Detection of differentially expressed transcripts in soybean mature pollen

Using the soybean GeneChip^®, we compared the transcript profiles of soybean pollen with that of sporophytic tissues consisting of an equal mix of RNA derived from leaves and stems of 10-day-old soybean seedlings. The raw intensity data generated from the microarray hybridization experiment were imported into AffylmGUI [7] and were analysed as outlined in Materials and Methods.

When the normalized data were visually displayed by scatter-plotting the log₂-transformed signal intensities of the two different samples, there was much complexity and differences on the transcript pattern between pollen and sporophytic tissues as indicated by the greater scatter of the points in the plot in comparison to a similar plot between sporophytic tissues [i.e.] stems, roots and leaves (this study) versus shoot apical meristem (Haerizadeh et al., unpublished) (Figure 1).

The soybean GeneChip^® used contains probe sets for 37,500 transcripts and the resulting analysis revealed that approximately 27% of these are expressed in the soybean pollen while 75% are being expressed in sporophytic tissues. This difference reflects the specialization of pollen as compared to other tissues with respect to providing a specific set of transcripts for specific functions such as germination, pollen tube growth, and the subsequent process of fertilization. Meanwhile, only 7.87% of the pollen-expressed genes are likely to be pollen-specific as no 'present' calls were detected for the corresponding probe sets in the sporophytic tissues. A total of 8,763 transcripts show statistically significant differential regulation in pollen as compared to sporophytic tissues with 1,686 of them showing higher expression levels in the pollen than the sporophytic tissues (at adjusted p-value < 0.05; Additional File 1 and Additional File 2). When the expression pattern for sporophytic tissue-expressed chlorophyll a/b binding protein family members were examined, none of these transcripts were represented in the pollen-expressed dataset and hence validate our experimental approach.

Functional categories of transcripts differentially expressed in pollen

The transcripts represented by the soybean GeneChip^® have been annoated as described in Materials and Methods. This allowed us to examine functional categories of transcripts that are up- or down-regulated in the pollen. As shown in Figure 2, although many of the genes fall into "unclassified" or "no homology to known protein" categories, the general distribution and over-representation of categories such as intracellular trafficking, signal transduction and transcription are evident. The up-regulated transcripts in the "no homology to known protein" category provide a valuable opportunity for the initiation of many functional analysis experiments toward an in-depth understanding of the pollen gene regulatory system and its components, which are presently incomplete.

It is interesting to note that none of the significantly up-regulated transcripts encode products that are related to the small RNA pathways (Additional File 1). A closer inspection of the expression values revealed that all of the small RNA pathways associated transcripts have signals below the detection threshold. This is consistent with a previous report [3] although a recent study by the same group has revealed the detection of 3 out of 15 genes of the ARGONAUTE family that were previously below the detection limit. The authors have attributed this discrepancy to "improved chemistry for sample processing, array hybridization, and staining that resulted in a better signal to noise ratio and thus a higher sensitivity" [8]. It is equally likely that the small RNA pathways are only active in the generative cells and hence further transcript profiling work on gametes shall resolve this issue.

Top 30 candidates up-regulated in the pollen

Meanwhile, 9 out of 30 highly abundant transcripts in mature soybean pollen are predicted to encode members of pectin esterase and pectate lyase families of cell-wall loosening enzymes (Table 1). Corresponding genes in Arabidopsis were among those with the highest expression in pollen [2, 4, 5]. It has been proposed that besides their possible involvement in pollen tube wall modification, these hydrolytic enzymes may be important for the penetration of the stigmatic tissues.

Transcription factors up-regulated in the soybean pollen

Zinc finger transcription factors are prominent in our differentially regulated gene data (25 genes). Although reported as pollen-specific genes in 1992 [11], zinc finger proteins act as master regulators (transcriptional repressors) in neuronal development, animal germ cells, and spermatogenesis [12]. For instance, Blimp1/Prdm1, a zinc finger transcriptional repressor, is the key regulator of early axis formation and primordial germ cell specification in animals [13]. Also, it has been shown that a targeted silencing of Ovol1 (also known as movo1), a zinc-finger transcription factor, leads to germ cell degeneration and defective sperm production in mice [14]. These proteins are also reported to be important regulatory molecules in various plant developmental processes, such as apical meristem development via chromatin remodeling process, anther development, and flowering.

