The Plant Cell, Vol. 3, 115-125, February 1991 © 1991 American Society of Plant Physiologists

Expression Patterns of myb Genes from Antirrhinum Flowers David Jackson,1 Francisco Culianez-Macia, Andy G. Prescott, Keith Roberts, and Cathie Martin Departments of Cell Biology and Genetics, John Innes Institute, John Innes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UH, United Kingdom

Six genes that contain sequence encoding the DNA binding domain of the Myb oncoproteins have been isolated from a cDNA library prepared from Antirrhinum majus (snapdragon) flowers using oligonucleotide probes directed against part of this domain. The derived amino acid sequences of these genes reveal acidic domains in their carboxy termini, suggesting that they might act as transcriptional activators. Analysis of their expression patterns with respect to organ specificity, floral differentiation, and response to light suggests that these genes are not involved in controlling anthocyanin biosynthesis, unlike the characterized myb-related genes C1 and PI from maize. One of the genes is expressed mainly in the nectary and the transmitting tract of the style, two major secretory tissues of the flower, suggesting that the function of this gene is related to active carbohydrate secretion. We conclude that plants contain a number of myb-related transcriptional activators involved in a diversity of gene regulation.

INTRODUCTION

Relatively few genes involved in transcriptional regulation in higher plants have been characterized at a molecular level. The first to be isolated was the C1 gene from maize (Cone et aI., 1986; Paz-Ares et aI., 1986), which regulates structural genes involved in the biosynthesis of anthocyanin pigment in a tissue-specific manner (Dooner and Nelson, 1979). The C1 gene contains a domain at the amino terminus of the deduced protein sequence that shows high similarity to the DNA binding domain of the c-myb proto-oncogene (Paz-Ares et aI., 1987). c-myb, which is very highly conserved in chicken, mouse, and humans (Klempnauer et aI., 1982; Gonda et aI., 1985; Majello et aI., 1986), is expressed in immature thymocytes (Westin et aI., 1982) and is thought to be involved in the regulation of hematopoiesis (Duprey and Boettiger, 1985; Gewirtz and Calabretta 1988). The protein encoded by c-myb is located in the nucleus (Klempnauer et aI., 1984). It can be divided into two parts with different functions: the amino-terminal region, which contains three imperfect repeats of 51 to 52 amino acids and which binds DNA in a sequence-specific manner (Biedenkapp et aI., 1988; Howe et aI., 1990), and a part of the carboxy terminus, which acts as a transcriptional activator when fused to a heterologous DNA binding domain or as part of the intact c-Myb protein (Weston and Bishop, 1989; Sakura et aI., 1989). The maize C1 gene also contains a carboxy-terminal domain that could form an acidic amphipathic a-helix 1

To whom correspondence should be addressed.

and act as a transcriptional activator (Paz-Ares et aI., 1990). The presence of this domain implies that C1 regulates anthocyanin production directly by activating transcription of biosynthetic genes. This idea is supported further by experiments showing that C1 is required for production of luciferase when the reporter gene is under the control of an anthocyanin biosynthetic gene promoter (Goff et aI., 1990). C1 controls anthocyanin biosynthesis in the maize aleurone. In other maize tissues, anthocyanin biosynthesis is regulated by the PI locus, which has high sequence identity with C1 and is thought to be a duplicated gene (Cone and Burr, 1988). In maize, therefore, regUlation of anthocyanin biosynthesis operates through at least two myb-related genes, depending on the developmental stage. The myb DNA binding domain has also been found in genes in humans (Nomura et aI., 1988), Drosophila (Katzen et aI., 1985), yeast (Tice-Baldwin et aI., 1989), and another two genes from maize and two from barley (Marocco et aI., 1989). The plant myb-related genes show no similarity to C1 outside the DNA binding domain, and their function(s) remain unknown. myb-related genes appear to perform a range of functions in different organisms through the transcriptional activation of different genes. We were interested in determining the extent to which myb gene homologs might be involved in controlling floral development and whether their role is exclusively concerned with the regulation of anthocyanin biosynthesis or extends to controlling other meta-

116

The Plant Cell

bolic or developmental processes in flowers. Toward this end we have used the snapdragon, Antirrhinum majus, in which many mutations that affect floral development have been identified, including some which, like C1, regulate anthocyanin production in a tissue-specific manner (Martin et aI., 1987). Some of these have been shown to affect the expression of specific genes of the anthocyanin biosynthetic pathway (Almeida et aI., 1989). We report that at least six myb genes are expressed in flowers, and, by investigating their patterns of expression, we have begun to infer which specific processes these genes may be regulating as well as drawing more general conclusions about the roles of myb-related genes in plants.

RESULTS

Isolation of myb-Related eDNA Clones from Antirrhinum Flowers

1

A.majus305 A.majus340 A.majus330 A.majus315 A.majus308 A.majus306

MDKKPCNSQDVE MDKKPCNSHDVE MGRSPC-CEKAH MERQPC-REKFG MGRSPC-CEKAH MGRPPC-CDKIG

Z.mays C1

MGRRAC-CAKEG 65

305 340 330 315 308 306

.RK.P .. ME .. LI. IN. IAN V.NSLARS K.T .RK.P .. ME .. LI. INFISN V. NTIARS K. T TNK KE .. QR. IN. IR C.. SL.KA L L P .. EE .. QK. TS .. LKN. IQG .. VI .KL S TNK KE R. V.. IR C.. SL.KA L .. K. P .. PE .. II. VS. IQE .. P.N .. AI. SNT .. L .. S

