Sex Plant Reprod (2003) 16:77–85 DOI 10.1007/s00497-003-0178-4
ORIGINAL ARTICLE
Filip Cnudde · Chiaraluce Moretti · Andrea Porceddu · Mario Pezzotti · Tom Gerats
Transcript profiling on developing Petunia hybrida floral organs
Received: 9 April 2003 / Accepted: 2 May 2003 / Published online: 27 June 2003 Springer-Verlag 2003
Abstract The cDNA-AFLP transcript profiling technique was used to analyse gene expression during flower development in Petunia hybrida. Reproductive and vegetative floral organs were sampled at five developmental stages and gene expression profiles were compared. This allowed us to assemble an inventory of genes expressed mainly in anthers during microspore development and in ovaries during macrosporogenesis. About 6,000 transcript tags were generated, 354 of which showed a modulated and/or organ-specific expression pattern. Stamen-specific transcripts exhibiting an upregulation in gene expression were well represented in our screening. Ovary-specific transcripts were less frequently observed and often displayed a constant level of gene expression. Of 194 fragments characterised further by sequencing, 35% showed homology with known genes in a database search. They belong to a wide range of gene classes, such as proteases, transcription factors and genes involved in metabolism, cell cycle and disease resistance. The usefulness of cDNA-AFLP transcript profiling as a tool to unravel complex developmental processes at the molecular level is discussed. F. Cnudde and C. Moretti contributed equally to this work F. Cnudde ()) · T. Gerats Department of Experimental Botany, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands e-mail:
[email protected] Fax: +31-24-3652787 C. Moretti Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Universit degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy A. Porceddu Institute of Plant Genetics—CNR sez Perugia, Via Madonna Alta 130, 01626 Perugia, Italy M. Pezzotti Dipartimento Scientifico e Tecnologico, Universit degli Studi di Verona, Strada Le Grazie 7, 37134 Verona, Italy
Keywords Sporogenesis · cDNA-AFLP · Flower development · Expression analysis · Petunia
Introduction Genetic studies have identified two classes of consecutively acting genes involved in the early steps of flower development. First, meristem identity genes are required for the regulation of the floral meristem formation. Second, homeotic genes are expressed in discrete domains of the flower meristem to specify the identity of floral organ types (Coen and Meyerowitz 1991). Little information is available on the downstream genes involved in subsequent processes of floral organ morphogenesis and tissue differentiation. These genes should include those that: (1) regulate floral organ compartment differentiation, (2) control floral shape and size, (3) initiate cell- and tissue-specification programs, and (4) activate organ-specific gene subsets (Meyerowitz 1997). The elucidation of genes belonging to the above categories is both an intriguing and a challenging task. Ovule development is particularly well described at the morphological level (Schneitz et al. 1995), but the underlying genetic and molecular mechanisms are just beginning to be unravelled (for reviews, see Drews et al. 1998; Gasser et al. 1998; Grossniklaus and Schneitz 1998; Schneitz et al. 1998). A number of interesting ovule identity mutants have been identified in Petunia hybrida and Arabidopsis thaliana, leading to the cloning of the first regulatory genes involved in ovule development (Angenent and Colombo 1996). Differential screening of cDNA libraries has also been used for the isolation of genes associated with ovule development (Gasser et al. 1989; Nadeau et al. 1996), although the inaccessibility of the ovule within the ovaries and the difficulty in harvesting adequate amounts of tissue have been, and still are, major obstacles. In contrast, much attention has been devoted to the genetic control of anther development (for reviews, see Koltunow et al. 1990; Scott et al. 1991b; Goldberg et al.