It has been recently reported that a class of MYB factors regulate sperm cell formation in plants [15]. We identified three members of the MYB family as up-regulated in soybean pollen (Table 2). Certain MADS box proteins have been identified as pollen-specific in Antirrhinum [16] and have also been reported as an important non-classical transcriptional factor family in Arabidopsis pollen. Pina et al reported the over-representation of MADS box genes in the Arabidopsis pollen transcriptome, with 17 genes expressed in pollen and nine showing enrichment in pollen [3].

Plant homeodomain (PHD) finger transcription factors are up-regulated in soybean pollen. The PHD finger may promote both gene expression and repression through interactions with trimethylated lysine 4 on histone H3 (H3K4), a universal modification seen at the beginning of active genes [17, 18]. PHDs are associated with chromatin condensation during mitosis or meiosis, general transcriptional machinery, and a transcriptional regulator required for proper development, flowering, and fertility of plants [19, 20].

Meanwhile, very little is known about the physiological and developmental roles of WRKY proteins, another family of transcription factor up-regulated in the soybean pollen. Although the DNA binding site of WRKY proteins is well-defined, determining the individual role of WRKY factors remains a challenge [21, 22]. Though the function of WRKY proteins in pollen is not clear, our data suggest an important and novel regulatory role for these proteins in soybean pollen.

A member of the basic helix-loop helix (bHLH) transcription factor also shows differential expression in soybean pollen; this group also shows a similar pattern of expression in Arabidopsis pollen. bHLH proteins are a family of transcription factors that bind to their DNA targets as dimmers [23, 24]. They have been characterized in non-plant eukaryotes as important regulatory components in diverse biological processes such as the control of cell proliferation and the development of specific cell lineages. It has been shown that Tcfl5, a testis-specific bHLH protein, interacts with the regulatory region of the Calmegin gene promoter as a testis-specific activator of this gene and other testis-specific genes in mouse spermatogenesis [25]. Whether pollen-expressed bHLH transcription factors regulate sperm cell specific gene expression remains to be determined. Two NAC transcription factor family members are up-regulated in soybean pollen, suggesting a role of this family of proteins in the regulation of pollen genes, a function that to the best of our knowledge has not been reported for this class of genes.

Transcripts associated with the ubiquitin system

Signal transduction and transporters

Higher plants possess two distinct families of sugar carriers: the disaccharide transporters that primarily catalyse sucrose transport and the monosaccharide transporters that mediate the transport of a variable range of monosaccharides [37]. The STP4 gene encodes a membrane located monosachharide H+ symporter that can catalyze the uptake of various monosaccharides [38]. High expression of monosachharide transporter in soybean pollen points towards glucose and fructose as preferred source of nutrition for pollen germination and tube growth. A similar pollen specific expression of a putative hexose transporter gene was reported in Arabidopsis and Petunia [39, 40]. It has been proposed that in species where monosachharides are taken up preferentially, sucrose might be hydrolysed to glucose and fructose by a cell-wall invertase before uptake by monosachharide transporters in the growing pollen tube.

High up-regulation of H⁺ATPases including those encoding AHA8 and AHA9 in soybean pollen points to an essential role similar to their Arabidopsis and Nicotiana counterparts. The expression of AHA8 and AHA9 has been shown to be pollen-specific in Arabidopsis [3]. Recently, a pollen H+ ATPases has been shown to be associated with the tip growth in Nicotiana pollen tubes [41]. Uptake and translocation of cationic nutrients play essential roles in plant growth, nutrition, signal transduction, and development [42]. The plant cation transporter gene families include potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels and cation diffusion facilitator proteins. Our data show that several of the members of cation/proton exchanger family proteins are expressed at a higher level in the soybean pollen in comparison to those of sporophytic tissues. Bock et al [36] reported that fourteen members of the cation/proton exchanger (CHX) gene family are expressed late in pollen development and also raised questions about their roles and multiplicity. The possibility that they are localized to different intracellular compartments was proposed. It is noteworthy that a similar multiplicity of cation/proton exchanger family genes that are up-regulated in the soybean pollen is apparent in our data.

WD-40 repeat proteins

Heat shock proteins

Heat shock proteins (HSPs)/chaperones) are divided in five major families: the HSP70, the HSP60, the HSP 90, the HSP 100 families and a small HSP family [46]. The accumulation of heat shock proteins (HSPs) under heat and other abiotic stresses has been suggested to play a key role in the acquisition of thermotolerance in plants and other organisms. At the cell level these proteins are responsible for protein folding, assembly, and translocation, and can assist in protein re-folding under stress conditions. Some studies could not detect heat shock response in developing microspores or mature pollen of various species [47, 48] while others have shown that many HSPs are expressed in microspores and mature pollen [49].

