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First published online March 23, 2007; 10.1104/pp.107.098723 Plant Physiology 144:155-172 (2007) © 2007 American Society of Plant Biologists Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development1,[W]Business Units Bioscience (R.J., J. Cordewener, E.D.J.S., O.V., M.L., T.Z., T.A., J. Custers, K.B.) and Biometry (C.M.), Plant Research International, 6700 AA Wageningen, The Netherlands; Research Center for Biotechnology, Bogor Agricultural University, Bogor 16610, Indonesia (E.D.J.S.); and Eastern Cereal and Oilseeds Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada K1A 0C6 (B.M.)
Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants.
Microspore embryogenesis describes the process in which the immature male gametophyte is induced to form a haploid embryo in vitro. Microspore embryo culture is a valuable tool for plant breeders because the haploid embryos that are produced (microspore-derived embryos [MDEs]) can be converted to homozygous doubled haploids using chromosome-doubling agents. The ability to obtain a homozygous population of plants in a single generation not only significantly reduces the time needed to develop inbred lines, but also facilitates selection for recessive and polygenic traits and speeds up the development of mapping populations and molecular markers for marker-assisted breeding (Forster and Thomas, 2005  ). Microspore embryogenesis has been described for more than 250 plant species and efficient protocols are available for about 20 of these (Maluszinsky et al., 2003). As with other regeneration processes, many species still remain recalcitrant for microspore embryogenesis and, even within a successful species, there are usually recalcitrant genotypes. There is, therefore, considerable interest in identifying the molecular genetic factors that define and control this process.
MDE culture is also receiving increasing attention as a model system to study cell totipotency and for studies on early embryo development (Boutilier et al., 2005
A large repository of data has been compiled concerning the early cellular and morphological changes that take place during the transition from male gametophyte to haploid embryo development in culture and commonalities among different species have been identified (for review, see Aionesei et al., 2005 Increasing use and availability of functional genomics tools have brought with it renewed interest in unraveling the molecular pathways that underlie cell totipotency. Identification of genes whose expression marks or controls cell totipotency is of major interest to the applied research community because such genes could be used to overcome recalcitrance for regeneration in crop species. Likewise, genes whose expression reflects the normal progression of embryo development can be used as markers to optimize critical points in the tissue culture process.
MDEs, although single cell in origin, do not exhibit the ordered cell divisions that occur during early zygotic embryogenesis. Multicellular embryos of 40 to 60 cells are formed through random (unorganized) cell divisions and only then develop into globular embryos with a distinct protoderm through a hypothetical self-organizing mechanism. Suspensor development, which takes on many forms in various plant species, is also absent in microspore embryos, although a short or malformed rudimentary suspensor is occasionally observed (Hause et al., 1994
Development of Rapeseed MDE Cultures
Conventional MDEs Differ from Zygotic Embryos
Optimized Protocol for Production of Zygotic-Like MDEs Based on the above observations, we optimized the culture conditions to obtain a high frequency of MDEs with long suspensors and zygotic embryo-like morphology. The critical aspects of the new culture system are a more accurate selection of the microspore starting population using narrower increments in floral bud size (Supplemental Table S1) and the use of a shorter and more accurate heat-stress treatment (Fig. 2 ). Cytological analyses showed that cultures that were most responsive in the development of normal-looking suspensors were always very uniform with respect to the stage of the starting microspore population, consisting of at least 50% to 60% late unicellular microspores and 30% to 40% early bicellular pollen. To better understand the relation between formation of MDEs with normal-looking suspensors and those with aberrant suspensors, we carried out a more thorough analysis of the duration of the 32°C heat-stress treatment (Fig. 2). Microspores from highly responsive buds were treated for various periods at 32°C and then transferred to 25°C. After 16 d of culture, the quality of suspensor formation was determined based on three morphological classes: (1) embryos with long, normal-looking suspensors (  30 µm uniseriate); (2) embryos with short, irregular-shaped suspensors (<30 µm uniseriate); and (3) embryos without a suspensor. As shown in Figure 2, a 32°C heat-stress treatment applied for 8 to 12 h led to production of a long suspensor in more than two-thirds of the embryos (Fig. 2A). Increasing the duration of 32°C heat stress from 8 h to continuous treatment resulted in a gradual decrease in the frequency of embryos with a long suspensor to close to 0 and a parallel increase in embryos lacking suspensors. The second class of embryos, those with short appendant structures, either uniseriate or swollen and irregular, were found in all treatments, but the frequencies of such embryos were significantly higher when the microspores were treated at 32°C for 24 or 48 h as compared to shorter or longer heat-stress treatments (Fig. 2A). Although the modified heat-stress treatment positively affected the production of embryos with long, normal-looking suspensors, it had a negative effect on the total number of embryos formed (Fig. 2B).
