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. 2003 Oct;133(2):713–725. doi: 10.1104/pp.103.028241

Transcriptional Profiling of Arabidopsis Tissues Reveals the Unique Characteristics of the Pollen Transcriptome1,[w]

Jörg D Becker 1, Leonor C Boavida 1, Jorge Carneiro 1, Matthias Haury 1, José A Feijó 1,*
PMCID: PMC219046  PMID: 14500793

Abstract

Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division.

Pollen has been the subject of intense studies not only for its importance as the male partner in plant reproduction, but also as a model for the study of cell growth and morphogenesis in a broader sense (Feijó et al., 2001). Whereas early studies focused on pollen physiology and biochemistry (for review, see Mascarenhas, 1975), the last 20 years have been marked by increasing efforts to decipher the genetic basis of pollen development and functions (for reviews, see Scott et al., 1991; McCormick, 1993; Preuss, 1995; Taylor and Hepler, 1997; Franklin-Tong, 1999; Hepler et al., 2001; Lord and Russell, 2002). These efforts led to the identification of more than 150 pollen-expressed genes from more than 28 species (Twell, 2002). These genes encode proteins thought or known to be involved in pollen development, pollen germination, and pollen tube growth, as well as in interactions with the stigma/transmitting tissue or the female gametophyte. Many of these studies were conducted in Lilium sp. or in Solanaceae species, because their pollen is easily collected in sufficient quantities, and methods for in vitro germination are robust. However, the available genetic information for these species is limited. The availability of the genome sequence of Arabidopsis (The Arabidopsis Genome Initiative, 2000) and the concomitant increase in available genomic tools (Wixon, 2001) make Arabidopsis a preferable model for large scale genetic studies of pollen germination and tube growth. The shift toward Arabidopsis is reflected in recent studies of the cellular organization and ultrastructure of in vivo- and in vitro-grown pollen tubes of Arabidopsis (Lennon and Lord, 2000; Derksen et al., 2002) and by efforts to improve in vitro germination of Arabidopsis pollen (Fan et al., 2001).

Studies in several plant species have indicated that the bulk of mRNAs needed for pollen germination and tube growth accumulates in pollen grains well before germination (Mascarenhas, 1989; Guyon et al., 2000). Thus, we expected that identifying transcripts that were enriched (up-regulated gene expression) or even selectively expressed in hydrated pollen grains on a genomic scale would increase significantly our knowledge of the genetic basis of pollen germination and tube growth. We chose an approach using pollen of Arabidopsis and overcame the limitations of small-scale transcriptional analysis applying oligonucleotide-based microarray technology. The Arabidopsis Genome GeneChip array (Affymetrix, Santa Clara, CA) has been used to examine the transcriptional regulation of genes in Arabidopsis by the circadian clock (Harmer et al., 2000) and to identify gene expression changes during shoot development (Che et al., 2002). As a prerequisite for high-quality transcriptional profiles of pollen grains, we developed a protocol using fluorescence-activated cell sorting (FACS) to obtain highly purified hydrated pollen grains of Arabidopsis. We obtained transcriptional profiles for hydrated pollen grains and for four types of vegetative tissues (leaves, roots, seedlings, and siliques) of Arabidopsis. By comparison of these data sets we show that the transcriptional profile of Arabidopsis pollen is clearly distinguishable from those of the vegetative tissues. About 1,500 genes (approximately 90%) expressed in pollen grains are also expressed in at least one of the vegetative tissues, whereas the remaining 10% (162 genes) are selectively expressed in pollen. Most of these 162 genes have not been described as selectively expressed in Arabidopsis pollen before, and their functional classification yields new insights into several aspects of the genetic program underlying germination and tube growth of Arabidopsis pollen.

RESULTS AND DISCUSSION

FACS Yields Highly Purified Arabidopsis Pollen Grains

An essential requirement for obtaining high-quality DNA array data is purity of the tissue or cellular source for RNA extraction, because any kind of impurity could result in an inaccurate transcriptional profile. Because the reported sensitivity of the Arabidopsis Genome GeneChip arrays is one transcript in 100,000 to one transcript in 300,000 (Zhu and Wang, 2000), we consider this requirement especially important for pollen. Therefore, we have developed a new protocol to obtain highly purified, hydrated pollen grains. Prehydrated pollen was washed out from flowers in buffer and subjected to filtering steps. The resulting solution contained fully hydrated pollen grains (oval shape), non-hydrated and/or destroyed pollen grains (round shape) as well as smaller impurities (Fig. 1A). For a final purification step, we used FACS using the size and autofluorescence properties of pollen. To separate hydrated from non-hydrated pollen, a two-dimensional histogram using the forward scatter versus pulse width parameters was used, and an additional gate was then applied on the 670/40 and 580/20 nm detection channels to further purify the pollen grains based on their characteristic autofluorescence properties (see Fig. 2). The characteristic oval shape of fully hydrated pollen grains caused a longer pulse when passing the laser, thus allowing a separation of the non-hydrated pollen grains and smaller impurities from the fully hydrated pollen grains (Fig. 1, B and C). The purity of the hydrated pollen grain fraction was routinely >99%. The viability of the hydrated pollen grains was assessed by fluorescein diacetate staining (Fig. 1D). The remaining impurities (<1%) consisted of very small debris, which most likely did not include organelles. One hundred-fifty thousand hydrated pollen grains yielded 1 μg of total RNA. More than 500,000 non-hydrated pollen grains resulted in the same yield, supporting our assumption that the majority of the non-hydrated pollen grains were not intact.

Figure 1.

Figure 1.

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Purification steps and viability of Arabidopsis pollen. Pollen and impurities before sorting (A) were separated into non-hydrated (B) and hydrated pollen (C) by flowcytometric sorting. D, A viability stain of the sorted, hydrated pollen grains.

Figure 2.

Figure 2.

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Flowcytometric sorting of Arabidopsis pollen. Arabidopsis pollen was identified through its size (A) and autofluorescence properties (B). A, Hydrated pollen was located in region R1 of the pulse width versus Forward Scatter (FSC) display, whereas non-hydrated pollen was mostly contained in R2. B, For sorting, a logical gate combination of regions R1 and R3 was used.