It is interesting to note that in our present study on mature soybean pollen transcriptome, there is significant up-regulation of transcripts encoding heat shock proteins as well as heat shock transcription factors HSFB2A and HSFA5 (Table 7; Figure 3). A recent study on transcriptome changes during pollen germination showed significant up-regulation of HSPs during pollen germination and tube growth, and many of these HSPs are undetectable at the expression level in mature pollen [50]. These authors proposed that these HSPs might function as molecular chaperones for protein modification processes during pollen germination and tube growth. Heat shock factors are the primary molecules responsible for activating genes responsive to both heat stress and other stressors [51]. The up-regulation of heat shock transcription factor HSFB2A and HSFA5 in soybean pollen matches similar up regulation of its counterpart in Arabidopsis pollen [51]. The plant HSF family has been reported to comprise more than 20 members with recent evidence pointing towards the unique functions of individual HSFs in signal transduction pathways activated in response to environmental stress and during development. Conserved up-regulation of HSFB2A and HSFA5 in both soybean and Arabidopsis pollen points towards unique role of these transcription factors in pollen development and possibly in gamete development. It is interesting to note that heat shock proteins are known for their role in animal spermatogenesis by acting as molecular chaperones to assist with protein folding [52].

Plant growth and pollen collection

Soybean plants [Glycine max. (L) Merr. Cv. Bragg] were used in this study. The plants used for pollen collection were grown in a temperature-controlled greenhouse with a 16 hour light/8 hour dark photoperiod at 30°C. They were grown in vermiculite with the addition of a slow release fertilizer (osmocote). When the plants had matured and developed significant biomass, flowering was induced by changing the photoperiod to 12 hours. Pollen was collected on coverslips by rubbing isolated anthers together, and anther tissue was removed from the coverslip prior to freezing at -80°C. Pollen purity and viability was assessed by microscopic observations and fluorescein diacetate test (Figure 4).

RNA isolation and microarray hybridization

Total RNA from pollen or sporophytic tissues (primary stem, primary roots and mature leaves of 10-day-old soybean seedlings) was isolated using the QIAGEN RNeasy Mini Kit (QIAGEN) and eluted with nuclease-free water. Subsequent cDNA labelling and Affymetrix Soybean GeneChip hybridization was carried out by AGRF (Australian Genome Research Facility, Melbourne, Australia) using 3 μg of total RNA according to protocols outlined in http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf.

Analysis of expression data

The GeneChip^® Soybean Genome Array (Affymetrix, Inc.) containing probe sets for 37,500 transcripts was used in this study. Three biological replicates for pollen and two biological replicates for sporophytic tissues were used. Raw numeric values representing the signal of each feature were imported into AffylmGUI (Affymetrix linear modeling Graphical User Interface [7] that uses the Empirical Bayes linear modeling approach of Smyth (2005)[53] for identifying differentially expressed genes in pollen. The data were normalized using Robust Multiarray Averaging (RMA) method and a linear model was then used to average data between replicate arrays and to look for variability between them [7]. The list of transcripts that were detected to be differentially expressed at adjusted p-value of < 0.05 were used for all subsequent analysis. All microarray data have been submitted to Gene Expression Omnibus (GEO) at NCBI http://www.ncbi.nlm.nih.gov/geo under the accession GSE 12286.

To obtain the number of pollen-expressed genes (expressed in pollen and sporophytic tissues), we collect the expression signals, average expression values, and present/absent calls from AffylmGUI (RMA data) and sorted the data in Excel. To find pollen-specific group of genes, we used the following criteria: 1) showed statistically significant differential expression at adjusted pvalue < 0.05; 2) possessed a signal greater than or equal to 100 on each replicate; 3) had a cut-off value of a 2-fold change; and 4) had "Absence" calls on all of the sporophytic replicates.

The annotation for the transcripts represented by the soybean GeneChip^® was downloaded from the Seed Development website http://estdb.biology.ucla.edu/seed/. The annotation is based on the best BLASTX match of the corresponding soybean sequences against TAIR Arabidopsis protein database or NCBI non-redundant protein database (expect value < 0.01). Functional categories for these transcripts were assigned based on the EU Arabidopsis sequencing project [54] as described at the Seed Development website http://estdb.biology.ucla.edu/seed/.