Plant Molecular Biology 49: 225–237, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
225
Spatial and temporal expression of the orchid floral homeotic gene DOMADS1 is mediated by its upstream regulatory regions Hao Yu, Shu Hua Yang and Chong Jin Goh∗ Plant Growth and Development Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Kent Ridge, Singapore 117543, Republic of Singapore (∗ author for correspondence; e-mail:
[email protected]) Received 20 November 2000; accepted in revised form 5 November 2001
Key words: DOMADS1, MADS-box gene, orchid, promoter, spatial and temporal regulation
Abstract The orchid floral homeotic gene, DOMADS1, is a marker gene specifically expressed in the transitional shoot apical meristem during floral transition in Dendrobium Madame Thong-In. DOMADS1 is not detectable in vegetative tissues except a weak expression in the stem. Its transcript is uniformly localized in both of the inflorescence meristem and floral primordia, and later expressed in almost all of the floral organs. We isolated and sequenced a 3.5 kb DOMADS1 promoter fragment upstream of the transcription start site, demonstrating the location of several putative DNA-binding sites, through which MADS-box and class 1 knox genes may modulate the DOMADS1 expression. To gain insight into the molecular basis of the regulation of DOMADS1, deletion analysis of the DOMADS1::β-glucuronidase (GUS) gene fusions was performed by means of the stable orchid transformation systems. The study shows that the full-length upstream promoter sequence confers the same spatial and temporal GUS staining pattern as that of the distribution of DOMADS1 RNA during orchid development. We also identified the distinct cis-acting regulatory regions required for the control of DOMADS1 expression in vegetative and reproductive tissues, as well as the shoot apical meristem during floral transition.
Introduction During plant development and differentiation, a wide range of endogenous and environmental signals are integrated to evoke a cascade of cellular processes leading to the regulation of downstream gene activities. As the initiation of transcription is the major regulatory control of gene expression, transcription factors act as key switches in plant development. Mutations in the cis-regulatory elements of these regulators may alter the expression patterns of a series of related genes, resulting in the generation of novel plant phenotypes (Doebley and Lukens, 1998). Plant MADS-box genes represent a large family of highly conserved transcription factors that play key roles in different phases of plant growth, especially in the flowering process and flower development. MADS-box genes have been isolated and characterized in ferns, gymnosperms and different types of angiosperms (Theissen et al.,
2000). The extensive genetic, molecular and evolutionary studies on this group of genes have not only demonstrated their ability to control specific plant developmental processes in complex networks, but also revealed their roles in the morphological evolution of plant forms (Theissen et al., 1996, 2000). It is of interest to clarify the molecular mechanisms responsible for the evolution of these orthologous genes and the corresponding plant organs. Therefore, the identification of functional promoters of MADS-box genes will not only provide important information concerning the environmental and developmental factors affecting the regulation of MADS-box gene expression, but also help to further elucidate the role of this group of genes in plant evolution. Whilst a considerable number of MADS-box genes have been studied in different plant species, analyses of their promoter functions remain lacking until recent reports of the AGAMOUS (AG) and APETALA3 (AP3)
226 gene promoters (Sieburth and Meyerowitz, 1997; Tilly et al., 1998; Hill et al., 1998). AG and AP3 are both Arabidopsis MADS-box genes required for the developmental regulation of floral organ identity (Bowman et al., 1989; Yanofsky et al., 1990; Jack et al., 1992). Molecular dissection of the AG regulatory elements revealed that the cis elements essential for normal regulation of AG expression are located in the intragenic region (Sieburth and Meyerowitz, 1997). However, normal expression of AP3 is shown to require only upstream regulatory sequences (Jack et al., 1994; Tilly et al., 1998; Hill et al., 1998). Detailed analysis of AP3 promoter region suggests that spatial and temporal control of its transcription is conferred by a few specific domains and several other MADS-domain proteins may regulate AP3 expression by acting through the CArG boxes (CC(A/T)6 GG) found in its promoter. These studies have shed light on the underlying molecular mechanisms by which the distinct developmental fate of floral organs are decided and maintained in the floral meristem. In our effort to study the molecular mechanisms of floral transition in orchids, we have identified three orchid MADS-box genes (DOMADS1, DOMADS2 and DOMADS3) specifically expressed in the transitional shoot apical meristem (TSAM) during in vitro floral transition (Yu and Goh, 2000). The expression patterns exhibited by these three genes suggest a role in regulating the switch from vegetative to reproductive growth in orchids (Yu and Goh, 2000). To further elucidate the molecular regulatory networks involved in this process, we have isolated a 3.5 kb promoter fragment immediately upstream of the DOMADS1 coding region and have undertaken a functional analysis of this region using GUS reporter gene fusions. In a wildtype orchid plant, DOMADS1 RNA is expressed early in the 12-week old TSAM, where its expression is mainly concentrated in the apical region of the TSAM and the floral primordium. At a later stage, the DOMADS1 transcript is uniformly present throughout the inflorescence meristem and the floral primordium. In the developing floral buds, DOMADS1 is detectable in all of the floral organs, including sepals, petals, column and also the basal floral meristem (Yu and Goh, 2000). In this study, we report that the 3.5 kb promoter region contains all the essential cis-regulatory elements required for the spatial and temporal control of DOMADS1 gene expression. By using deletion analysis, we identified the discrete regulatory elements responsible for controlling the specific DOMADS1 ex-
pression in individual organs or tissues during orchid development.
