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Plant Physiology 132:517-529 (2003) © 2003 American Society of Plant Biologists Use of Serial Analysis of Gene Expression Technology to Reveal Changes in Gene Expression in Arabidopsis Pollen Undergoing Cold Stress1,[w]Department of Life Sciences, Ewha Woman's University, Seoul 120–750, Korea
We have characterized the global gene expression patterns of Arabidopsis pollen using Serial Analysis of Gene Expression (SAGE). A total of 21,237 SAGE tags were sequenced and 4,211 unique tags were identified. Interestingly, the number of unique tags in pollen was low compared with the SAGE library of the leaf constructed on a similar scale. The transcript profiles of pollen reflect accurately the characteristics of pollen as a reproductive organ. Functional classification of the expressed genes reveals that those involved in cellular biogenesis such as polygalacturonase, pectate lyase, and pectin methylesterase make up more than 40% of the total transcripts. However, genes involved in energy and protein synthesis, which are prevalent in leaves, were expressed at a relatively low level. The expression level of the great majority of transcripts was unaffected by cold treatment at 0°C for 72 h, whereas pollen tube growth and seed production were substantially reduced. Interestingly, many genes thought to be responsible for cold acclimation such as COR, lipid transfer protein, and  -amylase, that are highly induced in Arabidopsis leaves, were only expressed at their normal level or weakly induced in the pollen. The expression patterns of the cold-responsive transcripts identified by SAGE were confirmed by microarray analysis. Our results strongly suggest that poor accumulation of proteins that play a role in stress tolerance may be why Arabidopsis pollen is cold sensitive.
The functional and biochemical features of specific cell types are determined by their particular gene expression profiles. Such global gene expression patterns can be represented by a "transcriptome", which reveals the identity and the level of expression of each expressed gene in a defined population of cells (Velculescu et al., 1997  ). The transcriptome can be modulated by both external and internal factors, and thereby provide not only information useful for the understanding of the basic cellular biology but also a global view of biological responses over environmental stimuli.
Gene expression profiles can be obtained and compared by various methods, such as RNA-DNA hybridization measurements, subtractive hybridization, subtraction libraries, and differential display. However, these methods have been limited in providing overall gene expression patterns due to their technical shortcoming. The recent DNA microarray technique allows large-scale quantitative gene expression analysis. Especially, it has been possible to cover most of the genome in GeneChips for several model systems. However, in many experimental systems, it is still limited by the fact that it only analyzes arbitrarily chosen genes. Another technology, Serial Analysis of Gene Expression (SAGE), in part overcomes this limitation. SAGE allows simultaneous, comparative, and quantitative analysis of gene-specific, 9- to 10-bp sequence tags (Velculescu et al., 1995 The male gametophyte of flowering plants, represented by the pollen grain, contains all the genetic information required to unite with the female gamete at fertilization and form a new sporophyte. Because of this crucial function in the plant's reproductive cycle, pollen has been the object of energetic cytological, biochemical, and molecular biological research. In recent years, insight into processes underlying pollen development and function has been extended by studying pollen-specific gene expression.
Male gametogenesis in flowering plants, from archesporal cells to mature pollen, involves a series of complicated events (Goldberg et al., 1993
Temperature is one of the most important environmental factors affecting plant development and crop productivity. Most plants, such as Arabidopsis, develop tolerance to freezing after being exposed to low nonfreezing temperatures. This adaptive process, known as cold acclimation, involves a number of biochemical and physiological changes. The most notable include reduction or cessation of growth, reduction of tissue water content (Levitt, 1980
In recent years, several comprehensive genomic studies based on DNA microarray performed in the field of plant cold acclimation, identified large amount of cold-responsive genes, and discussed their signal pathways (Seki et al., 2001 In this study, a comprehensive transcript profile of Arabidopsis pollen was analyzed by SAGE and compared with that of cold-treated pollen. Additionally, the transcript profiles of the Arabidopsis leaf, which is thought to be more cold resistant than pollen, were used as references to understand the molecular behavior of pollen. Information on gene expression profiles in pollen and gene regulation by cold stress provides valuable information for understanding why pollen cells are so sensitive to cold stress as well as yielding extensive knowledge of the characteristics of pollen itself. In addition, studies in these areas may reveal new strategies for improving the freezing tolerance of agronomic plants by genetic engineering.
