Transposon Insertions in the Promoter of the Zea mays a1 Gene Differentially Affect Transcription by the Myb Factors P and C1

Corresponding author: Erich Grotewold, Plant Biotechnology Center, The Ohio State University, 206 Rightmire Hall, 1060 Carmack Rd., Columbus, OH 43210., grotewold.1{at}osu.edu (E-mail)

Communicating editor: V. SUNDARESAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The understanding of control of gene regulation in higher eukaryotes relies heavily on results derived from non-in vivo studies, but rarely can the significance of these approximations be established in vivo. Here, we investigated the effect of Mutator and Spm insertions on the expression of the flavonoid biosynthetic gene a1, independently regulated by the transcription factors C1 and P. The a1-mum2 and a1-m2 alleles carry Mu1 and Spm insertions, respectively, in a cis-element (ARE) of unknown function located between the P- and C1-binding sites. We show that the insertions of Mu1 and Spm similarly influence the expression of a1 controlled by C1 or P. The P-controlled a1 expression in a1-m2 is Spm dependent, and the mutant phenotype of a1-mum2 is suppressed in the pericarp in the absence of the autonomous MuDR element. Footprints within the ARE affect the regulation of a1 by C1 and P differently, providing evidence that these factors control a1 expression using distinct cis-acting regulatory elements. Together, our findings contribute significantly to one of the best-described plant regulatory systems, while stressing the need to complement with in vivo experiments current approaches used for the study of control of gene expression.

THE maize a1 gene encodes the enzyme dihydroflavonol reductase (DFR; O'REILLY et al. 1985 ; SCHWARZ- SOMMER et al. 1987 ), which participates in the formation of the red phlobaphene and purple anthocyanin pigments. Loss of a1 function results in no anthocyanin or phlobaphene pigmentation. While plant and aleurone tissues remain green or colorless, respectively, in the absence of a1 function, the pericarp accumulates a brown pigment of unknown composition if the other genes in the pathway are expressed (STYLES and CESKA 1989 ).

In maize, the accumulation of the anthocyanin and phlobaphene pigments is regulated independently by the transcription factors C1 and P, respectively (reviewed in MOL et al. 1998 ). C1 and P are members of the large maize R2R3 Myb family of regulatory genes (RABINOWICZ et al. 1999 ). C1 is responsible for the anthocyanin pigmentation of the aleurone, while its close relative Pl (CONE et al. 1993 ) leads to pigmentation with anthocyanins in other plant organs such as green tissues and the pericarp (COE and NEUFFER 1989 ). The regulatory activity of C1 is modulated by the physical interaction of the Myb domain of C1 with members of the R/B class of helix-loop-helix factors (GOFF et al. 1992 ; GROTEWOLD et al. 2000 ). Thus, C1 activates a1 expression and anthocyanin accumulation only when expressed together with R or B. In contrast, P activates transcription independently of the R/B proteins (GROTEWOLD et al. 1994 ), and no coactivators necessary for P function have yet been identified. The a1 promoter has a modular organization (Fig 1) with a proximal cis-acting regulatory element bound in vitro with moderate high affinity by P (^haPBS) and by a distal element bound by P with low affinity (^laPBS). Between the ^haPBS and ^laPBS, the a1 promoter contains the anthocyanin regulatory element (ARE; Fig 1), which is present in the promoters of other anthocyanin biosynthetic genes (TUERCK and FROMM 1994 ; LESNICK and CHANDLER 1998 ). In vitro, C1 binds both the ^haPBS and ^laPBS with low affinity (SAINZ et al. 1997 ). In transient expression experiments using maize suspension cells, either C1 and R (C1/R) or P can direct high levels of expression from promoters containing the ^haPBS (GROTEWOLD et al. 1994 ) or the ^laPBS together with the ARE (TUERCK and FROMM 1994 ). However, mutations in any one of these three cis-acting regulatory regions (^haPBS, ARE, or ^laPBS) have modest effects on the activation of a1 by P or by C1/R in transient expression experiments. For example, mutations in the ^haPBS reduce the P-regulated expression of a1 to 15% of wild-type levels, while the activation by C1/R is reduced to only 35% (GROTEWOLD et al. 1994 ). Similarly, mutations of the ^laPBS or the ARE reduce a1 expression to 20% by P and to 35% by C1/R (SAINZ et al. 1997 ), and mutations of the ARE site (in a promoter lacking the ^haPBS) reduce a1 expression to 30% by P and to 10% by C1/R (TUERCK and FROMM 1994 ). Only mutations in both the ^haPBS and ^laPBS resulted in >95% inhibition of a1 activation by P and in >80% inhibition by C1/R (SAINZ et al. 1997 ). These findings indicate that the cis-acting regulatory elements in the a1 promoter can largely compensate for each other in these assays, suggesting a high level of redundancy in the regulation of a1.