Materials and methods Plant materials and growth conditions All plant materials were self-pollinated F1 progeny of orchid Dendrobium Madame Thong-In, a hybrid between Dendrobium Somsak × Dendrobium Suzie Wong. Plants were cultured under the conditions described in our previous study (Yu and Goh, 2000). Isolation of DOMADS1 promoter by GenomeWalker DNA walking The promoter region of the DOMADS1 gene was isolated according to the protocol of Universal GenomeWalker kit (Clontech, Palo Alto, CA) with some modifications. Genomic DNA was extracted from young leaves by the method described in the previous study (Yu and Goh, 2000). Five GenomicWalker libraries containing uncloned genomic DNA fragments were produced by respective digestion of genomic DNA with EcoRV, ScaI, StuI, DraI and PvuII and subsequent ligation of each batch of digested DNA fragments with GenomeWalker adaptor. The 3.6 kb DOMADS1 promoter region (−3483 to +92, GenBank/EMBL/DDBJ accession number AJ288901) was isolated by three successive PCRbased rounds of screening from GenomeWalker libraries (Table 1). The advantage 2 PCR Enzyme System (Clontech) was adopted to offer high fidelity and great consistency in PCR amplification of promoter regions. For each round of walking, the primary PCR products amplified by a gene-specific primer (GSP1) and the outer adaptor primer (AP1) were diluted and used as templates for the secondary ‘nested’ PCR with a nested gene-specific primer (GSP2) and the nested adaptor primer (AP2). The secondary PCR products were analyzed on agarose gels. The major bands were purified from gels with QIAEXII Gel Extraction Kit (Qiagen, Valencia, CA), cloned into pGEM-T Easy Vector (Promega, Madison, WI) and sequenced as mentioned in under ‘Sequencing and sequence analysis’. Identification of transcription start site by 5 -RACE Total RNA extracted from 12-week old TSAM, by the method of Yu and Goh (2000), served as starting mate-
227 Table 1. Oligonucleotide primers used to amplify 5 flanking regions of DOMADS1 gene. Walking step
Primer namea
Sequence
1
DOM1-GSP1 DOM1-GSP2
5 -CTTCATCTCCACCCTTCCTCTCCCCATT-3 5 -CTCTTTCTCTCCCTTCGTTCTCAGC-3
2
DOM2-GSP1 DOM2-GSP2 DOM3-GSP1 DOM3-GSP2
5 -GCCGAGGCGGGAGCCATATTATTGAA-3 5 -ATGAAAGGACACGTGTTGGTAGTGGA-3 5 -GTACCCTTGAGAAGAAGGATATCCAC-3 5 -GACCCCTCAACAAGCACATGCCAATA-3
3
Walking sizeb
bp 202 306 415 969 1045 1309 2360
Position of walking products in genomic DNA sequencec +92 to −110 +92 to −214 +92 to −323 −246 to −1214 −246 to −1290 −1159 to −2467 −1159 to −3518
a A gene-specific primer (GSP1) and a nested specific primer (GSP2) were used with the outer adaptor primer (AP1) and the nested adaptor
primer (AP2) respectively for each walking process. b For every walking step, several PCR products of different molecular weight were obtained and the walking sizes were calculated from
GSP1 primers to the genomic binding sites of the walking adaptor. c Numbers represent the distances relative to the transcription start sites (+1) described in Figure 1.