SAGE Analysis of Arabidopsis Pollen A total of 21,237 tags from Arabidopsis pollen, consisting of 4,211 unique tags, were obtained and analyzed (Table I). Of these, tags at an abundance level of 2 or greater represented 1,507 genes. Of 4,211 unique tags, only 45% of the tags matched the entries of the National Center for Biotechnology Information Arabidopsis SAGE tag-to-gene mapping database. Currently, the proportion of no match is high, probably because a large fraction of the low-abundance class of transcripts might not be reported yet. Even among the reported sequences in the UniGene DB, the 5' expressed sequence tags (ESTs) are excluded because it is not proper target sequences considering that the tag sequences result from the 10 base sequences 3' adjacent to the 3'-most NlaIII sites (CATG) of the transcripts.
It has been estimated that the total number of average-sized transcripts ranges from 100,000 to 500,000 per cell in higher plants (Kiper et al., 1979
The 50 most highly expressed genes are listed in Table II. An extended list is available as supplemental material (Supplemental Table I; supplementary data can be viewed at http://www.plantphysiol.org). As expected, a large proportion of these corresponded to well-characterized enzymes required for cell wall metabolism such as polygalacturonase, pectate lyase, and pectin methylesterase. The growth of the pollen tube is extremely rapid and is associated with the presence of numerous vesicles in the apical tip. The principle component of the vesicles is pectin (Drashek and Rosen, 1966
Furthermore, several of the genes highly represented in Arabidopsis pollen reflect the characteristics of pollen itself. For example, Agp1 is supposed to be related to Bcp1, an anther-specific cDNA isolated from Chinese cabbage (Brassica campestris; Theerakulpisut et al., 1991
In addition to pollen-specific genes, nectarin-like protein is abundantly expressed in this library. Nectarin I, a protein that accumulates in the nectar of Nicotiana sp. (Carter et al., 1999 Predicted or known genes were classified according to function, which was assigned essentially by applying the Munich Information Center for Protein Sequences (http://mips.gsf.de) analysis system as well as additional publications. The proportion of genes in each category is shown in Figure 1. Of the 437 genes analyzed, the most highly enriched functional categories are those of cellular communication/signal transduction (23%), metabolism (21%), and transcription (10%). There is no great difference in the overall pattern of gene distribution between pollen and total genome (Fig. 1A). However, if the tag abundance of all transcripts corresponding to each category is considered, genes involved in cellular biogenesis are the most abundant class, indicating that the average copy number per gene of this category is significantly higher than that of any other group of genes (Fig. 1B). This finding suggests that transcripts encoding proteins required for cellular biogenesis represent high copy genes, even if there are not many such genes. Compared with leaves, genes involved in energy and protein synthesis were expressed at lower levels.
To examine the physiological changes caused by cold stress, its effects on pollen tube growth in vitro and on seed production were measured. The germination rate of pollen was reduced to a greater or lesser extent in all conditions tested (Fig. 2). Exposure of pollen grains to 0°C for 72 h had an especially marked effect, leading to a 60% reduction. Although the effect on pollen germination at –2°C and 0°C was similar, the effect on other parts of plants was quite different. Plants undergoing 0°C treatment re-initiate normal growth after transfer to normal conditions, whereas plants treated at –2°C for long periods tend to wilt due to freezing injury. Plants maintained at 0°C for 72 h and –2°C for 24 h were chosen for seed production assay. As shown in Figure 3, the seed number per silique in unstressed plants is about 47, and this number is reduced in cold-treated plants. Worst of all, some siliques produced abnormal withered seed. Reduced seed production is likely to depend on the stage of flower development. The floral stage of the flower primordia at the time of cold treatment varied from 9 to 16 depending on their location in the plant. Generally, two different stages of flower primordia, that is, stages 11 to 12 and stage 16, suffered severe effects of cold treatment. At stage 16, petals and sepals are starting to wither, and siliques form subsequently. Therefore, the effect on stage 16 is unlikely to be related to pollen development but may instead affect silique formation. However, floral stage 9 to 12 is an important phase in the formation of mature pollen grains (Bedinger, 1992
To generate a profile of relative gene expression patterns in cold-treated pollen, RNA was extracted from pollen cells obtained from plants exposed to 0°C for 72 h, where pollen tube growth and seed production were greatly affected, and used for constructing a SAGE library. With 21,075 tags, similar to the number of tags analyzed from untreated pollen, 4,927 different genes were obtained. The 50 most highly expressed genes are shown in Table III. An extended list is available as supplemental material (Supplemental Table II). The expression levels of 57 genes were not much altered, and these ranked among the top hundred most highly expressed genes in both experimental conditions. The finding that the most abundantly expressed genes in cold-treated pollen were also highly expressed in untreated pollen suggests that the highly abundant genes responsible for pollen function are not greatly affected by cold stress.