C1

VKRQAW~SKtPDALAAYVKAHGEGKWREVPQKAGLRRCG~S¢RL~WLijY~RgN

I M I T

D D D D D G

118

305 340 330 315 308 306

V TPE. QL .. ME .. AKW V TPE. QL .. ME .. AKW LK FTEE. DEI . . K • • S LKK.PLTEM .. NQ .. E .. AH LK FTEE. DE K• •S .K .. DFTEH .. KM .. H.QA

C1

IRRGNISYDtEDLtIRLaRLtG~NRWSLt~G~~m9~~pijEt~Y~§WLG~

K

K.. KT K.. KH A K.. LHI

RTRIQ.!ili RTRIQ.!ili THIK.K THIKKK THIR.K

K AA .. SY

H

D

TH.~

173

The maize C1 gene has sequence similarity to animal myb genes over a stretch of about 100 amino acids at the amino-terminal end of the deduced protein sequence. We constructed two 42-mer oligonucleotides corresponding to a part of this domain that is particularly highly conserved: one oligonucleotide (02) corresponding to the C1 sequence (Paz Ares et aI., 1987), and the other (01) to an myb consensus sequence. Each oligonucleotide was used to probe duplicate filters lifted from a >..gt10 cDNA library prepared from mRNA from developing flower buds. Using these, we were able to isolate six different types of cDNA clones: one (308) using oligonucleotide 01 , and the other five using oligonucleotide 02. The longest clone from each class was sequenced together with some of the shorter clones. Three of the cDNA clones (305,306, and 315) were incomplete at the 3' end because of the presence of unmethylated EcoRI sites during preparation of the library. Therefore, we used 3' polymerase chain reaction (peR) race amplification to isolate fUll-length cDNAs in these cases (Frohman et aI., 1989). In addition, clones 305 and 308 were incomplete at the 5' end, and we used the genomic clones of these to deduce the full protein sequence. The complete amino acid sequence derived for each gene is shown in Figure 1. The sequences are aligned according to the 51 to 53 amino acid imperfect repeats at the amino terminus of the protein (boxed in Figure 1), as suggested by Frampton et al. (1989). As is the case for C1 (Paz-Ares et aI., 1987), all six genes have two of these imperfect repeats, unlike animal and Drosophila myb genes, which have a third repeat at the amino-terminal end. These repeats in c-myb interact with DNA in vitro in a sequence-specific manner (Biedenkapp et aI., 1988; Howe et aI., 1990). The degree of amino acid identity over these repeats

MEQGDQSSSTTFNNGQMNLDHSCNDQ~SSSQMSACGPVVDHTAVDQS§¥SPHS~

305 340 330 315 308 306

IKQ EASFIG INPEHSNEQi(§TSLLSSSCHADHAVE~¥$sj3li'~gW1GNNvQYPN LVS IDP T SLNSATTTATATPTVNNSCLDFRTSPSNSKNICMPTTDNNNNS LKL IDPNN PFEHKGNVDETKIESDTKESNSQDMKQIVNEVSRQGNNDQITE LLS IDPTT SINDGTASQDQVTTISFSNANSKEEDTKHKVAVDIMIKEENSP LEKLQSPENGKCQDGNSSVDSDKSVSKGQWERRLQTDIHMAKQALCDALSLDKTS

C1

AGAGAGAGGSWVVVAPDTGSHATPAATSGACETGQNSAAHRADPDSAGTTTTSAA 228

305$ijDliTFg~pli'ii~Dg$l{p~::i~~J:l~WSI1QI;Ll{$D*

340 330 315 308 306

Hrl'~E~NI>YF~~l-tEI),L~~l-t9APN~~G*

SSSTDDTKCNSSTTEESQSLITPPPKEEEKSVPLVDLELSLGLPSQSQCNKSVSL STSPEIKDEIVTSCOSDYLMHNNDLMSNRSSNYYSPSFSMEESLSNPKSTGQTSF

VQEREpDLNLDLKISP~QQQINYHQENLKTGGRNGSSTLCFVCRLGIQNSKDCS

SSTDDPKLSTVQTTQPRPFQASTYSSAENIARLLENWKKKSPVNASSTSQAGSSE

C1

AVWAPKAVRCTGGLFFFHRDTTPAHAGETATPMAGGGGGGGGEAGSSDDCSSAAS

330 315 308 306

NSSSSGFYDLFRPPAKVAQRMCVCKWTLGLQKGEQFCNCQSFNGFYRYC* AVSIHEESMKQWVQSVDSKLPWDCFNQLDEQLYLSFQQNQSNS* CSDGVGN* STTTSFNYPSVCLSTSSPSEGAISTNFISFNSSNSDILEDHDQAKFEAATNGVNF

C1

VSLRVGSHDEPCFSGDGDGDWMDDVRALASFLESDEDWLRCQTAGQLA*

306

QDESKPILDNQMPLSLLEKWLLDDSAAVAQGQDVDF*

283

Figure 1. Complete Amino Acid Sequences of the Six Antirrhinum myb Genes.