Comparison of Conventional and Suspensor-Bearing MDE Cultures The major developmental events that take place in the conventional and suspensor-bearing MDE cultures that were used in our transcriptome and proteome analyses are summarized in Figure 1. Conventional MDE cultures were induced from freshly isolated microspores (Fig. 1A) by continuous 32°C heat-stress treatment. MDE development begins with swelling of the microspore, followed by symmetric division of the vegetative nucleus. Approximately 15% to 25% of the microspores undergo embryonic divisions within the first 2 d of culture (Fig. 1B). By the fifth day of culture, randomly oriented cell divisions in a subset of these few-celled embryos generate preglobular-stage embryo clusters that can be seen breaking out of the microspore exine wall (Fig. 1C). Globular- to heart-stage embryos are usually present after 10 d of culture (Fig. 1D). Suspensor-bearing MDEs were induced by culturing microspores for 1 d at 32°C, followed by transfer to 25°C. Suspensor-bearing MDEs do not originate by random division of the microspore, but rather by formation of a suspensor-like filament, followed by embryo-proper development from the distal cell. The first cell divisions take place at around the fourth day of culture (Fig. 1E) with a nose-like suspensor emerging from the swollen microspore around the seventh day of culture (Fig. 1F). The suspensor develops as a single file of cells, elongating by transverse divisions, with one end connected to the microspore. Around the eighth day of culture, the distal cell of the three- to eight-celled suspensor undergoes transverse divisions to form the embryo proper (Fig. 1G). Thereafter, cell division and histodifferentiation of the embryo proper follow the same regular pattern as in zygotic embryos, and preglobular-stage embryos with a defined protoderm and hypophysis can be seen after 10 d of culture (Fig. 1H). Cell division in the suspensor generally ceases once the embryo reaches the late-globular stage. Suspensor-bearing MDE cultures develop more slowly than conventional cultures, with the result that two- to four-celled embryos are found at around day 2 in conventional cultures and at day 8 in suspensor-bearing cultures (compare Fig. 1, B and G), whereas preglobular embryos are found at around day 5 in conventional cultures and day 10 in suspensor-bearing cultures (compare Fig. 1, C and H).
The heat-stress treatment used to induce conventional and suspensor-bearing MDE development is not sufficient to block gametophyte development in all of the donor microspores. As a result, both pollen and embryo developmental pathways initially occur side by side in the same cultures. The large amount of developing pollen in MDE cultures can confound attempts to identify early embryo-expressed genes and proteins; therefore, we used control pollen cultures to discriminate between embryo and pollen expression profiles in MDE cultures. Cultures with up to 90% viable tricellular pollen can be obtained by culturing isolated microspores at 18°C instead of 32°C (Fig. 1, I and J, gametophytic pathway; Custers et al., 1994
Microarray Enriched for MDE Culture cDNAs The custom cDNA microarray was hybridized to targets derived from two independent microspore cultures. The developmental time points that were analyzed are shown in Figure 1. For each of the two cultures, one sample was harvested immediately (0d; Fig. 1A); one sample was cultured to promote pollen development (5p; Fig. 1J), and five samples were cultured to induce embryo development. Of the five samples cultured for embryo development, three samples were induced for conventional MDE development (two- to four-celled stage [Fig. 1], preglobular stage [Fig. 1C], and globular to heart stage [Fig. 1D]), and two samples were induced for suspensor-bearing MDE development (two- to four-celled embryo-proper stage [Fig. 1G] and preglobular embryo-proper stage [Fig. 1H]). The percentage of embryo and viable pollen present in each culture at the successive sampling time points is shown in Table I . Each of the amplified targets was hybridized using a common reference strategy ("Materials and Methods"). Array hybridizations with amplified leaf and flower bud targets were performed to obtain an indication of the relative expression levels of the probes in non-embryo samples.