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1993). This exceptional interest can be attributed partly to (1) the synchronicity of anther development, and (2) the strong allometric relationship between anther development and bud length (Koltunow et al. 1990; Scott et al. 1991a). In early DNA/RNA hybridisation experiments it was estimated that approximately 25,000 genes are expressed in the tobacco anther, of which 10,000 are thought to be anther-specific (Kamalay and Goldberg 1980, 1984). Anther-specific genes have been cloned from a number of plant species such as tobacco (Koltunow et al. 1990), Brassica napus (Scott et al. 1991a), Arabidopsis (Rubinelli et al. 1998) and rice (Tsuchiya et al. 1992). Less genomic information is available on the morphogenesis of vegetative floral organs. P. hybrida is considered as a model system for studying morphogenesis of floral organs because, among other advantages, it has a large flower and a strong allometric relation between bud size and developmental processes in reproductive organs. The identification of expressed sequence tags (ESTs) has proven to be a powerful and rapid approach to identify genes that are preferentially expressed in certain tissues or cell types (Adams et al. 1995). The expression profile of these sequences can then be studied using microarray-based technologies (Brown and Botstein 1999). Though powerful, this approach is expensive and can be readily applied only to model species for which significant genomic information is available (Baldwin et al. 1999). Present additional limitations include the sensitivity of microarrays and reduced specificity because of potential cross-hybridisation problems with genes belonging to the same family (Breyne and Zabeau 2001). Since relatively little genomic sequence information is available for P. hybrida, we employed cDNA-AFLP transcript profiling technology to identify genes that exhibit a modulated expression during male and female sporogenesis as compared to vegetative tissues. This sensitive and reproducible method does not require prior sequence information and allows the identification of as yet unidentified genes. Moreover, small amounts of RNA are sufficient as starting material, allowing the analysis of small samples (Breyne et al. 2002). Direct comparison of 6,000 amplicons yielded 354 candidate cDNA fragments displaying a modulated and/or organ-specific gene expression pattern. Of these, 35 ovary-fragments and 159 anther-fragments were further analysed by direct sequencing. The majority of the cDNA fragments yielded no significant homology in a database search, although homologies with diverse classes of known sporogenesis genes were retrieved, indicating that our screening was basically effective.
Materials and methods Plant material P. hybrida (line W115) plants were grown under standard greenhouse conditions (21C; 16 h photoperiod). Flowers were
collected at five different developmental stages recognised on the basis of petal length and corresponding to stages 4–8 as published in Porceddu et al. (1999). Samples were taken separately from sepals, petals, anthers, style and stigma, and ovaries. Root and leaf samples were also collected and frozen in liquid N2. mRNA isolation and cDNA synthesis Total RNA was isolated from about 2 g each of floral or vegetative organs using the TRIZOL method (Gibco-BRL, Paisley, UK). Total RNA concentration was determined spectrophotometrically and adjusted to a final concentration of 1 mg/ml. A poly(A)+ RNA fraction was isolated from 1 mg total RNA using an mRNA purification kit from Amersham Pharmacia (Uppsala, Sweden) according to the manufacturer’s instructions. First and second cDNA strands were synthesised according to standard protocols (Sambrook et al. 1989). The resulting doublestranded cDNA was subsequently purified by phenol extraction and ethanol precipitation. cDNA-AFLP analysis The cDNA-AFLP-based transcript profiling procedure was performed according to Breyne et al. (2002). Double-stranded cDNA (500 ng) was used for cDNA-AFLP analysis. The restriction enzymes used were BstYI and MseI (New England Biolabs, Beverly, Mass.). For preamplifications, an MseI-primer without selective nucleotides was combined with a BstYI-primer containing a T at the 30 -end. The amplification mixtures obtained were diluted 600-fold and 5 ml was used for final selective amplifications following a described procedure (Breyne et al. 2002). BstT- and MseI-primers with two and one selective nucleotides, respectively, were used for the cDNA-AFLP analysis, and all 64 possible primer combinations were performed. The 33P-ATP-labelled fragments were separated on a 5% polyacrylamide gel. Dried gels were exposed to Kodak Biomax films in addition to scanning in a Phospho-imager (Molecular Dynamics). Characterisation of AFLP fragments Selected fragments were excised from the polyacrylamide gel, suspended in H2O and eluted DNA was reamplified using the same PCR conditions and the same primer combination as for selective amplification. Reamplified products were checked on a 1% agarose gel. Sequence information was obtained by direct sequencing using the selective BstYI-primer as a sequencing primer. The sequences obtained were compared to those in the GenBank database using BLAST sequence alignments (Altschul et al. 1997).
Results Developmental staging of Petunia flower buds Flowers of P. hybrida line W115 were harvested and classified into five developmental stages according to petal length. These stages correspond to stages 4–8 as defined by Porceddu et al. (1999). At each stage, we harvested samples from sepals, petals, stamens, ovaries and style/stigma. During these developmental stages, Petunia sepals increase in length (from 9 to 14 mm), almost exclusively through cell expansion. Petals show a 5-fold increase in length (5.75–24.75 mm) through cell division and expansion (Martin and Gerats 1993; Reale et al. 2002).