The maize 01 gene sequence is shown for comparison. The region recognized by oligonucleotides 01 and 02 corresponds to amino acids 98 to 111. The two 51 to 53 amino acid imperfect repeats are boxed. Dots within these boxes denote residues in the Antirrhinum sequences that are identical to those in C1. Shading in the 01 sequence denotes residues identical to murine c-myb. Basic residues at the end of the second repeat are underlined. Outside the boxed repeats, shading of the sequences of clones 305 and 340 denotes identity between these in the carboxy half of the proteins. Other short stretches of identity between the Antirrhinum myb genes and those from maize and barley (discussed in the text) are boxed. Numbers refer to the 305 and 340 sequences.

myb Genes in Antirrhinum

between the Antirrhinum genes and animal myb genes is in the same range as C1 (42% to 51 % identity compared with 42% for Ct). As expected, there is greater amino acid identity between the myb repeats of C1 and the Antirrhinum genes (57% to 67%). Furthermore, if one considers only those residues identical between animal c-myb and C1, between 81 % and 91 % of these are also conserved in the different Antirrhinum genes. In particular, the regularly spaced tryptophan residues are conserved, except for the first tryptophan of the second repeat, which is replaced by another hydrophobic residue (isoleucine, leucine, or phenylalanine) in Antirrhinum myb genes. In C1, the residue at this position is isoleucine. The tryptophan residues have been implicated in the binding of DNA (Anton and Frampton, 1988). The sequences are most similar in the carboxy half of each repeat, where it has been proposed that a helix-tum-helix motif may occur (Frampton et ai., 1989). Although there is considerable variation at the end of the second repeat, the positively charged nature of this region is maintained in all the Antirrhinum myb genes. Outside the myb repeats, there is little similarity between any of the Antirrhinum genes except between clones 305 and 340 (marked in Figure 1). Clones 305 and 340 are also almost identical over the myb domain, suggesting that they share a very similar function or are related in the same way as the duplicated C1 and PI genes of maize. No similarity was found between the Antirrhinum myb genes and C1 or c-myb, and, in a search of the EMBL data base (Devereux et ai., 1984), we failed to find any sequences significantly similar to the carboxy termini of any of the Antirrhinum myb genes. However, close to the carboxyterminal end of the myb domain, the sequence GIPDXH is found in several of the Antirrhinum clones (308, 315, and 330) and in myb genes from barley (Hv1 and Hv33) and maize Zm38). In addition, a sequence identified as conserved between myb genes Hv1 and Zm38, CPDLNLDLXISPP (Marocco et ai., 1989), is also found in the carboxy-terminal portion of the 308 sequence. These two conserved domains, which are not found in any other protein in the EMBL data base, may indicate some functional similarity between these genes. All six Antirrhinum myb genes have domains in the carboxy half of the protein that have overall negative charge and show a-helical tendencies in a Chou-Fasman secondary structure prediction (Devereux et ai., 1984). For 305, 306, 315, and 340, this domain is at the carboxy terminus, as is the case for C1, whereas for 308 and 330, the acidic domain is more central, as is found in c-myb (Weston and Bishop, 1989).

Expression of the Antirrhinum myb Genes

Each gene gives rise to a major transcript of between 0.9 kb and 1.3 kb, as shown in Figure 2. In addition, we

117

305 340 330 308 315

.-255 .-185

Figure 2. Floral Transcripts of Five of the Antirrhinum myb Genes.

Autoradiographs of RNA gel blots are overexposed to show additional larger transcripts as well as each major transcript. Five micrograms of poly A+ RNA was loaded per track.

detected one or more larger transcripts of low abundance for each gene. We believe these to be processing intermediates because RNA gel blots and PCR-amplified cDNAs probed with an intron from one gene, 308, showed hybridization only to the larger transcripts for this gene. We have examined PCR-amplified cDNA for each myb gene from a number of different tissues but have found no evidence that differential splicing plays a role in controlling myb gene products in different tissues. All the genes were expressed at relatively low levels, and the frequency of obtaining cDNA clones suggested that in flowers they may represent less than 0.01 % of the mRNA. Expression of 306 was very low such that transcripts could only just be detected after a 2-week exposure of poly A+ RNA gel blots.

11 8

The Plant Cell

We predicted that if any of the floral myb genes were involved in controlling transcription of anthocyanin biosynthetic genes, they would be expressed in pigmented tissue at the same time as or slightly before the structural genes. Consequently, we examined organ-specific expression as well as temporal expression during floral development. The pattern of expression for five of the genes in different organs, studied by RNA gel blotting, is shown in Figure 3A. The expression of 306 was extremely low; therefore, we used PCR amplification of cDNA prepared from different organs, followed by DNA gel blotting to ascertain the expression pattern of this gene (Figure 38). Under the conditions used (see Methods), the PCR was semiquantitative. On the basis of this analysis, the genes could be grouped into three classes: 308 and 315 showed expression in all plant organs, although 315 was noticeably enhanced in the stem; 306 and 330 were expressed in flowers and some, but not all, of the other organs tested; and 305 and 340 were expressed specifically in flowers. All of the differences observed on RNA gel blots were confirmed in independent experiments using PCR amplification of cDNA (results not shown). The expression of the two flowerspecific myb genes (305 and 340) was compared with that of flavanone 3-hydroxylase (F-3-H), a biosynthetic gene in the anthocyanin pathway that is controlled by many of the genetically characterized anthocyanin regulatory loci (Martin et aI., 1987; Almeida et aI., 1989). The temporal expression during floral development is shown in Figure 4 and differed between the two flower-specific myb genes. Expression of 340 started, like that of F-3-H, at the earliest stage tested (0 mm to 5 mm buds), whereas expression of 305 was not detectable until the buds were 15 mm to 20 mm in length (Figure 4). This suggests that, whereas 340 could be a regulator of anthocyanin biosynthesis, it is unlikely that this is the case for 305, at least not in the earliest stages of anthocyanin expression. The expression of 305, 306, 308, 330, and 340 all increased as flowers developed (Figure 3). Although flowers are complex organs with many different tissues, this expression pattern suggests that these myb-related genes are involved in the regulation of processes occurring in mature tissues rather than processes involved in the differentiation of the floral organs themselves. In Situ Hybridization

To examine the possibility that 340 is a regulator of anthocyanin biosynthesis, we hybridized tritium-labeled riboprobes to sections of whole flower buds of 25 mm to 30 mm length, the stage at which expression of 340 is at a maximum. Hybridization over the flower was localized in discrete cell or tissue types, as shown in Figure 5. Expression was strongest in tissues at the base of the carpel (Figure 5A), and in transverse sections, this was seen as

...