Principal Component Analysis Principal component analysis (PCA) was used to analyze gene expression profiles obtained after microarray hybridization of amplified pollen and MDE culture samples. PCA helps to interpret overall patterns in gene expression by dimension reduction. A plot of the samples on the first two principal components, together explaining 82% of the observed variability, is shown in Figure 3A . Corresponding distribution of the individual genes is shown in the inset. Information on the composition of the culture samples in terms of the relative contribution of pollen and embryo-like structures, as shown in Table I, is added in the plot. The first principal component (PC1), which explains 70% of the variance, can be interpreted as describing the transition in the cultures from a pollen-dominated gene expression state to an embryo-dominated gene expression state. The 2-d conventional embryo culture samples (2e), which contain both embryogenic cells and a high proportion of viable pollen, are clustered toward one side of the axis together with the 5-d pollen culture samples, whereas the conventional (10e) and suspensor-bearing embryo or culture samples that do not contain viable pollen (8se and 10se) are clustered at the other end of the axis (Table I; Fig. 3). Microarray analysis of Arabidopsis (Arabidopsis thaliana) pollen gene expression profiles show that mature pollen grains contain proportionately more abundant mRNAs than do sporophytic tissues (Honys and Twell, 2003 ). The relative contribution of embryo-expressed genes to the entire mRNA pool in 2-d conventional embryo cultures might therefore be diluted by abundant pollen mRNAs, giving these cultures an overall pollen-like identity despite the presence of a large amount of developing embryos.
Identification of Differentially Expressed Probes Cluster analysis of the microarray expression data was used to identify groups of similarly expressed genes in different culture samples. As shown in Figure 4 , two main gene expression clusters were identified: a large cluster comprising genes primarily up-regulated in the 5-d pollen and 2-d conventional embryo culture samples, and a smaller cluster comprising genes up-regulated in the remaining conventional and suspensor-bearing embryo samples. A large number of known late pollen-expressed genes (Twell, 2002 ) are up-regulated in the 5-d pollen and conventional 2-d embryo culture samples, suggesting, as observed in the PCA plots, that the mRNA pool in the 2-d conventional MDE culture is dominated by abundant pollen transcripts. We refer to the larger gene expression cluster as pollen up-regulated and to the smaller gene expression cluster as embryo up-regulated. We made use of the ANOVA test statistic (F value) and LSD values ("Materials and Methods") to identify the pollen and embryo up-regulated probes in these two clusters. The dominance of pollen-expressed genes in 2-d conventional embryo cultures confounds microarray-based attempts to identify embryogenesis-associated transcripts in this sample; therefore, the data for this time point, although presented, was omitted from this analysis. We identified 220 probes that are significantly up-regulated during one or more of the embryo cultures as compared to 5-d pollen cultures and 363 probes that are significantly up-regulated in 5-d pollen cultures as compared to all of the embryo cultures (Supplemental Table S3). Expression profiles of four selected embryo up-regulated probes and one pollen up-regulated probe were validated by quantitative reverse transcription (RT)-PCR (Supplemental Fig. S1; Supplemental Table S4).
Analysis of the identities of the pollen and embryo up-regulated probes and their accompanying expression patterns in freshly isolated microspores, pollen, and embryo cultures provides insight into the developmental processes that take place as microspores develop into either pollen grains or haploid embryos (Supplemental Fig. S2; Supplemental Table S3). The progression from microspores to tricellular pollen grains is accompanied by a dramatic increase in the expression of cell wall-loosening enzymes needed for pollen germination. There is also a switch from expression of genes for glyocolysis-based carbohydrate metabolism in microspores to genes that play key roles in the glyoxylate cycle and gluconeogenesis in pollen (Supplemental Fig. S2; Supplemental Table S3). The corresponding enzymes are likely to be involved in the mobilization of lipid reserves by glyoxysomes to produce sugars to sustain pollen growth (Zhang et al., 1994 ). There is also a sharp decrease in the expression of probes for components of protein synthesis machinery (Supplemental Fig. S2; Supplemental Table S3). In contrast, expression of probes coding for glycolytic enzymes is maintained or increased during the switch from microspore to embryo development (Supplemental Table S3), whereas the majority of probes encoding components of the protein synthesis machinery are expressed at the same level during the transition from microspore to haploid embryo development (Supplemental Table S3).
