. 1991). Similar duplications and rearrangements of chalcone synthase genes have been observed in dominant inhibitory alleles at the i locus (seed coat color) in soybeans (Todd and Vodkin 1996). The inhibition appears to be due to gene silencing, although the exact mechanism is unknown.

The function of the in1 locus in maize is unknown, although its product, a myc-related protein, appears to down-regulate the structural genes in the anthocyanin biosynthetic pathway (Burr et al. 1996). The dominant inhibitory allele, In1-D, carries an imperfect duplication of nonmutant In1 sequences, and it is expressed at a higher level than the nonmutant In1 allele (Scheffler et al. 1999). The aleurone color inhibition observed in In1-D lines is due to overexpression of a down-regulator of the anthocyanin biosynthetic pathway.

Some modifiers have been reported to enhance anthocyanin pigmentation of vegetative parts in plants carrying specific r1 haplotypes. The term haplotype rather than allele is used in reference to variations at the r1 locus because it is a complex locus composed of one to several genes that affect anthocyanin pigmentation of various plant parts (Panavas et al. 1999). The dominant modifier Vr enhances the weak plant color pigmentation of the dilute R1-d haplotypes, conferring mature plant coloration that is more intense than that of other r1 haplotypes (Stadler 1951). Vr apparently has no effect on aleurone color. Unfortunately, all known Vr stocks have been lost. The recessive modifier a3 is a more general enhancer of plant color and seems to enhance and extend anthocyanin pigmentation of plant parts in combinations of b1 and r1 variants that would be expected to produce at least some plant color (Styles and Coe 1986).

While there is anecdotal information as to the existence of r1 haplotype-specific inhibitors of aleurone color in maize (Sastry 1969; Sastry and Kurmi 1970), none has been characterized in detail. The present study reports the characterization of two r1 haplotype-specific aleurone color inhibitor loci in maize, inr1 (inhibitor of r1 aleurone color 1) and inr2 (inhibitor of r1 aleurone color 2).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Genetic Stocks
All stocks are homozygous for alleles at the anthocyanin loci necessary for purple aleurone color (A1 A2 C1 C2 R1) unless otherwise indicated. All stocks have been deposited with the Maize Genetics Cooperation Stock Center, Urbana, IL.

Inr1-Ref (the reference allele) was identified in the Maize Genetics Cooperation Stock Center stock 908F C1 wx1 da1 and outcrossed for at least seven generations to W23 R1-Randolph. Inr1-JD and Inr2-JD were identified in an open-pollinated green dent variety "John Deere," so called for the resemblance of the green aleurone pigmentation to the green color of a John Deere tractor. John Deere was obtained by Dr. George F. Sprague from Carl Barnes of Corns, Turpin, OK, and given as a gift to the authors. The John Deere line may be identical to an open-pollinated variety known as Oaxacan Green Dent. The haplotype R1-Randolph was substituted for the R1-r haplotype originally present in John Deere by genetic crosses, and Inr1-JD and Inr2-JD were separated and outcrossed for at least seven generations to W23 R1-Randolph. In1-D was obtained from the Maize Genetics Cooperation Stock Center stock 701B In1-D and outcrossed for six generations to W23 R1-Randolph.

All r1 haplotype lines are free of the inhibitors under study and were obtained or derived as follows:

R1-r:standard—purple aleurone, red plant color, susceptible to r1 aleurone mottling (Kermicle 1970, Walker et al. 1995). The line used in this study is a W22 conversion obtained from Oliver Nelson, University of Wisconsin, Madison, WI.
R1-Randolph—purple aleurone, weakly susceptible to r1 aleurone mottling. The line used in this study is a W23 conversion of a haplotype originally described as R1-g and obtained by the Maize Genetics Cooperation Stock Center from L. F. Randolph, Cornell University, Ithaca, NY, during the 1950s. Later it was found that R1-Randolph conditions dilute plant color during early seedling stages and so may belong to the R1-d group of r1 haplotypes. For this reason, the green plant color designation "-g" has been dropped from the name.
R1-ch:Stadler—purple aleurone, variable red plant color, susceptible to r1 aleurone mottling; confers a strongly purple pericarp color in the presence of the plant color allele Pl1 (Sastry 1970). The line used in this study is a W22 pl1 conversion obtained from Ron Okagaki, University of Minnesota, St. Paul, MN.
R1-d:Catspaw—purple aleurone, dilute plant color, not susceptible to r1 aleurone mottling (Stadler 1948; Walker and Panavas 2001). The line used in this study is a W22 conversion obtained from Jerry Kermicle, University of Wisconsin, Madison, WI.
R1-d:Arapaho—purple aleurone, dilute plant color, not susceptible to r1 aleurone mottling (Stadler 1948). The line used in this study is a W22 conversion obtained from Ron Okagaki.
R1-nj:Cudu—Navajo aleurone color pattern (a patch of purple aleurone on the crown), red plant color, not susceptible to r1 mottling (Kyle and Styles 1973). This is a W23 conversion obtained from Jack Beckett, USDA/ARS, Columbia, MO.
R1-scm2—purple aleurone, not susceptible to r1 aleurone mottling (Panavas et al. 1999; Styles 1993). The line used in this study is a W22 conversion obtained from Ed Coe, USDA/ARS, Columbia, MO.
R1-sc:124—purple aleurone, not susceptible to r1 aleurone mottling (Eggleston et al. 1995; McWhirter and Brink 1962). The line used in this study is a W22 conversion obtained from Hugo Dooner, Waksman Institute, Rutgers University, Piscataway, NJ.
R1-sup—R1-suppressible: generic designation for any otherwise uncharacterized r1 haplotype that is suppressed by inhibitory alleles of inr1 or inr2, for example, the r1 haplotype present in the Maize Genetics Cooperation Stock Center's 908F C1 wx1 da1 line.
r1-{Delta}902—colorless aleurone, green plant. r1-{Delta}902 (also called r1-Del902) represents a deletion of r1 coding sequences (Alleman and Kermicle 1993). The line used in this study is a W22 conversion obtained from Jerry Kermicle.
r1-g—colorless aleurone, green plant (Brink 1958). The line used in this study is a W23 conversion obtained from Jerry Kermicle.
r1-r—colorless aleurone, red plant color (Brink 1958). This haplotype was obtained from E. G. Anderson.
B1-Peru r1-g—purple aleurone, green plant, not susceptible to mottling (Radicella et al. 1992; Styles et al. 1973). B1-Peru is a duplicate factor with respect to r1 for aleurone color. The line used in this study is a W22 conversion obtained from Jay Hollick, University of California at Berkeley.

All B-A and A-A translocation stocks were obtained from the Maize Genetics Cooperation Stock Center. All other genetic mapping stocks were obtained from the Maize Genetics Cooperation Stock Center and were converted to an R1-Randolph background as needed.

Tests of allelism and linkage were done in accordance with the protocols established by Coe (1994) and are described in detail below. Mapping through the use of B-A translocations was done as described by Beckett (1994). Mapping through the use of reciprocal A-A translocations was performed as described by Patterson (1994).

For purposes of comparing degrees of aleurone color inhibition under various combinations of inr1 and inr2 alleles and r1 haplotypes, a five-point system for scoring aleurone color was used. On a scale of one to five, colorless aleurone kernels were given a score of one and full-color aleurone kernels were given a score of five. Intermediate standards were assigned as two (light pale purple), three (medium pale purple), and four (dark pale purple). Ears were individually scored without regard to genotype and the range of color intensities representing the kernel phenotypes on each ear determined.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Identification and Characterization of Inr1-Ref
Inr1-Ref was identified as a pale to colorless aleurone mutant from a dilute aleurone1 (da1; Eyster 1931) chromosome 9 marker stock maintained at the Maize Genetics Cooperation Stock Center in Urbana, IL (stock number 908F). From a two-point coupling backcross linkage test of inr1 and the chromosome 9 marker wx1, we obtained the following ratios: 100 inr (dark purple aleurone) Wx (starchy endosperm):98 Inr (pale purple endosperm) wx (waxy endosperm):118 inr wx:97 Inr Wx. This does not differ significantly from a 1:1:1:1 ratio ({chi}2 = 2.855), and indicates that the two mutants are not linked. The lack of linkage of inr1 with wx1 indicates that Inr1-Ref is not identical to the da1 mutant originally described on chromosome 9 (da1 and wx1 are separated by 21 cM according to Emerson et al. [1935]). It is possible that da1 and Inr1-Ref are the same factor and that the original mapping and characterization of da1 were incorrect. Alternatively, da1 and Inr1-Ref might be independent factors present in the same stock and over the years da1 was lost from the stock during propagation due to the confounding pale aleurone color phenotype of these two factors. We are presently trying to isolate the original da1 mutant from early sources of stock 908F.

Since inr1 did not show linkage with wx1 as would be expected if it were identical to da1, complementation tests were conducted between inr1 and other aleurone color factors. Crosses of Inr1-Ref to the standard a1, a2, c1, c2, bz1, and bz2 aleurone color tester lines all yielded kernels with full purple aleurone color, indicating complementation (the r1 haplotype of these aleurone testers is R1-r:standard). However, crosses to r1-r (a colorless aleurone haplotype of the r1 locus located on the long arm of chromosome 10) gave a range of pale to colorless kernels, indicating an interaction, possibly allelism.