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It has already been demonstrated that bud length is tightly linked to the stages of postmeiotic microspore development and to megasporogenesis in Petunia (Porceddu et al. 1999); at stage 4, most of the ovules harbour a megaspore mother cell, while at developmental stages 5, 6 and 7, dyads, triads and tetrads, respectively, are predominant. At stage 8 only tetrads and megaspores are detected (data not shown). At the beginning of stage 4, the first phase of anther development has already been completed (Porceddu et al. 1999): the anther has acquired its characteristic four-lobed shape; cell differentiation has occurred and all major anther tissues have been specified; the microspore mother cells have undergone meiosis and tetrads of haploid microspores are present in the pollen sac. During the developmental range monitored in our work (stages 4–8), microspores differentiate into pollen grains, the anther enlarges and is pushed upwards in the flower by filament extension, tissue degeneration occurs and the anther enters a dehiscence program ending with the release of pollen grains. cDNA-AFLP transcript profiling cDNAs were synthesised from 1 mg poly(A+) RNA of different floral organs sampled at the five developmental stages described above. We focused on the analysis of gene expression in anthers and carpels, while samples from sepals, petals and style/stigma served as a reference. cDNA was also prepared from roots and leaves as a second control for vegetative tissues. The transcript profiling technology used in this experiment is based on the original cDNA-AFLP method described by Bachem et al. (1996). The method was adapted such that theoretically only one unique restriction fragment is obtained from each cDNA (Breyne and Zabeau 2001). The radiolabelled cDNA fragments were separated on high-resolution polyacrylamide gels. We primarily analysed fragments that ranged in length from 50 to 400 basepairs. For the majority of the gels, the preamplification products of separate developmental stages were pooled for the reference samples (sepals, petals and style/stigma) to reduce the number of lanes per primer combination. When BstYI/MseI templates were amplified using primers with two selective nucleotides, a high number of fragments per lane was generated, mostly corresponding to abundant mRNAs. The addition of a third selective nucleotide decreased the number of fragments to an average of 90 for each primer combination and enhanced the sensitivity, allowing detection of less highly expressed genes. In a check for the reliability of the transcript profiling method (Fig. 1), we could confirm that each transcript tag amplified by primers with two selective nucleotides (+1/ +1) reappeared more intensely in one of the four lanes of the +2/+1 or the +1/+2 primer combinations.
Fig. 1A–E Sensitivity and selectivity of cDNA-AFLP. B, C Transcript profiles from anthers and ovaries, respectively, sampled at five developmental stages (stages 4–8 as defined by Porceddu et al. 1999). Two selective nucleotides were used: one for the BstTprimer and one for the Mse-primer (+1/+1). A, D Profiles of the ten pooled preamplification products from anthers and ovaries and using a third selective nucleotide; this third nucleotide was added to the BstT-primer in panel A (+2/+1) and to the Mse-primer in panel D (+1/+2). An extra nucleotide increases the sensitivity and the selectivity of cDNA-AFLP transcript profiling: each transcript tag from a +1/+1 combination reappears more intensely in one of the four lanes of the +2/+1 and +1/+2 primer combinations. Panel E represents the +1/+1 profiles for some of the reference samples: St/ St style-stigma, R roots, L leaves