Pollen Grains Have a Unique Transcriptional Profile

Gene expression patterns of approximately 8,200 genes, representing roughly one-third of the Arabidopsis genome, were obtained for Arabidopsis pollen grains and for several vegetative tissues: seedlings, leaves, root, and siliques. The results were highly reproducible as underlined by the high correlation coefficients of the replicates, which ranged from 0.977 to 0.992. For each vegetative tissue, a similar percentage of genes were called Present by the MAS 5 algorithm, with a mean of 59% in seedlings, 56% in leaves, and 64% in roots and siliques. In contrast, only 21% of the genes represented on the arrays were called Present in the pollen samples (1584 unique genes). Normalization reduces variation of non-biological origin and is therefore a prerequisite for the direct comparison of expression profiles from different arrays. The large differences in the transcriptional profiles in this study, i.e. the sample-specific differences in Presence calls, precluded the use of global scaling/normalization methods. Instead, we employed a sample-wise normalization to the median median probe cell intensity of all arrays, implemented into version 1.3 of the dChip software (http://www.dchip.org; Wong laboratory, Harvard, Boston). This method works independently from the overall intensity of an array. After normalization and model-based computation of expression values, we excluded genes called Absent in all arrays and genes with inconsistent expression levels within the replicate arrays. Thus, for further analysis, our data set contained 6,459 genes.

We developed a new graphical tool to visualize the striking differences between the transcriptional profiles of the vegetative tissues and pollen (Fig. 3). This tool, “Snail View”, compares and displays the changes in expression levels of thousands of genes simultaneously, but still allows meaningful interpretations of the overview obtained (the software can be downloaded at http://eao.igc.gulbenkian.pt/ti/Soft/SnailView/). The average expression value in the seedling samples were chosen as reference, considering that seedlings probably contain cell types found in roots, leaves and, most likely, in siliques. The high correlation of data derived from the replicates is exemplified by the comparison of expression values obtained for the single-seedling replicates. This high correlation is visualized by the small deviations from the line representing the average value of the seedling replicates. The expression values of leaves and of seedlings are strongly correlated, especially for those genes with the highest expression level in seedlings. The cotyledons contribute the largest part to the biomass of 4-d-old seedlings, so this similarity was anticipated. Expression values obtained for siliques and root samples show high deviations from the seedling reference. Genes highly expressed in seedlings are down-regulated (most considerably in roots), and those with low expression levels in seedlings are up-regulated (most considerably in siliques). The most dramatic differences are seen in the pollen to seedling comparison. Most genes with high or medium expression values in seedlings show low expression values in pollen. This trend is reversed for genes with low expression values in seedlings, because a high proportion of these genes are highly expressed in pollen and reach expression values comparable with those of genes with the highest expression in seedlings. The correlation coefficients (0.032, 0.029, 0.040, and 0.067) of the expression values of pollen relative to expression values of seedlings, leaves, siliques, and roots, show that pollen has a transcriptional profile that is clearly distinguished from that of vegetative tissues.

Figure 3.

Figure 3.

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“Snail view” representation of tissue-dependent gene expression patterns. The expression for 5,999 genes is represented in angular coordinates, in which angle encodes gene rank (clockwise from top) and radius encodes the logarithm of gene expression (values <1 were set to 1 for better visualization). Genes were ranked according to increasing mean expression in seedlings. For each tissue, the mean pattern in seedlings (black line) is coplotted with the mean pattern of specific tissue (gray dots), except for seedling where one replicate is coplotted. The values are the correlation coefficient between the gene expression patterns in the two tissues (for seedlings between the replicates).

Ten Percent of the Genes Expressed in Pollen Are Not Expressed in the Sporophyte

We assumed that gene products of transcripts that were highly enriched or even selectively expressed in pollen grains might be of major importance, if not crucial for successful pollen germination and tube growth. To identify such genes, we followed two complementing approaches. First, we measured enrichment in gene expression as the lower limit (lower confidence bound) of a 90% confidence interval for the fold change in gene expression. Gene expression values in pollen were compared with those in each of the vegetative tissues, and a score above 1.2 was used as the criterion to select genes enriched in pollen; 469 genes met this criterion in all four comparisons (supplemental Table I). Second, we used Affymetrix MAS 5 Present calls to sort genes; 5,775 genes were reproducibly called Present in at least one of the four vegetative tissues. Of the 1,584 genes called Present in pollen (for hierarchical clustering, see Fig. 4A), 1,422 (90%) showed an overlap with the genes detected in vegetative tissues (Fig. 4B). The remaining 10% (162 genes) were called Present only in pollen and are referred to as selectively expressed from hereon. The two methods yielded an overlap of 150 genes that we characterized in more detail.

Figure 4.

Figure 4.

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Analysis of genes enriched or selectively expressed in pollen. A, Hierarchical cluster analysis of the 1,584 genes called Present in pollen. Default parameters in dChip were used (standardization and clustering methods follow Golub et al. [1999] and Eisen et al. [1998]). Each column represents a single gene, and each row a single tissue type. Genes mainly enriched in pollen are found in the right cluster, whereas those expressed in pollen but with higher expression values in at least one of the vegetative tissues are found in the left cluster. P, Pollen; Se, seedling; L, leaf; Si; silique; R, root. B, Venn diagram depicting total numbers of genes expressed in vegetative tissues and in pollen. Genes were scored as Present in the vegetative tissues, if they received at least one Present call (MAS 5) in the seedling, leaf, root, or silique sample (5,775 genes). In pollen, 1,422 genes of these were also called Present. A set of 162 genes was exclusively called Present in pollen. Genes recognized by more than one probe set in the Affymetrix array were only counted once. C, Overview of the functional classification of 150 pollen selectively expressed genes listed in Table I. Total number of genes in each category is given in brackets. Functional classification is based on established as well as putative functions.

Thus, our expression analysis of roughly one-third of the annotated genes of Arabidopsis shows that 10% of the genes expressed in pollen are selectively expressed in pollen, whereas the other 90% are also expressed in one or more vegetative tissues. This substantial overlap of genes active in the sporophyte and in the male gametophyte had been predicted earlier based on isoenzyme studies in pollen and vegetative tissues in barley (Hordeum vulgare; Pederson et al., 1987), heterologous hybridizations of pollen cDNA with shoot poly(A) RNA and shoot cDNA to pollen poly(A) RNA in maize (Zea mays; Willing et al., 1988), as well as colony hybridizations of maize pollen cDNA libraries with cDNAs from pollen and vegetative tissues (Stinson et al., 1987).

Functional Classification of Selectively Expressed Genes

We sorted the 150 selectively expressed genes into functional categories (Table I; Fig. 4C), taking into consideration several aspects of current knowledge about the pollen grain and tube physiology. This categorization is based on known functions of the gene products as well as gene ontology annotations derived from homologies. Our analysis confirmed the expression of some genes already known to be expressed in pollen, but it also led to the identification of several genes not known to be expressed in the male gametophyte so far.

Table I.