When the heterozygous Inr1-Ref/r1-r kernels from the complementation test ears were planted and the resulting plants crossed again by r1-r, the ears that were obtained bore a consistent, but low percentage of fully colored kernels (around 10%; see Table 1). This percentage was much too high to be accounted for by intragenic recombination at the r1 locus, which encompasses a length of about 0.16 cM if one considers only the R1-r:standard complex (Dooner and Kermicle 1974), or 2 cM if one considers displaced r1 sequences in the vicinity of r1 such as lc1 (Dooner and Kermicle 1976) and sn1 (Tonelli et al. 1994); and there were no signs of somatic instability that would indicate that the purple kernels were the result of the excision of a transposable element at the locus.


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  		Table 1.. Counts of full-colored (Cl) and pale or colorless kernels (cl) on ears from the cross [inr1 r1-r x Inr1-Ref R1-sup] x inr1 r1-r and its reciprocal.

 
The behavior of Inr1-Ref was explained after it was found to inhibit aleurone color in crosses to lines carrying certain suppressible r1 aleurone color haplotypes (R1-sup; e.g., R1-Randolph and R1-ch:Stadler; see below for a more thorough analysis). The original Inr1-Ref line was homozygous for one such suppressible haplotype. Therefore R1-sup aleurone color expression was suppressed in the presence of Inr1-Ref, resulting in an apparent lack of complementation in crosses to the inr1 r1-r tester. Inr1-Ref subsequently recombined and segregated away from the susceptible r1 haplotype in the backcross to inr1 r1-r tester, yielding full purple kernels. If inr1 were not linked to r1, one would expect 25% full-color kernels in such backcrosses: 25% inr1 r1-r (colorless; all genotypes indicated are heterozygous with inr1 r1-r), 25% Inr1-Ref R1-sup (colorless), 25% Inr1-Ref r1-r (colorless), and 25% inr1 R1-sup (full color). The lower frequency of purple kernels (only about 10%) indicates linkage of inr1 with r1 with approximately 20 cM of separation, which was borne out in subsequent linkage tests (see below). This linkage places inr1 to the long arm of chromosome 10.

inr1 10L Mapping Data
Preliminary tests described above placed inr1 on the long arm of chromosome 10, linked to r1. This placement was confirmed through the use of B-A translocations. Crosses of homozygous Inr1-Ref R1-sup stocks by the B-A translocation TB-10L19 homozygous for the R1-scm2 haplotype (Beckett 1994) yielded ears segregating for small, pale, and colorless kernels with purple scutellum (putative hypoploid Inr1-Ref R1-sup endosperms with hyperploid embryos) as would be expected if inr1 were located on the long arm of chromosome 10. Furthermore, hypoploid plants grown from purple kernels of the B-A translocation cross proved to be hemizygous for both Inr1-Ref and R1-sup, confirming the placement of inr1 to the long arm of chromosome 10. Control crosses of Inr1-Ref R1-sup by an R1-scm2 line not carrying a B-A translocation gave full-color kernels.

Additional linkage tests with multiple chromosome 10 markers were conducted in order to map inr1 more precisely. The results of a three-point linkage test for inr1, g1, and r1 on chromosome 10 are presented in Table 2. The linkage test was set up as a series of modified backcrosses as indicated in Table 2. The kernels from the first cross (inr1 g1 R1-g x Inr1-Ref G1 R1-sup) were all purple since the particular R1-g haplotype present in the inr1 g1 R1-g stock is not inhibited by Inr1-Ref. Kernels from the second cross [(inr1 g1 R1-g/Inr1-Ref G1 R1-sup) x inr1 g1 r1-g] bore full-purple kernels and pale purple kernels in an approximate ratio of 6:4. Full-purple kernels from this cross were grown in our summer nursery, scored for g1, and crossed as males onto an Inr1-Ref G1 R1-sup tester in order to evaluate which haplotypes were present at the r1 locus, and were crossed as females by inr1 G1 R1-sup to evaluate whether Inr1-Ref was present. Pale purple kernels were grown, scored for g1, and crossed as females by inr1 G1 R1-sup to confirm that Inr1-Ref was present. The following linkage relationship was established: inr1-12.1 cM-g1-12.1 cM-r1. These data are consistent with the g1-r1 distance (14 cM) from the most recent genetic map of chromosome 10 (Neuffer et al. 1997). These data place inr1 about 12 cM proximal to g1 on the long arm of chromosome 10.


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  		Table 2.. Three-point linkage data for inr1, g1, and r1.

 
A three-point linkage test involving the 10L markers inr1, du1, and g1 was set up as indicated in Table 3. Kernels from these crosses with the crossover phenotypes plump solid-colored (Du inr) and dull pale (du Inr) were planted and the resulting plants scored for phenotypes at the g1 locus in order to provide partial three-point linkage data. Crosses were made in order to confirm the du1 and inr1 scoring as well. Complete scoring of the parental classes for g1 was not done because of the large populations involved, and because complete scoring was not necessary to determine the gene order since the linkage relation between du1 and g1 is already well established (these two loci are approximately 19 cM apart; Neuffer et al. 1997). The partial three-point linkage data are presented in Table 3. These data indicate that inr1 and du1 are very tightly linked, with approximately 0.14 cM between them. These data also determine the global order of the three loci on chromosome 10 as du1 inr1 g1.