View larger version (8K): In this window In a new window Download PPT slide   Figure 1. Modular structure of the maize A1 promoter. The high-affinity (haPBS) and low-affinity P-binding sites (laPBS) and the anthocyanin regulatory element (ARE) and the TATA box are shown as boxes. The start of transcription in the wild-type A1 allele (SCHWARZ-SOMMER et al. 1987 ) is indicated as +1. The positions of the Spm transposon in the a1-m2 allele (SCHWARZ-SOMMER et al. 1987 ) and of the Mu1 transposon in the a1-mum2 allele are shown as triangles.

The results derived from the transient expression and in vitro binding experiments described above are contrasted by the striking effect upon aleurone pigmentation in a1-m2 and a1-mum2 alleles of a1. a1-m2 alleles, originally isolated and described by MCCLINTOCK 1961 , carry the autonomous Spm transposon or nonautonomous dSpm derivatives inserted in the ARE element (MASSON et al. 1987 ; Fig 1). a1-m2 belongs to the Spm-dependent class of alleles (MASSON et al. 1987 ), in which the expression of the a1 gene (evidenced by anthocyanin accumulation) happens only in the presence of a trans-acting nondefective Spm. The a1-mum2 allele, originally isolated by Robertson (described in CHOMET et al. 1991 ), carries the nonautonomous Mu1 transposable element inserted in the ARE element, two nucleotides 5' of the site of Spm insertion in the a1-m2 allele (Fig 1). a1-mum2 belongs to the Mu-suppressible mutant class (reviewed in MARTIENSSEN 1996 ), in which the mutant phenotype (i.e., no anthocyanin pigmentation) is suppressed (i.e., results in pigmentation) in the absence of the autonomous MuDR element. In the presence of MuDR, excision of Mu1 in the aleurone results in the formation of frequent revertant sectors on a colorless background, because of the inhibitory effect of MuDR on the expression of a1. In this regard, a1-mum2 is similar to the hcf106-mum1 allele, a well-described suppressible mutation in maize (BARKAN and MARTIENSSEN 1991 ; SETTLES et al. 2001 ). In hcf106-mum1, transcription in phenotypically suppressed plants (i.e., no MuDR) initiates within the Mu1 element inserted in the promoter of the hcf106 gene. The presence of MuDR is associated with an extensive hypomethylation of Mu1 and adjacent hcf106 promoter sequences (reviewed in MARTIENSSEN 1996 ) and may interfere with the activation of a regulatory element present in Mu1 that is responsible for the expression of hcf106-mum1. However, the suppression of the mutant a1-mum2 phenotype in the absence of MuDR appears to be tissue specific, as it was reported to occur in the plant body, but not in the aleurone (MARTIENSSEN 1996 ).

The dramatic effects on anthocyanin pigmentation caused by the Mu1 and Spm insertions in the promoter of the a1 gene are in striking contrast to transient expression and in vitro DNA-binding experiments results, in which no single cis-regulatory element completely abolished a1 activity when mutated (GROTEWOLD et al. 1994 ; TUERCK and FROMM 1994 ; SAINZ et al. 1997 ). Here, we investigated the effect that these transposon insertions have on the phlobaphene pigmentation specified by the P gene and compared it with the effect that they have on the anthocyanin pigmentation regulated by C1/R. We show that the a1-m2 allele is Spm dependent for the pericarp pigmentation specified by P, and that a1-mum2 mutant phenotype is partially suppressible in the pericarp. In contrast with previous findings, our studies suggest that the a1-mum2 mutant phenotype is also suppressible in the aleurone, although this suppression takes more than one generation after the removal of MuDR function. The study of somatic and germinal excisions of Mu1 and Spm from the a1-m2 and a1-mum2 alleles, respectively, provides the first evidence that specific cis-acting regulatory elements in the a1 promoter important for the regulation of a1 by P or C1/R differ. Together, our findings suggest that these two transposon insertions provide a unique opportunity to correlate results derived from transient expression and in vitro DNA-binding experiments with the in vivo control of gene expression.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic stocks:
Unless otherwise noted, all the stocks used in this study contain the dominant functional alleles for all the structural and regulatory genes necessary for anthocyanin pigment formation (Table 1). Alleles of the P1 gene (indicated as P in this study to avoid confusion with the Pl regulator of anthocyanin biosynthesis) are named according to the red phlobaphene pigmentation in the pericarp and cob glumes. For example, P-rr specifies red pericarp and red cob, P-wr white pericarp and red cob, and P-ww white pericarp and white cob. In the pericarp, P-rr is dominant over P-wr and P-ww. The P-rr allele used in this study corresponds to the P-rr-4B2 allele previously described (GROTEWOLD et al. 1991 ), unless otherwise indicated. The P-ww allele corresponds to P-ww-1112 resulting from a deletion of the entire transcribed region of P (ATHMA and PETERSON 1991 ). The a1-mum2 alleles used in this study were obtained from the Maize Cooperation Stock Center (Maize Coop, Urbana, IL) and correspond to accessions 330I (a1-mum2; A2 C1 C2 MuDR R1) and 330J (a1-mum2; A2 C1 C2 R1). The a1-m2 and derivative alleles were obtained from the Maize Coop and correspond to a1-m2-7991A::Spm-s (accession 317K), a1-m2-8010A::Spm-s (accession 317M), a1-m2-7991A-p2 (accession 315L), a1-m2-7991A-p4 (accession 315N), and a1-m2-7991A-p5 (accession 315P). These alleles were all homozygous and linked to the dominant Sh2 allele. The a1::r-dt sh2 stock was obtained from Dr. Patrick Schnable (Iowa State University).