Q,)

'0

- 0 0

a:

E Q,)

en

ro

Q,)

..J

0

a. '0 Q,) Q,)

en

~

Q)

...::J

~

~

Q,)

n;

E

.E

~

...::J

Q,)

ro

~

A

305

340 330 308 315 B

306

NT

Figure 3. Organ-Specific Expression Patterns of the Antirrhinum myb Genes.

(A) Expression elucidated by RNA gel blotting. Five micrograms of poly A+ RNA extracted from root, stem, leaf, seedpod, immature flower (buds 0 mm to 5 mm long), or mature flower (buds >30 mm long) was loaded per track. (8) Expression of gene 306 (which is expressed at levels too low to detect by RNA gel blotting), elucidated by DNA gel blot of PCRamplified cDNA prepared from the different organs. Under the conditions used, the PCR is semiquantitative. NT, not tested.

a ring of expression that extended around the base of the carpel (data not shown). These tissues make up the nectarsecreting organ of the flower, the nectary. Expression was also evident in the style; this is clearest in transverse sections where expression is localized in a layer of cells of the transmitting tract at the boundary between this tissue

myb Genes in Antirrhinum

305

340

F-3-H

Figure 4. Temporal Expression of the Two Floral-Specific myb

119

levels of 330 were found in dark-grown seedlings. Expression of 306 was significantly higher in dark-grown than in light-grown tissue, and this difference was observed in three different Antirrhinum lines. The higher expression of 306 in the dark suggests that this myb gene may be involved in controlling the expression of genes active in dark-grown tissue. There is no evidence that any of these Antirrhinum myb genes are strongly induced in response to light in contrast to the anthocyanin biosynthetic genes of Antirrhinum such as chalcone synthase (Figure 6). The influence of light on anthocyanin biosynthesis may occur partly through regulatory genes such as R (Taylor and Briggs, 1990) and C1. For example, an allele of C1 in which expression is induced by light has been characterized (Hsu, 1970; Wienand et aL, 1989). The absence of light-induced expression of the Antirrhinum myb genes argues against a role for them in floral pigmentation because pigmentation is light responsive.

Genes. RNA gel blots of poly A+ RNA (5 Jig per track) were prepared from developing flower buds of the sizes indicated. Expression was compared with that of F-3-H, a structural gene of the anthocyanin biosynthetic pathway.

and the cortical tissue (Figure 5B). Expression of 340 was also evident in petals, although at a lower level, and there was an even distribution of expression over the epidermis and mesophyll (Figure 5C). To avoid the possibility of cross-hybridization with other myb transcripts, a probe containing only the 3' half of 340 was also used. Although the level of signal obtained with this shorter probe was lower than with the full-length probe, the same localization was observed, showing that the pattern of expression observed was indeed specific for 340. This expression is in contrast to expression of structural genes in the anthocyanin biosynthetic pathway, which in flowers is strongest in the petals and here is epidermal specific (D. Jackson, unpublished data). Effect of Light on Expression of myb Genes

We examined the expression of the myb genes in response to light by using developing seedlings because flowers do not develop in complete darkness. Antirrhinum seedlings were germinated in the dark, and after 1 week, half were exposed to light for 1 week. RNA was prepared from lightgrown and dark-grown tissue, and the expression of the myb genes was examined by peR amplification of cDNA, as shown in Figure 6. Transcripts of 305 and 340 were not detected, supporting our observations that expression of these genes was flower specific. No difference in the expression of clones 308 or 315 was observed in lightgrown and dark-grown material, whereas slightly higher

DISCUSSION

Isolation of myb-Related Genes from Antirrhinum

We have isolated six genes expressed in Antirrhinum flowers, all of which contain sequences encoding the myb DNA binding domain. The primary sequence of each gene shows that, as in C1 (Paz-Ares et aL, 1987), the first repeat of the three 51 to 53 amino acid repeats found in animal and Drosophila Myb proto-oncoproteins is absent. Deletion of this first repeat in c-myb, however, has no effect on sequence-specific DNA binding in vitro (Howe et aL, 1990), and indeed the protein encoded by v-myb, which exhibits sequence-specific DNA binding, contains a truncation of part of the first repeat (Biedenkapp et aL, 1988). These findings suggest that, despite lacking the first repeat, the plant myb-related genes are likely to bind DNA in a sequence-specific manner. It has been postulated that toward the end of each repeat there may be a helix-turn-helix motif similar to that found in A cro-Iike proteins and the Antennapedia gene product from Drosophila (Frampton et aL, 1989; Qian et aL, 1989). In addition, the regularly spaced tryptophan residues have been implicated in DNA binding (Anton and Frampton, 1988), and their conservation in the plant myb genes suggests that they playa specific role in addition to constituting the hydrophobic residues of a helix-turn-helix motif. In fact, it is unlikely that a helix-turn-helix could form in the first repeat because in most of the Antirrhinum myb genes and in C1 there is a proline residue in the middle of a predicted helix, as illustrated in Figure 7. However, a helix-turn-helix motif could form in the second repeat. If this were so, then the residues thought to be involved in intimate contact with the DNA and in regulating sequencespecific binding activity would be those associated with

___- - - - s t y l e

~_~~___7:...+_~..:.......----st ame n

;;,.~~---;...~

carpel -----ll e ct a r y

Figure 5. Expression of myb Gene 340 in the Mature Flower Shown by in Situ Hybridization.