Functional classification of genes selectively expressed in pollen

One hundred-fifty genes identified as selectively expressed in pollen are classified based on established as well as putative functions. The first column gives The Arabidopsis Information Resource (TAIR) annotation and the second gives the expression value of the gene in pollen (weighted average of triplicates). In the third column, the lower confidence bound fold change (FC; average of the comparisons of pollen to the four vegetative tissues) is given. The fourth and fifth column depict the Affymetrix probe set (Probes set) and, if available, the TAIR locus (AGIID) assigned to this probe set. Within each category, genes are ranked by highest signal.

Annotation Signal Probe Set Arabidopsis Genome Initiative Identification (AGI ID)
Signal transduction (37)
    Putative protein kinase 4,728 76.8 17803_at At1g61860
    Putative receptor-like protein kinase 4,061 78.4 18205_s_at At4g39110
    Putative Pro extensin-like receptor kinase PERK7 4,010 20.3 17812_at At1g49270
    Putative calcium-dependent protein kinase CPK24 3,538 160.8 16806_s_at At2g31500
    Putative 4,5 PIP kinase 3,425 45.9 19788_at At1g01460
    Putative calmodulin 3,326 214.9 13415_at At4g03290
    Putative Leu-rich repeat transmembrane protein kinase AtPRKc 3,051 80.7 19047_at At2g07040
    Putative phosphatidylinositol/phophatidylcholine transfer protein 1,887 15.0 19052_at At2g18180
    Putative calcium-dependent protein kinase CPK14 1,511 250.6 20236_s_at At2g41860
    Putative protein kinase 1,083 16.9 18152_at At2g24370
    Putative Leu-rich repeat transmembrane protein kinase AtPRKd 1,026 17.2 19103_at At5g35390
    Calcium-dependent protein kinase-like protein CPK26 854 26.4 20241_at At4g38230
    Putative Ser/Thr protein kinase 725 21.7 14364_at At1g04700
    Putative protein kinase (cdc2 kinase homolog cdc2MaC, M. sativa) 670 48.9 16824_at At4g10010
    Putative GTP-binding protein 652 11.6 18107_at At2g33870
    Putative inositol polyphosphate 5′-phosphatase 636 8.8 19795_at At2g43900
    Putative proline extensin-like receptor kinase PERK5 611 29.0 16876_at At4g34440
    Putative protein (GTPase-activating protein, Yarrowia lipolytica) 574 4.9 16758_at At4g27100
    Putative inositol polyphosphate 5′-phosphatase 563 17.1 19582_at At2g31830
    Putative protein (various predicted protein kinases, Arabidopsis) 532 11.0 18193_at At5g26150
    AK20 gene protein kinase catalytic domain (fragment) 519 6.1 19111_at
    Putative receptor-like Ser/Thr kinase RKF2 483 10.4 17496_at At1g19090
    Putative Pro extensin-like receptor kinase PERK12 335 14.7 17359_at At1g23540
    Hypothetical protein (similar to wall-associated kinase 1, Arabidopsis) 324 10.8 18186_at At1g78940
    Putative Ptα kinase interactor (similar to Ptα kinase interactor 1, tomato) 300 3.8 18825_at At4g13190
    Putative GTP-binding protein 292 16.3 12489_at At2g22290
    Protein phosphatase 2C (PP2C) 275 4.8 19345_at At2g40180
    Putative calcium-dependent protein kinase CPK20 241 8.8 12311_at At2g38910
    Putative calmodulin (calmodulin PIR1:MCKM, Chlamydomonas reinhardii) 229 4.7 19274_i_at At4g12860
    Protein kinase-like protein 214 5.7 19556_at At4g31220
    Putative protein (various predicted protein kinases, Arabidopsis) 159 2.2 19071_at At5g35380
    Putative Ser/Thr protein kinase 110 4.0 16866_at At1g79250
    Putative STE20/PAK-like protein kinase (similar to yeast STE20) 74 4.7 18999_at At1g70430
    Putative receptor-like kinase 65 3.4 14275_at At4g31230
    Putative protein kinase 62 1.8 16848_at At2g20470
    Putative phosphatidylinositol-4-phosphate 5-kinase 55 4.3 19310_at At2g41210
    Calcium-dependent Ser/Thr protein kinase CPK18 44 1.8 16849_at At4g36070
Cell wall biosynthesis and regulation (16)
    Pectinesterase family (nearly identical to pollen-specific BP10 protein, canola) 7,180 110.3 17753_at At1g55570
    Exopolygalacturonase 7,059 51.9 18923_g_at At3g07850
    Extensin-like protein 6,221 71.5 15765_at At4g33970
    Putative polygalacturonase 5,660 91.2 15716_at At2g23900
    Putative cellulose synthase (AtCalD1—homolog of NaCalD1) 4,911 58.9 20243_at At2g33100
    Glycosyl hydrolase family 10 (xylan endohydrolase isoenzyme X-I, barley) 4,536 40.1 13393_at At4g33860
    Polysaccharide lyase family 1 (similar to pectate lyase P59, tomato) 4,226 184.1 16837_at At2g02720
    Polysaccharide lyase family 1 (At59) 3,914 83.0 16857_at At1g14420
    Putative cellulose synthase (AtCalD4—homolog of NaCalD1) 3,489 28.5 16782_at At4g38190
    Putative protein (bp4A protein, Brassica napus) 1,196 51.4 15360_at At4g11760
    COBRA-like protein COBL-11 929 13.1 15331_at At4g27110
    Cellose synthase (1,3-β-glucan synthase) family (AtGal2, homolog of NaGal1) 925 2.7 18515_at At2g13675
    Pectinesterase family 662 19.2 18519_at At4g33230
    Glycosyl hydrolase family 17 (BETAG5, β-1,3-glucanase bg5) 438 5.9 18539_at At5g20340
    Putative xyloglucan endotransglycosylase 214 5.8 19530_at At4g18990
    Glycosyl hydrolase family 17 (BETAG4, β-1,3-glucanase bg4) 214 2.3 18120_i_at At5g20330
Stress response (11)
    Putative plant defensin protein PDF2.6 9,739 53.2 19928_at At2g02140
    Putative peroxiredoxin (similar to type 2 peroxiredoxin, Brassica rapa) 3,640 7.3 15966_i_at At1g60740
    17.6-kD heat shock protein (AA 1-156) 1,637 26.5 13276_at At1g53540
    Heat shock protein 17.6A (At-HSP17.6A) 731 17.2 13278_f_at At5g12030
    Heat shock protein 22.0 (At-HSP22.0) 431 8.6 17076_s_at At4g10250
    Putative protein (similar to Mlo proteins, barley) 402 4.8 14639_at At2g33670
    Heat shock protein 18 299 3.1 13280_at At5g59720
    Putative stress protein 130 6.1 13378_at At2g01330
    Putative peroxidase 108 4.4 17828_at At4g17690
    Mitochondrion-localized small heat shock protein (AtHSP23.6-mito) 69 2.0 13282_s_at At4g25200
    Putative disease resistance protein (TIR-NBS-LRR class) 61 2.8 12258_at At4g14370
Metabolism (9)
    Glycosyl hydrolase family 35 (β-galactosidase) BGAL11 6,152 194.9 16874_at At4g35010