Hand pollination was used for all genetic crosses in the field and greenhouse experiments. To create the P-rr a1::r-dt sh2 stock (referred to in this study as P-rr a1 sh2), a1::r-dt sh2 plants were crossed to P-rr A1 Sh2 pollen. Seeds were planted and the resulting plants were self-pollinated. Kernels carrying the recessive sh2 allele providing the typical shrunken phenotype in the endosperm were planted and backcrossed as females to the original a1::r-dt sh2 stock to score for pericarp pigmentation.

Determination of the site of Mu1 insertion in the a1-mum2 allele:
The 5' and 3' regions flanking the Mu1 element in the a1-mum2 allele were PCR-amplified using a1-specific primers based on the published sequence of A1 (SCHWARZ-SOMMER et al. 1987 ) and the LIR50 Mutator primer previously described (DAS and MARTIENSSEN 1995 ). PCR products were cloned into the pT-AdvanTAge vector (CLONTECH, Palo Alto, CA) and sequenced.

Analysis of somatic and germinal excision products:
To characterize the stable footprint alleles derived from a1-m2, maize genomic DNA was extracted from 2- to 3-week-old seedlings (CONE 1989 ). PCR was performed using two independent genomic DNAs with high-fidelity Platinum Taq polymerase (Bethesda Research Laboratories, Gaithersburg, MD) and the a1 promoter-specific primers, AP-FG-1 and AP1. AP-FG-1 corresponds to the sequence 5'-CACTGAGTACTGCTGGTTGTTCAA-3' located between positions -262 and -239 (Fig 1) and AP1 to the sequence 5'-TCTCAGCTGCTCCAGTTCCG-3' located between positions +13 and -7 (Fig 1). The 275-bp PCR products were excised from agarose gel and purified using a Qiaquick PCR purification kit (QIAGEN, Germany). The PCR products were directly sequenced.

Analysis of pericarp pigments by spectrophotometric methods:
The extraction method was modified from DAS et al. 1994 . A total of six kernels were soaked in water for at least 2 hr. Pericarps were peeled manually, ground with liquid nitrogen, and placed in a tared tube. Tissue was dried by lyophilization overnight and its dried weight was measured (50 mg). The dried ground pericarps were extracted with 0.1 ml concentrated HCl and 0.4 ml dimethylsulfoxide (DMSO) sequentially with vigorous vortexing after each addition. Extracts were vortexed for another 20 min, after which tubes were centrifuged for 1 min to clarify the suspension. Clear extracts were diluted with methanol (20% final concentration) and centrifuged briefly to clarify the suspension. Absorbance spectra of the clear supernatants were determined within the 380–650 nm range using a CALY3E spectrophotometer (Varian, San Fernando, CA). The -max of phlobaphene pigment was determined at 510 nm.