rials for 5-RACE using SMART RACE cDNA amplification kit (Clontech). 5 -RACE PCR was successively performed with two nested antisense primers (5 AGAAGATGATTAGTGCAACCTCGACAT-3 and 5 AGTGCAACCTCGACATCACAGAGGAC-3) specific to the DOMADS1 gene. The resulting PCR products were subsequently cloned and sequenced as described in the analysis of GenomeWalker DNA walking products. Sequencing and sequence analysis Both strands of DNA clones were sequenced by the dideoxy chain-termination method using the Big Dye Terminator Cycle Sequencing Ready Reaction kit and the model 377 automated sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). The promoter sequence was compared with all known DNA sequences using the default settings of BLASTN (Altschul et al., 1997) from the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov). Sequence analyses were performed with the BioPortal software package from the Bioinformatics Center at the National University of Singapore. Construction of chimeric reporter gene fusions In-frame translational fusions between the DOMADS1 promoter deletion derivatives and the β-glucuronidase (GUS) reporter gene were constructed by cloning the various 5 deletion promoter fragments into the multiple cloning sites upstream of the GUS gene present in the pBI101 binary vector (Clontech). For all of these
constructs, the fusion point was at +92 nucleotide of the DOMADS1 gene. Constructs pBI-PM3, pBI-PM5 and pBI-PM6 were generated using the native restriction enzyme sites found in the promoter region. The 3.6 kb promoter fragment (−3483 to +92) with blunt ends was produced by PCR-amplification with the Vent R DNA Polymerase (New England Biolabs, Beverly, MA) using a forward primer (5 GGTGGATCCTATATGCTATCTCTGTCATC-3) and a reverse primer (5 -CTTCATCTCCACCCTTCCTCT CCCCATT-3 ). The 1.7, 1.0, and 0.6 kb deletion fragments with 5 cohesive ends were obtained by digestion of the resulting 3.6 kb PCR fragments with XbaI, HindIII and BamIII, respectively. These fragments were subsequently cloned into the corresponding restriction sites (Xba1 and SmaI, HindIII and SmaI, and BamIII and SmaI, respectively) upstream of the GUS coding region of the pBI101 binary vector (Clontech) to produce the constructs of pBI-PM3, pBI-PM5 and pBI-PM6, respectively. Several 5 deletion fragments with blunt ends were yielded by direct PCR amplification from GenomeWalker generated pGEM-T clones (containing the inserts of the walking products 202, 306, and 415 bp in size) using SP6 primer and the gene specific primer (5 -CTTCATCTCCACCCTTCCTCTCCCCATT-3 ). These resulting PCR products were excised with SalI to produce the 5 cohesive ends, and subsequently cloned into the SalI and SmaI sites upstream of the GUS gene in the pBI101 vector (Clontech) to create the constructs of pBI-PM7, pBI-PM8 and pBI-PM9.
228 The 3.6, 2.5 and 1.3 kb promoter fragments were obtained by PCR amplification with a forward sequencing primer (5 -GGTGGATCCTATATGCTATCT CTGTCATC-3 , 5 -GGATGGTCCACTTGGCAACC AAGG-3, or 5 -GGCATGTGCTTGTTGAGGGGTC TA-3 , respectively) and a reverse primer (5 CTTCATCTCCACCCTTCCTCTCCCCATT-3 ). We cloned the resulting PCR products into pGEM-T Easy vector (Promega) to introduce a new SalI site at the 5 end of these deletion fragments. The subsequent construction of fusion plasmids of pBI-PM1, pBIPM2 and pBI-PM4 were produced using the cloning strategy as described in the generation of pBI-PM7, pBI-PM8 and pBI-PM9. The sequences of all promoter constructs were analyzed to eliminate possible PCR-introduced mutations. The pBI101 vector containing a promoterless GUS cassette and the pBI121 vector containing the cauliflower mosaic virus (CaMV) 35S promoter-GUS fusions were used as negative and positive controls, respectively. Orchid transformation by microprojectile bombardment GUS activity for each of the DOMADS1 promoter construct described above was analyzed using the stable orchid transformation systems. DOMADS1 promoter deletion-GUS fusion plasmids were isolated by Wizard Plus SV Minipreps DNA Purification System (Promega) and coated on gold particles 1.1 µm in diameter by coprecipitation as described by Klein et al. (1987). Before microprojectile bombardment, thin section explants (1 mm thickness and 4–5 mm diameter) from protocorm-like bodies (PLBs) were pre-cultured for 3 days in liquid modified Knudson C (KC) medium (Yu and Goh, 2000), and then placed on a central core 2 cm in diameter on solid modified KC medium. These sections were bombarded using a Model Biolistic PDS-1000/He (BioRad) at 2.6 MPa helium gas pressure, 93 kPa vacuum and 9 cm target distance. For the stable transformations, 1.6 µg of plasmid DNA of each promoter deletion construct was delivered into the thin section explants. Under these conditions, the cells in thin section explants were uniformly transformed, which was indicated by the uniform spread of blue spots with histochemical detection of GUS activity. For stable transformation, the bombarded thinsection explants were maintained for 4 days on solid KC medium and subsequently cultured on the same
medium supplemented with 200 mg/l kanamycin for selection of drug-resistant calli. Repeated screenings were performed to avoid obtaining chimeric plants. The selected calli were excised and cultured on the same KC medium supplemented with 250 mg/l kanamycin for two more rounds of screening. After the third screening of kanamycin-resistant calli, the selected calli were subcultured every fortnight. Transgenic orchid plantlets proliferated from calli were further selected by PCR and Southern blot analysis and maintained by culturing on solid KC medium containing 300 mg/l kanamycin. For each DOMADS1 promoter-GUS fusion, GUS staining pattern was analyzed in at least eight independent transgenic lines. GUS histochemical assays Histochemical detection of GUS activity was performed as described by Jefferson (1987) and Topping and Lindsey (1997). Tissues for GUS staining were vacuum-infiltrated in staining solution (50 mM sodium phosphate pH 7.0, 10 mM EDTA, 2 mM 5-bromo-4-chloro-3-indoyl glucuronide, 1 mM potassium ferricyanide, and 1 mM potassium ferrocyanide) and incubated at 37 ◦ C overnight. Stained tissues were cleared of chlorophyll in an ethanol series. Except for the tissues used directly for observation, other tissues were fixed, dehydrated and embedded in paraffin via the butanol series. These tissues were then sectioned at 15 to 25 µm, briefly incubated in xylene, and photographed on a microscope (TMS-F, Nikon) using bright or dark-field illumination. RNA gel blot analysis Total RNA was separated on 1% glyoxal-agarose gels and transferred onto positively charged nylon membranes (Boehringer Mannheim, Mannheim, Germany). RNA gel blots were hybridized overnight in DIG Easy Hyb buffer at 50 ◦ C with the digoxigeninlabeled DNA probe. The detection of blots and synthesis of DNA probe were performed as described previously (Yu et al., 2000)
Results Isolation and sequence analysis of the DOMADS1 5 -upstream region The orchid DOMADS1 promoter was isolated by genomic DNA walking strategy using a series of gene
229
Figure 1. 5 promoter sequence of the orchid DOMADS1. Three transcription start sites, as determined by 5 -RACE analysis, are designated with asterisks. Bases are numbered with reference to the most likely transcription start site (+1) that is the first upstream nucleotide of the major band displayed in 5 -RACE analysis. Open reading frame of the DOMADS1 cDNA is shown in uppercase letters and the amino acid sequences are given below the corresponding nucleotide sequences. The start site of each 5 deleted fragment used in promoter analysis is indicated by a horizontal arrow. Six CArG boxes within the promoter region are shown in bold and labeled consecutively. Five putative DNA binding sites (TGAC) of class 1 knox genes are indicated in bold by wavy lines. The putative CAAT box, TATA box, and the ATG translational start sites are underlined.