The distribution of fold-changes in tag number between normal and cold-treated cells is shown in Figure 4A. The great majority of transcripts were expressed at similar levels in cold-treated and normal pollen: approximately 92% of the genes showed less than a 3-fold difference in expression. The similarities in the global expression profiles of the two samples can be readily visualized using a scatterplot (Fig. 4B). Twenty-six tags were expressed at least 10-fold more in the cold-treated than in the normal cells, and 28 tags were expressed at least 10-fold more in normal pollen (Table IV). Interestingly, most of the known genes whose products are thought to be responsible for cold acclimation were not cold-induced. Even of the genes whose expression was increased more than 8-fold, only two are known to be involved in stress responses or defense: aluminum tolerance-associated protein and glutathione S-conjugate transporting ATPase (AtMRP1), a multidrug-resistance-associated protein homolog. However there is no direct evidence that the function of these genes is related to the cold stress.
According to our SAGE data, most known genes thought to be responsible for cold acclimation were not induced in Arabidopsis pollen. To confirm this result, a specialized cDNA microarray, constructed in this laboratory to study cold stress, was used. Total RNA from normal and cold-treated pollen was hybridized to the cDNA microarray to examine the expression of hundreds of cold-responsive genes. Among them, the expression patterns of 20 well-known cold-induced genes (Seki et al., 2002a
No comprehensive study of gene expression in pollen cells has been reported previously. In this study, in an effort to better understand the development and function of the pollen cell at the molecular level, SAGE analysis was performed, and a comprehensive gene expression profile of Arabidopsis pollen was obtained. SAGE data from Arabidopsis pollen shed light on the transcriptional profile of pollen as a reproductive tissue and hence reveal the classes of proteins and metabolic pathways that are probably used during pollen development. It has been surmised that the early genes might encode cytoskeletal proteins and proteins needed for wall synthesis or starch deposition, whereas the late genes whose mRNAs accumulates throughout microsporogenesis might be required during maturation or pollen tube growth. The primary synthetic events that occur during pollen tube growth are concerned with pollen tube wall synthesis and the synthesis of the cell membrane of the elongating tube. Accordingly, one might expect that many of the pre-existing mRNAs and proteins in the mature pollen grain code for enzymes required in these two processes. By analyzing the SAGE cDNA tag library, many genes involved in cell wall metabolism and other pollen-specific genes were identified. In addition, numerous novel genes not identified previously were also detected.
Comparison of the tags present in the pollen cells with those identified in other tissues throws light on the similarities and differences in gene expression among different tissue types. Two SAGE tag sets, one from Arabidopsis leaves constructed in this laboratory (Jung et al., 2003
Pollen is a specialized organ for accomplishing reproduction. Therefore, its gene expression pattern differs from that of normal vegetative growing leaves. Generally, genes involved in energy production were strongly expressed in leaves (Fig. 1). On the other hand, in pollen, a high level of expression of genes responsible for cellular biogenesis was reveled. The gene expression patterns identified by SAGE reflect well the characteristics of leaves as photoautotrophic organs and pollen as reproductive organs. Thus, the specific composition of a SAGE tag set from a tissue can provide a useful fingerprint representing the tissue-specific pattern of gene expression. One aim of functional genome analysis is to understand the temporal and spatial expression patterns of all genes with roles in the developmental processes of an organism, and to understand how they function in response to biotic and abiotic cues such as pathological conditions and environmental stresses. In the present work, SAGE was applied to analyze the gene regulation pattern in response to cold stress in Arabidopsis pollen. Because pollen is an important reproductive organ, it is highly likely that the reduced productivity induced by stress originates from malfunction of the pollen. Although cold stress in plants has been extensively studied, there has been little investigation into the effect of stress on pollen development and function. In this study, as a step in unraveling the complexities of gene expression in pollen cells, the SAGE profile of transcripts derived from pollen cells before and after cold treatment was compared. In addition, comparison of transcriptomes between leaf and pollen has yielded interesting information on the dynamics of total genome expression attributable to a change in environmental conditions, and provides clues to the function of those genes whose contribution to cold acclimation is still unknown.
