bHLH transcription factor mediates organ,region and flower type specific signals

The Plant Journal (1998)16(1),93–99

SHORT COMMUNICATION

A bHLH transcription factor mediates organ,region and ?ower type speci?c signals on dihydro?avonol-4-reductase (dfr )gene expression in the in?orescence of Gerbera hybrida (Asteraceae)

Paula Elomaa*,Merja Mehto,Mika Kotilainen,Yrjo ¨Helariutta,Leena Nevalainen and Teemu H.Teeri Institute of Biotechnology,PO Box 56,FIN-00014University of Helsinki,Helsinki,Finland

Summary

The angiosperm family Asteraceae is characterized by composite in?orescences,which are highly organized structures consisting of different types of ?owers.In order to approach the control of ?oral organ differentiation in Asteraceae at molecular level,we are studying regulation of ?avonoid biosynthesis in Gerbera hybrida .Dihydro?a-vonol-4-reductase (dfr )expression is regulated according to anthocyanin pigmentation patterns in all tested gerbera varieties at several anatomical levels.We have isolated a promoter for one of the dfr genes,Pgdfr2.Gerbera plants transgenic for a Pgdfr2-uidA construct reveal that the activity of the Pgdfr2promoter from one variety follows the pigmentation in other varieties which have different color patterns.It is thus evident that the observed complex regulation of dfr expression occurs in trans .In order to identify the trans -acting regulators,we isolated a cDNA (gmyc1)homologous to the previously characterized genes encoding bHLH-type regulators of the anthocyanin pathway in plants.The expression of gmyc1in different varieties suggests that it has a major role in regulating dfr activity in corolla and carpel,but not in pappus and stamen.Speci?cally in gerbera,the identical patterns of gmyc1and dfr expression in corolla tissue suggest that GMYC1also regulates dfr expression in a region and ?ower type speci?c manner.Our studies show that in gerbera GMYC1–dfr interaction is part of several develop-mental processes characteristic for Asteraceae (such as speci?cation of ?ower types across the composite in?or-escence),whereas in other processes (such as differenti-ation of sepal as pappus)other regulators control dfr expression to determine the spatial speci?city.

Received 29May 1998;revised 5August 1998;accepted 13August 1998.*For correspondence (fax ?358970859570;e-mail Paula.Elomaa@Helsinki.Fi).

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Introduction

The composite in?orescence of Asteraceae,the capitulum,is a highly organized structure of different types of ?owers which may vary in sexuality,symmetry and anthocyanin pigmentation.Also common to Asteraceae are the ?oral organs which form specialized structures like pappus hairs (sepals),and organ fusion which takes place within the petals and anthers (for details see Bremer,1994).We are interested in studying gene expression related to ?oral organ differentiation in Asteraceae,starting from the early events of organ determination and ?nally leading to differ-entiation of the complex in?orescense using an ornamental plant,Gerbera hybrida .At the molecular level,organ speci?c gene expression related to differentiation is con-trolled by distribution of common and spatially speci?c regulatory molecules,i.e.the pre-pattern (Coen et al .,1988).As an approach to understanding the molecular control of capitulum development we have identi?ed genes that sense the pre-pattern in different ways.

Different anthocyanin pigmentation patterns in gerbera varieties follow the anatomy of the capitulum and thus provide a good marker trait for our studies.In breeders’collections,varieties where pigmentation is ?ower organ speci?c,?ower type speci?c,or varies according to different regions of an organ can be found.We have previously shown that the expression of dfr ,a late gene of the anthocyanin pathway,is regulated precisely according to the anthocyanin pigmentation patterns of different gerbera varieties (Helariutta et al .,1995b).Through learning how dfr expression is controlled in gerbera at different anatomical levels,we will gain an important insight into how differen-tial gene expression is regulated in the composite in?or-escence.