Analysis of aleurone pigments by spectrophotometric methods:
A total of 8–10 kernels were soaked in water for at least 2 hr. Pericarps and embryos were removed manually. The aleurone and endosperm were ground in liquid nitrogen and placed in a tared tube. Tissue was dried by lyophilization overnight and its dried weight was measured (80 mg). The dried ground tissues were extracted with 2 ml of acidic (1% HCl) methanol by shaking at room temperature for at least 3 hr. The tube was centrifuged for 1 min to clarify the suspension. Clear extracts were diluted with methanol (50% final concentration) and the absorbance spectra were determined within the 380–650 nm range using a CALY3E spectrophotometer (Varian). The -max of anthocyanin pigment was determined at 520 nm.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Transposon insertions in the a1-mum2 and a1-m2 alleles have similar effects upon P- or C1/R-regulated a1 expression:
Aleurones expressing dominant alleles of C1 and R accumulate purple anthocyanins (Fig 2A) that are absent in the presence of the homozygous recessive mutant a1 allele (Fig 2B). P-rr A1 pericarps (Fig 2F) accumulate the brick-red phlobaphene pigments, while P-rr pericarps are brown in the presence of the recessive mutant a1 allele (Fig 2G). To investigate the effect of Mu1 and Spm insertions (Fig 1) on the regulation of A1 by P, we crossed plants carrying the P-rr allele of P with plants carrying the a1-mum2 or a1-m2 alleles. Because the pericarp is a maternal tissue (modified ovary wall), pigmentation in the pericarp was scored in the subsequent generation. Fig 2 shows that the Spm insertion present in the a1-m2-7991A::Spm-s allele has a similar effect upon pigmentation in the pericarp (Fig 2H), controlled by P, and in the aleurone (Fig 2C), controlled by C1 and R. P-rr a1-m2-7991A::Spm-s pericarps display frequent variegation with some large red revertant sectors (see Fig 2H, inset) as expected from the excision properties of these Spm elements from the aleurone tissue (MASSON et al. 1987 ; FEDOROFF 1989 ). Pericarps carrying the a1-m2-7991A:: Spm-s allele also have significant red pigmentation (compare background color between Fig 2G and Fig 2H), suggesting that as for the aleurone pigmentation, phlobaphene accumulation is also Spm dependent. Indeed, P-rr pericarps containing a defective Spm (a1-m2-7977B:: dSpm, Table 1) show, in the absence of the autonomous Spm element, a pigmentation indistinguishable from P-rr a1 pericarps (data not shown). Unlike a1-m2-7991A:: Spm-s, the a1-m2-8010A::Spm-s allele shows infrequent aleurone spotting and colorless background pigmentation (Fig 2D; MASSON et al. 1987 ) in the presence of C1/R. Similarly, almost no variegation was detected in a1-m2-8010A::Spm-s pericarps, which accumulate only brown pigments (Fig 2I) in the presence of P-rr. Thus, the Spm insertions present in the different a1-m2 alleles affect similarly aleurone pigmentation controlled by C1 and R and pericarp pigmentation specified by P.

View larger version (133K): In this window In a new window Download PPT slide   Figure 2. Pigmentation provided by various a1 alleles in pericarp and aleurone tissues. All the top ears (A–E) express the C1 and R (R-g) genes, but lack pericarp pigmentation because they contain either the P-wr or P-ww P alleles. (A) Anthocyanin accumulation in the presence of the wild-type A1 allele. The presence of purple and pink kernels reflects the segregation of the recessive pr allele (COE and NEUFFER 1989 ). (B) Absence of a1 function in the aleurone results in no anthocyanin pigmentation. (C) The a1-m2-7991A::Spm-s allele provides frequent spotting as well as pink background anthocyanin pigmentation. (D) The a1-m2-8010A::Spm-s allele shows less frequent aleurone spotting and no background pigmentation. (E) Ear resulting from the self-pollination of a a1-mum2 Sh2/a1 sh2, MuDR plant. The frequent spotting in the aleurone reflects the somatic excision of Mu1 from the a1-mum2 allele. The shrunken kernels lack anthocyanin pigmentation because of the close linkage between the recessive sh2 and a1 alleles. All the bottom ears (F–J) express the P-rr allele in the pericarp that specifies phlobaphene pigmentation, but unless otherwise specified, lack aleurone anthocyanins because the functional regulators C1 or R are not expressed. (F) Phlobaphene accumulation in the A1 pericarps. (G) Brown pigments accumulate in a1 pericarps. The segregation of shrunken kernels reflects the fact that this ear was obtained from a self-pollination of a a1 Sh2/a1 sh2 plant. (H) Pericarps carrying the a1-m2-7991A::Spm-s allele are variegated and show intense red background pigmentation. The segregation of shrunken kernels reflects that this ear was obtained from a self-pollination of a a1-m2-7991A::Spm-s Sh2/a1 sh2 plant. Darker kernels correspond to aleurones expressing R and C1 to verify Spm activity. (I) Pericarps carrying the a1-m2-8010A::Spm-s allele show no evident variegation and brown pigmentation. The segregation of shrunken kernels reflects that this ear was obtained from a self-pollination of a a1-m2-8010A::Spm-s Sh2/a1 sh2 plant. Darker kernels correspond to aleurones expressing R and C1. (J) Pericarps carrying the a1-mum2 allele are brown and display almost no variegation (see text). The segregation of darker kernels reflects that the underlying aleurone segregates for the a1-mum2 allele displaying the frequent purple spotting.