specific primers derived from the DOMADS1 cDNA sequence and its upstream promoter region (Figure 1). By searching the gene databases, the isolated DOMADS1 promoter fragment showed no significant homology with any other reported genes. Sequence analysis of the promoter region revealed the presence of some cis elements thought to be the binding sites of certain important regulatory proteins in plant development. As shown in Figure 1, six putative CArG box motifs have a nine out of ten match with the core consensus binding site CC(A/T)6 GG, which are potential binding sites of MADS-box proteins (Dolan and Fields, 1991; Treisman, 1992). Five putative DNAbinding core sequences (TGAC) of the class 1 knox
genes (Krusell et al., 1997) are also present in the DOMADS1 promoter region (Figure 1). GUS staining patterns in intact tissues from orchid stable transformants In a wild-type plant, DOMADS1 was strongly expressed in flowers, not in vegetative tissues (PLBs, roots, and leaves) except for a weak expression in stems. In situ hybridization showed that DOMADS1 transcript was first detected in the apical region of TSAM. At a later stage, DOMADS1 RNA was uniformly expressed in both of the inflorescence meristem and the floral primordia and in all of the floral organs
230
Figure 2. Construction of chimeric reporter gene fusions. Different deletions from the 5 end of DOMADS1 promoter were cloned as translational fusions to the GUS reporter gene in pBI101 and analyzed for the GUS activity after stable transformation. The numbers in the left panel represent the position relative to the transcription start site (+1) as described in Figure 1. The plasmids containing a promoterless GUS gene (pBI101) and a CaMV 35S-GUS fusion (pBI121) were used as negative and positive controls, respectively.
(Yu and Goh, 2000). To further investigate the regulatory elements responsible for the observed temporal and spatial expression of DOMADS1 gene, stable orchid transgenic plants were histochemically analyzed for GUS activity. The full-length DOMADS1 promoter and a series of eight 5 deletions containing the 5 untranslated sequence and the initial part of the coding region were translationally fused to the GUS reporter gene (Figure 2). The vectors containing a CaMV 35SGUS fusion (pBI121) and a promoterless GUS gene (pBI101) served as positive and negative controls, respectively. The distribution of GUS activity for each construct was similar in the different lines analyzed (at least 8) despite the slightly varied intensity of staining. As controls, no significant GUS activity was observed in plants transformed with the promoterless pBI101 vector, while all tissues in transformants with the 35S
promoter-GUS fusion gene showed constitutive GUS activity (data not shown). A summary of activity of all deletion constructs is given in Table 2. Moderate GUS staining in roots was displayed in plants harboring the pBI-PM8 and pBI-PM9 constructs containing the minimal 214 bp and 110 bp promoter fragments (Table 2). The transformants with pBI-PM4, pBIPM5, pBI-PM6, and pBI-PM7 constructs showed a weak activity in the root, whereas GUS activity was completely absent in roots from plants containing the pBI-PM1, pBI-PM2, and pBI-PM3 constructs (Table 2). The different patterns of GUS staining between the pBI-PM3 and pBI-PM4 lines (Table 2) suggested that the promoter region from −1663 to −1189, with a CArG box motif, was required to negatively regulate the DOMADS1 expression in the root. The important regulatory feature of this region was also reflected on the dramatically reduced GUS staining pattern in the
231 Table 2. Summary of activity of DOMADS1 promoter-GUS fusions in transgenic orchid plants. Construct
Leaves
Roots
Stems
Vegetative shoot apicesa
Transitional shoot apicesb
Inflorescence apicesc
Flowers
pBI-PM1 pBI-PM2 pBI-PM3 pBI-PM4 pBI-PM5 pBI-PM6 pBI-PM7 pBI-PM8 pBI-PM9
− − − − − − − + +
− − − + + + + ++ ++
+ + + +++ +++ +++ +++ +++ +++
− − − − − − − − +
+++ +++ + + + + − − −
+++ +++ +++ +++ ++ + + + +
+++ ++ ++ ++ ++ ++ + + +
Different GUS activity is denoted by relative staining levels (+ + +, high, ++, moderate; + low; −, no detactable expression). a 6-week old culture. b 12-week old culture. c 15-week old culture.