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  		Table 3.. Partial three-point linkage data for du1, inr1, and g1.

 
Identification of Inr1-JD and Inr2-JD
In order to determine the cause of the green aleurone pigmentation in John Deere, an open-pollinated green dent heirloom variety, it was crossed to various colored aleurone stocks for reextraction. In crosses of John Deere corn to stocks carrying specific susceptible haplotypes of r1, dominant inhibition of aleurone color was observed in a pattern similar to that found for Inr1-Ref. After several generations of backcrossing the John Deere line to a W23 conversion of R1-Randolph, a pattern emerged. Plants were obtained that produced self-pollinated ears giving a 15:1 ratio of colorless and pale:full-color aleurone kernels (Figure 1), and whose outcrosses to R1-Randolph gave a 3:1 ratio of colorless and pale:full-color aleurone kernels. The ratios produced from these crosses are indicative of independent segregation of two dominant aleurone color inhibitors. Through several generations of self-pollination we were able to obtain lines homozygous for each dominant inhibitor separately.



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  		Figure 1.. Photograph of a self-pollinated ear segregating for Inr1-JD and nr2-JD in a homozygous R1-Randolph background. Note the 15:1 segregation of colorless and pale purple kernels (carrying either Inr1-JD , Inr2-JD , or both) to full-purple kernels (inr1 inr2).

 
Complementation tests of these inhibitors with inr1 were done by crossing each inhibitor line to a homozygous Inr1-Ref line, and then crossing the resulting F1 by R1-Randolph. One of the inhibitors produced only colorless kernels in a population of thousands of kernels, indicating likely allelism. (There is always the remote possibility that the inhibitor is not allelic to inr1, but instead represents a second locus extremely tightly linked to inr1—these tests cannot rule out that possibility). The other inhibitor produced ears segregating 3:1 for colorless to colored kernels in these crosses, indicating nonallelism with inr1. The inhibitor allelic to inr1 was named Inr1-JD and the second unlinked inhibitor was named Inr2-JD.

inr2 Maps to 9L
Because Inr2-JD exhibits dominant inhibition of aleurone color, easily scored on the ear in the presence of appropriate r1 haplotypes, we chose to map inr2 using a set of wx1-marked A-A translocations (Patterson 1994). Plants homozygous for Inr2-JD and R1-Randolph were crossed to a series of wx1-marked translocations in a colorless aleurone (r1) background free of inhibitors. F1 plants were backcrossed by a homozygous inr2 wx1 R1-Randolph line and the resulting ears were scored for colorless (Inr) versus colored (inr) and waxy (wx) versus starchy (Wx) kernels (Table 4). In crosses involving wx1 y1 T6-9e, some of the backcrosses were made by plants homozygous for inr2, wx1, y1, and R1-Randolph, so three-point linkage data for wx1, y1, and inr2 were obtained (Table 5). All crosses demonstrated linkage of inr2 with wx1, indicating that inr2 is located on chromosome 9. The distance between wx1 and inr2 showed variability (from 7.6 to 30.3 cM) depending upon which A-A translocation was used in the linkage cross. This kind of linkage variability is not unusual in crosses involving translocation stocks (Patterson 1952). Furthermore, the production of viable duplicate-deficient eggs by adjacent disjunction when plants heterozygous for certain translocations are used as females in linkage crosses can also distort linkage data somewhat (Patterson 1952).


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  		Table 4.. Two-point linkage data for wx1 inr2 in crosses involving various A-A translocations.

 

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  		Table 5.. Three-point linkage data for wx1 y1 inr2 in crosses involving T6-9e.

 
The linkage data obtained from the crosses with T6-9e (breakpoints 6L.18, 9L.24) provide sufficient information to fix the location of inr2 on chromosome 9 with respect to wx1. Because wx1 is located on the short arm of chromosome 9 (Neuffer et al. 1997) and y1 is located on the 9-6 chromosome very close to the breakpoint in T6-9e (Buckner and Reeves 1994), and the gene order determined from the linkage data is clearly wx1 y1 inr2, inr2 has to be located on 9L distal to the T6-9e 9L breakpoint (9L.24). If we take the T6-9e linkage data for the wx1–inr2 distance (25.2 cM) as a minimum value for the distance between wx1 and inr2, inr2 is located near or distal to the brittle stalk2 (bk2) locus on 9L. Additional tests using B-A translocations and 9L linkage markers are being conducted to confirm and refine the location of inr2 on 9L.