In this paper we have isolated from gerbera a cDNA (gmyc1)that is homologous to the bHLH-type regulatory genes of anthocyanin pathway (Goodrich et al .,1992;Ludwig et al .,1989;Quattrocchio et al .,1998).Transient assay using particle bombardment indicates that GMYC1,together with a heterologous MYB regulator from petunia (Quattrocchio,1994),is able to activate a gerbera dfr promoter.The expression of gmyc1suggests that it has a major role in regulating anthocyanin biosynthesis in corolla and carpel,including regional as well as ?ower type levels

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during corolla development.However,in pappus and sta-men dfr ,expression is controlled by factors other than GMYC1.Results

Dfr expression is under trans regulation in gerbera We isolated a promoter from a dfr gene using the previously characterized gdfr1cDNA as a probe (Helariutta et al .,1993).The 150bp ?rst exon of the genomic clone was 98%identical to gdfr1cDNA.In line with the presence of a small gene family in gerbera genome (Helariutta et al .,1993),we concluded that this gene represents a member closely related to gdfr1,and was named Pgdfr2.Genetic analysis (see Experimental procedures)indicates that gdfr1and gdfr2are non-allelic.We transformed the 1195bp Pgdfr2promoter,fused with the uidA reporter gene,into the red gerbera variety Terra Regina.Based on ?uorometric measurements of 11individual transformants that expressed the transgene (data not shown),high GUS activity was detected in all pigmented organs (petiole,?ower scape,corolla and carpel).No or very low activity was detected in unpigmented parts (leaf,pappus,tube and stamen),indicating that the gdfr2promoter contains all essential elements that mediate the spatial and temporal speci?city needed for transcriptional regulation of dfr activ-ity in the Regina variety (Helariutta et al .,1993,1995b).In order to study the nature of dfr regulation (cis or trans )in gerbera,we crossed the Pgdfr2-uidA transgene into other genetic backgrounds.Transgenic Regina lines con-taining low copy numbers (1–2)of the transgene were crossed with the Nero and Parade varieties which have different ?oral anthocyanin pigmentation patterns (Helariutta et al .,1995b).Regina has pigmented corolla and carpel tissues.In Nero,only pappus bristles are strongly anthocyanin pigmented,while in Parade all ?oral organs except carpels are pigmented.As expected,due to the heterozygotic nature of gerbera,a range of novel pigmenta-tion patterns were displayed in the progeny both at ?ower organ and ?ower type level.From Regina ?Parade cros-sings (P-progeny)we obtained varieties with segregating stamen pigmentation (1:1;red:yellow),while carpel pig-mentation appeared to depend on multiple genes and various forms,with unpigmented to strongly pigmented carpels being found.In the P-progeny,corollas were always anthocyanin pigmented as in the parental varieties.In the N-progeny (Regina ?Nero),segregating pappus pig-mentation (1:1)was observed.Interestingly,differential pigmentation of corollas of distinct ?ower types was also observed.Often,the trans ?ower corollas were unpig-mented while the ray and disc ?ower corollas showed anthocyanin pigmentation (e.g.N5in Figure 1a).Northern analysis of selected progeny showed that uidA expression,

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under the control of the gdfr 2promoter,spatially follows the new pigmentation pattern and new dfr expression instead of the Regina pattern in all cases (Figure 1).This indicates that pigmentation patterns at the ?oral organ and ?ower type levels in these varieties result from differential spatial expression of regulatory molecules that control dfr expression,rather than from different structures in the dfr promoters.

Isolation of a gerbera cDNA homologous to bHLH -type regulatory genes of anthocyanin pathway

In order to approach the nature of the trans -acting signals,we isolated a cDNA (gmyc1)from gerbera corolla cDNA pool,based on homology between the bHLH-type anthocy-anin regulatory genes,lc from maize (Ludwig et al .,1989),delila from snapdragon (Goodrich et al .,1992),and jaf13from petunia (Quattrocchio et al .,1998).gmyc1cDNA encodes a protein of 533amino acids that is highly similar to DEL,JAF13and LC at the amino acid sequence level (Figure 2).The highest similarity is shared in the N terminal regions of the proteins as well as in the C terminal ends,while the intermediate regions are less conserved.Unique to gerbera GMYC1,compared to the other regulators,is that it is approximately 100amino acids shorter and speci?cally lacks a 12amino acids long region in the conserved basic region of the bHLH motif that is suggested to be involved in DNA binding (Prendergast and Ziff,1989).Southern analysis,using the full length cDNA as a probe,under low washing stringency,showed only one hybridiz-ing fragment in Regina genomic DNA,digested with enzymes that are not cutting within the cDNA (data not shown).This indicates that in variety Regina,gmyc1is a single copy gene.