stem of the pBI-PM3 transformant, as compared with the staining patterns in both of the pBI-PM4 transgenic line and the transformants containing shorter 5 deletion constructs (Table 2). A low level of staining in the leaf (Table 2) was only detected in transformants carrying two minimal promoter deletion constructs (pBI-PM8 and pBI-PM9), thus indicating the presence of crucial cis-acting element(s) in the region from −323 to −214 to repress the DOMADS1 expression in the leaf. In vegetative tissues (root, stem, and leaf), the GUS staining patterns exhibited by transformants containing the three longest promoter fragments (−3483, −2442, and −663) matched well with our previous results of the DOMADS1 expression in wild-type orchid plants, where this gene was not detectable in vegetative tissues except for the weak expression in stems (Yu and Goh, 2000). Therefore, the minimum promoter region necessary for normal expression of DOMADS1 in vegetative tissues was within the 1663 bp fragment directly upstream of the transcription start site. Since GUS staining in floral buds or flowers was observed in all transgenic lines and the staining intensity was gradually reduced during the progressive deletions of 5 -upstream regions (Table 2), we deduced therefore that the essential elements required for the initiation of DOMADS1 expression in flowers were located within the minimal 110 bp promoter region and the promoter between −3483 and −110 should contain one or more enhancers in promoting the gene expression in flowers. In a wild-type plant, the DOMADS1 transcript was first detected in the 12-week-old TSAM, but not in the
VSAM (Yu and Goh, 2000). To examine the regulatory elements controlling the temporal expression of DOMADS1, we investigate the GUS staining patterns in shoot apices (SA) during orchid development. As presented in Table 2, weak GUS activity in vegetative SA was only detected in the pBI-PM9 line, while weak to intense staining in 12-week-old transitional SA was conferred by pBI-PM6 and the longer promoter constructs (Table 2). These results suggested that two discrete cis-regulatory regions involved in temporal regulation of DOMADS1 expression in SA during floral transition were located within the regions from −519 to −323 and −214 to −110, respectively. The altered pattern of GUS staining in inflorescence apices exhibited by the successive 5 deletions is similar to that in flowers (Table 2). However, the elements for enhancing the DOMADS1 expression in inflorescence apices seemed to lie in a more limited region (i.e. −1189 to −519). RNA gel blot analyses were performed to examine whether the levels of the GUS transcript accumulation are consistent with the GUS activities observed. Two examples are shown in Figure 3A and 3B. In the pBIPM1 transformant, GUS gene was expressed weakly in stems, and strongly in transitional shoot apices, inflorescence apices, and flowers (Figure 3A). In the transitional shoot apices, the GUS transcript was accumulated at high levels in the pBI-PM1 and pBI-PM2 transformants, and at rather low levels in the pBI-PM3 to pBI-PM6 transformants (Figure 3B). These results indicated that the expression of the GUS gene was well
232
Figure 3. RNA gel blot analysis of GUS reporter gene expression in transgenic orchid plants harboring different reporter gene constructs. RNA gel blots containing 10 µg of total RNA in each lane were hybridized with the digoxigenin-labeled GUS DNA probe. The rRNAs stained by methylene blue indicate the amount of total RNA loaded in each lane. A, Expression of GUS gene in pBI-PM1 transformants. Total RNA was isolated from leaves (L), roots (R), stems (S), vegetative shoot apices (V, 6 week old), transitional shoot apices (T, 12 week old), inflorescence apices (I, 15 week old ), and flowers (F). Total RNA extracted from the whole part of wild-type (WT) and pBI121 (P) transgenic plants were used as negative and positive controls, respectively. B. Expression of GUS gene in transitional shoot apices in orchid plants harboring different deletion constructs (pBI-PM1 to pBI-PM9). Total RNA extracted from wild-type (WT) plants was used as a negative control.