inr1 and inr2 Alleles Show the Same Patterns of r1 Haplotype-Specific Inhibition of Aleurone Color
In order to assess the relative abilities of Inr1-Ref, Inr1-JD, and Inr2-JD to inhibit aleurone color in the presence of susceptible and nonsusceptible r1 haplotypes, reciprocal crosses were done between lines homozygous for R1-Randolph and either homozygous or heterozygous for Inr1-Ref, Inr1-JD, or Inr2-JD and lines homozygous for a series of r1 haplotypes (Table 6). Self-pollinations and reciprocal crosses of the r1 haplotype series with the recessive colorless aleurone r1-g haplotype or a deletion of the r1 locus, r1-{Delta}902, were made to serve as inr1 inr2 controls. Self-pollinations and reciprocal crosses of lines homozygous for R1-Randolph and either homozygous or heterozygous for Inr1-Ref, Inr1-JD, or Inr2-JD with lines homozygous for r1-g or r1-{Delta}902 were done to establish a baseline of aleurone color for the inhibitor lines. Reciprocal crosses of lines homozygous for R1-Randolph and heterozygous for In1-D with the r1 haplotype series were made for purposes of comparison. The colors of the kernels resulting from these crosses were assessed on an ear-by-ear basis and ranked on a five-point color scale as described in the Methods section. The results of these crosses are presented in Table 6.


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  		Table 6.. Kernel aleurone color scores of individual ears from reciprocal crosses of Inr1-Ref, Inr1-JD, Inr2-JD, and In1-D alleles with various r1 haplotypes.

 
Inr1-Ref, Inr1-JD, and Inr2-JD all showed the same patterns of r1 haplotype aleurone color suppression. The r1 haplotypes R1-Randolph and R1-ch:Stadler are almost fully suppressed when crossed as males onto the Inr1-Ref, Inr1-JD, and Inr2-JD lines and give color scores similar to those for crosses of the Inr1-Ref, Inr1-JD, and Inr2-JD lines to the colorless aleurone haplotypes r1-{Delta}902 and r1-g (colorless to light pale purple coloration). The r1 haplotypes R1-d:Catspaw and R1-d:Arapaho show an intermediate level of suppression, with R1-d:Catspaw being slightly more suppressible (medium pale purple coloration) than R1-d:Arapaho (dark pale purple coloration). There was little or no color suppression observed in crosses with R1-r:standard, R1-nj:Cudu, R1-scm2, or R1-sc:124. Some variation in kernel cap size was observed in the crosses involving R1-nj:Cudu, but it did not seem to correlate with the presence or absence of inhibitory inr1 or inr2 alleles in the reciprocal crosses. Crosses with B1-Peru r1-g also showed little or no suppression, indicating that the inr genes do not seem to interact with B1-Peru. Inr1-Ref does not appear to alter plant coloration conferred by the standard B1 allele either, since the da1 stock in which Inr1-Ref was identified carried B1, and all Inr1-Ref B1 plants had full sun-red color. Crosses of Inr1-Ref, Inr1-JD, and Inr2-JD as males onto the r1 haplotype lines showed the same patterns of suppression indicated above, with a slight overall increase in the amount of aleurone coloration in crosses involving R1-d:Catspaw and R1-d:Arapaho. Whether the observed differences are due to dosage differences at the r1 locus, dosage differences at the inhibitor loci, or transmission-dependent imprinting phenomena (Alleman and Doctor 2000; Kermicle 1970) could not be determined from this experiment. There seemed to be no major reciprocal differences in crosses of the inhibitor lines with R1-Randolph and R1-ch:Stadler; aleurone color suppression was nearly complete for these latter two haplotypes in both reciprocal directions. Control crosses of the colored aleurone r1 haplotypes lacking inhibitors with r1-g and the null r1-{Delta}902 haplotype showed little or no aleurone color suppression and the expected patterns of r1 mottling. Therefore, the reduction in aleurone coloration observed in crosses of the inhibitory inr lines with the suppressible haplotypes R1-ch:Stadler, R1-d:Catspaw, and R1-d:Arapaho is real, and not merely the result of the suppression of the seed color component(s) of the R1-Randolph haplotype carried in these crosses by inhibitory inr lines. The genetic backgrounds of the r1 haplotypes used (W23 versus W22) did not seem to affect the results of these crosses. However, this will be tested more thoroughly in future studies.

The aleurone color inhibitor In1-D produced the same general degree of inhibition (medium pale aleurone color) when crossed to all r1 haplotypes (except R1-Randolph) and B1-Peru r1-g. Crosses to R1-Randolph showed a high degree of inhibition similar to that of inhibitory inr alleles. Thus In1-D does not show the same r1 haplotype specificity as Inr1-Ref, Inr1-JD, and Inr2-JD, and seems to be a more general aleurone color inhibitor.