    Glycosyl hydrolase family 32 (invertase) 3,759 54.4 16369_s_at At3g52600
    Glycosyl hydrolase family 35 (β-galactosidase) BGAL13 3,074 69.7 17316_at At2g16730
    FAD-linked oxidoreductase family 2,481 126.4 15302_at At1g11770
    Putative thioredoxin 2,236 21.2 19937_at At2g33270
    Putative acyl-CoA:1-acylglycerol-3-phosphate acyltransferase 656 10.4 18423_at At1g51260
    3-hydroxy-3-methylglutaryl CoA reductase HMG1 isoform HMGR1L 550 3.0 20160_at
    Glycosyl hydrolase family 1 101 18.6 19601_at At2g25630
    Putative CDP-diacylglycerol synthetase 53 1.7 19016_at At4g26770
Ion transport (8)
    Putative cation/H+ antiporter AtCHX8 3,259 17.1 17246_at At2g28180
    Putative protein (anion exchange protein 2, Homo sapiens) 3,208 67.1 18837_at At4g32510
    Shaker potassium channel SPIK 1,489 51.8 16783_at At2g25600
    Putative cation/H+ antiporter AtCHX15 618 4.3 14334_at At2g13620
    Ion transporter-like protein AtNRAMP5 404 30.2 18118_at At4g18790
    Putative cyclic nucleotide-regulated ion channel AtCNGC16 205 8.3 14291_at At3g48010
    G subunit of vacuolar-type H+-ATPase (vag1) 78 5.3 16226_at At4g25950
    Putative cation/H+ antiporter AtCHX13 60 2.4 15704_at At2g30240
Cytoskeleton (8)
    Actin 4 3,262 110.7 14356_at At5g59370
    Actin depolymerizing factor-like protein 3,092 37.4 12529_at At4g25590
    Actin 12 1,815 35.8 15697_at At3g46520
    Prolilin 4 2,691 25.8 12411_s_at At2g19770
    Myosin heavy chain-like protein AtVIIIB 341 19.7 12257_at At4g27370
    Putative myosin heavy chain AtXID 224 2.7 16327_at At2g33240
    Putative myosin II heavy chain (Naegleria fowleri) 179 6.2 15329_at At4g33390
    Kinesin-related protein 97 3.3 16794_at At1g09170
Transcriptional regulation (5)
    Putative protein (NorM, Vibrio parahaemolyticus) 1,471 33.5 14992_at At4g21900
    MYB97 (similar to anther-specific myb-related protein 2, tobacco) 309 10.4 17979_at At4g26930
    MADS-box protein AGL30 (homolog of MADS1;11, tobacco) 167 5.2 18443_at At2g03060
    MADS-box protein AGL29 154 15.8 19309_at At2g34440
    CONSTANS B-box zinc finger family protein 23 1.9 15698_at At4g15250
Proteolysis (5)
    Putative Ser carboxypeptidase II 7,644 55.0 17765_at At2g05850
    Ser-type carboxypeptidase-like protein 4,000 117.4 18137_at At3g52010
    Subtilisin-like Ser protease 1,348 26.8 16349_at At4g21326
    Ser-type carboxypeptidase-like protein 721 12.1 17814_at At3g52000
    Ser-type carboxypeptidase-like protein 64 6.4 18461_at At3g52020
Vesicle trafficking (5)
    Syntaxin AtSYP124 1,243 10.7 15275_at At1g61290
    ARF GTPase-activating domain-containing protein 829 15.2 12639_at At2g35210
    Clathrin protein family 631 8.9 13499_at At1g05020
    ARF GTPase-activating domain-containing protein 598 4.4 17446_at At2g14490
    Unknown protein (similar to yeast Sec7p protein) 280 7.9 18381_at At2g30690
Sugar and protein transport (3)
    Monosaccharide transporter 4,441 129.6 19270_at At5g23270
    Putative ABC transporter AtNAP12 437 4.6 13049_at At2g37010
    Putative vacuolar sorting receptor 182 2.8 18509_at At2g30290
Transposition (3)
    Ac-like transposase (related to Ac/Ds transposon family of maize) 198 4.8 16730_at At2g16040
    Putative Mutator-like transposase 118 2.1 12987_s_at At2g30640
    Putative TNP2-like transposon protein 60 2.6 19339_i_at At2g10140
Water channel (2)
    Aquaporin-like protein TIP5;1 (aquaporin, Vernicia fordii) 1,497 13.6 18856_at At3g47440
    Putative water channel protein—major intrinsic protein (MIP) TIP1;3 544 6.1 17798_at At4g01470
Translational regulation (2)
    Putative protein (similar to polyadenylate-binding protein PABP) 429 10.9 16836_at At3g16380
    Putative translation initiation factor 65 5.2 18609_at At2g39820
Cell cycle regulation (1)
    Cyclin A2;1 89 1.8 16851_s_at At5g25380
Unknown and hypothetical (35)
    Arabinogalactan-protein AGP6 16,351 50.8 12003_at At5g14380
    Nectarin-like protein (nectarin I precursor, Nicotiana plumbaginifolia) 11,576 81.8 19014_at At5g26696
    Hypothetical protein 9,322 89.6 16201_at At1g49490
    Unknown protein (homolog of protein ARPL 1;1) 9,259 181.8 19385_at At1g54070
    Putative LIM-domain protein (AtPLIM2) 8,733 82.0 20478_at At2g45800
    Unknown protein 8,161 92.0 19301_at At4g36490
    Unknown protein 7,359 35.4 12568_at At2g33420
    Putative pistil-specific protein (similar to potato pistil-specific protein STS15) 7,018 149.3 14936_at At4g02250
    Hypothetical protein 4,933 47.1 13912_at At1g04540
    Unknown protein 3,744 101.5 12638_at At1g04470
    Hypothetical protein 3,186 61.3 18426_at At2g13350
    Unknown protein 1,225 42.7 17230_at At2g40990
    Unknown protein 962 96.2 20549_i_at At2g26850
    Hypothetical protein 818 33.4 17687_at At1g60240
    Putative GDP-dissociation inhibitor 753 28.9 19790_at At1g12070
    Hypothetical protein 466 5.4 15925_at At1g43630
    Unknown protein 420 13.4 13836_at At1g06750
    Unknown protein 379 27.4 15838_at At2g29790
    Hypothetical protein 377 2.7 13304_at At1g24330
    Putative protein (MSP1, Saccharomyces cerevisiae) 348 21.5 19604_at At4g28000
    Unknown protein (related to MO25, early mouse development protein family) 329 28.7 17711_at At2g03410
    RALF-LIKE 10 313 7.0 18385_at At2g19020
    Hypothetical protein 258 6.9 13814_at At2g29040
    Unknown protein 254 5.8 15277_at At2g41440
    Glu-rich protein 238 22.1 16656_at At4g20160
    Putative protein 227 18.3 15322_at At4g24580
    Unknown protein 221 7.6 19849_at At2g18080
    Unknown protein 219 4.3 14496_at At2g45610
    Hypothetical protein 155 4.7 14379_at At2g33320
    Unknown protein 128 2.6 18454_g_at At4g08605
    Unknown protein 75 1.9 15016_at At1g04090
    Putative protein 51 2.1 12076_at At4g36600
    Hypothetical protein 49 2.3 15266_at At4g08630
    Hypothetical protein 45 3.5 15262_at At2g33190
    Putative protein, (BRCA1-associated RING domain protein, H. sapiens) 32 1.4 17790_g_at At4g21070
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Cytoskeleton