Robust Markers for MDE Induction
The vast majority of these markers encodes proteins of unknown function or corresponds to functionally annotated proteins for which neither an expression profile nor a function during embryogenesis have been assigned. Twelve of these probes encode the BNM2 BURP domain protein, which was previously identified in two independent screens for embryo markers in MDE cultures (Boutilier et al., 2002 ; Tsuwamoto et al., 2006). BURP domain proteins are an abundant class of secreted proteins of unknown function that are defined by their modular structure and conserved C-terminal BURP domain (Hattori et al., 1998). The BNM2 BURP domain gene, although expressed in 0-d cultures and 5-d pollen cultures, is expressed at a 100 times higher level in two- to four-celled embryos (Fig. 5; Supplemental Table S4). We analyzed the temporal and spatial expression pattern of BNM2 in independent MDE cultures, as well as a range of seed and nonseed samples. RNA gel-blot analysis of BNM2 expression confirmed our microarray and RT-PCR data (Fig. 6 ) and further showed that BNM2 is temporally regulated in a similar manner during MDE and seed development, with expression peaking around the midcotyledon stage of embryo development (Fig. 6, A and B). Messenger RNA in situ hybridizations on rapeseed MDEs and seeds showed that spatial expression of BNM2 is also conserved in MDEs and zygotic embryos; BNM2 is initially expressed throughout the preglobular embryo and becomes confined to the epidermal and ground tissues as the embryo differentiates (Fig. 6, D, E, and G). BNM2 is not an embryo-specific gene because it is expressed in flower buds (Fig. 6C) and in the integument and endosperm of developing seeds (Fig. 6, F and G).
Probes encoding proteins involved in transcriptional regulation and signaling were also identified as robust markers for the transition to haploid embryo development (Supplemental Table S3; Fig. 5). Among the transcriptional regulators is the MADS-box transcription factor AGL53, which is also expressed in somatic embryo cultures (Lehti-Shiu et al., 2005 ). Two probes encoding a PHYTOSULFOKINE protein and an arabinogalactan protein, AGP15, are noteworthy due to the role of such proteins in promoting cell proliferation and in vitro embryogenesis (Yang et al., 2000; Matthys-Rochon, 2005; Letarte et al., 2006).
We identified a number of glutathione S-transferases (GSTs) that are up-regulated in embryos as compared to pollen and freshly isolated microspores (Supplemental Table S3; Fig. 5). GSTs have been identified as embryo markers in barley (Hordeum vulgare) MDE cultures (Vrinten et al., 1999
We also identified 23 lipid transfer proteins (LTPs)/LTP-like proteins corresponding to six different groups among the robust MDE markers (Supplemental Table S3; Fig. 5). LTP/LTP-like proteins are small, basic, abundant proteins with an eight-Cys residue region that is also found in a larger group of structurally related proteins (Jose-Estanyol et al., 2004
Comparison of Suspensor-Bearing and Conventional MDE Gene Expression Profiles
We used the same developmental time points studied in the microarray analysis to compile proteome maps of rapeseed MDE cultures. A larger amount of starting material was needed for proteome analysis using 2-D gel electrophoresis as compared to microarray analysis; therefore, biological replicates were made for each of the developmental time points by pooling samples derived from multiple independent cultures. The composition of these cultures, in terms of the percentage embryo development as measured at the end of the culture period, is shown in Table II . Protein extracts from each of these cultures were run on 2-D gels and analyzed with PDQuest software. Table II shows the number of spots detected per gel, which varies between, on average, 528 spots in the 0-d starting culture gels to 1,160 spots in the 10-d suspensor embryo culture gels. The same amount of protein was loaded for each sample; thus, the large increase in the number of detected protein spots in the embryogenic cultures after day 5 might reflect the increase in complexity of the developing embryos.
PCA PCA was used to identify and give biological meaning to the protein expression profiles that are characteristic for the pollen and MDE cultures. A plot of the pollen and MDE culture samples and the corresponding distribution of protein spots (inset) are shown in Figure 2B. The first two principal components explain 38% of the observed variability in protein expression between the different culture samples, with PC1 explaining 22% and PC2 16% of the variability. As observed for the microarray data, PC1 appears to describe a transition in the culture material from a pollen-dominated to an embryo-dominated state, whereas the 0-d culture is assigned a unique position PC2. Distribution of the pollen and embryo culture samples along PC1 is more gradual than was observed for the mRNA expression profiles (Fig. 3B). Together, these observations point to the greatly reduced contribution of pollen-expressed proteins as compared to pollen-expressed mRNAs to the sample identities. Distribution of the expression profiles for the individual protein spots is centered on the intersection of the PC1 and PC2 axes, a position that reflects proteins with low variance or low information among the different samples. Thus, although PCA uncovered the most important structure in the 2-D gel data, the amount of variance not explained by the first two principal components is large (62%). A combination of technical and biological factors may contribute to the low variance of the protein expression data. For example, the levels of lower abundance proteins may change more than those of higher abundance proteins, but will not be detected by 2-D gel electrophoresis. Other factors contributing to the low variance of the protein dataset might include the use of pooled samples.