In the presence of the autonomous MuDR element, a1-mum2 aleurones display very frequent small revertant spots on a colorless background (Fig 2E), characteristic of the frequent and late excision of Mutator (CHANDLER and HARDEMAN 1992 ; BENNETZEN et al. 1993 ). In contrast, pericarps with the same a1 allele are brown in color and display none or very little of the variegation expected for the excision of Mu1 (WANG and GROTEWOLD 1999 ; Fig 2J). Thus, like the aleurone pigmentation specified by C1 and R, the Mu1 insertion interferes with the normal expression of a1 in response to P, in the presence of MuDR.

The mutant phenotype associated with a1-mum2 is partially suppressible in the pericarp:
To investigate whether the mutant phenotype of P-rr a1-mum2 pericarps (Fig 2J) is suppressed by the absence of MuDR, we carried out the genetic crosses shown in Fig 3A. The presence of shrunken kernels, a phenotype provided by the recessive mutant sh2 allele closely linked to the recessive mutant a1 allele, allowed us to distinguish ears containing the a1-mum2 allele from sibling ears carrying the mutant a1 allele. In the absence of MuDR, the a1-mum2 allele confers a reddish color upon the pericarp and cob glumes (Fig 3B, left ear) absent in a1 P-rr pericarps, which are solid brown (Fig 3B, right ear). However, the reddish color of the P-rr a1-mum2 pericarp is weaker than the red color provided by the dominant A1 allele in P-rr pericarps (Fig 2F). The degree of suppression of the mutant phenotype was investigated by comparing the accumulation of the pigments present in P-rr A1, P-rr a1, and P-rr a1-mum2 pericarps (Fig 3C). Quantification of the absorption peak at 510 nm, characteristic of the red phlobaphene pigments (DAS et al. 1994 ) and absent in P-rr a1 pericarps (data not shown), indicated that P-rr a1-mum2 accumulate 14% of the level of phlobaphenes present in P-rr A1 pericarps. In the presence of MuDR, P-rr a1-mum2 pericarps are completely brown (Fig 2J), with no detectable phlobaphene accumulation (Fig 3C). Thus, as previously described for other vegetative plant tissues (MARTIENSSEN 1996 ), the mutant phenotype of the a1-mum2 allele is partially suppressible in the pericarp.

View larger version (35K): In this window In a new window Download PPT slide   Figure 3. The mutant phenotype of a1-mum2 is partially suppressible in the pericarp. (A) Pedigree outlining the genetic cross that allows the direct comparison of P-rr a1 and P-rr a1-mum2 (-MuDR) ears. (B) Phenotypes of a1-mum2/a1 P-rr (left ear) and a1/a1 P-rr pericarps (right ear). (C) Absorption spectra of the pericarp pigments extracted in the conditions described (DAS et al. 1994 ) from ears carrying several different a1 alleles.

Suppression of the mutation a1-mum2 in the aleurone:
The reported differences in the suppression of the a1-mum2 mutant phenotype in vegetative tissues (suppressible) and in the aleurone (not suppressible; MARTIENSSEN 1996 ) prompted us to investigate in more detail the behavior of this allele in the aleurone. Self-pollination of a1-mum2 Sh2/a1 sh2, MuDR plants resulted in an ear with 244/426 (57%) plump spotted kernels, 71/426 (17%) plump colorless kernels, and 111/426 (26%) shrunken colorless kernels (Fig 4A). This segregation is consistent with a single MuDR element unlinked to the a1 locus, lost in the plump colorless kernels containing the a1-mum2 allele and the linked dominant Sh2 allele. Plump colorless kernels were planted and the ears resulting from the self cross showed variable amounts of aleurone pigmentation (Fig 4B). In contrast to a1-mum2 aleurones containing MuDR (Fig 4A), the a1-mum2 (-MuDR) aleurones are not spotted; rather they display a variable amount of pigmentation at the top of the kernel (Fig 4B). The pigmentation in the aleurone increases even further in the next generation (data not shown). Interestingly, however, not all the aleurones are equally pigmented (Fig 4B), a phenomenon found in many ears derived from similar experiments (not shown). Similarly, ears derived from planting seeds obtained from the Maize Coop carrying the a1-mum2 allele but no MuDR (stock 330J) show, after self-pollination, a variable pigmentation in the top region of the kernels (Fig 4C). Similar findings with different Mutator stocks are reported elsewhere (WALBOT and RUDENKO 2002 ). Thus, contrary to previous reports (MARTIENSSEN 1996 ), the mutant phenotype of a1-mum2 is also suppressible in the aleurone, although this suppression is also partial and requires at least an additional generation after the removal of MuDR.