A fundamental aspect of tip growth of pollen tubes is the continuous deposition of new cell wall and plasma membrane at the tube apex. Vesicles delivering this material are transported by the actin cytoskeleton. In addition to components of the cytoskeleton in pollen tubes, which have been described as such, i.e. the actin genes ACT4/12 (Huang et al., 1996) and profilin PRF4 (Kandasamy et al., 2002), we have identified a previously uncategorized actin-depolymerizing factor-like protein (At4g25590). Furthermore, three potential motor proteins are selectively expressed. The myosins AtVIIID and AtXID might function in cargo movement along actin filaments (Reddy and Day, 2001), whereas the kinesin-related protein At1g09170 might be involved in movement along microtubules. The already described members of the myosin family, MYA1 to -3 (Kinkema et al., 1994), are not pollen selectively expressed but are pollen-enriched (supplemental Table I). We confirmed that profilin PRF3 (At4g29340) is highly expressed in pollen grains but is otherwise only called Present in roots, which is in disagreement with its reported constitutive, strong expression in all vegetative tissues (Kandasamy et al., 2002).

Vesicle Trafficking

Exo- and endocytosis are required to release the contents of the transported vesicles and to reincorporate excess membrane material. The syntaxin AtSYP124 (At1g61290) and a homolog of the yeast Sec7p protein (At2g30690) fall into the large group of genes presumably involved in vesicle trafficking. Other potential SNAREs that might be required for vesicle fusion in pollen are encoded by the pollenenriched genes AtBET12 (At4g14450) and AtVAMP725 (At2g32670). The clathrin family protein At1g05020 is selectively expressed, and a clathrin assembly protein (At1g03050), which is highly enriched in pollen, might have endocytic functions. Furthermore, two putative ARF GTPases (At2g35210 and At2g14490) are selectively expressed.

Cell Wall Biosynthesis and Regulation

The pollen tube wall is a bipartite structure with an inner sheath of 1,3-β-glucan (callose) covered by an outer fibrillar layer of 1,4-β-glucan (cellulose) and α-linked pectic polysaccharides. Recently, NaCslD1 and NaGsl1 were identified as pollen-expressed genes, potentially encoding the major β-glucan polysaccharide synthases in Nicotiana alata pollen tubes (Doblin et al., 2001). Here, we show that AtCslD4 (putative cellulose synthase; At4g38190) and AtGsl2 (callose synthase; At2g13675) are selectively expressed homologs in Arabidopsis pollen, as assumed in that study. However, AtCslD1 (At2g33100) is also selectively expressed in pollen, and it shows a higher expression than AtCslD4.

Arabidopsis pollen grains express a whole range of cell wall hydrolytic and cell wall-loosening enzymes such as polygalacturonases, pectate lyases, pectin esterases, glycosyl hydrolases, and expansins. The genes encoding these proteins are among those with the highest expression levels in pollen grains in our study, e.g. the putative polygalacturonase At3g07820. Besides their putative roles in modifications of the pollen tube wall, they may be important for the penetration of the stigmatic tissue.

Glycosylphospatidylinositol (GPI)-anchored proteins are targeted to the cell surface and presumably are involved in remodeling of the extracellular matrix and/or in signaling (Borner et al., 2002). The putative GPI-anchored COBL11 (At4g27110), a member of the recently described COBRA family (Roudier et al., 2002), is selectively expressed in pollen in our study. COBRA is implicated in the regulation of oriented cell expansion in the root (Schindelman et al., 2001), and we propose that COBL11 might fulfill a similar function in pollen tubes. Two other putatively GPI-anchored proteins identified in this study are the arabinogalactan proteins AGP6 (At5g14380) and AGP23 (At3g57690), with the highest expression values of the selectively expressed and the pollen-enriched genes, respectively. Although the exact function of AGPs in pollen tubes and styles remains to be determined, it is interesting that AGP23 encodes an AG-peptide (Schultz et al., 2002) of only 61 amino acids length. If cleaved and thus released from its lipid anchor, it might serve as a diffusible signal molecule.

Ion Dynamics

Besides calcium fluxes, several studies indicate the involvement of polarized internal gradients and/or external fluxes of protons, potassium, and chloride in pollen tube growth (for review, see Hepler et al., 2001). However, channels and transporters accounting for the observed ion fluxes across the plasma membrane in pollen tubes remain largely unknown. Thus, the identification of ion transporters that are selectively expressed or enriched in pollen could open new avenues (Feijó et al., 2001). The important role of the selectively expressed, inwardly rectifying K+ channel SPIK (At2g25600) for pollen tube development and competitive ability has been shown by Mouline et al. (2002) and the pollen enriched, outwardly rectifying K+ channel SKOR (At3g02850) has been characterized extensively in root stelar tissues (Gaymard et al., 1998; Lacombe et al., 2000). Interestingly, SKOR is permeable to both monovalent cations and Ca2+ and decreasing cytoplasmic pH reduces SKOR-mediated currents. The selectively expressed, putative cyclic-nucleotide-gated ion channel AtCNGC16 (At3g48010) could be involved in the control of [Ca2+]cyt, too, because it has been shown that an increase in cytoplasmic cAMP or cGMP elicits both a Ca2+ influx and a rise in [Ca2+]cyt in plant cells (Kurosaki et al., 1994; Volotovski et al., 1998). Furthermore, an overlap of the cyclic nucleotide-binding domain and a calmodulin-binding site in AtCNGC1 and -2 (Kohler and Neuhaus, 2000) suggests a regulation of the channels by cyclic nucleotides and calmodulin. Because it has been demonstrated that cAMP can modulate pollen tube growth and reorientation (Moutinho et al., 2001), AtCNGC16 could constitute a possible link between increases in cAMP concentrations and increases in [Ca2+]cyt in pollen tubes.