Protein Sequence Identification
Sequenced proteins were selected based on their preferential expression in MDE cultures as compared to pollen cultures. Although many of the corresponding protein spots are still detected in pollen cultures, their expression is up to 20 times higher in the earliest embryo culture samples analyzed (2-d sample) as compared to pollen cultures, and their expression generally increases during subsequent stages of development. Most of the proteins that we identified are expressed during multiple stages of embryo development, as was observed for the embryo up-regulated probes identified in the transcriptome analysis; however, two of the identified protein spots show stage-enhanced expression (Fig. 7). Protein spot 29 corresponds to a tau class GST that is preferentially expressed in 2-d conventional embryo cultures. A second protein (spot 16) encodes a dehydroascorbate reductase and is preferentially expressed in suspensor-bearing MDE cultures. The identified proteins are involved in a range of processes; however, proteins active in carbohydrate metabolism and redox processes are particularly abundant among sequenced proteins. Three consecutive enzymes of the glycolytic pathway (glyceraldehyde-3-P dehydrogenase, 3-phosphoglycerate kinase, and phosphoglycerate mutase) were up-regulated at various stages of embryo development (Fig. 7). Genes and proteins involved in protecting cells against oxidative stress caused by reactive oxygen species (ROS) are well represented in both the pollen and embryo transcriptome; however, proteome data suggest a specific role for ascorbate metabolism during early embryo development. Ascorbate peroxidase, as well as ascorbate-recycling enzymes, dehydroascorbate reductase, and monodehydroascorbate reductase, are expressed (Fig. 7). The presence of these three enzymes suggests that high levels of ascorbate are required during the early stages of embryo development.
Performing expression analysis at both the mRNA and protein level can provide insight into the relationship between the timing of gene expression and protein function during development. Here, we examined the relationship between the temporal expression patterns of matched probes on the microarray and sequenced protein spots to identify coregulated transcript-protein pairs that can be used as robust markers and candidates for functional analysis. Matching the probe and protein sequences had to be performed indirectly because many of the probes on the array correspond to partial cDNA fragments, especially those obtained using SSH, making them difficult to align with the peptides derived from protein sequencing. Matched transcript-protein pairs were therefore identified by using BLASTX to compare all of the cDNA probe sequences on the microarray with the best hit obtained for each of the protein spots. The Pearson correlation of the transcript and protein expression profiles was then calculated for each of the resulting links (Supplemental Table S5). The resulting network is represented by the graph in Figure 8 in which cDNA probes (squares) and protein spots (circles) form the nodes, whereas the sequence similarity and the correlation between the mRNA and protein expression profiles across the different culture samples are encoded by, respectively, the thickness and the color of the connecting lines. Of the 32 proteins identified, 11 proteins did not show sequence similarity to any of the translated cDNA probes. The remaining 21 proteins showed sequence similarity to one or more of the translated cDNA probes. The sequence similarity between 14 of these 21 proteins and their corresponding translated cDNA probes was very high. Differences in the strength and direction of the correlation between the expression profiles of the matched protein-cDNA probes were observed. The expression profiles of the majority of the matched protein-transcript pairs showed either no correlation or only weak correlation. A strong negative expression correlation, which corresponds to an opposite expression pattern at the mRNA and protein level, was observed for cDNAs matched to proteins 3, 18, and 27b. The corresponding cDNA probes are all pollen expressed (Supplemental Tables S2 and S3). Of particular interest for this study are the expression patterns of four of the highly similar transcript-protein pairs that show a strong positive expression correlation, namely, those of a glyoxylase (spot 15, probes 157, 840, 1,365), a CCAAT-box binding factor (CBF) HAP5a subunit (spot 5, probes 19 and 1,877), a 14-3-3 protein (spot 3, probe 164), and a phosphoglycerate kinase (spot 20, probe 907). All of these matched DNA probes, with the exception of the two probes encoding the CBF HAP5a subunit, are among the list of embryo up-regulated probes identified in the microarray analysis (Supplemental Table S3). The CBF HAP5 subunit probes were not identified among the embryo up-regulated probes due to missing data points; however, independent validation by quantitative RT-PCR confirms that the corresponding gene is expressed in embryogenic cultures (Supplemental Fig. S1).