in agreement with the staining activities detected in transgenic plants (Table 2). Localization of GUS activity in SA and developing floral buds Localization of GUS activity in cellular details was performed to more precisely define the specific regulatory regions in the promoter. Figure 4 shows the GUS staining pattern in orchid plants harboring the pBIPM1 construct during floral transition. GUS activity was first detected faintly in the apical region of the 11-week-old TSAM and in both of the emerging bract primordia (Figure 4A). The staining was more intense in the 12-week-old TSAM, where the first floral primordium had just protruded on one flank of the apical meristem (Figure 4B). Abundant staining was clearly concentrated in the apical region of the TSAM along with small amount of staining in both of the bract primordia. At the subsequent stage of inflorescence
development, heavy staining was distributed throughout the whole region of the 15-week-old inflorescence meristem and the developing floral primordia at different stages (Figure 4C). The same staining pattern was also observed in the 16-week-old inflorescence apex (Figure 4D). In the young developing floral bud, the incipient floral organ primordia (sepal, petal, and column) and the basal floral meristem showed uniform patterns of GUS staining (Figure 4E). Stronger staining was detected in the sepal primordia than in other organ primordia. At a later stage, a moderate level of GUS activity was uniformly detected in the developing petal primordium and the floral meristem, while a high level of staining was clearly observed in the developing sepal and column primordia (Figure 4F). In the fully developed floral bud of 19-week-old culture, GUS activity was visible in all of the floral organs and relatively intense signals were detected in the maturing pollinarium (pollinium apparatus), rostellum (a platform located below the pollinarium), and the column (Figure 4G). All the above results showed that the pBI-PM1 construct containing the full-length promoter region produced a spatial and temporal pattern of GUS activity almost indistinguishable from the expression pattern demonstrated by DOMADS1 gene in a wild-type orchid plant (Yu and Goh, 2000). This fact rules out all possible post-transcriptional effects caused by the 5 -untranslated region of DOMADS1 in the promoter fusions. One interesting aspect of our present results was the earlier onset of GUS activity in the 11-week-old TSAM of the pBI-PM1 transgenic plants at the stage when the first floral primordium had yet to be differentiated. This suggested that the start of DOMADS1 expression during floral transition may be earlier than revealed in our previous study (12 weeksold; Yu and Goh, 2000). Our recent examination of DOMADS1 expression by RT-PCR method has also revealed the presence of DOMADS1 transcripts in the 11-week-old TSAM, which still could not be detected by Northern blot and in situ hybridization analysis (data not shown). Thus, the slightly different results obtained by the methods of GUS staining and in situ localization may be due to their different sensitivity in detecting weak signals. Assays of GUS staining patterns in plants transformed with the pBI-PM2, pBI-PM3, pBI-PM4, and pBI-PM5 constructs demonstrated the spatial and temporal expression pattern identical to that conferred by the pBI-PM1 construct (data not shown). These results indicated that 930 bp promoter region 5 to the transcription start site could spatially and temporally
233
Figure 4. The localization of GUS activity in SA and developing floral buds from orchid plants carrying different deletion constructs. Sections A to G are from the pBI-PM1 transformant, and sections H and I are from the pBI-PM9, and pBI-PM6 transformants, respectively. All sections were photographed under dark-field illumination. Gus staining is localised in: A. The 11-week-old SA, in the apical region of the TSAM and the bract primordia (× 100). B. The 12-week-old SA, in the apical region of the inflorescence meristem and the first floral primordium as well as the bract primordium (× 100). C. The 15-week-old inflorescence apex, in the inflorescence meristem and the developing floral primordia at different stages (× 20). D. The 16-week-old inflorescence apex, in the inflorescence meristem and the emerging floral primordium (× 40). E. The 16-week-old developing floral bud, in all the emerging floral organ primordia (× 40). F. The 17-week-old young floral bud, in all the developing floral organ primordia (× 50). G. The 19-week-old floral bud, in the maturing pollinarium, the rostellum, and the column (× 50). Diffuse GUS staining is visible in H and I. H. The 6-week-old vegetative SA, in the VSAM and the incipient leaf primordia (× 150). I. The 15-week-old inflorescence apex, in the whole inflorescence apical region (× 30). am, apical meristem; b, bract; c, column; fm, floral meristem; fp, floral primordium; im, inflorescence meristem; lp, leaf primordium; p, petal; pl, pollinarium; r, rostellum; s, sepal.
regulate the DOMADS1 expression in the SAM during floral transition. The staining patterns of transformants carrying other constructs were also investigated to further delimit the elements for other aspects of DOMADS1 expression. Of all the vegetative apices, a low level of diffuse staining was observed only in the plants carrying the pBI-PM9 construct in the apical region and the
emerging leaf primordia, as in the 6-week-old SAM (Figure 4H). In intact tissues, the different GUS activity between the pBI-PM5 and pBI-PM6 transgenic plants was primarily reflected by the staining levels in individual tissues (Table 2). However, localization of GUS activity in tissue sections revealed a significant difference in the spatial distribution of staining in distinct tissues between these two lines. Compared
234 with the pattern in the pBI-PM5 transformant (which is similar to that of pBI-PM1), in the pBI-PM6 one, a spotty and irregular GUS staining was observed in the inflorescence apex (Figure 4I), as well as the developing floral bud, the fully developed floral bud, and the transitional shoot apex (data not shown). This irregular staining pattern was also displayed by the plants carrying other shorter promoter constructs. Thus, the promoter region between −930 and −519 could contain the most important cis-acting elements required for the spatial regulation of DOMADS1 expression in the SAM during floral transition as well as in the floral organs.