GMYC1activates a Pgdfr2-luc reporter construct in transient assay

To assess the functionality of the gmyc1cDNA as a tran-scriptional activator,we used particle bombardment for transient expression of the regulator and its putative target promoter,Pgdfr2,which was fused to the ?re?y luciferase reporter gene.Co-bombardment of gmyc1with the hetero-logous myb partner an2from petunia (Quattrocchio,1994),both under the CaMV 35S promoter,together with the Pgdfr2-luc construct into leaf tissue,activated the reporter gene (Table 1).The affector or reporter constructs alone did not give signi?cant LUC activity.The results suggest that gmyc1encodes an active regulator,and that the Pgdfr2promoter contains the sequence elements responding to the regulatory protein encoded by the gmyc1cDNA.

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Figure1.Northern analysis of the crossing progeny with novel pigmentation patterns indicates that the promoter itself is not responsible for the pattern but reacts to trans-acting signals.

uidA expression driven by the gdfr2promoter consistently follows the new anthocyanin pigmentation pattern(and dfr expression pattern)in differentially pigmented?ower types(a),stamens(b),pappus bristles(c)and carpels(d).

Expression pattern of gmyc1in gerbera suggests a regulatory role in corolla and carpel tissues

Northern analysis was performed to study the expression pattern of the putative anthocyanin regulator in gerbera. At the level of whole plant,gmyc1expression was detected in tissues where anthocyanin pigmentation is also detected: in developing in?orescence buds,in?oral stems and very faintly in bracts.No expression was detected in leaves or roots(Figure3a).Expression during ray?ower develop-ment was studied in more detail.Stages2–9correspond to stages from small buds of5–10mm in size to the completely open?owers(see Helariutta et al.,1993). Expression of gmyc1is already detectable at stage2and continues rather uniformly until stage7and fades out thereafter(data not shown).Temporally,it precedes the expression of anthocyanin biosynthetic genes,whose expression peak at stages6–7,just before anthesis (Helariutta et al.,1993,1995a).

Spatial distribution of gmyc1expression was studied in ?oral organs of different varieties.Figure3(b)shows gmyc1 expression in corolla tissue.Ray?ower corollas(stages3,5 and7)show strong gmyc1expression in the red pigmented Regina and Parade and much lower expression in Nero (yellow variety),each in accordance with the dfr expression in them(Helariutta et al.,1995b).Similarly,in carpel tissue, gmyc1expression follows the pigmentation pattern,as shown in Figure3(c).In variety Regina,carpels are clearly pigmented,in variety Nero only faint pigmentation is detected,and in variety Parade there is no pigmentation at all.Within corolla tissue,in variety Mix,pigmentation is restricted to the basal part of the corolla while the distal part

?Blackwell Science Ltd,The Plant Journal,(1998),16,93–99is unpigmented(Helariutta et al.,1995b).gmyc1expression follows the distribution of anthocyanin pigmentation,indic-ating that gmyc1is probably responsible for the regulation imposed on the dfr gene also in the different regions within the corolla ligule(Figure3b).Among the crossing progeny described above,we obtained varieties with differentially pigmented corollas in distinct?ower types.In N1with equally pigmented ray and trans?ower corollas gmyc1 expression is also detected in both?ower types,while in N5with unpigmented trans?owers gmyc1expression is detected in ray but not in trans?owers(Figure3b).

In general,gmyc1expression pattern follows the pigmentation in corolla and carpel tissues of gerbera sug-gesting a major regulatory role in these organs.Especially in corolla,gmyc1and dfr expression patterns coincidence at all anatomical levels that characterize the composite in?orescence,including the levels of?ower type and regions within a single corolla.The different gerbera variet-ies studied also vary in pigmentation of pappus and anthers.However,gmyc1expression in these organs is very low,irrespective of their pigmentation phenotype (data not shown),suggesting that pappus and stamen pigmentation is not determined by GMYC1.

Discussion

Asteraceae is characterized by complex in?orescences (capitula)where differentiation occurs not only at the level of?oral organs and their substructures,but also at the level of the capitulum,which may carry different types of ?owers(e.g.Bremer,1994).In Gerbera hybrida,spatial

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al.

Figure 2.Amino acid comparison of the (b)HLH-type regulator of gerbera,GMYC1with DEL of snapdragon,JAF13of petunia and LC of maize.