The small size and inaccessibility of the developing zygotic embryo make it technically challenging to perform large-scale, stage-specific analyses of the transcripts and proteins associated with few-celled embryos. Laser capture microdissection has been used for transcript profiling of specific cell types, but this technique has only been applied starting from the globular stage of embryo development (Casson et al., 2005 ). Currently, most of our knowledge about the genes and developmental programs that operate during the very early stages of embryo development comes from mutagenesis screens aimed at identifying gametophytic and embryo-defective mutants in model species. However, loss-of-function mutations in many genes have little or no phenotypic effect due to compensation of gene function by related family members, and genes for transcription factors and other signaling components appear to be underrepresented among embryo-defective mutants (Tzafrir et al., 2004). Here, we have taken an alternative approach, namely, transcriptome and proteome analysis of B. napus MDE cultures, to identify approximately 220 genes and 32 proteins that are expressed between the two- to four-cell and globular/heart stages of embryo development.
Analysis of the proteome and transcriptome datasets identified a number of metabolic processes associated with the establishment of embryo development in MDE cultures that can be used to differentiate between cells undergoing pollen or embryo development in the same culture. The shift from microspore to pollen development is accompanied by a sharp decrease in expression of probes coding for components of the protein synthesis machinery, whereas the majority of probes coding for the same class of proteins are not differentially expressed between microspores and the earliest stages of haploid embryo development. A number of studies have shown a decrease in mRNAs involved in protein synthesis in mature pollen grains relative to sporophytic tissues (Honys and Twell, 2003
Both the MDE transcriptome and proteome datasets are enriched for mRNAs/proteins involved in glycolysis, a process that uses Glc or other monosaccharides to generate ATP or metabolites for the biosynthesis of storage products (Plaxton, 1996
The MDE proteome dataset also suggests an important role for ascorbate metabolism during early embryo development. Ascorbate peroxidase is a H2O2-scavenging enzyme that utilizes ascorbate to reduce H2O2 to water with the generation of monodehydroascorbate (Asada, 1992
One of the goals of this study, in addition to identifying a collection of early embryo-expressed genes, was to identify gene expression profiles associated with the initial transition from microspore to embryo development. Genes that are transiently expressed in embryogenic cells shortly after the heat-stress treatment could play an active role in the induction of embryogenesis, for example, in promoting stress tolerance or in the initial reprogramming of microspores to a new developmental state. Genes that are transcribed shortly after the initial heat-stress application but that are also transcribed at other stages of embryo development may play a role in the establishment of embryo identity, as was shown for the rapeseed BABY BOOM gene (Boutilier et al., 2002
Identification of probes expressed as early as 2 d after the induction of conventional MDE cultures proved to be difficult due to the large contribution of pollen transcripts to the total mRNA pool at this time point. These pollen gene expression profiles could be correlated with the proportion of viable pollen grains in the cultures; however, we cannot rule out the possibility that pollen and embryo genes are coexpressed in the same embryogenic structures. The influence of the pollen genome at the proteome level was greatly reduced compared to the transcriptome, allowing us to identify a number of embryo up-regulated proteins in conventional 2-d embryo cultures. The reduced effect of the pollen proteome on the embryo culture identities as compared to the transcriptome may be due, in part, to an inverse correlation between the abundance of late pollen transcripts and their corresponding proteins (i.e. for some proteins, a large number of mRNAs may be present, but their corresponding proteins are less abundant in mature pollen [Holmes-Davis et al., 2005
One protein encoding a GST (spot 29) was identified as being transiently expressed in 2-d embryo cultures and is a good candidate for further analysis. GSTs are a family of enzymes that catalyze the conjugation of glutathione to lipophilic compounds. They are expressed at all stages of development and are involved in a range of processes, including detoxification of xenobiotics and protection against oxidative damage, and may also function as reversible ligands during hormone signaling (for review, see Dixon et al., 2002