Using BstYI and MseI primers carrying two or one selective nucleotides, respectively, gives a total of 128 possible primer combinations. With half of these combinations performed, around 6,000 transcripts could be detected. In silico analysis of full-length cDNAs from A. thaliana has shown that 80% of all transcripts are cut by both BstYI and MseI restriction enzymes and that 68.8% are represented as BstYI–MseI 50 –30 fragment (P. Breyne, personal communication). The collection of available full-length cDNA sequences in P. hybrida is too limited for such an analysis, but if we assume that a comparable number of messenger RNAs is tagged by this enzyme combination and that 10% of the restriction fragments generated are either too small or too large to be analysed on polyacrylamide gels (Breyne et al. 2002), we may estimate that approximately 30% of the Petunia transcriptome has been analysed by applying half of the BstYI/MseI primer combinations. This would lead to an estimate of around 20,000 transcripts for the whole transcriptome. Analysis of cDNA-AFLP expression patterns and characterisation by sequencing Of the 6,000 AFLP tags analysed, 354 gene fragments (6%) were found to be organ-specific or modulated during the developmental range tested. As our study focuses on
80 Table 1 Classification of differentially expressed cDNA fragments
Ovary fragments (89) Anther fragments (247) Anthers and ovaries (18)
Specific Non-specific Specific Non-specific
Induced
Repressed
Non-modulated
Total
4 9 72 74 7
7 2 40 21 7
19 48 26 14 4
30 59 138 109 18
Table 2 Relevant homologies of sequences of AFLP fragments to sequences in the database AFLP tag
Length (bp)
Expression patterna
Homologyb
BLAST scorec
4 5, 7, 12 13 22 25, 27 29 38 40 41 43 50 62, 66 67 71 72 80 81 83 94 95 99 100 107 108 110 115 123 124 128 135 138 140
158 142, 125 89, 85 218 52 120 168 167 468 131 413 534 304 288 96 407 253 315 240 418 193 203 315 139 226 175 135 87 250 377 469 170 181 162 209 162
O, = O, = O, + O, = A, O, + AS, + AS, + AS, + AS, + AS, + A, AS, = A, = AS, = A, + OS, = A, + O, = A, + A, = A, O, + AS, = A, + A, + A, + A, + AS, = AS, + AS, + AS, + AS, = A, + A, + AS, = A, + A, +
6e–12 3e–19, 4e–11 2e–8 2e–10 5e–5 7e–4 7e–10 4e–21 1e–35 8e–18 2e–9 6e–60 6e–14 5e–23 8e–10 2e–21 0.001 6e–30 1e–29 2e–32 2e–26 1e–4 1e–16 0.001 1e–26 5e–18 3e–10 5e–5 2e–23 1e–59 5e–68 4e–11 2e–10 8e–7 3e–26 3e–13
142 145 146 148 151 152 153 154 155 157 158 169 170 173 174 175 188
128 162 399 279 329 251 230 223 327 279 276 161 121 258 247 243 169
AS, = O, = A, + AS, + A, O, + AS, + AS, + F, = AS, = A, O, AS, + AS, + AS, + A, = A, AS, = A,
189
168
OS, =
Unknown Arabidopsis (AC012680) Histone H3.2 from Medicago sativa (U09460) Proline-rich protein from Brassica napus (S42552) Tumour-related protein MR4 from tobacco (D26454) Cf-9 resistance gene from tomato (AJ002236) Glycine hydroxymethyltransferase from Arabidopsis (AL034567) Globulin-like protein from Daucus carota (U47078) Betaine aldehyde dehydrogenase from Sorghum bicolor (U12195) Subtilisin-like proteinase from Arabidopsis (X85974) Hexameric polyubiquitin from tobacco (M74101) Unknown Arabidopsis (AC007357) Allyl alcohol dehydrogenase from tobacco (AB036735) Meiotic asynaptic mutant asy1 from Arabidopsis (AF157556) ABC transporter protein from Arabidopsis (AC074360) Actin from tobacco (U60491) Putative bHLH transcription factor from Arabidopsis (AAF488634) Putative ripening-regulated protein from Vitis vinifera(AJ237994) Knolle from Capsicum annuum (AJ249504) Dof zinc finger protein from tobacco (AJ009594) Actin from tobacco (U60494) Putative protein from Arabidopsis (AC012188) Subtilisin-like proteinase from Arabidopsis Cysteine synthase from tobacco (AJ299249) Probable wound-induced protein from Arabidopsis (T01970) Probable pectate lyase precursor LAT59 from tobacco (S27098) 6-phosphogluconate dehydrogenase from Arabidopsis (AC068900) Unknown from Arabidopsis (AL021889) Orthophosphate dikinase from rice (D87745) Plastidial phosphoglucomutase from Arabidopsis (AJ242601) GDP Dissociation inhibitor from tomato (AJ401079) 110 kDA protein HMP fromPisum sativum (AB055904) Inorganic pyrophosphatase from tobacco (X83729) Hexokinase-related protein 1 from potato (AF118134) Unknown Arabidopsis (AC002311) Putative protein kinase from Arabidopsis (NM_1282561) Delta-1-pyrroline-5-carboxylate synthetase from Solanum lycopersicum (U60267) Calmodulin CAM53 from Petunia hybrida (M80831) Unknown