(*)indicates amino acids that are fully conserved in each of the proteins and (.)amino acids that GMYC1shares with at least one of the other regulators.The basic helix-loop-helix region is indicated in bold.Highest amino acid similarity (66%)is shared with snapdragon DEL.

Table 1.Co-bombardment of the gerbera gmyc1and petunia an2,both under CaMV35S promoter,activate the Pgdfr2-luc reporter construct in gerbera leaf tissue.35S-ruc was used as an internal control and was co-bombarded in each case.Ten leaves were bombarded in each case Constructs ?35S-ruc

LUC/RUC activity (SD)Pgdfr2-luc

0.023(0.011)Pgdfr2-luc ?35S-gmyc10.021(0.010)Pgdfr2-luc ?35S-an2

0.072(0.025)Pgdfr2-luc ?35S-gmyc1?35S-an2

0.382

(0.138)

patterns of anthocyanin pigmentation are regulated,in the different varieties,in diverse ways along several of these morphological units.In our earlier work we showed that dihydro?avonol-4-reductase gene expression,as a repres-entative of late biosynthetic gene expression of the antho-

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Figure 3.Expression of gmyc1in gerbera follows very precisely the anthocyanin pigmentation pattern at the level of the whole plant and in corolla and carpel tissues of individual ?owers.

The presence of anthocyanin pigmentation is indicated as (?)and absence as (–).

(a)gmyc1expression in developing in?orescences (I),bracts (B),?ower stems (S),leaves (L)and roots (R).

(b)In corolla tissue of varieties Regina,Nero,Parade,Mix,N1and N5,gmyc1expression follows the pigment distribution at ?oral organ,regional and ?ower type levels.

(c)gmyc1expression follows pigmentation in carpel tissue of varieties Regina,Nero and Parade.

cyanin pathway,precisely coincides with the pigmentation patterns,possible providing a tool for studying perception of the signals differentiating the morphological levels of the capitulum (Helariutta et al .,1995b).

We report here on a cis -and trans -acting element of dfr expression that represents part of the previously identi?ed regulatory processes.Transgenic analyses with the gdfr2promoter indicate that the promoter activity is fully consist-ent with the dfr activity previously detected by Northern analysis in different tissues of variety Regina (Helariutta et al .,1993,1995b),as well as in a crossing progeny displaying novel patterns of anthocyanin accumulation at ?oral organ and ?ower type levels.This indicates that the isolated promoter contains sequences that are responsive to various spatially speci?c signals during capitulum devel-opment.

Previous studies in maize,snapdragon and petunia on

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the transcriptional regulation of(late)biosynthetic genes of anthocyanin pathway have shown that the trans-acting elements share remarkable conservation both in structure and function(reviewed by Dooner et al.,1991;Koes et al., 1994;Martin and Gerats,1993;Mol et al.,1996).Therefore, in order to approach the trans-acting signals controlling dfr expression,we isolated a gene encoding a putative bHLH-type regulator from corolla tissue of gerbera.The deduced amino acid sequence of GMYC1closely resembles those of the previously isolated regulators,suggesting that it may be functionally homologous.Indeed,although most of the conserved basic region is lacking from GMYC1,the protein is able to activate the Pgdfr2promoter in transient gene expression system,if a(heterologous)MYB partner is provided.Furthermore,previously Goff et al.(1992)have shown that only the N-terminal region of the maize bHLH type protein B,a close homologue of LC,is critical for interaction with the corresponding MYB partner,C1.Fur-ther support for the regulatory role of GMYC1is provided by its expression in corolla tissue of variety Regina,which temporally precedes and partly overlaps with the biosyn-thetic gene expression,and is thus analogous to delila expression in snapdragon(Goodrich et al.,1992)and to jaf13expression in petunia(Quattrocchio et al.,1998). These results suggest that a bHLH regulator/dfr promoter interaction analogous to one previously identi?ed in sev-eral other species plays a role during corolla development in gerbera.This will be?nally veri?ed by homologous transformation of gmyc1both in sense and antisense orientation into gerbera.However,support for the conser-vation of the regulatory interaction is provided by our other analyses.We have earlier transformed the homologous gene from snapdragon,delila,under CaMV35S promoter into gerbera and obtained transgenic lines with strongly enhanced pigmentation in?ower scapes and leaf tissue due to activation of dfr expression(Elomaa,1996).Conser-vation of the regulatory mechanism is further supported by bombardment of the maize regulators,lc(Ludwig et al., 1989)and c1(Paz-Ares et al.,1987),which together with the Pgdfr2-luc construct lead to activation of the reporter construct in leaf tissue(M.Mehto,unpublished results).In addition,transformation of the Pgdfr2-uidA construct into petunia(V26)showed that the promoter activity followed the petunia pigmentation pattern(data not shown). Spatially during corolla development,gmyc1expression follows the anthocyanin pigmentation pattern very pre-cisely in different varieties,and speci?cally in gerbera, both regionally within a single corolla(tip/base axis)and at the level of?ower type(Figure3b).This indicates that the variation in corolla pigmentation in these plants would be due to differences either in the gmyc1promoters, or higher level spatially speci?c trans-acting signals.To approach this,we are proceeding with the isolation and characterization of the promoter of gmyc1and its equiva-