View larger version (42K): In this window In a new window Download PPT slide   Figure 4. The suppression of the mutant phenotype of a1-mum2 aleurones takes several generations. (A) Ear resulting from self-pollinating a a1-mum2 Sh2/a1 sh2, + MuDR plant. Colorless plump kernels have lost MuDR but retain the a1-mum2 allele. Such kernels were planted to give a plant that, when self-pollinated, gave a a1-mum2 Sh2/a1-mum2 Sh2 ear shown in B, which displays weak purple pigmentation in the aleurone, but no spotting indicative of the absence of MuDR. (C) Ear obtained by self-pollination of a a1-mum2 Sh2/a1 sh2 (no MuDR) plant showing the appearance of pigmentation in the aleurone after MuDR has been lost for several generations.

Stable derivative alleles derived from a1-m2 affect P- and C1/R-specified pigmentation differently:
Several stable a1 alleles obtained by excision of Spm from the unstable a1-m2-7991A::Spm-s were analyzed (see MATERIALS AND METHODS). The footprints left by Spm excision in these alleles were determined by PCR amplification and direct sequencing. The stable alleles were also introduced into P-rr maize stocks (see MATERIALS AND METHODS), and the phenotype of the pericarp (specified by P-rr) and aleurone (specified by C1 and R) were compared (Fig 5). Derivative alleles from a1-m2-7991A:: Spm-s frequently have pale pigmentation in the aleurone, as previously reported (MASSON et al. 1987 ). Allele a1-315L contains the three-nucleotide target site duplication characteristic of Spm (AAT), as well as the insertion of the ATT trinucleotide sequence, present in the original a1-m2-7991A::Spm-s allele flanking the right end of the transposable element (MASSON et al. 1987 ). The a1-315L allele shows pale pigmentation in the aleurone (7% of wild-type levels), yet the pigmentation of the pericarp is visually indistinguishable from wild-type A1 alleles, with 67% of wild-type levels of phlobaphenes (Fig 5). Thus, the six-nucleotide insertion that distinguishes a1-315L from the wild-type A1 allele dramatically affects a1 activation by C1/R, but not by P. The excision of Spm that resulted in the a1-315M allele left behind a footprint, which contains just the ATT insertion (Fig 5). Like a1-315L, this allele shows pale aleurone pigmentation (36% of wild-type levels), but pigmentation in the pericarp is only minimally affected by this insertion (Fig 5). The a1-315P allele contains a four-nucleotide insertion when compared with wild-type A1 alleles (Fig 5). This footprint results in normal aleurone and pericarp pigmentation (Fig 5), and the anthocyanin and phlobaphene levels are above wild-type levels (160 and 110%, respectively). Together, these findings indicate that the footprints left in the ARE by Spm excision affect the pigmentation specified by P or C1/R in different ways.

View larger version (25K): In this window In a new window Download PPT slide   Figure 5. Stable excision alleles derived from a1-m2. The sequence of the footprints left by the excision of Spm from a1-m2-7991A::Spm-s is shown, with the phenotype of the corresponding kernels in the presence of the C1 and R (aleurone) or P-rr (pericarp) regulatory alleles. The approximate amount of phlobaphenes in the pericarps is derived from Fig 3C. The quantitation of anthocyanins is described in MATERIALS AND METHODS. The black text denotes the sequence of the wild-type A1 allele in the region of the ARE element, and the red text indicates extra nucleotides left as footprints by the excision of Spm. The red triangle indicates the position of the Spm transposon in the a1-m2-7991A::Spm-s allele.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Study of control of gene expression is largely dependent upon the results of in vitro (e.g., DNA binding) and semi-in vivo (e.g., transient expression) studies to investigate the participation of trans-acting factors and cis-acting elements in gene regulation. Few cases are available to directly compare results derived from these approximations with the relevance that particular cis-acting regulatory elements have for gene expression in vivo. We have utilized two previously described transposon insertions in the promoter of the maize a1 gene to highlight the in vivo significance of a cis-acting element central in the regulation of a1 by the related R2R3 Myb-domain transcription factors, P and C1.