protein from Arabidopsis (NM_106465) Ethylene responsive element binding protein fromFagus sylvatica(AJ420195) Phospholipase D from tomato (AY013256) Sucrose-phosphate synthase 2 from Craterostigma plantagineum(T09837) Petunia germinating pollen (PGPS/D9) from Petunia hybrida (AF049925) Unknown protein from Arabidopsis (AY088669) Receptor kinase-like protein from Arabidopsis (NM_119888) Unknown protein from Arabidopsis (AB024031) Probable transposase from Mycobacterium tuberculosis(H70582) Male sterility-2 protein from Arabidopsis (S33804) Inorganic pyrophosphatase from tobacco (X83728) Phenylalanine ammonia-lyase from tobacco (X78269) Squalene epoxidase from Arabidopsis (AB008021) Putative beta-galactosidase from Arabidopsis (AY045791) Protein similar to seed maturation protein fromArabidopsis(AC005397) Protein with homology to Vicia sativa early nodulation binding protein 1, from Arabidopsis (AC008113) Hypothetical protein from Arabidopsis (NM_105395)
181 8
113
305
4e–10 6e–13 5e–28 5e–36 0.001 3e–12 2e–13 3e–4 6e–21 4e–8 3e–23 2e–15 6e–13 7e–36 2e–18 1e–18 2e–7 3e–5
81 Table 2 (continued) AFLP tag
Length (bp)
Expression patterna
Homologyb
BLAST scorec
205 206 209 210 211 214
86 112 307 254 175 276
A, + AS, + O, = AS, + AS, = AS, +
1e–24 5e–13 1e–39 8e–9 8e–10 1e–36
216
137
223 232 245 248 249 260 271 300 321 328 329 332 337 342 351 352
196 109 135 292 230 113 160 240 204 429 251 236 125 197 364 228
Sepals, Style/Stigma A, = A, O, + A, + AS, + A, = A, = AS, A, AS, = A, + AS, + AS, = O, + A, + A, + AS,
Glycine-rich protein from Petunia hybrida Profilin from tobacco (X93466) Putative carboxyl-terminal peptidase from Arabidopsis (AY087706) Scenescence-associated protein (SAG29) from Arabidopsis (AC391711) Nucleoside diphosphate kinase 3 from Arabidopsis (AF044265) ATPase beta subunit, nuclear gene encoding mitochondrial protein from Nicotiana sylvestris (U96499) RNA polymerase sigma factor from Burkholderia cepacia (AF095748) Mitochondrial genome DNA from Arabidopsis (Y08501) Inorganic pyrophosphatase from potato (Z36894) Hypothetical protein from Arabidopsis (AC006341) S-locus F-box-S2 protein from Antirrhinum hispanicum (AJ297974) AMP-binding protein from Arabidopsis (AB026636) UDP glucose-4-epimerase from Arabidopsis (Z54214) bHLH transcription factor from Arabidopsis (AC005167) Flavone synthase II from Perilla frutescens (AB045592) Putative metal ion transporter (nramp6) from Arabidopsis (AJ291831) Putative transcription factor MYB24 from Arabidopsis (AF175987) Na+/H+antiporter from Arabidopsis (AF510074) Pectinesterase-like protein from Arabidopsis (AB010700) Novel secreted protein of tobacco BY2 cells PR-protein (AB024600) Putative phospholipase D from Arabidopsis (AL078606) SNF1-like protein kinase from tomato (AF322108) Fertilization-independent endosperm protein from Arabidopsis (AF129516)
1e–12 1e–19 3e–5 4e–6 2e–20 4e–7 7e–8 2e–4 5e–19 1e–34 6e–17 4e–14 8e–5 1e–15 7e–16 1e–5
2e–4
a
A Anther non-specific, AS anther-specific O ovary non-specific, OS ovary-specific, F flower-specific, + induction, repression, = nonmodulated, higher expression level b GenBank accession numbers of sequences homologous to AFLP fragments are in parentheses c All are BLASTX scores except for 71, 110, 135, 154, 205, 223 and 232, which are BLASTN scores
changes in gene expression in developing reproductive organs, we report a classification of fragments based on their expression profile in these organs (Table 1). Fragments expressed exclusively in anther (or ovary) are denoted as anther (or ovary)-specific. Fragments found also in other organs but with a clearly different expression pattern in anthers or ovaries were denoted as anther (or ovary)-non-specific. Fragments classified as non-modulated (and specific or non-specific) typically exhibited a constant and high expression level in the stages tested. Most of the ovary-modulated fragments were also present in the style and stigma; moreover, most of the ovary fragments overall were non-modulated. Also, 72 of the anther-specific and 74 of the anther-non-specific fragments were highly induced. Finally, 