?Blackwell Science Ltd,The Plant Journal,(1998),16,93–99lents in different varieties.Analogously to corolla develop-ment,gmyc1expression follows the pigmentation pattern in carpel.This suggests that gmyc1has a key role in regulating dfr expression during differentiation of both corolla and carpel.However,in pappus and stamen,where Pgdfr2is active when anthocyanins accumulate,the activa-tion is probably not mediated by GMYC1,as evidenced by the invariantly faint expression pattern.An alternate, independent regulation mechanism must exist in these tissues which still acts on the same promoter.Crossing experiments demonstrate that while corolla and carpel pigmentation are complex,polygenic traits,the inheritance of stamen and pappus pigmentation are more simple and suggests involvement of single,dominant genes.Whether the latter factors are other members of the bHLH family, or whether they are different factors that react with different parts of the Pgdfr2promoter remains to be studied.In either case,dfr regulation,which takes place along diverse developmental units in the Asteraceae capitulum,is branched and taken care of at least by two separate regulatory systems.One of these systems acts in corolla and carpel and is mediated by a(b)HLH regulatory protein.

Experimental procedures

Plant material

Gerbera hybrida varieties Regina,Nero,Parade and Mix were obtained from Terra Nigra BV,Holland.Developmental stages of the in?orescence are described by Helariutta et al.(1993) and anthocyanin accumulation patterns of different varieties in Helariutta et al.(1995b).

Isolation of plant DNA and RNA

Plant DNA was isolated using the methods of Dellaporta et al. (1983)or Doyle and Doyle(1990).Total RNA was isolated as described in Jones et al.(1985).Poly(A)?RNA was isolated using oligo(dT)cellulose af?nity chromatography(Sambrook et al., 1989).

Screening of the genomic library

Genomic library was constructed from partially Sau3A digested DNA into lambda GEM11vector.3.4?106plaques were screened according to standard methods(Sambrook et al.,1989)using gdfr1cDNA(Helariutta et al.,1993)as a probe.Washing conditions were2?SSC,0.1%SDS at68°C.Two positive clones were detected,one of which contained a gdfr promoter region(Pgdfr2). Genetic analysis of the allelic relationship between gdfr1 and gdfr2

Comparison of the150bp region common to the genomic clone and the previously isolated gdfr1cDNA indicated that the isolates differed in three nucleotides(data not shown).Using information from both the genomic clone(Pgdfr2)and the cDNA(gdfr1)we

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ampli?ed by PCR two major products from variety Regina,a longer fragment that corresponds exactly to the gdfr1cDNA and a shorter product similar to the genomic clone (M.Mehto,unpublished results).The same two PCR products were also obtained from a dihaploid Regina derivative produced via ovule culture,proving that gdfr1and gdfr2are non-allelic and correspond to two highly homologous dfr genes in gerbera.

Transformation of gerbera and crossing of the primary transformants

Transgenic gerbera plants containing the gdfr2promoter fragment (1195bp)in front of the uidA marker gene encoding for β-glucuronidase (GUS)were obtained by agrobacterium mediated transformation (Elomaa et al .,1993).The transformation construct (pHTT441)was basically similar to our previously published vec-tors (Elomaa et al .,1993;Elomaa et al .,1996)except that an intron was inserted into the Pvu II site of nptII (Elomaa,1996).Transformation was con?rmed by Southern analysis and ?uoro-metric GUS assay was performed according to Jefferson and Wilson (1991).Some of the primary transformants were reciproc-ally crossed with varieties Nero and Parade.Seeds were collected,surface sterilized and germinated on MS-medium (Murashige and Skoog,1962)in the presence of kanamycin (100mg l –1).