Possible Mechanisms of Action of inr1 and inr2
The suppression of aleurone color expression of certain r1 haplotypes in the presence of inhibitory inr1 or inr2 alleles is reminiscent of the behavior of suppressible alleles of Suppressor-mutator (Spm; McClintock 1956; also called Enhancer [En]; Peterson 1965) transposable element-induced mutants in the presence of Spm. In this system, certain mutant alleles of maize loci containing nonautonomous (defective) dSpm (also called Inhibitor [I]; Peterson 1960) insertions are capable of a certain background level of gene expression. When an autonomous Spm element is brought into the system in trans, gene expression is suppressed, resulting in a null phenotype (the suppressor function of Spm). Excision of the dSpm element is also induced, resulting in revertant sectors of nonmutant tissue appearing in the affected maize plant parts (the mutator function of Spm). Some suppressible Spm-induced alleles are defective in the mutator function (McClintock 1961, 1965a,b). Alternatively some Spm elements can elicit the suppressor response without the mutator response (McClintock 1961, 1965a,b). It is these suppressible, nonmutable responses that bear similarity to the interaction of inr1 and inr2 with suppressible r1 haplotypes.

Molecular analysis of suppressible alleles of maize loci containing dSpm inserts indicates that affected alleles are capable of producing functional transcripts in the absence of autonomous Spm. However, in the presence of Spm, gene transcription is suppressed (Schwarz-Sommer et al. 1985). This is correlated with the binding of Spm transposase A (TNPA) to subterminal repeat elements (SREs) of dSpm (Grant et al. 1990). The precise mechanism of suppression is not known, but it has been hypothesized that TNPA molecules bound to dSpm SREs physically block transcription reading through dSpm (Schwarz-Sommer et al. 1985).

The sigma region of inverted repeat r1 haplotypes such as R1-r:standard has been shown to confer aleurone-specific expression to the seed color (S) component of the R1-r:standard haplotype (May and Dellaporta 1998). The sigma region contains fragments of a Doppia transposable element that has apparently visited the r1 locus during the course of its evolution (Walker et al. 1995). Doppia transposable elements are members of the CACTA family of transposable elements, as is Spm, and share a high degree of homology of structural features, such as terminal inverted repeats, SREs, and coding regions, with Spm (Bercury et al. 2001). The fractured Doppia element in the sigma region of R1-r:standard is flanked by duplicate transcriptional units, which are referred to as S1 and S2 (subcomponents of the S complex of r1), inverted with respect to each other (Walker et al. 1995). Mutants of R1-r:standard having a deleted sigma region are incapable of producing aleurone anthocyanins (Walker et al. 1995). Therefore, sigma seems to act as a promoter for S1 and S2 expression. The fact that sigma contains Doppia SREs and is required for aleurone expression of R1-r:standard S components suggests the possibility that the R1-r:standard S complex may be capable of behaving as a suppressible gene complex with respect to aleurone color. Genetically active Doppia elements have not yet been identified (Bercury et al. 2001). However, a functional cDNA for the hypothetical Doppia transposase DOPA has been constructed using sequence information obtained from genomic Doppia fragments in conjunction with reverse transcriptase polymerase chain reaction (RT-PCR), and DOPA protein has been produced in vitro using expression vectors. This DOPA protein has been found to bind to Doppia SREs (Bercury et al. 2001). A genetically active Doppia element might have the effect of regulating transcription of r1 seed color components that have Doppia sequences as promoters. The inhibitory alleles of the inr1 and inr2 loci do not seem to suppress aleurone color in R1-r:standard lines. However, that does not rule out the possibility that they might be interacting directly or indirectly with Doppia sequences in certain r1 haplotypes to inhibit transcription of S components. This possibility needs further testing.

Variations in the structure of the r1 locus have been correlated to the phenomenon of paramutation. Walker and Panavas (2001) categorized a wide geographic collection of r1 haplotypes as paramutable, paramutagenic, or neutral, based on classic tests outlined by Van Der Walt and Brink (1968). They then performed PCR analysis of these haplotypes with respect to five distinct promoter types known to exist in various haplotypes of r1. Without exception, members of each paramutation class shared structural similarities with each other based on the presence, absence, or fragment size of amplified promoter sequences. This suggests some possible avenues to pursue with respect to assessing which aspects of the r1 locus might affect the susceptibility of specific r1 haplotypes to inhibition by alleles of inr1 and inr2. Unfortunately, not enough is known about some of the r1 haplotypes used in this study to be able to place them in r1 structural categories and draw conclusions based on r1 haplotype structure. However, the haplotypes described in the studies of Walker and Panavas (2001) have been made available to us, and the appropriate crosses will be made in order to assess the susceptibility of these haplotypes to inr1 and inr2 color inhibition.


   Acknowledgments
 
This work was supported by the USDA/ARS. We thank Jerry Kermicle, Mary Alleman, and Elsbeth Walker for their discussions, which helped guide this research. We also thank the members of the maize genetics community who shared their knowledge and seed stocks with us.