18 fragments were (non)-modulated in both anthers and ovaries. In total, 354 transcript fragments were isolated from gels, reamplified by PCR and sequenced. Direct sequencing of PCR products gave high quality sequence for 194 fragments (55%). For the remaining transcript tags no clear sequence information was obtained, presumably because these cDNAs were a mixture of co-migrating fragments. These were not further characterised. The success rate of direct sequencing can be increased by using cDNA-AFLP primers containing an additional selective nucleotide. This reduces the complexity of the amplification mixture 4-fold and will lead to less comigrating fragments. As an alternative, PCR products can
be cloned and individual colonies can be sequenced (Breyne et al. 2002). The sequences obtained were compared to those present in the GenBank database using the BLAST program (Altschul et al. 1997); 71 fragments (36%) show significant homology (maximum E-value 0.001) to genes with known function, while another 10 (5%) match genes without allocated function (ESTs or putative proteins; Table 2). The remaining 59% either represent sequences of previously uncharacterised genes, or share no homology, presumably because the AFLP tags are too short and/ or originate from the 30 -end region of the transcripts, which is often assumed to be less conserved. Of the fragments with ovary-specific or (non)-modulated expression, 42 were successfully sequenced. Only 11 (25%) displayed significant homology with known genes in a database search (Table 2). This could reflect the backlog in the molecular analysis of megasporogenesis compared to the more extensively studied anther development. Homologies were found with proline-rich proteins, a peroxidase, a tobacco tumour-related protein eliciting the hypersensitive response and a PR-protein. They can be added to a growing list of abundantly expressed pistil genes that encode defence-related proteins. Pistils are particularly vulnerable to pathogen infection because they are rich in precursors of macromolecules required for supporting pollen tube growth (Harris et al. 1984). A fragment was found that exhibited homology with the Capsicum KNOLLE gene, which
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encodes a cytokinesis-specific protein related to syntaxins, a protein family involved in vesicular trafficking. KNOLLE mRNA has previously been shown to accumulate preferentially in organs with actively dividing tissues, such as inflorescence and floral meristems (Lukowitz et al. 1996). A total of 142 anther-fragments were successfully sequenced; for 62 (49%) of these a significant homology was found. Based on sequence homology we could relate some of the genes identified to physiological processes occurring during anther development. A first group of genes that could be discerned is involved in desiccation tolerance and was upregulated during pollen maturation. P. hybrida has pollen with a water content of 10% of its fresh weight at dispersal. Reserves consist of cytoplasmic mono-, oligo- and polysaccharides, whereas starch is absent (Baker and Baker 1979). A cDNA fragment with an induced expression profile in anthers showed homology to a delta 1-pyrroline-5carboxylate synthetase, the rate-limiting enzyme in the biosynthesis of proline (Yoshiba et al. 1999). Proline represents about 50% of the amino acid pool of Petunia pollen and is utilised for protein synthesis during pollen germination (Zhang et al. 1982) and as a protectant against desiccation (Zhang and Croes 1983). Fragments homologous to sucrose phosphate synthase, Na+/H+ antiporter and betaine aldehyde dehydrogenase also indicate the occurrence of drought tolerance processes in developing anthers. Sucrose has been shown to play a role in the acquired tolerance to severe dehydration in the resurrection plant Craterostigma plantagineum (Ingram et al. 1997). Na+/H+ antiporter anther-specific induction has already been described during desiccation in Oryza sativa (Fukuda et al. 1999) and the involvement of betaine aldehyde dehydrogenase in