The putative cation/H+ antiporters AtCHX8, -13, and -16 (At2g28180, At2g13620, and At2g30240) are possible regulators of proton fluxes observed during pollen tube growth (Feijó et al., 1999), although their specificity and membrane localization are still to be determined (Maser et al., 2001). In addition, the identification of a pollen selectively expressed G subunit of the vacuolar type H+-ATPase underlines the possible importance of V-ATPases for proton homeostasis in growing pollen tubes. Recently, Cl– fluxes have been shown to play a role in growth and cell volume regulation in pollen tubes (Zonia et al., 2002). The chloride channel CLC-c (At5g49890) is pollen enriched, and its function in cation homeostasis has been demonstrated by suppression of the Gef1 mutant phenotype in yeast (Gaxiola et al., 1998). Thus, CLC-c might fulfill a similar function in pollen. Furthermore, we identified two tonoplast intrinsic proteins (TIPs), TIP5;1 (At3g47440) and TIP1;3 (At4g01470), as selectively expressed in pollen.

Signal Transduction

Inhibitor studies with pollen of several plant species (for review, see Mascarenhas, 1975) indicate that pollen germination and early tube growth are largely independent of transcription, but strictly dependent on translation. Thus, signal transduction and translational control might play a more important role than transcriptional control. In fact, 25% of the selectively expressed genes fall under the signal transduction category. Most of these genes (26 of 37) encode putative protein kinases. In addition to the Arabidopsis receptor-like kinase RKF1 (At1g29750; Takahashi et al., 1998), the Leu-rich repeat receptor-like kinases LePRK1-3 and ZmPRK1 were characterized as examples of pollen-specific receptor kinases possibly involved in these signaling events (Muschietti et al., 1998; Kim et al., 2002). Here, we show that the Arabidopsis homologs AtPRKc (At2g07040) and AtPRKd (At5g35390) are not only expressed in pollen, but that their expression is restricted to pollen. For RKF2 (At1g19090), which was reportedly expressed at low levels in several organs (Takahashi et al., 1998), we found that it was selectively expressed in pollen. Moreover, we identify the putative Pro-rich, extensin-like receptor kinases AtPERK5, -7, -12, and -4 (At4g34440, At1g49270, At1g23540, and At2g18470) as selectively expressed and pollen enriched, respectively. PERK1 is a canola (Brassica napus) homolog of this novel family of plant receptor-like kinases and is rapidly induced by wounding (Silva and Goring, 2002). Sequence similarities of its extracellular domain to extensins might indicate an involvement of the Arabidopsis pollen homologs in a signal transduction pathway, signaling changes in the mechanical properties of the pollen tube cell wall to target proteins in the pollen cytoplasm.

Potential ligands to pollen receptor kinases might be expressed by the pistil or by the pollen itself as shown for LePRK2 and its pollen-expressed ligand LAT52 (Tang et al., 2002). The potential signaling peptide RALF-LIKE 10 (At2g19020; Olsen et al., 2002) is selectively expressed in pollen. Its tobacco (Nicotiana tabacum) homolog rapid alkalinization factor (RALF) encodes a ubiquitous 115-amino acid prepro-protein, which is processed into a 5-kD signaling peptide (Pearce et al., 2001). RALF causes a rapid alkalinization of tobacco cell cultures and an arrest of root growth and development when supplied to germinating tomato (Lycopersicon esculentum) and Arabidopsis seeds. RALF-LIKE 10, encoding a 73-amino acid protein with a potential N-terminal signal peptide for export, might be a putative ligand, e.g. for Leu-rich receptor-like kinases in the plasma membrane of pollen or the pistil. The importance of intracellular signaling for pollen tube growth has been demonstrated by studies covering several types of molecular switches, e.g. Rop/Rac GTPases (Gu et al., 2003) and the MAP kinase kinase kinase AtMAP3Kγ (Jouannic et al., 1999). We confirm the pollen-enriched expression of the Rho-related GTPases At-Rac1 (At2g17800) and AtRac6 (At4g35950) and of AtMAP3Kγ (At5g66850), as well as the pollen-enriched expression of the G-protein AtRAB2 (At4g17170; Moore et al., 1997). The Rho-related GTPase Rop1At (At3g51300), reported to be specifically expressed in anthers (Li et al., 1998), is highly enriched in pollen but was called Present in roots and siliques. Moreover, our study enlarges this list significantly, including several putative protein kinases, a putative STE20/PAK-like protein kinase (MAP4K-At1g70430), and two putative G-proteins (At2g33870 and At2g22290). Considering the established link between elevation of cytosolic Ca2+ at the pollen tube tip and its growth (for review, see Franklin-Tong, 1999) our identification of two selectively expressed putative calmodulins (At4g03290 and At4g12860) and the five calcium-dependent protein kinases CPK14, -18, -20, -24, and -26 (At2g41860, At4g36070, At2g38910, At2g31500, and At2g31500) points out six new potential players in Ca2+-mediated signaling in Arabidopsis pollen. In addition, several genes presumably involved in phosphoinositide signaling are selectively expressed in pollen (At2g18180, At2g43900, At2g31830, and At2g41210).

Translational and Transcriptional Regulation

Although only two genes encoding potential regulators of translation were selectively expressed (At3g16380 and At2g39820), there are several more potential regulators of translation showing an enriched expression in pollen (supplemental Table I). Protein turnover in pollen might also contribute to regulation, and therefore the five genes involved in proteolysis are notable. Three percent of the genes that are selectively expressed encode proteins potentially involved in transcriptional regulation. Among these are transcription factor MYB97 (At4g26930) and the MADS-box proteins AGL29 and AGL30 (At2g34440 and At2g03060). AGL30 is a homolog of MADS1;11 of tobacco, which is thought to be a regulator of gene expression during early pollen tube growth (Steiner et al., 2003). Several more potential transcription factors are pollen enriched, including the response regulator ARR2 (At4g16110), as described previously (Lohrmann et al., 2001). The expression of several genes encoding transcription factors in hydrated pollen grains is surprising, because it might indicate that more de novo synthesis of RNA takes place in germinating pollen and during tube growth than had been anticipated by early inhibitor studies (for review, see Mascarenhas, 1975).