PCR cloning of gmyc1

First-strand cDNA was synthesized from poly(A)?RNA from ray ?oret corolla (stages 5–9,Helariutta et al .,1993)using Promega’s Riboclone cDNA synthesis kit,and used as a template for PCR ampli?cation.Partially degenerate primers GGAGGGATCCTA(T/C)AA(T/C)GGIGAI(A/G)TIAA(A/G)AC and TCTCGGATCC(A/G)TGIGC(A/G)TT(A/G)CAIA(G/A)CCA (including Bam HI sites)cor-responding to the peptides YNGDIKTRK and WLCNAH,respect-ively,were used.DNA-polymerase Dynazyme (Finnzymes)was used for ampli?cation and the conditions were:94°C/75sec,46°C/2min,72°C/3min for 30cycles.The PCR product (300bp)was cloned into pUC19(Yanisch-Perron et al .,1985)and sequenced.For 3?/5?RACE,?rst-strand cDNA was synthesized from corolla poly(A)?RNA from stages 2–4,sequence speci?c primers were designed based on the PCR fragment,and the ampli?cation of the cDNA ends was performed according to the Boehringer Mannheim Kit (no.1734792).To ensure that the fragments obtained using the RACE method originate from the same transcript,the whole cDNA (2268bp)was ampli?ed once more from corolla cDNA using Expand High Fidelity enzyme (Boehringer)and sequenced in both directions.For sequence similarity comparisons,amino acids were grouped by their chemical similarity as in Manninen and Schulman (1993)and GMYC1,DEL,JAF13and LC sequences were analyzed using PC/Gene protein analysis package (Intelligenetics Inc.)

DNA and RNA blot analyses

Ten μg of digested DNA or 10–20μg of total RNA was loaded per lane.For studies of gmyc1expression,RNA was isolated from different developmental stages of ray ?oret corollas.Pappus RNA was pooled from stages 2,3and 4,and carpel RNA from stages 5,6and 7.Stamen RNA was isolated from samples covering stages 7and 8.Electrophoresis and hybridizations were undertaken using standard protocols (Sambrook et al .,1989).Full-length cDNA of gdfr1was used as a probe for detection of dfr expression

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(Helariutta et al .,1993).This probe probably detects all dfr genes active in ?oral tissue.uidA expression was detected using Sac I-Bam HI fragment (1940bp)isolated from pBI221(Jefferson,1987)as a probe.gmyc1expression was detected using a 419bp (Sal I fragment)from the 3?end as a probe.

Particle bombardment of gerbera leaf tissue and luciferase assay

For particle bombardment,BioRad PDS-1000/He equipment was used.Five mg of 0.6μm gold particles were precipitated with maximum 5μg of plasmid DNA using CaCl 2and spermidine.Plasmids containing Pgdfr2-luc ,35S-gmyc1and 35S-an2con-structs were used.35S-ruc ,encoding Renilla luciferase (Mayer-hofer et al .,1995),was used as an internal control.Leaves from in vitro plantlets were placed on water agar and bombarded on their upper epidermis using 900psi pressure plates.Leaves were kept in dark for 20h and measured for ?re?y and Renilla luciferase activities according to the protocol of the Promega Dual Luciferase Kit,except that the protein extraction buffer was the ‘modi?ed lux buffer’as described in Herrera-Estrella et al .(1994).

Acknowledgements

We thank Dr Francesca Quattrocchio for the petunia jaf13sequence and for the petunia an2plasmid,Prof.Susan Wessler for the maize lc and Prof.Udo Wienand for the c1cDNAs.Dr Anne Uimari is thanked for helpful comments on the manuscript.Eija Takala,Marja Huovila and Anu Immonen are acknowledged for excellent technical assistance.We also thank Anne Aaltonen for taking care of the plants and Eija Saarikko for the crossing experiments.The tissue culture and greenhouse facilities were provided by Kemira Agro Oy.

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