desiccation has been demonstrated in sorghum (Wood et al. 1996). Another group of genes could be related with tapetum degradation. Inorganic pyrophosphatase (V-PPase) might supply the driving force for loading sugars from the cytosol into the vacuole (Mitsuda et al. 2001). Yet another group of fragments identified genes expressed in the pollen grain that may play a role during pollen germination. An “early” anther-specific cDNA fragment expressed up to the time of microspore mitosis showed homology to the Arabidopsis FERTILIZATION INDEPENDENT ENDOSPERM (FIE) gene. FIE encodes a WD polycomb group protein that represses endosperm development in the absence of pollination and fertilisation (Ohad et al. 1999). Luo et al. (2000) demonstrated that FIE::GUS fusion proteins were expressed in the female gametophyte and transiently in developing microspores. A “late” anther-specific cDNA was homologous to the MALE STERILITY 2 (MS2) gene, encoding a fatty acyl reductase involved in the formation of the pollen wall (Aarts et al. 1997). Its importance in pollen development was demonstrated by the isolation of a transposon-tagged male-sterile mutant in Arabidopsis (Aarts et al. 1993). Flavonoids and other phenylpropanoids are present in large amounts in the exine layer of pollen grains
(Mascarenhas 1990). A key enzyme in phenylpropanoid metabolism, phenylalanine ammonia lyase, was induced upon pollen maturation. Anther-specific cDNA fragments were also isolated that show homology to genes functioning during pollen tube growth. Their mRNA accumulates during pollen maturation and is translated upon germination. Calmodulin, a Ca2+-interacting protein and profilin, an actin-binding protein, both play a role in signalling pathways involving protein kinase and phosphatase cascades that may regulate pollen tube growth by acting on the actin-based cytoskeleton organisation and assembly (Franklin-Tong et al. 1999). Homologues of LAT59, a tomato pectate lyase (Wing et al. 1990) and a pectin esterase-like protein (Mu et al. 1994) may be involved in pectin degradation during germination and pollen tube growth. Maybe not unexpectedly, anther-specific induction was observed for a cDNA fragment homologous to an S-locus F-box protein involved in self-incompatibility. Finally, the isolation of cDNA fragments exhibiting homology to transcription factors such as a zinc finger transcription factor and a bHLH transcription factor are an indication of the high sensitivity of our transcript profiling experiment. An interesting expression pattern was observed for a protein kinase homologue present in all floral organs but not in roots and leaves.
Discussion The advantages of cDNA-AFLP transcript profiling The combination of developmentally staged floral organ samples with the cDNA-AFLP transcript profiling technique allowed us to perform a large screening for genes exhibiting a modulated and/or organ-specific expression pattern during gametogenesis in P. hybrida flower buds. The identification of hundreds of transcript tags together with the generation of reliable expression data prove that cDNA-AFLP transcript profiling is a valuable tool for high-throughput expression analysis. This is certainly true for non-model species, where it can be used as a valid alternative to microarrays. Compared to subtractive hybridisation library screening, cDNA-AFLP transcript profiling allows a more thorough analysis because of the direct comparison of gene expression levels in different organs during a range of developmental stages. Further characterisation of the cDNA fragments showed that they corresponded to diverse classes of known gametogenesis genes, indicating that our screening was effective. Genes active in the same pathways are often coexpressed, allowing the identification of ongoing biological processes and their mutual coordination. In agreement with recent studies (Durrant et al. 2000; Breyne et al. 2002) a large number of as yet unknown genes was identified, proving that cDNA-AFLP transcript profiling is an efficient technique to identify previously unknown transcripts.