Cell Cycle

The cell cycle in the vegetative cell of pollen is believed to be arrested. However the sperm cells continue through the S phase of the cell cycle after pollination and are deposited into the embryo sac with a 2C content of DNA in G2 (Friedman, 1999). According to our results, cyclin A2;1 (At5g25380) is selectively expressed, and cyclin B3;1 (At1g16330) is enriched in pollen with an additional Present call only in roots. Both genes were described recently as showing a peak of expression in M phase of the cell cycle in synchronized cell suspensions of Arabidopsis (Menges et al., 2002). Interestingly, the mitotic cyclin gene CycA1;1 of maize was found to be expressed in isolated sperm and zygotes, although sperm of maize remain in G1 until fertilization (Sauter et al., 1998). We cannot exclude that the cyclin transcripts we have detected are derived from expression in the sperm cells, but whatever their origin, their expression in pollen might be important for fertilization or post-fertilization events rather than for pollen tube growth.

Stress Response

Stress response-related genes, such as the small heat shock protein gene At-HSP17.6A (At5g12030), represent 7% of the pollen selectively expressed genes. The expression of At-HSP17.6A was shown to be induced by heat and osmotic stress (Sun et al., 2001). Thus, the expression of this gene could be explained by osmotic stress during dessication and rehydration of the pollen grain, or the flowcytometric sorting might have caused the induction of stress response-related genes.

Hypothetical proteins and proteins with unknown function account for 23% of the 150 selectively expressed genes. The high expression levels for some of them indicate their potential importance for the male gametophyte. Furthermore, our data confirm the expression of 10 genes that had hypothetical status so far (i.e. no representation in EST databases existing), which makes them interesting candidates for a functional characterization of their encoded gene products.

One-Third of Constitutively Expressed Genes in Vegetative Tissues Are Not Expressed in Pollen

Our main goal was to identify enriched or selectively expressed genes in pollen to gain insight into the genetic basis of pollen germination and tube growth, but identifying genes that are specifically down-regulated in pollen is also informative. For this purpose, we used a list of constitutively expressed genes from samples from 5-week-old Arabidopsis (leaves, roots, inflorescence stems, and flowers; Zhu et al., 2001). Comparing the analysis of Zhu et al. (2001) with our data yielded 283 genes that showed constitutive expression in vegetative tissues in our study as well. We used the lower confidence bound 1.2 criterion and MAS 5 calls to identify those of the 283 genes that were down-regulated or called Absent in pollen with high confidence, resulting in a list of 104 genes, which were functionally characterized (Table II; supplemental Table II). Thus, 37% of 283 genes constitutively expressed in vegetative tissues are not expressed in pollen grains. The largest set (27%) of transcripts is functionally related to protein biosynthesis; all of the 29 genes in this category encode putative or known ribosomal proteins. Labeling experiments of lily and Tradescantia sp. pollen tubes indicated transcriptional inactivation of rRNA genes in immature pollen grains and during pollen tube growth (for review, see Mascarenhas, 1975), and our data is in accordance with these early observations. This supports the view that in many species, the majority of rRNAs and ribosomal proteins as well as tRNAs, mRNAs, and other proteins are already stored in the mature pollen grain to ensure rapid germination and initial tube growth on the stigma (Mascarenhas, 1989). Thus, in most cases, the transcript abundance that we have measured in Arabidopsis pollen grains most likely reflects the accumulation and storage of transcripts during earlier stages of pollen development.

Table II.

Functional classification of constitutively expressed genes not expressed in pollen

Of the 283 genes identified as constitutively expressed in the vegetative tissues, 104 are called Absent in pollen. These 104 genes are classified based on established as well as putative functions. For each functional category, the number of genes is given in brackets, and each gene is given by its TAIR locus identifier (if no locus is associated, only the Affymetrix probe set is given. Genes in each category were ranked by highest negative fold change (see Supplemental Table II for more details).

Protein biosynthesis (29)
    At2g34480, At1g43170, At4g11050, At2g09990, At4g27090, At1g34030, At1g67430, At1g09690, At1g70600, At2g39460, At4g34670, At2g19730, At4g16720, At3g49010, At2g25210, At3g52580, At1g27400, At4g14320, At2g18020, At3g47370, At4g00100, At2g36160, At1g72370, At2g19750, At4g26230, At2g33370, At2g40510, At2g31610, At4g18730
Metabolism (28)
    At2g25450, At1g04410, At3g61440, At2g26670, At4g21960, At5g35630, At2g28000, At5g43940, At2g30860, At2g31570, At3g58610, At4g23860, At5g04590, At2g30490, At1g70310, (12881_s_at), At1g53240, At1g50480, At2g13360, At4g09320, At5g39950, At2g30870, At2g43090, At5g60920, (20709_s_at), At1g65960, At1g75330, At2g36580
Water channel (8)
    At2g37170, At3g53420, At3g26520, At3g61430, At4g35100, At2g45960, At4g23400, At2g36830
Signal transduction (6)
    At5g10450, At2g42590, At5g55190, At5g42080, At1g02130, At5g38480
Stress response (5)
    At1g47128, At4g24190, At1g20620, At1g20440, At2g02130
Transcriptional regulation (5)
    At3g16770, At2g32080, At4g40060, At2g33340, (18032_i_at)
Translational regulation (4)
    At3g04730, At1g30230, At1g09640, At2g22670
Others
    At2g27020, At1g13060, At4g26840 (proteolysis); At5g49720, At4g19410 (cell wall biosynthesis and regulation); At3g06720, At2g14720 (protein transport); At4g30270 (senescence); At5g12250 (cytoskeleton); At1g04400 (phototropism); At5g40890 (ion transport)
Unknown (8)
    At2g41430, At3g15353, (12847_at), At1g13930, At4g36040, At2g35810, At4g16520, At1g14910
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The next largest set (26%) of transcripts encodes proteins involved in diverse functions related to metabolism. Surprisingly, the third largest group (8%) consists of genes encoding membrane intrinsic proteins. Besides two TIPs (TIP1;1 and TIP1;2), six of these encode plasma membrane intrinsic proteins (PIPs; PIP1;1, PIP1;2, PIP1;5, PIP2;1, PIP2;2, and PIP2; 7). This observation prompted us to study the expression levels and calls of the remaining seven annotated Arabidopsis PIPs (Johanson et al., 2001). Probe sets for five of these genes (PIP1;3, PIP1;4, PIP2;5, PIP2;6, and PIP2;8) were found on the version of the Arabidopsis array used in this study. Taking all our available data about PIPs together, we identified PIP1;3 (At1g01620) as the only PIP expressed in pollen. It has been assumed that water fluxes might be linked to the flux of Cl– (Zonia et al., 2001). Plant major intrinsic proteins have been reported to be enriched in zones of fast cell division and expansion or in areas where water flow or solute flux density would be expected to be high (for review, see Johanson et al., 2001; Tyerman et al., 2002). Our data indicate the sole expression of PIP1;3 (old name TMP-B) in pollen, although 13 PIPs are annotated in the Arabidopsis genome. Interestingly, it has been shown that PIP1;3 expression is turgor responsive (Shagan et al., 1993). The selectively expressed TIPs, TIP1;3 and TIP5;1, might be involved in cytosolic osmoregulation, too. The subcellular localizations indicated by the TIP and PIP labels should be taken as putative (Barkla et al., 1999) because they are solely based on sequence data in most cases.