Discussion In this study, we have extended our investigation on the DOMADS1 expression in orchid to analyze the possible regulatory elements in its promoter region. The 5 -upstream sequence of the DOMADS1 gene was isolated, sequenced and characterized by the analysis of DOMADS1 promoter-GUS fusions in the stable orchid transformation systems. As the first step towards the understanding of essential elements supporting DOMADS1 gene expression, we used the stable orchid transformation system to carry out in vivo investigation of the cis-acting sequence motifs necessary for the specific regulation of DOMADS1 expression in orchid development. In wild-type orchid plants, DOMADS1 transcripts are absent in almost all of the vegetative tissues except the stem, where very low expression can be detected. Analyses of the GUS staining patterns in stable transformants have revealed the respective minimal promoter region required for normal DOMADS1 expression in distinct vegetative tissues (Figure 5). Because plants carrying the pBI-PM3 construct showed the same pattern of GUS staining in the root and stem as the DOMADS1 RNA accumulation pattern in wild-type plants, the spatial regulation of DOMADS1 expression in both of the vegetative organs appears to depend on the 1663 bp promoter region upstream of the transcription initiation site (Figure 5). One or more cis-acting regulatory elements that are necessary for the repression of DOMADS1 activity in roots and stems must be located from −1663 to −1189. The progressive deletion analyses also suggest that negative regulation of GUS staining in leaves is at least conferred by the pBI-PM7 promoter construct and the −323 to −214 promoter fragment could contain
cis-acting motif(s) responsible for recruiting transacting repressors functioning in the spatial regulation of DOMADS1 expression in orchid leaves. Both spatial and temporal control of DOMADS1 expression in the SAM have been shown to require the upstream 2442 bp promoter fragment (Figure 5). Temporal regulation of DOMADS1 expression in SAM during the switch from vegetative to reproductive growth depends on at least two discrete promoter regions. The first region maps to the position from −214 to −110, where the negative cis-acting element(s) may exist to inhibit the DOMADS1 expression in the vegetative SAM. The other region located at −519 to −323 could contain one or more positive regulatory elements participating in the initiation of the DOMADS1 expression in the TSAM during floral transition. The cis-acting elements essential for the spatial pattern of the DOMADS1 expression in the TSAM are present within the region from −930 to −519, because the diffuse GUS staining pattern revealed by the pBI-PM6 transformants contrasts greatly to that observed in the pBI-PM5 transformants and also the DOMADS1 RNA distribution pattern in wild-type plants. Distantly located (−442 to −1663) enhancer(s) are also present to dramatically up-regulate the DOMADS1 expression in the TSAM. The regulatory regions required for conferring the full spectrum of DOMADS1 expression in the reproductive organs are also defined using 5 deletion analyses (Figure 5). The weak GUS activity in the inflorescence meristem and flower was observed in plants carrying the shortest promoter region (pBIPM9). However, the pattern of the diffuse GUS activity (Figure 4I) is in great contrast to the definite pattern of DOMADS1 expression revealed by in situ hybridization in wild-type plants. In the inflorescence apex or flower, the −930 promoter construct is capable of providing a normal spatial pattern of GUS staining at a moderate level that is enhanced by the addition of the enhancer elements in the sequence from −1189 to −519 (in inflorescence apices) or from −3483 to −2442 (in flowers). Taken together, these results suggest that the 3483 bp promoter region upstream of the transcription start site contains multiple cis-acting elements that are sufficient for accurate regulation of DOMADS1 expression in orchid reproductive organs. The modulation of the expression of MADS-box genes via the CArG boxes by themselves or other MADS-box genes may reflect an important or possibly fundamental aspect of the transcriptional regulation of MADS-box genes (Riechmann and Meyerowitz
235
Figure 5. Schematic diagram summarizing the required regulatory elements for the normal DOMADS1 expression. The full-length promoter region is shown on the top with the labeled sequence position corresponding to 5 end of each promoter fragment relative to the transcription start site (+1). The minimal promoter regions required for the normal expression of DOMADS1 in individual tissues are represented by horizontal boxes, where the regions containing cis-acting elements for spatial and temporal regulation of DOMADS1 expression in individual tissues are marked by diagonally and vertically lined boxes, respectively. The shaded boxes denote the region with enhancer elements necessary for promoting the level of the gene expression in individual tissues. This figure does not exclude the possibility that other potential cis-acting elements also exist in certain regions, which are not detected due to the limited constructs.