Previously, we showed that the ^haPBS cis-element (Fig 1) fused to a minimal CaMV 35S promoter is sufficient to drive transcription by P and C1/R in maize transient expression experiments (GROTEWOLD et al. 1994 ). Thus, the more distally located Mu1 and Spm insertions present in the a1-mum2 and a1-m2 alleles, respectively (Fig 1), would not have been predicted to affect the expression of a1 by these regulators. In contrast, we show here (Fig 2) that the phlobaphene pigmentation specified by P in the pericarp and the anthocyanin pigmentation specified by C1 and R in the aleurone are dramatically affected by these insertions.

a1-m2-7991A::Spm-s aleurones are spotted due to the frequent excision of Spm and a1-m2-7991A::Spm-s pericarps are variegated (Fig 2). As previously shown for the aleurone anthocyanin pigmentation (MASSON et al. 1987 ), the a1-m2-7991A::Spm-s allele is Spm dependent for phlobaphene pigmentation in the pericarp. The mechanism by which transcription of a1 becomes dependent on a gene product encoded by the autonomous Spm element is not yet understood. However, our results provide evidence that this phenomenon is not restricted to the control of a1 by the C1/R regulators of anthocyanin biosynthesis, but that the participation of components of the Spm transposon on the regulation of a1 extends as well to the independent P regulatory system. The insertion of Spm into the a1 promoter in the a1-m2-7991A::Spm-s is characterized by the 3-bp target site duplication characteristic of Spm (AAT, Fig 5). In addition, the a1-m2-7991A::Spm-s has another three-nucleotide insertion that probably arose during the selection of novel a1-m2 alleles, derived from the original a1-m2 identified by McClintock (MASSON et al. 1987 ). In contrast, the a1-m2-8010A::Spm-s allele contains only the three-nucleotide insertion from the target site duplication (MASSON et al. 1987 ). Unlike a1-m2-7991A::Spm-s, a1-m2-8010A::Spm-s shows much less frequent aleurone spotting and no obvious pericarp variegation (Fig 2). Moreover, a1-m2-8010A::Spm-s does not display the Spm-dependent background pigmentation in the aleurone (MASSON et al. 1987 ) or in the pericarp tissues (Fig 2). The frequent generation of unpigmented derivative excision alleles from the a1-m2-8010A::Spm-s allele (MASSON et al. 1987 ) suggests that the three-nucleotide ATT insertion present in a1-m2-7991A::Spm-s is responsible for the different properties of these two a1-m2 alleles. The insertion of Mu1 in the a1-mum2 allele is 2 bp closer to the transcription start site from the Spm insertion site in a1-m2 (Fig 1). In spite of the proximity of these insertion sites, the Mu1 and Spm insertions affect transcription of a1 in very different ways. The a1-mum2 allele is a member of the growing class of suppressible mutations, in which the mutant phenotype, in this case lack of pigmentation, is evidenced only in the presence of the autonomous MuDR element (MARTIENSSEN 1996 ). We show here that the lack of phlobaphene pigmentation in the pericarp is partially suppressible (Fig 3B), suggesting that the presence of MuDR interferes with the activation of a1 by P. This is similar to what is described for other vegetative tissues (CHOMET et al. 1991 ; MARTIENSSEN 1996 ), although we observe only a reduced (14% of wild type) accumulation of phlobaphenes in P-rr a1-mum2 pericarps (Fig 3B). Thus, even in the absence of MuDR, the Mu1 insertion present in a1-mum2 impairs the activation of a1 by P, further indicating that in vivo, the ^haPBS are not sufficient for high-level activation of a1 by P or that the presence of Mu1 inhibits the interaction of P with the ^haPBS. In contrast to previous studies that indicated that the mutant phenotype is not suppressible in the aleurone (MARTIENSSEN 1996 ), we observed significant accumulation of anthocyanin pigments in this tissue in the absence of MuDR. Interestingly, however, this suppression of the mutant aleurone phenotype does not occur immediately after the removal of MuDR, but takes at least one additional generation (Fig 4). Different from the hcf106-mum1 Mu-suppressible allele (BARKAN and MARTIENSSEN 1991 ), in which Mu1 is inserted 3' of the regulatory elements that control normal hcf106 expression, the location of Mu1 in the a1-mum2 allele is upstream of the ^haPBS cis-regulatory elements required for the control of a1 by P and C1 (Fig 1). Thus, MuDR must be interfering with the control of a1 by C1/R and P in different ways than proposed for hcf106-mum1 (MARTIENSSEN 1996 ).