The top panel shows a section through a mature flower bud (25 mm to 30 mm) as used for in situ hybridization, stained with toluidine blue. The lower panels contain magnified views of those parts of the flower that show expression of 340. (A), (B), and (C) refer to the

myb Genes in Antirrhinum

L D

CHS

L D LDLDLD

330

308

315

121

in the EMBL data base. However, each gene contains a stretch of amino acids with net negative charge and which, according to a Chou-Fasman secondary structure prediction, could form an a-helical structure. This type of structure has been implicated in transcriptional activation (Ptashne, 1988; Sigler, 1988), and this suggests that the Antirrhinum myb genes, which were selected as containing the particular DNA binding domain, are also likely to be transcriptional activators. Furthermore, it is interesting to note that the carboxy termini of several of the Antirrhinum myb genes are rich in serine and threonine residues, which are potential sites for phosphorylation. Phosphorylation could provide a means by which transcriptional activation is post-translationally modified because this would provide the acidity required for activation. Such a mechanism has indeed been proposed for c-fos, which is also rich in serine and threonine (Lech et ai., 1988).

306

Figure 6. Influence of Light on myb Gene Expression in Antirrhinum, Seedlings. cDNA prepared from light-grown (L) or dark-grown (D) seedlings was PCR amplified using specific oligonucleotides for each myb gene or for a gene of the anthocyanin biosynthetic pathway, chalcone synthase (CHS). The PCR products were separated on an agarose gel, blotted, and probed with the relevant cDNA.

the recognition helix of the helix-tum-helix motif (Figure 7). The end of the predicted recognition helices of the second repeat and the patches of basic amino acids that adjoin them differ significantly between the Antirrhinum genes (Figure 7). Although it has been reported that the C1 protein binds to a DNA sequence similar to that to which the c-myb protein binds (Wienand et ai., 1989), these differences imply that the Antirrhinum myb genes may bind to different recognition sequences. Our preliminary data indeed show that two of the Antirrhinum myb genes do not bind to either the c-myb or the C1 recognition sequence. Outside the DNA binding domain, there is only limited similarity between the Antirrhinum myb genes and other myb genes from plants and none with any other sequence

Expression of myb Genes The organ-specific and temporal patterns of expression show differences between each of the six myb genes.

Repeat 1

.. +G. WT. eED .. L#. y# •. hG. g. w .. #p+. agL. RcgKSCRLRW. NyLRPd

Repeat 2

#++G.#t .. E- .. I#.Lh .. IG N+ws.iA .• IP gRTDNeIKNyWnt+#+++

I

helix 1

H

helix 2

H

helix 3

I

recognition helix

Figure 7. Consensus Sequence Derived from 14 Plant mybRelated Genes. The sequences included are the six Antirrhinum genes reported in this paper: C1 (Paz-Ares et aI., 1987), Zm1 and Zm38 from maize, Hv1 and Hv33 from barley (Marocco et aI., 1989), and three genes from petunia (sequences kindly provided by J. PazAres). The consensus is shown over the two 51 to 53 amino acid repeats. Also shown is the position of the proposed helix-turnhelix motif. Upper-case letters denote 100% conservation. Lowercase letters and + (basic residue), - (acidic residue), and # (hydrophobic residue) denote> 10/14 residues conserved. Dots denote < 10/14 residues conserved.

Figure 5. (continued). panels marked on the whole flower bud section; the upper panels are bright-field micrographs, and the lower panels are the corresponding dark-field images showing silver grains corresponding to expression. (A) Section through the lower part of the carpel. n, nectary; 0, ovule; s, stamen; c, carpel wall. Expression is specifically in the nectary. (B) Transverse section through the style. t, transmitting tissue; c, cortex. Expression is in a ring of cells of the transmitting tissue next to the cortex (denoted by arrows); the inner layers of cells of the transmitting tissue are not expressing but appear bright under dark-field microscopy because of reflection of light by the walls of these cells. This distinction is clear when sections are observed at higher magnification. (C) Section through the petal. e, epidermis; m, mesophyll. Expression is not localized in the petal but is seen evenly over mesophyll and epidermal cells. Note that the outer epidermis is highly reflective and appears bright under dark-field microscopy.

122

The Plant Cell

Expression in organs other than floral remains consistent with a proposal that, like C1, these genes may be regulating anthocyanin biosynthesis because anthocyanin is present also in leaves and stems. However, anthocyanin production in roots is only sporadic (C. Martin, unpublished data), which argues against 308, 315, or 330 being transcriptional activators of anthocyanin production because these genes are expressed in roots. It is possible, however, that an additional factor is required for anthocyanin biosynthesis and that it is this factor that is absent from roots. Of the other three myb genes, 305, 306, and 340, the temporal expression of 340 mimics exactly that of F-3-H, one of the genes involved in anthocyanin biosynthesis (Forkmann and Stotz, 1981; Martin et aI., 1987). In situ hybridization showed that the spatial and tissue-specific expression of 340 is very different from that of F-3-H, which matches precisely the observed epidermal-specific distribution of anthocyanin in petals (D. Jackson, unpublished results). Expression of 340 is strongest in the nectary, an organ that encircles the base of the carpel and is involved in the secretion of nectar through modified stomatal pores. It is also expressed in a layer of cells of the transmitting tract of the style, which is the tissue through which the pollen tube grows; this tissue is also active in carbohydrate secretion. We postulate that this gene may be involved in regulation of extracellular carbohydrate secretion. It is interesting to note that the mim 1 gene, which is regulated by c-myb, encodes a secreted protein (Ness et aI., 1989), suggesting that one of the functions of c-myb in animals is to regulate secretion in specific cell types. The maximum expression of the two flower-specific myb genes (305 and 340) and also 306, 308, and 330 occurs late in flower development and suggests that these genes are involved in the regulation of metabolic pathways rather than in primary developmental events such as floral induction. In this respect, they are similar to the maize C1 gene, and it may be that there are a large number of myb-related genes in plants involved in regulation of metabolism in this way. We are currently investigating the regulatory role of these floral myb genes by expressing antisense constructs in transgenic plants.