   Footnotes
 
Corresponding Editor: Susan Gabay-Laughnan Back

Received July 15, 2002
Accepted October 15, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

    Alleman M, Doctor J, 2000. Genomic imprinting in plants: observations and evolutionary implications. Plant Mol Biol. 43:147-161.[CrossRef][Web of Science][Medline]

    Alleman M, Kermicle JL, 1993. Somatic variegation and germinal mutability reflect the position of transposable element Dissociation within the maize R gene. Genetics. 135:189-203.[Abstract]

    Beckett JB, 1994. Locating recessive genes to chromosome arm with B-A translocations. In: Maize handbook (Freeling M and Walbot V, eds). New York: Springer-Verlag; 315–327.

    Bercury SD, Panavas T, Irenze K, Walker EL, 2001. Molecular analysis of the Doppia transposable element of maize. Plant Mol Biol. 47:341-351.[CrossRef][Web of Science][Medline]

    Brink RA, 1958. Paramutation at the R locus in maize. Cold Spring Harbor Symp Quant Biol. 23:379-391.[Abstract/Free Full Text]

    Bruce WB, Folkerts O, Garnaat C, Crasta O, Roth B, Bowen B, 2000. Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P. Plant Cell. 12:65-79.[Abstract/Free Full Text]

    Buckner B, Reeves SL, 1994. Viability of female gametophytes that possess deficiencies for the region of chromosome 6 containing the Y1 gene. Maydica. 39:247-254.

    Burr FA, Burr B, Scheffler BE, Blewitt M, Wienand U, Matz EC, 1996. The maize repressor-like gene intensifier1 shares homology with the r1/b1 multigene family of transcription factors and exhibits missplicing. Plant Cell. 8:1249-1259.[Abstract]

    Coe EH, 1994. Genetic experiments and mapping. In: Maize handbook (Freeling M and Walbot V, eds).New York: Springer-Verlag; 189–197.

    Coe EH, Neuffer MG, Hoisington DA, 1988. The genetics of corn. In: Corn and corn improvement, 3rd ed (Sprague GF and Dudley JW, eds).Madison, WI: American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America; 81–258.

    Cone KC, Cocciolone SM, Burr FA, Burr B, 1993. Maize anthocyanin regulatory gene pl is a duplicate of c1 that functions in the plant. Plant Cell. 5:1795-1805.[Abstract]

    Dooner HK, Kermicle JL, 1974. Reconstitution of the R-r compound allele in maize. Genetics. 78:691-701.[Abstract/Free Full Text]

    Dooner HK, Kermicle JL, 1976. Displaced and tandem duplications in the long arm of chromosome 10 in maize. Genetics. 82:309-322.[Abstract/Free Full Text]

    Dooner HK, Robbins TP, Jorgensen RA, 1991. Genetic and developmental control of anthocyanin biosynthesis. Annu Rev Genet. 25:173-199.[CrossRef][Web of Science][Medline]

    Eggleston WB, Alleman M, Kermicle JL, 1995. Molecular organization and germinal instability of R-stippled maize. Genetics. 141:347-360.[Abstract]

    Emerson RA, Beadle GW, Fraser AC, 1935. A summary of linkage studies in maize. Cornell Univ Agric Exp Stn Memoir. 180:1-83.

    Eyster WH, 1931. Heritable characters of maize: XLI—dilute aleurone. J Hered. 22:225.[Free Full Text]

    Goff SA, Cone KC, Fromm M, 1991. Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins. Genes Dev. 5:298-309.[Abstract/Free Full Text]

    Grant SR, Gierl A, Saedler H, 1990. En/Spm encoded tnpA protein requires a specific target sequence for suppression. EMBO J. 9:2029-2035.[Web of Science][Medline]

    Kermicle JL, 1970. Dependence of the R-mottled aleurone phenotype in maize on mode of sexual transmission. Genetics. 66:69-85.[Free Full Text]

    Kyle D, Styles ED., 1973. The effects of light on pigmentation in developing maize seeds. Maize Genet Coop Newslett. 47:181-184.

    Ludwig SR, Habera LF, Dellaporta SL, Wessler SR, 1989. Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci. 86:7092-7096.[Abstract/Free Full Text]

    May BP, Dellaporta SL, 1998. Transposon sequences drive tissue-specific expression of the maize regulatory gene R-s. Plant J. 13:241-247.[CrossRef]

    McClintock B, 1956. Intranuclear systems controlling gene action and mutation. Brookhaven Symp Biol. 8:58-74.[Medline]

    McClintock B, 1961. Some parallels between gene control systems in maize and in bacteria. Am Nat. 95:265-277.[CrossRef]

    McClintock B, 1965a. Components of action of the regulators Spm and Ac. Carnegie Inst Washington Year Book. 64:527-536.

    McClintock B, 1965b. The control of gene action in maize. Brookhaven Symp Biol. 18:162-184.