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Number of anther-specific genes identified lower than expected Previous estimations of the number of genes specifically expressed during gametogenesis have focused on the male gametophyte. Early DNA/RNA hybridisation experiments indicated that about 25,000 genes are expressed in the tobacco anther at stage 6 (Kamalay and Goldberg 1980). Approximately 10,000 of these genes were estimated to be anther-specific because their mRNA was not detectably expressed in other floral or vegetative organs (Kamalay and Goldberg 1980, 1984). Based on these figures, and taking the smaller genome content into account, Sanders et al. (1999) predicted 8,000 genes to be expressed during the equivalent stage of anther development in Arabidopsis, of which 3,500 exhibit an antherspecific expression pattern. Stage 6 in tobacco anther development (Koltunow et al. 1990) corresponds to stage 3 in Petunia (Porceddu et al. 1999); most specialised cell types are still present within the anther and the microspore nuclei divide in the locules. It is likely that many additional genes might be active immediately after meiosis and during the early stages of microspore development. Similar experiments on mature pollen of Tradescantia and maize indicate that 20,000 and 24,000 genes, respectively, are transcribed, about 10% of which are considered to be pollen-specific (Willing and Mascarenhas 1984; Willing et al. 1988). Looking at our transcript profiling experiment with the choice of restriction enzymes and half of all possible primer combinations carried out, we estimate that 30% of the transcriptome has been visualised on gels, resulting in 6,000 AFLP tags. Since each expressed gene is theoretically represented by one fragment, approximately 20,000 genes are transcribed in anthers of P. hybrida during postmeiotic microspore development and pollen maturation. The number of generated fragments did not differ significantly between all organs tested, suggesting that an overall equal number of genes is expressed. In our screening, 138 anther-specific cDNA fragments were identified. This suggests that no more than 500 genes with an anther-specific expression pattern can be expected in a genome-wide screening. How can this difference from the 10,000 genes proposed by Kamalay and Goldberg (1980, 1984) be explained? First, the sensitivity of the cDNA-AFLP transcript profiling method is higher than that of a hybridisation-based technique (Breyne and Zabeau 2001); up to one mRNA copy per cell can be visualised on a gel (Bachem et al. 1998). This leads to a more stringent definition of anther-specificity because it allows the detection of low levels of expression in other floral and vegetative organs. Second, 10,000 genes corresponds to the number of anther mRNAs that were not shared with leaf. Although it was shown that this mRNA set is distinct between anthers and ovaries, the selection of anther-specific genes was more precise in our experiment, which allows a direct comparison of expression levels for each individual anther-expressed gene with
ovary, style-stigma, sepal, petal, root and leaf. Third, with cDNA-AFLP transcript profiling, gene numbers are determined unambiguously by counting fragments on a polyacrylamide gel, whereas in the RNA/DNA hybridisation experiments of Kamalay and Goldberg, gene numbers were estimated in an indirect way based on mRNA complexities. Gene expression patterns during male gametophyte development With our approach, it is possible to group genes based on their expression pattern and to search for a possible function based on their co-expression with already defined genes. Mascarenhas (1990) distinguished two patterns of pollen gene expression. “Early” genes are transcribed soon after meiosis and are reduced or undetectable in mature pollen. Transcripts of the “late” genes are first detected around the time of microspore mitosis and continue to accumulate as pollen matures. In our transcript profiling experiment, we screened for genes expressed in anthers displaying a modulated or antherspecific expression pattern. Of these, 60% belong to the class of “late” genes, showing an increase in gene expression starting from stage 6 and 7 up to the end of pollen maturation. These accumulating mRNAs belong to genes involved in pollen maturation. A set of mRNAs is stored in mature pollen for mobilisation upon pollen germination and pollen tube growth. Around 25% of anther genes are repressed; in most cases the gene expression level decreases from stage 4 to stage 6 (microspore mitosis). They belong to the class of “early” genes that encode cytoskeletal proteins and proteins needed for pollen wall biosynthesis and energy production; most of these genes are expressed in the tapetum. We propose a third class of gene expression, comprising the remaining 15% of anther-specific genes showing a non-modulated gene expression pattern, but one that displayed anther-specificity or a high level of expression compared to other plant organs. Genes involved in signalling and cell-cell interactions such as subtilisin-like serine protease and calmodulin belong to this class. Conclusion We have demonstrated that cDNA-AFLP transcript profiling is a suitable method for the identification of genes involved in male and female gametogenesis in P. hybrida. By comparing gene expression profiles of floral organ-specific samples during several stages of flower development, modulated and anther- or ovary-specific cDNA fragments were encountered, including many as yet unidentified genes. The generated cDNA tags can readily be used as probes for further in situ hybridisation experiments or for mutational analysis. Further characterisation of these sets of genes will help to unravel the
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control mechanisms and genetic networks underlying male and female gametogenesis in plants. Acknowledgements Filip Cnudde was funded partly by the Flanders Fund for Scientific Research (FWO), partly by the Flemish Institute for Biotechnology (VIB) and partly by a grant from the subfaculty of Biology, University of Nijmegen.
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