tin and Northcote, 1981). cDNA was prepared and cloned into i\gt10 (in accordance with the instructions in the Amersham cDNA synthesis-plus and cDNA cloning kits). Two 42-mer oligonucleotides containing sequences from the most highly conserved region of the myb domain were synthesized for screening. Oligonucleotide 01 represented a "consensus" sequence taking into account the amino acid sequence of c-myb from human and Drosophila as well as the C1 sequence (TTGCCIGGICGIACIGATAATGAIATTAAIAATTATTGGAAT), and oligonucleotide 02 represented the nucleic acid sequence from C1 encoding the equivalent amino acid sequence (CTGCCTGGCCGAACAGACAATGAAATCAAGAACTACTGGAAC) (Paz Ares et aI., 1987). Each oligonucleotide was end-labeled using polynucleotide kinase in the presence of 32 ),- p-dATP (Maniatis et aI., 1982) and used to screen one set of duplicate filters lifted from a library of approximately 50,000 plaques. Filters were hybridized in 6 x SSC, 0.1% SDS, 0.02% Ficoll, 0.02% PVP, 50 JIg/mL salmon sperm DNA, and washed in 3 x SSC, 0.5% SDS, both at 54°C. Four clones containing the sequence of myb 305 were isolated, two containing the 308 sequence and one each for 306, 315, 330, and 340. Only 330 and 340 were found to be full length. Despite the similarity between the myb domains, at high stringency (0.1 x SSC 0.5% SDS, 65°C) no cross-hybridization was found between the cDNA clones except between 340 and 305 on plasmid DNA gel blots. No cross-hybridization was found between the carboxy termini of 305 and 340 on genomic DNA gel blots, indicating that under these conditions they could be used as gene-specific probes. RNA Gel Blotting Poly A+ RNA was prepared from developing flower buds and other plant organs as described by Martin and Northcote (1981), and RNA gel blots were performed as in Martin et al. (1985) using approximately 5 Jig of poly A+ RNA per track. Isolated cDNA fragments were nick translated in the presence of a- 32 P-dCTP for use as probes (Maniatis et aI., 1982). Filters were washed at high stringency (0.1 x SSC, 0.5% SDS at 65°C). Under these conditions, no cross-hybridization was noted between any of the genes, even between 305 and 340, which could be distinguished by different transcript size. A cDNA clone of the gene encoding F-3H (pJAM 239) (Almeida et aI., 1989) was used for comparison of myb gene expression with that of genes involved in anthocyanin biosynthesis.

In Situ Hybridization

myb gene cDNAs and genomic clones were isolated from Antirrhinum majus line JI:522, which has fully pigmented flowers.

In situ hybridization was performed as described by Jackson (1991). Sense and antisense probes were transcribed from either full-length cDNA clones in a pGEM-Z vector or from clones containing the carboxy terminus without the myb domain to avoid possible cross-hybridization between the different myb transcripts. Autoradiographic exposure was for 6 weeks at 4°C. In all cases, no signal over background was observed using control sense-strand probes.

Construction and Screening of the cDNA Library

PCR Amplification

RNA was isolated from developing flower buds and purified by oligo(dT)-celiulose chromatography, as previously described (Mar-

cDNA was amplified by PCR according to Frohman et al. (1989). First-strand cDNA was synthesized from 10 Jig of total RNA using

METHODS

Stocks

myb Genes in Antirrhinum

the (dT)-17 adaptor as a primer. To stop the reaction, the sample was diluted to 1 mL with water. A 10-,uL sample was taken for amplification using either oligonucleotide 01 or 02 as the 5' primer or specific 25-mers made for each myb gene sequence from less highly conserved regions of the myb domain. The best results were obtained with the 3' primer at 50 nM and the 5' primer at 500 nM. Amplification involved 40 cycles with a denaturation time of 2 min at 94°C, an annealing time of 2 min at 55°C, and an extension time of 3 min at 72°C. To obtain sufficient quantities of 315 and 306 cDNA for subcloning, reamplification was necessary. The products from the first round of amplification were size fractionated on 2% agarose (1: 1 Nieuwsieve agarose FMC:Sigma agarose), and the DNA (approximately 1 ,ug) was isolated and resuspended in 20 ,uL of TE (10 mM Tris-HCI, 1 mM EDTA, pH 8.0). An aliquot (5 ,uL) was then used for reamplification. Amplified cDNA was size fractionated on agarose gels and isolated, and the ends were made flush using T4 DNA polymerase (Maniatis et aI., 1982). Where possible, cDNA was subcloned using Clal, Sail, or Xhol, which cut in the (dT)-17 adaptor, and a known enzyme close to the myb domain primer. Where this was not possible, cDNA was blunt ended and subcloned into the EcoRV site of pK8.2 (a gift from P.T. Jones), which contains the m13K11 polylinker in pUC8 and allows positive selection against nonrecombinants (Waye et aI., 1985). In each case where PCR was used to obtain full-length cDNA clones, at least two clones from each gene were sequenced. No sequence discrepancies were found for 305 or 315. Only two full-length cDNA clones were obtained for 306. These showed two differences in the sequence, both being single-base substitutions. Both differences involved the presence or absence of a restriction enzyme site. The sequence was verified, therefore, by digesting PCR-amplified cDNA with the relevant restriction enzymes and determining whether the majority of amplified molecules did or did not contain each site.