    McWhirter KS, Brink RA, 1962. Continuous variation in level of paramutation at the r locus in maize. Genetics. 47:1053-1074.[Free Full Text]

    Neuffer MG, Coe EH, Wessler SR, 1997. Mutants of maize. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

    Panavas T, Weir J, Walker EL, 1999. The structure and paramutagenicity of the R-marbled haplotype of Zea mays. Genetics. 153:979-991.[Abstract/Free Full Text]

    Patterson EB, 1952. Studies on crossing over in homozygous and heterozygous chromosome rearrangements in Zea mays (PhD dissertation). Pasadena: California Institute of Technology.

    Patterson EB, 1994. Translocations as genetic markers. In: Maize handbook (Freeling M and Walbot V, eds).New York: Springer-Verlag; 361–363.

    Paz-Ares J, Ghosal D, Saedler H, 1990. Molecular analysis of the C1-I allele from Zea mays: a dominant mutant of the regulatory C1 locus. EMBO J. 9:315-321.[Web of Science][Medline]

    Paz-Ares J, Ghosal D, Wienand U, Peterson PA, Saedler H, 1987. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J. 6:3553-3558.[Web of Science][Medline]

    Peterson PA, 1960. The pale green mutable system in maize. Genetics. 45:115-133.[Free Full Text]

    Peterson PA, 1965. A relationship between the Spm and En control systems in maize. Am Nat. 99:391-398.[CrossRef]

    Radicella JP, Brown D, Tolar LA, Chandler VL, 1992. Allelic diversity of the maize B regulatory gene: different leader and promoter sequences of two B alleles determine distinct tissue specificities of anthocyanin production. Genes Dev. 6:2152-2164.[Abstract/Free Full Text]

    Radicella JP, Turks D, Chandler VL, 1991. Cloning and nucleotide sequence of a cDNA encoding B-Peru, a regulatory protein of the anthocyanin pathway in maize. Plant Mol Biol. 17:127-130.[CrossRef][Web of Science][Medline]

    Sastry GRK, 1969. A description of R-ch alleles. Maize Genet Coop Newslett. 43:201-204.

    Sastry GRK, 1970. Paramutation and mutation of R-ch in maize. Theor Appl Genet. 40:185-190.[Web of Science]

    Sastry GRK, Kurmi SL, 1970. Spotted-dilute and the instability of R-r. Maize Genet Coop Newslett. 44:101-105.

    Scheffler BE, Rojek R, Pusch I, Wienand U, 1999. Molecular analysis of In-D; a semi-dominant allele of the anthocyanin regulatory gene Intensifier. Maize Genet Coop Newslett. 73:35.

    Schwarz-Sommer Z, Gierl A, Berndtgen R, Saedler H, 1985. Sequence comparison of "states" of a1-m1 suggests a model of Spm (En) action. EMBO J. 4:2439-2443.[Web of Science][Medline]

    Stadler LJ, 1948. Spontaneous mutation at the R locus in maize. II. Race differences in mutation rate. Am Nat. 82:289-314.[CrossRef][Web of Science]

    Stadler LJ, 1951. Spontaneous mutation in maize. Cold Spring Harbor Symp Quant Biol. 16:49-63.[Abstract/Free Full Text]

    Styles ED, 1993. Extending the expression of self-color R1-scm alleles in maize. Maydica. 38:127-133.

    Styles ED, Ceska O, Seah KT, 1973. Developmental differences in action of R and B alleles in maize. Can J Genet Cytol. 15:59-72.[Web of Science]

    Styles ED, Coe EH, 1986. Unstable expression of an R allele with a3 in maize. J Hered. 77:389-393.[Abstract/Free Full Text]

    Todd JJ, Vodkin LO, 1996. Duplications that suppress and deletions that restore expression from a chalcone synthase multigene family. Plant Cell. 8:687-699.[Abstract]

    Tonelli C, Dolfini S, Ronchi A, Consonni G, Gavazzi G, 1994. Light inducibility and tissue specificity of the R gene family in maize. Genetica. 94:225-234.[CrossRef]

    Van Der Walt WJ, Brink RA, 1968. Geographic distribution of paramutable and paramutagenic R alleles in maize. Genetics. 61:677-695.

    Walker EL, Panavas T, 2001. Structural features and methylation patterns associated with paramutation at the r1 locus of Zea mays. Genetics. 159:1201-1215.[Abstract/Free Full Text]

    Walker EL, Robbins TP, Bureau TE, Kermicle J, Dellaporta SL, 1995. Transposon-mediated chromosomal rearrangements and gene duplications in the formation of the maize R-r complex. EMBO J. 14:2350-2363.[Web of Science][Medline]

    Wienand U, Weisskirchen I, Franken P, Saedler H, 1991. Molecular analysis of the dominant inhibitory C2 allele C2-Idf. Maize Genet Coop Newslett. 65:51.


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