CONCLUSIONS

We have identified transcripts of 1,584 genes in Arabidopsis pollen, of which 30% are pollen enriched and 10% pollen selectively expressed. Thus, our study significantly increases the current knowledge of genes expressed in the male gametophyte of Arabidopsis. The specific down-regulation of otherwise constitutively expressed genes emphasizes that a particular genetic program underlies the unique growth of pollen tubes. T-DNA insertion lines are available for the majority of the 150 genes selectively expressed in pollen, and their characterization might support the ongoing efforts to combine genetic and physiological evidence into a model for pollen germination and pollen tube growth. We show here that pollen possesses a significantly lower amount of expressed genes compared with other vegetative tissue and yet retains remarkable self-organized regulatory mechanisms of growth. This makes pollen an excellent model for the study of cell growth and morphogenesis on apical growing cells because it seems to be using a “minimal” set of genes encoding a mechanism with obvious evolutionary success.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis ecotype Col-0 was used in this study. To minimize interplant variability, tissues from a minimum of 12 plants were pooled for each RNA extraction. For seedling and root, seeds were surface-sterilized and then spread on petri dishes containing B-5 medium (Duchefa, Haarlem, The Netherlands) solidified with 0.8% (w/v) phytagar (Duchefa). The seeds were cold-treated for 3 d at 4°C to ensure uniform germination. The plates were transferred to short-day conditions (8 h of light at 21°C–23°C) and grown in a horizontal (for harvest of seedlings) or vertical position (for harvest of roots). For the seedling samples more than 25 seedlings from five petri dishes were collected after 4 d of growth. For the root samples more than 25 roots from 10 petri dishes were harvested after 13 d.

Plants for the leaf, silique, and pollen samples were grown on soil for 12 weeks in short-day conditions (8 h of light at 21°C–23°C) and then changed to long-day conditions (16 h of light) to induce flowering. After bolting, more than 12 rosette leaves from different plants per leaf sample were collected. Young siliques were harvested 2 weeks later. Old parts of the flower, especially stamens, were removed from the siliques to ensure pollen-free silique samples, and more than 25 siliques from different plants were pooled per sample.

Isolation and FACS Sorting of Pollen Grains

A detailed protocol of the purification of hydrated pollen grains can be found in the supplemental “Materials and Methods”. In brief, flower heads were cut and placed in a humid chamber for 2 h. Then the flower heads were agitated three times in 500 mL of pollen-sorting buffer (10 mm CaCl2, 1 mm KCl, 2 mm MES, and 5% [w/v] Suc, pH 6.5 with NaOH, in double-distilled water). After consecutive filtration and centrifugation steps, the resulting pellet, highly enriched in pollen, was re-suspended in 10 mL of pollen-sorting buffer. Hydrated pollen grains were separated from non-hydrated and/or destroyed pollen grains and other impurities in a final purification step using FACS based on size and autofluorescence criteria of pollen. Pollen viability was assessed by enzymatically induced fluorescence using fluorescein diacetate according to Heslop-Harrison and Heslop-Harrison (1970).

RNA Isolation, Target Synthesis, and Hybridization to Affymetrix GeneChips

Total RNA was extracted from the tissue and cell samples, respectively, using the RNeasy Mini Plant Kit (Qiagen, Hilden, Germany). RNA quality was assessed by agarose gel electrophoresis and spectrophotometry. RNA was processed for use on Affymetrix Arabidopsis Genome GeneChip arrays, according to the manufacturer's protocol. In brief, 7 μg of total RNA was used in a reverse transcription reaction (SuperScript II, Invitrogen, Paisley, UK) to generate first-strand cDNA. After second-strand synthesis, double-strand cDNA was used in an in vitro transcription reaction to generate biotinylated cRNA. After purification and fragmentation, 15 μg of cRNA was used in a 300-μL hybridization containing added hybridization controls. Two hundred microliters of mixture was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols were used on an Affymetrix GeneChip Fluidics Station 400. Arrays were scanned on an Affymetrix GeneChip Scanner 2500.

Data Analysis

Scanned arrays were analyzed first with Affymetrix MAS 5.0 software to obtain Absent/Present calls and to assure that all quality parameters were in the recommended range. For subsequent analysis, dChip 1.3 (http://www.dchip.org; Wong laboratory, Harvard) was used. The following conditions were applied to ensure reliability of the analyses (for details, see supplemental “Materials and Methods”): First, each GeneChip experiment was performed with biological replicates and triplicates in the case of pollen, respectively. Second, we used a sample-wise normalization to the median median probe cell (CEL) intensity of all arrays. Third, normalized CEL intensities of the 11 arrays were used to obtain model-based gene expression indices based on a Perfect Match-only model (Li and Hung Wong, 2001). Finally, all genes compared were considered to be differentially expressed if they were called Present in at least one of the arrays and if the 90% lower confidence bound of the -fold change between experiment and baseline was above 1.2.

To achieve a higher stringency for the identification of constitutively expressed genes in the vegetative tissues, we combined our data with a set identified by Zhu et al. (2001). Because a proprietary pre-version of the Arabidopsis Genome GeneChip array was used by these authors, 81 of the 346 probe sets they identified as constitutively expressed were not included in our study, resulting in an overlap of 283 probe sets.

Gene Annotation

For gene annotation, we used the updated TAIR (The Arabidopsis Information Resource) annotation (October 2002 release) for the Arabidopsis Genome GeneChip array (http://www.arabidopsis.org). Genes were classified into functional categories using the Gene Ontology information available from TAIR as of October 2002. Genes represented by two or more probe sets on the array were analyzed manually, and only the most significant probe set for this gene was included in the final tables.

Supplementary Material

Supplemental Data
plntphys_133_2_713__index.html (1KB, html)

Acknowledgments

We thank Philip Benfey (New York University [now at Duke University, Durham, NC]) for initial support and critical reading of the manuscript, Pedro Coutinho (Instituto Gulbenkian de Ciência) for his help to update the gene annotation, and Sheila McCormick (Plant Gene Expression Center, Albany, CA) for critical reading of the manuscript.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.028241.

1

This work was supported by the Fundação para a Ciência e a Tecnologia (FCT; project nos. POCTI/BCI/41725/2001, POCTI/BIA/34772/1999, and POCTI/BCI/46453/2002). J.D.B. and L.C.B. were supported by FCT fellowships SFRH/BPD/3619/2000 and SFRH/BD/1128/2000.

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