1997; Hill et al., 1998; Tilly et al., 1998). Although in vitro binding of the CArG box sequence by plant MADS-box genes have been widely reported (Schwarz-Sommer et al., 1992; Huang et al., 1993; Shiraishi et al., 1993; Savidge et al., 1995; Huang et al., 1996; Mizukami et al., 1996; Riechmann et al., 1996), the in vivo function of the CArG box was investigated first in the AP3 promoter region by the assay of the GUS staining patterns produced by different deletion or mutation constructs in transgenic Arabidopsis plants with different background (Hill et al., 1998; Tilly et al., 1998). These detailed analyses demonstrate that the CArG boxes in the AP3 promoter can function in both positive and negative ways in mediating the distinct phases of AP3 expression in the petal and stamen. Compared with the Arabidopsis floral organ identity gene AP3, the orchid DOMADS1 was identified as a marker gene specifically expressed in the TSAM during floral transition (Yu and Goh, 2000), indicating the different regulatory contexts required for the establishment and maintenance of the two distinct expression patterns. It is interesting to
note the presence of six CArG boxes within the orchid DOMADS1 promoter. As shown in Figure 5, these CArG boxes lie within the spatial regulatory region (CArG2 and CArG3), the temporal regulatory region (CArG4 and CArG5), and the enhancer region (CArG1, CArG3, CArG4, and CArG5), respectively. The location of the CArG boxes in these important regions implies that the basic mechanism of autoor cross-regulation of CArG box-binding MADS domain transcription factors as described in Arabidopsis may be conserved in the taxonomically distant orchid species during the flowering process and flower development. Since the detailed definition of the CArG boxes in the promoters of early-acting MADS-box genes involved in floral transition has never been reported in any other plant species, our present description of these important cis-regulatory elements in the DOMADS1 promoter will spur more efforts in further characterization of functionally active promoter regions. In our previous study, we have isolated and characterized the first orchid class 1 knox gene, DOH1, from
236 Dendrobium Madame Thong-In (Yu et al., 2000). In wild-type plants, the expression of DOH1 and DOMADS1 demonstrates an antagonistic, reciprocal pattern during floral transition. The early onset of DOMADS1 expression is also shown in the earlyflowering phenotype exhibited by DOH1 antisense transformants. Furthermore, the upstream full-length DOMADS1 promoter drives the GUS expression in the same temporal and spatial pattern as DOMADS1 expression in DOH1 antisense transformants. These results suggest that DOH1 functions as a negative regulator of DOMADS1. Examination of the DOMADS1 promoter has revealed the presence of five putative DNA-binding sites of class 1 knox genes in the important regulatory regions responsible for spatial and temporal control of the DOMADS1 expression in specific tissues (Figure 5). Interestingly, the region between −519 and −323 crucial to the initiation of DOMADS1 expression in the TSAM simultaneously contains a set of the linked or overlapped sequences including the CArG4 box, the CArG5 box, and the class 1 knox gene binding site. The spatial organization of these three important elements indicates that during floral transition, DOH1 may directly bind to the specific DNA sequence to participate in the regulation of DOMADS1 expression by the mediation of closely linked MADS-box proteins or vice versa. The mutual interactions between MADS-box proteins and homeodomain proteins in the specific developmental stages have been demonstrated with the yeast proteins MCM1 and MATα2 and human proteins SRF and Phox1 (Grueneberg et al., 1992). Further elucidation of the potential correlation between DOH1 and DOMADS1 as well as other MADS-box genes would provide valuable insight into the mechanisms underlying the combinatorial control of eukaryotic transcriptional regulation by multiple transcription factors (Singh, 1998). The distinct expression patterns in the SAM exhibited by the early acting members of MADS-box gene family and other flowering genes during floral transition suggest that a subtle hierarchy of regulatory genes may exist for controlling the successive development in the switch from vegetative to reproductive growth (Ma, 1994; Yu and Goh, 2000). In Arabidopsis, LEAFY, the meristem identity gene, which itself is mediated by many flowering time genes, has been identified as an immediate upstream regulator of another meristem identity gene AP1 during floral transition (Parcy et al., 1998; Busch et al., 1999; Wagner et al., 1999). As the orchid LEAFY homologs have not
been isolated, it is not clear if the same mechanism of floral identity switch is present in orchids. However, based on the previous comparison of the expression patterns of a group of orchid MADS-box genes with those in Arabidopsis (Yu and Goh, 2000), we suggest that a unique regulatory process may exist in orchids during floral transition. The present study has demonstrated that the DOMADS1 upstream promoter sequences contain specific temporal and spatial cis-acting elements required for providing the normal regulatory features of the DOMADS1 expression during orchid development. Several important elements with similarity to the binding sites of trans-acting factors, which may be involved in the mediation or activation of DOMADS1 activity during floral transition, were also identified within the essential regulatory regions. These results will facilitate the further characterization of functionally active sequence motifs and their attached regulatory proteins, leading to the clarification of the molecular mechanisms underlying the regulation of the transition from vegetative to reproductive growth in orchids. As we employed only 5 deletion constructs in this study, it is possible that same other potential or redundant regulatory elements are also present. Internal deletions and further dissection of the identified regulatory regions will help to define exactly the regulatory elements sufficient and necessary for the normal DOMADS1 expression. Also, place of certain potential promoter elements 5 to a minimal promoter and the subsequent detection of particular expression patterns will test sufficiency of those positive acting factors. Future comparison of the promoters in orchid with those from other plant species will undoubtedly provide insight into the mechanisms underlying the evolution of earlyacting MADS-box genes as well as their associated plant organs.
Acknowledgments We are grateful to our colleagues in the department for their collaboration and assistance in this research. We thank Dr Prakash Kumar and Dr Sanjay Swarup for critically reading our manuscript. We also thank Ms Chooi Lan Lee for help in plant tissue culture. H.Y. and S.H.Y. are supported by postgraduate scholarships from the National University of Singapore. This research was supported by a research grant (R154-000-095-112) from the National University of Singapore.
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