Approximately 20,000 transcripts were analyzed, and numerous genes showing differential expression between cold-treated and untreated cells were identified. The availability of the transcriptome of the cold-treated leaf (Jung et al., 2003
Expression of several genes thought to be cold inducible, such as nonspecific lipid transfer protein and
The expression levels of many genes responsible for pollen germination and tube growth are not influenced by cold stress. In particular, genes that are strongly expressed under normal condition retain their level of expression in cold-treated samples (Table II). This result may imply that the physiological changes induced by cold stress are not caused by transcriptional repression of genes responsible for pollen function but rather by the inhibition of protein function at the translational or posttranslational level. It is possible that absence of proteins involved in protein stabilization, such as molecular chaperones and heat shock proteins, causes the failure of normal cellular homeostasis. In addition, the fact that many cold-inducible genes that promote membrane stability such as COR15a (Steponkus et al., 1998 In conclusion, SAGE was performed on normal and cold-treated Arabidopsis pollen, and permitted the identification of their characteristic gene expression profiles. The databases of over 20,000 SAGE tags may be useful resources for investigators interested in the relative expression level of transcripts in the pollen of Arabidopsis. In addition, such SAGE libraries offer important data sets for analyzing comparative gene expression patterns among different tissue types. The present study suggests that an inability to induce certain genes that are important for cold acclimation is responsible the cold sensitivity of pollen and is directly linked to pollen function.
Growth Conditions and Preparation of Plant Materials
Arabidopsis cv Columbia plants were grown for 4 weeks in an environmentally controlled growth chamber at 23°C under constant light (
The pollen from 20 mL of flowers was repelleted by centrifugation in an Eppendorf microcentrifuge at 12,000g for 2 min. The supernatant was removed, and the pellet was resuspended in 200 µL of Tri-reagent (Molecular Research Center, Cincinnati). The pollen cells were disrupted with a pellet pestle motor (Thomas Scientific, Swedesboro, NJ) and 200 µL of Tri-reagent was again added. After homogenizing twice, total RNA was prepared following the manufacturer's protocol. mRNA was purified using the Oligotex mRNA mini kit (Qiagen USA, Valencia, CA) according to the manufacturer's instructions. Poly(A+) RNA was converted to double-stranded cDNA as directed by the SuperScript Choice System for cDNA synthesis (catalog series 18090, Invitrogen, Carlsbad, CA).
For construction of the SAGE tag library from normal pollen, SAGE was performed as previously described (SAGE protocol v1.0c) with minor modifications. In brief, double-stranded cDNA was digested with NlaIII, and biotin-containing 3' cDNA fragments were collected with Streptavidin beads (Roche Diagnostic, Mannheim, Germany). After ligation of oligonucleotides containing recognition sites for BsmFI, the linked cDNA was released from the beads by digestion with BsmFI. The released tags were ligated to one another, blunt-ended, amplified, and redigested with NlaIII. The ditag pellets were resuspended in 3 mM Tris-HCl, pH 7.5, and 0.2 mM EDTA, pH 8.0 containing 25 mM NaCl to prevent denaturation. The ditags were then concatemerized at their NlaIII overhangs using DNA ligase. The ligation mixture was heated at 65°C for 15 min and separated on an 8% (w/v) polyacrylamide gel. DNA fragments between 500 and 1,000 bp in size were isolated and cloned into the SphI site of the pZero-1 vector (Invitrogen). Sequencing was carried out at GENE Inc. (Suwon, Korea). In the case of cold-treated pollen, a modified SAGE method for microscale RNA sample preparation (Lee et al., 2001