In contrast to the two- to four-celled conventional embryo cultures, we were able to identify a large number of probes that are up-regulated at the same stage of embryo development using the equivalent suspensor-bearing embryo cultures, most likely due to the absence of viable pollen grains in these cultures. The collection of probes expressed in two- to four-celled suspensor embryo cultures represents some of the earliest embryo-expressed genes identified in plants to date. Only a few of these probes encode functionally annotated proteins, making it difficult to identify underlying processes associated with the commitment to haploid embryo development. However, the robustness and utility of a number of the markers identified in this study are illustrated by their earlier identification as markers for embryogenic cells in tissue culture. The BURP domain protein BNM2 was independently identified here and in two subtractive screens of 4-d heat-stressed microspore embryo cultures (Boutilier et al., 2002
We identified 23 LTPs belonging to six different classes as robust markers for the commitment to MDE development. LTPs have been previously identified as markers for embryogenic cells both in carrot (Daucus carota) somatic embryo cultures (Sterk et al., 1991
The new suspensor-bearing microspore embryogenesis culture system is strikingly similar to the rapeseed zygotic embryogenesis pathway in that a short suspensor is formed prior to the establishment of the embryo-proper cell, followed by development of the embryo proper from the apical cell (Tykarska, 1976
Key functions have been attributed to suspensors during seed development, such as the positioning of the embryo in close proximity to sources of nutrition in the embryo sac, absorption and transport of nutrients to the embryo, and the synthesis of hormones to support embryo growth (Raghavan, 2001
Microspore Isolation and Culture The procedures for microspore isolation and culture are provided as Supplemental Materials and Methods S1. Two independent microspore isolations, yielding 70- and 150-mL suspension, respectively, were performed for the microarray analysis. The microspores from each isolation were divided over three temperature treatments: (1) continuously at 18°C ± 1°C to allow pollen maturation; (2) 24 h at 32°C ± 0.2°C followed by transfer to 25°C ± 1°C for suspensor-bearing MDE development; and (3) continuously at 32°C ± 0.2°C for production of conventional MDE development. For microarray analysis, each of the two starting cultures was divided into seven samples. For each of the starting cultures, one sample was harvested immediately (0d), one sample was cultured for 5 d to obtain mature pollen (5e), two samples were cultured for 8 d (8se) and 10 d (10se) to obtain suspensor-bearing MDEs, and three samples were cultured for 2 d (2e), 5 d (5e), and 10 d (10e) to obtain conventional MDEs. The 10-d conventional embryo cultures were sieved (>50 and <200 µm) so that the sample contained only embryos, whereas all other samples were prepared directly from the crude culture suspensions. The percentages of embryo-forming microspores and pollen were determined at the points of harvest. For the 2-D gel electrophoresis analyses, similar microspore cultures were initiated and samples were harvested at the indicated time points from pooled isolates. A small aliquot of each isolate was cultured continuously at 32°C ± 0.2°C to induce standard MDE development and used to determine the embryogenicity of the culture by counting the number of embryos that had developed after 10 d.
The majority of the probe DNAs spotted on the array correspond to MDE cDNAs isolated from six different cDNA libraries: 24U, a 24-h 32°C heat-stressed MDE culture library in HybriZAP (Stratagene); 4U, a 4-d 32°C heat-stressed MDE culture library; 4F, a forward-subtracted SSH (CLONTECH) library made using 4-d 32°C heat-stressed MDE culture RNA as the tester and 0-d microspore and 4-d, 18°C pollen culture RNA as the driver; 4R, a reverse-subtracted SSH library made using 0-d microspore and 4-d, 18°C pollen culture RNA as the tester and 4-d, 32°C heat-stressed MDE RNA as the driver; 9U, a 9-d globular-stage MDE culture library from the SSH unsubtracted tester control; and 9F, a forward-subtracted SSH library from the 9-d-old globular-stage MDE RNA as the tester and leaf RNA as the driver. All clones were single-pass sequenced and queried against The Arabidopsis Information Resource (TAIR) AtH1seq and Brassica databases using BLASTN algorithms (Altschul et al., 1990
A smaller number of probe DNAs comprised previously identified cDNA clones isolated from MDE cultures (Custers et al., 2001 Information on microarray spotting, target amplification, labeling and hybridization, and data acquisition are provided in Supplemental Materials and Methods S1.
Spot intensities were background corrected by subtracting the average over the 12 background control spots containing nonhybridizing yeast (Saccharomyces cerevisiae) DNA, as described above. Normalization for dye effects was performed using the intensities of the spiked Luciferase control spots described above. All the data are presented as log2-transformed ratios. For array 72-16, a pipetting error was made with the Luciferase spike control, resulting in a shift of the median normalized log2 ratio from 0 to –1.4. This shift was manually corrected.