123

Quantification of cDNA Amplification by PCR The concentration of primers was checked to give maximum amplification of the myb genes compared with non-myb artifacts. The degree of amplification was exponential up to and beyond 40 cycles. The PCR amplifications were compared with the estimated transcript level obtained from RNA gel blotting, and in all cases these were similar, indicating that the PCR was semiquantitative and could be used to infer presence or absence of a particular myb transcript in different organs.

Sequencing Sequences were determined according to Sanger et al. (1977) by subcloning into M13mp18 and M13mp19 or by double-stranded plasmid sequencing in Bluescript using Sequenase (United States Biochemicals).

ACKNOWLEDGMENTS

Special thanks are owed to Javier Paz-Ares for sharing of results before publication and for constructive and stimulating discussion on myb genes. We also thank Zsusanna Schwarz-Sommer and Udo Wienand for the gift of the C1 cDNA from maize, Steve MacKay for patient technical assistance, David Cutler and Mervyn Bibb for helpful discussion and advice on plant anatomy and design of oligonucleotides, respectively. We thank David Hopwood and Brian Scheffler for critical reading of the manuscript and Peter Scott, Andrew Davis, and Nigel Hannant for photography. D.J. is supported by the John Innes Foundation, F.C.-M. by the Spanish Ministry of Education and Science, and A.G.P. by the Agriculture and Food Research Council Plant Molecular Biology Fund.

Isolation of Genomic Clones DNA was purified from Antirrhinum line JI:522 as described by Martin et al. (1985). The DNA was partially digested with EcoRI, size-fractionated to give fragments between 15 kb and 20 kb, and then ligated into the EcoRI site of AEMBL4. Approximately 200,000 plaque-forming units were screened with cDNA clones 308 and 305. One positive clone was isolated hybridizing to 308 and two to 305. The genomic clones were mapped with reference to the cDNAs, and the sequences 5' to the cDNAs were determined. In the case of 308, the sequence of the genomic clone immediately 5' to the 308 cDNA revealed an open reading frame that, when translated, gave a derived amino acid sequence showing strong homology to the 5' region of the C1 and c-myb DNA binding domain. No introns were detected between the 5' end of the cDNA and the deduced first methionine residue. The genomic sequence of 305 showed one intron between the end of the cDNA and deduced 5' end of the gene. This intron was positioned at exactly the same point as the first intron of C1. A second intron in 305 and the only intron in 308 were positioned at exactly the same point as the second intron of C1. The translated regions of 305 were deduced by searching the sequence for open reading frames showing similarity to the myb DNA binding domain, and by hybridization with clone 340, which is very similar to 305 within the myb domain.

Received October 19, 1990; accepted December 4, 1990.

REFERENCES

Almeida, J., Carpenter, R., Robbins, T.P., Martin, C., and Coen, E.S. (1989). Genetic interactions underlying flower colour patterns in Antirrhinum majus. Genes Dev. 3,1758-1767. Anton, I.A., and Frampton, J. (1988). Tryptophans in myb proteins. Nature 336, 719. Biedenkapp, H., Borgmeyer, U., Sippel, A.E., and Klempnauer, K.-H. (1988). Viral myb oncogene encodes a sequence-specific DNA-binding activity. Nature 335, 834-837. Cone, K.C., and Burr, B. (1988). Molecular and genetic analyses of the light requirement for anthocyanin synthesis in maize. In The Genetics of Flavanoids, D.E. Styles, GA Gavazzi, and M.L. Racchi, eds (Milan: Edizioni Unicopli), pp. 143-146. Cone, K.C., Burr, F.A., and Burr, B. (1986). Molecular analysis of the maize anthocyanin regulatory locus C1. Proc. Natl. Acad. Sci. USA 83, 9631-9635.

124

The Plant Cell

Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehensive set of sequence analysis programmes for the VAX. Nucl. Acids Res. 12,387-395. Dooner, H.K., and Nelson, O.E. (1979). Interaction among C, R and Vp in the control of the Bz glucosyltransferase during endosperm development in maize. Genetics 91, 309-315. Duprey, S.P., and Boettiger, D. (1985). Developmental regulation of c-myb in normal myeloid progenitor cells. Proc. Natl. Acad. Sci. USA 82, 6937-6941 . Forkmann, G., and Stotz, G. (1981). Genetic control of flavanone 3-hydroxylase activity and flavanoid 3' -hydroxylase activity in Antirrhinum majus. Z. Naturforsch. 36c, 411-416. Frampton, J., Leutz, A., Gibson, T., and Graf, T. (1989). DNAbinding domain ancestry. Nature 342, 134. Frohman, M.A., Dush, M.K., and Martin, G.R. (1989). Rapid production of full length cDNAs from rare transcripts: Amplification using single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85,8998-9002. Gewirtz, A.M., and Calabretta, B. (1988). A c-myb antisense oligonucleotide inhibits normal human hematopoiesis in vitro. Science 242, 1303-1306. Goff, S.A., Klein, T.M., Roth, B.A., Fromm, M.E., Cone, K.C., Radicella, J.P., and Chandler, V.L. (1990). Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. EMBO J. 9, 2517-2522. Gonda, T.J., Gough, N.M., Dunn, A.R., and de Blaquiere, J. (1985). Nucleotide sequence of eDNA clones of the murine myb proto-oncogene. EMBO J. 4, 2003-2008. Howe, K.M., Reakes, C.F.L., and Watson, R.J. (1990). Characterization of the sequence-specific interaction of mouse c-myb protein with DNA. EMBO J. 9, 161-169. Hsu, S.M. (1970). The development of pigments in germinating colourless seeds. Maize Genet. Coop. Newslett. 44, 309-319. Jackson, D.P. (1991). In-situ hybridization in plants. In Molecular Plant Pathology: A Practical Approach, D.J. Bowles, S.J. Gurr, and M. McPhereson, eds (Oxford: Oxford University Press), in press. Katzen, A.L., Kornberg, T.B., and Bishop, J.M. (1985). Isolation of the proto-oncogene c-myb from D. me/anogaster. Cell 41, 449-456. Klempnauer, K.-H., Gonda, T.J., and Bishop, J.M. (1982). Nucleotide sequence of the retroviralleukemia gene v-myb and its cellular progenitor c-myb; the architecture of a transduced oncogene. Cell 31, 453-463. Klempnauer, K.H., Symonds, G., Evan, G.I., and Bishop, J.M. (1984). Subcellular localization of proteins encoded by oncogenes of avian myeloblastosis virus and avian leukemia virus E26 and by the chicken c-myb gene. Cell 37, 537-547. Lech, K., Anderson, K., and Brent, R. (1988). DNA-bound Fos proteins activate transcription in yeast. Cell 52, 179-184.