For germination assays, flowers were submerged in Tris-EDTA solution (10 mM Tris and 1 mM EDTA, pH 8.0), shaken twice for 15 min, and filtered through a Nitex filter. The pollen in the filtrate was plated on in vitro pollen germination medium (20% [w/v] Suc, 2 mM calcium chloride, and 1.65 mM boric acid) and incubated at 25°C for 4 h. Pollen tube growth was observed with a light microscope. Four different conditions (temperature of 0°C or –2°C, and growth period of 24 or 72 h) were used for this analysis. Total numbers of germinated pollen were monitored at times up to 4 h. Approximately 500 pollen grains harvested from flowers in the B4 phase (corresponding to stage 14 [Smyth et al., 1990
A specialized cDNA microarray generated in this laboratory for cold stress research was used. This microarray contains 712 cDNAs selected on the basis of SAGE and SSH (suppression subtractive hybridization) data, as well as additional reference genes. Pollen RNA was labeled by an indirect method with the 3DNA Array 50 Expression Array Detection Kit (Genisphere, Hatfield, PA). In brief, 25 µg of total RNA was reverse transcribed using reverse transcription primers tagged with either Cy3- or Cy5-specific 3DNA capture sequences. The synthesized tagged cDNAs were then fluorescently labeled by Cy3–3DNA or Cy5–3DNA based on the complementarity of capture sequences with 3DNA capture reagents. Hybridized microarrays were scanned using an ArrayWoRx (Applied Precision, Issaquah, WA). Intensity values were quantified from the resultant pairs of TIFF files using ImaGene image analysis software (BioDiscovery, Los Angeles) and analyzed using the GeneSight software package (BioDiscovery, Los Angeles). Analyses were performed using mean signal intensity values for each spot. Local background was subtracted from the signal intensity and minimum intensity was raised to 20 using "floor" function. The ratio of mean hybridization intensity for each element was normalized by dividing them by the mean of the selected subset (
We thank Dr. Kenneth W. Kinzler (Johns Hopkins University, Baltimore) for the SAGE protocol and analysis program. We are also grateful to Dr. Sanggyu Lee (University of Chicago) for providing the modified SAGE protocol and technical support. Received January 16, 2003; returned for revision February 25, 2003; accepted March 19, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.020511.
1 This research was supported in part by Korea Science and Engineering Foundation (grant no. 98–0401–06–01–3), by the Brain Korea 21 Project in 2001, and by the 21st Frontier R&D Program (grant no. CG1122).
[w] The online version of this article contains Web-only data. The supplemental material is available at http://www.plantphysiol.org. * Corresponding author; e-mail lee{at}ewha.ac.kr; fax 82–2–3277–2385.
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-8-spingolipid desaturase, was detected in pollen. However, their expression was not significantly increased by cold stress. Although it is possible that additional genes may be expressed at levels below the present detection threshold, it seems unlikely that such genes would contribute to cold tolerance. Microarray-based analysis of several genes showing strong induction in cold-treated Arabidopsis leaves also supports the observations that these genes are not strongly induced in cold-treated pollen. It seems probable that the genes induced or repressed by cold stress in pollen merely reflect general cellular changes unrelated to cold acclimation, and it is possible that the inability to induce expression of genes important in cold acclimation may be responsible for the cold sensitivity of pollen. 
100 µmol m–2 s–1). Flower stages from 9 to 14 were excised and collected in 50-mL tubes. Approximately 70% of the samples were at stage 12 to 14 and the rest were at stage 9 to 11. Pollen was harvested by shaking a tube full of flowers with 25 mL of Tris-EDTA solution (10 mM Tris and 1 mM EDTA, pH 8.0) for 15 min. Two such suspensions were filtered through a Nitex filter (40-µm pore size; Tetko, Elmsford, NY), and the pollen was pelleted at 4,000 rpm for 10 min with a clinical centrifuge. It was then resuspended in 100 µL of RNAlater (Ambion, Austin, TX) and stored at –80°C until use. For cold treatment of the plants, plants grown as above were moved to a growth chamber at the desired temperature under the same light condition and grown for the time period as specified time. 
-tubulin and actin-2). The mean of the two elements was used to combine duplicate spots on one slide. 
-3 fatty acid desaturase in transgenic tobacco. Plant Physiol 105: 601–605