ANOVA was used for analysis, per clone, of the background-corrected and normalized log ratio values. In the ANOVA model, a nested structure was used with the two cultures (biological replicates), arrays within cultures (the swapped dye pair or technical replicates), and duplicated spots within each array as the blocking factors (factors generating random variation), and the different samples (each sample is a combination of a time point and a biological tissue) as the single treatment factor. A weight factor was introduced in the ANOVA so that, in the averaging over the two cultures, each culture received equal weight despite the fact that two arrays were available for the samples from culture 1 (the swapped dye pair) and only one array was available for the samples from culture 2. For each clone, an F probability was separately calculated to test for significant differences between any of the samples. The F statistic is used to help identify whether the differences between treatment means are due to background noise or the actual treatment. If there are no differences between treatments, the F statistic will be less than or near 1. A maximum of 42 log-ratio values (seven samples x three arrays x two replicate spots within arrays) was available per clone if there were no missing values. LSD ( LSD values were used to identify differential gene expression patterns among the probes that showed a significant expression pattern across the treatments based on the F-statistic analysis (P = 0.005, 577 probes). Two samples are considered statistically different if they differ by as much or more than the LSD. Pair-wise comparisons were made between the expression ratios of the signal intensities for each probe in the different cultures to identify probes that are differentially expressed. Probes that are differentially expressed will exhibit an absolute difference in expression ratios between the samples that is greater or equal to the LSD. The normalized mean log2 signal ratios for all the microarray probes (excluding the controls) for each culture sample were used for PCA. Data were centered by subtracting the average of every row/column from every signal ratio. Composition of the analyzed samples in terms of the percentage of gametophytic and embryogenic structures was incorporated into the transcriptome plot using the position of the samples as weights.
Real-time RT-PCR experiments are described in Supplemental Materials and Methods S1. The RT-PCR primer sequences and threshold cycle values are available as Supplemental Table S4 and Supplemental Figure S2.
RNA gel-blot analysis and mRNA in situ hybridization was performed using a full-length BNM2 cDNA as described by Boutilier et al. (2002).
Protein isolation and subsequent 2-D gel electrophoresis were performed as described in Cordewener et al. (1994)
Digitized gel images were analyzed using PDQuest software, version 7.1.0 (Bio-Rad). In PDQuest, there are no default settings for spot detection so the spot detection parameters (sensitivity, minimum peak, size scale, large spot size, background removal, smoothing) were optimized for each group of replicates. Selection of protein spots of interest for MS analysis was performed by a combination of pairwise t-test analyses. From the combined set of the differential protein spots, a subset was chosen on the basis of spot intensity and quality of differential behavior. PCA was performed after normalization as follows. Total spot volume per gel was used to normalize spot intensities to compensate for the considerable variation in the total volume of all matched spots per gel. For each gel, 0 values (representing absent spots) were replaced with 0.03 percentiles. The log-transformed data matrix was then used for PCA in GeneMaths.
Protein spots of interest were excised from the gel, dried, and subjected to in-gel trypsin (sequence grade modified porcine trypsin; Promega) digestion according to Shevchenko et al. (1996)
All partial sequences from the cDNA fragments on the microarray were queried against a full-length protein database (n = 32) based on the protein identities derived from the 30 sequenced differentially expressed protein spots using the BLASTX program (at e value < 0.001 and a maximum of three hits). The Pearson correlation of the probe and protein expression profiles was determined using the mean log expression ratios (microarray) and the mean log intensities (2-D gel analysis). The resulting network was visualized using Cytoscape 1.1.1 (www.cytoscape.org). Sequence data from this article can be found in Supplemental Table S2.
The following materials are available in the online version of this article.
We thank Ruud de Maagd for comments on the manuscript. We are grateful to Claudio Stasolla and Yves Gibon for providing insight into ascorbate metabolism and glycolysis, respectively. Received March 2, 2007; accepted March 13, 2007; published March 23, 2007.
1 This work was supported by the Dutch Ministry of Agriculture, Nature, and Food Quality (program no. DWK 281–392), a Natural Science and Engineering Research Council visiting fellowship in a Canadian government laboratory, and the Biotechnology Research Indonesia-Netherlands research program, with financial aid from the Royal Netherlands Academy of Arts and Sciences and the fellowship program Quality for Undergraduate Education, Bogor Agricultural University, Indonesia.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kim Boutilier (kim.boutilier{at}wur.nl).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.098723 * Corresponding author; e-mail kim.boutilier{at}wur.nl; fax 31–317–423110.
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= 0.05) for between-sample means were calculated per probe using the SE at the level of biological replicates. 