The unexpected pigmentation patterns provided by the Mu1 and Spm insertions could be a consequence of the disruption by the transposons of cis-regulatory elements important for the regulation of a1. Alternatively, the insertion of the transposons could be interfering with the activities of the regulatory factors controlling a1 in an indirect fashion. To distinguish between these two possibilities, we examined the effect of germinal footprints left by the excision of the transposons on the P- and C1-specified pigmentation. Three germinal revertant alleles from a1-m2-7991A::Spm-s were molecularly characterized and their effects on anthocyanin and phlobaphene pigmentation were compared (Fig 5). Interestingly, while a1-315P has normal C1- and P-regulated pigmentation, it does contain an insertion of four nucleotides when compared with wild-type a1 alleles. In contrast, both a1-315N and a1-315L dramatically reduce anthocyanin accumulation, without significantly affecting phlobaphene pigmentation. In previous studies, promoter mutations that impaired a1 transcription controlled by P also affected transcription by C1/R (GROTEWOLD et al. 1994 ; TUERCK and FROMM 1994 ; SAINZ et al. 1997 ), consistent with the very similar DNA-binding preferences of P and C1 (SAINZ et al. 1997 ). Thus, the footprints present in a1-315N and a1-315L provide the first evidence indicating that promoter elements important for the control of a1 by C1/R are not important for the activation of a1 by P in vivo.

P-rr a1-mum2 pericarps almost completely lack variegation (WANG and GROTEWOLD 1999 ), compared with the frequent spotting of underlying aleurones (Fig 2). Although we cannot conclusively rule out the possibility that Mu1 does not transpose at a high frequency from a1-mum2 pericarps, it is more likely that the very low frequency of variegation is a consequence of the footprints left by the excision of Mu1 from this allele. These footprints would rarely result in sequences that allow the a1 promoter to be activated by P. In contrast, the frequent spotting of a1-mum2 aleurones suggests that similar footprints are not affecting transcription of a1 by C1/R. While Mu1 somatic footprints largely resemble those left by other transposons, larger insertions and deletions that may result from recombinational events have been identified as a result of the somatic excision of Mu1 from the bz-mum9 allele (DOSEFF et al. 1991 ). Together with the results obtained from the Spm germinal excision alleles, we conclude that the ARE element plays a much more important role in the regulation of a1 by P or C1/R than predicted from previous studies. Moreover, within the ARE there appear to be subelements that can be physically separated, which are important for the control of a1 by each regulator, P or C1/R.

The absolute in vivo requirement of the ARE for the regulation of a1 is strikingly different from previous transient expression studies indicating that the ^haPBS element is sufficient for activation by P and C1/R in maize cells (GROTEWOLD et al. 1994 ). Although a potential Myb consensus binding site is present slightly upstream of the defined ARE site (TUERCK and FROMM 1994 ), neither P nor C1 recognizes this site on the basis of in vitro DNA-binding experiments (GROTEWOLD et al. 1994 ; SAINZ et al. 1997 ). Thus, it is possible that other factor(s) involved in the regulation of a1 by C1/R and P recognize this element that is present in the promoters of several flavonoid biosynthetic genes. Alternatively, the presence of the ARE element in multiple flavonoid biosynthetic genes could reflect the participation of this element in other aspects of gene regulation, such as maintaining a particular chromatin structure. Such a participation of the ARE would explain some of the inconsistencies between the transient expression experiments (probably using nonchromatin templates) and the in vivo effects of mutations in this element.

Together, the findings described here stress the importance of the ARE element in the control of a1 by the P and C1/R regulatory systems. The importance of this element in the regulation of a1 by two different regulators was determined only because of the presence of transposable element insertions. These studies emphasize the limitations associated with in vitro DNA-binding and transient expression experiments and indicate that transposable elements provide a powerful tool to probe promoter function in vivo.

	ACKNOWLEDGMENTS

E.G. is grateful to Venkatesan Sundaresan (Sundar) for the original finding that P-rr a1-mum2 pericarps lack obvious variegation, to Rob Martienssen for sharing his extensive knowledge on Mutator, and to Tom Peterson for his guidance in maize genetics. We thank the Maize Coop for the maize stocks and Iris Meier and Ed Braun for helpful comments on the manuscript. We acknowledge the excellent technical assistance provided by J. Marcela Hernandez, Kevin Johnson, Amber Pollack, and Huili Wang. This project was funded by a National Science Foundation grant (no. MCB-9974474) to E.G.

Manuscript received July 13, 2001; Accepted for publication February 18, 2002.

	LITERATURE CITED

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