Marocco, A., Wissenbach, M., Becker, D., Paz-Ares, J., Saedler, H., Salamini, F., and Rohde, W. (1989). Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA-binding domain of the Myb oncoproteins. Mol. Gen. Genet. 216,183-187. Martin, C., and Northcote, D.H. (1981). Qualitative and quantitative changes in mRNA in castor beans during the initial stages of germination. Planta 151, 189-197. Martin, C.R., Carpenter, R., Sommer, H., Saedler, H., and Coen, E.S. (1985). Molecular analysis of instability in flower pigmentation in Antirrhinum majus following the isolation of the pal/ida locus by transposon tagging. EMBO J. 4, 1625-1630. Martin, C., Carpenter, R., Coen, E.S., and Gerats, T. (1987). The control of floral pigmentation in Antirrhinum majus. In Developmental Mutants in Higher Plants, H. Thomas and D. Grierson, eds (Cambridge: Cambridge University Press), pp. 19-52. Ness, S.A., Marknell, A., and Graf, T. (1989). The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene. Cell 59, 1115-1125. Nomura, N., Takahashi, M., Matsui, M., Ishii, S., Date, T., Sasamoto, S., and Ishizaki, R. (1988). Isolation of human eDNA clones of myb-related genes, A-myb and B-myb. Nucl. Acids Res. 16, 11075-11089. Paz-Ares, J., Wienand, U., Peterson, P.A., and Saedler, H. (1986). Molecular cloning of the c locus of Zea mays: A locus regulating the anthocyanin pathway. EMBO. J. 5, 829-833. Paz-Ares, J., Ghosal, D., Wienand, U., Peterson, P.A., and 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. Paz-Ares, J., Ghosal, D., and Saedler, H. (1990). Molecular analysis of the C1-1 allele from Zea mays: A dominant mutant of the regulatory C1 locus. EMBO J. 9, 315-321. Ptashne, M. (1988). How eukaryotic transcriptional activators work. Nature 335,683-689. Qian, V.Q., Billeter, M., Otting, G., Muller, M., Gehring, W.J., and Wuthrich, K. (1989). The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: Comparison with prokaryotic repressors. Cell 59, 573-580. Sakura, H., Kanei-Ishii, C., Nagase, T., Nakagoshi, H., Gonda, T.J., and Ishii, S. (1989). Delineation of three functional domains of the transcriptional activator encoded by the c-myb protooncogene. Proc. Natl. Acad. Sci. USA 86,5758-5762. Sanger, F., Nicklen, S., and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Sigler, P.B. (1988). Acid blobs and negative noodles. Nature 333, 210-212.

Majello, B., Kenyon, L.C., and Dalla-Favera, R. (1986). Human c-myb proto-oncogene: Nucleotide sequence of eDNA and organization of the genomic locus. Proc. Natl. Acad. Sci. USA 83, 9636-9640.

Taylor, L.P., and Briggs, W.R. (1990). Genetic regulation and photocontrol of anthocyanin accumulation in maize seedlings. Plant Cell 2, 115-127.

Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).

Tice-Baldwin, K., Fink, G.R., and Arndt, K. (1989). BAS1 has a Myb motif and activates HIS4 transcription only in combination with BAS2. Science 246, 931-935.

myb Genes in Antirrhinum

125

Waye, M.M.Y., Verhoeyen, M.E., Jones, P.T., and Winter, G. (1985). EcoK selection vectors for shotgun cloning into M13 and deletion mutagenesis. Nucl. Acids Res. 13, 8561-8571.

Weston, K., and Bishop, J.M. (1989). Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb. Cell 58,85-93.

Westin, E.H., Gallo, R.C., Arya, S.K., Eva, L., Souza, L.M., Baulda, M., Aaronson, S.A., and Wong-Staal, F. (1982). Differential expression of the amv gene in human hematopoietic cells. Proc. Natl. Acad. Sci. USA 79, 2194-2198.

Wienand, U., Paz-Ares, J., Scheffler, B., and Saedler, H. (1989). Molecular analysis of gene regulation in the anthocyanin pathway of Zea mays. In Plant Gene Transfer, UCLA Symposia on Molecular and Cellular Biology, Vol. 129, C.J. Lamb and R.N. Beachy, eds (New York: Alan R. Liss, Inc.), pp. 111-124.

Expression patterns of myb genes from Antirrhinum flowers. D Jackson, F Culianez-Macia, A G Prescott, K Roberts and C Martin Plant Cell 1991;3;115-125 DOI 10.1105/tpc.3.2.115 This information is current as of March 13, 2015 Permissions

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298 X

eTOCs

Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain

CiteTrack Alerts

Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain

Subscription Information

Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm

© American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY