Comparative transcriptional profiling analysis of the two daughter cells from tobacco zygote reveals the transcriptome differences in the apical and basal cells
© Hu et al; licensee BioMed Central Ltd. 2010
Received: 28 June 2010
Accepted: 11 August 2010
Published: 11 August 2010
In angiosperm, after the first asymmetric zygotic cell division, the apical and basal daughter cells follow distinct development pathways. Global transcriptome analysis of these two cells is essential in understanding their developmental differences. However, because of the difficulty to isolate the in vivo apical and basal cells of two-celled proembryo from ovule and ovary in higher plants, the transcriptome analysis of them hasn't been reported.
In this study, we developed a procedure for isolating the in vivo apical and basal cells of the two-celled proembryo from tobacco (Nicotiana tabacum), and then performed a comparative transcriptome analysis of the two cells by suppression subtractive hybridization (SSH) combined with macroarray screening. After sequencing, we identified 797 differentially expressed ESTs corresponding to 299 unigenes. Library sequence analysis successfully identified tobacco homologies of genes involved in embryogenesis and seed development. By quantitative real-time PCR, we validated the differential expression of 40 genes, with 6 transcripts of them specifically expressed in the apical or basal cell. Expression analysis also revealed some transcripts displayed cell specific activation in one of the daughter cells after zygote division. These differential expressions were further validated by in situ hybridization (ISH). Tissue expression pattern analysis also revealed some potential roles of these candidate genes in development.
The results show that some differential or specific transcripts in the apical and basal cells of two-celled proembryo were successfully isolated, and the identification of these transcripts reveals that these two daughter cells possess distinct transcriptional profiles after zygote division. Further functional work on these differentially or specifically expressed genes will promote the elucidation of molecular mechanism controlling early embryogenesis.
Embryo development from one-celled zygote to mature embryo is a critical part of the life cycle in higher plants. During double fertilization, one sperm cell from pollen grain fuses with an egg cell from embryo sac, and the resultant zygote undergoes a series of precise cell divisions and develops into an embryo [1, 2]. In most angiosperms, the first zygotic cell division is transverse and asymmetric, resulting in the formation of a two-celled proembryo with a small apical cell and a large basal cell. The small apical cell with dense cytoplasm develops into embryo proper, and the large vacuolated basal cell differentiates into hypophysis and suspensor. The hypophysis contributes to the formation of root meristem within the embryo proper . The suspensor, a terminally differentiated embryonic region, connects the embryo proper to the surrounding maternal tissues, serves as a conduit for nutrients and growth regulators supporting embryo development, and degenerates in the late embryo development . In Arabidopsis, the mutations of gnom (gn), root-shoot-hypocotyl-defective (rsh) and yoda (yda) alter the asymmetric division of zygote, and result in the formation of two nearly equal-sized daughter cells and subsequent defect of embryonic axis establishment [5–7]. It suggests that the asymmetric division of zygote producing the apical and basal cells is a crucial event of early embryogenesis.
Previous researchers adopted various techniques and experiment systems to investigate embryogenesis mechanism. In lower plant, the zygote and embryo of brown alga (Fucus) have long been served as a cellular model to investigate early embryogenesis because of their development free of maternal tissue [8–10]. However, embryo sac in higher plants is typically surrounded by the sporophytic tissues of ovule and ovary, thus access to the embryo is hampered. To overcome these difficulties, the researchers utilize some in vitro culture systems to study the early embryo development mechanism [11–15]. Compared with embryogenesis in vivo, there are some differences in the way of embryos originate and develop, therefore, the results obtained in vitro fail to explain all the questions.
Since specific gene expression is usually linked directly to different developmental process, many techniques are exploited to identify genes expressed in the developing embryo, including cDNA library construction , promoter/enhancer trapping  and mutational screens [18, 19]. Several embryo essential genes, such as gn, twin (twn), monopteros (mp), bodenlos (bdl), topless (tpl) and yda, were successfully identified by the mutant analysis in Arabidopsis [6, 7, 20–23]. cDNA libraries from complex tissues such as ovule are not efficient in identifying genes expressed at low level or only in the early several-celled proembryos. Recently, the development of laser capture microdissection (LCM) makes it possible to analyze the transcriptional profiles in specific embryo domains [24, 25], but the single egg cell, zygote or early several-celled embryo are still too small to be isolated. Fortunately, micromanipulation, a powerful skill, is used successfully to isolate single cells from the embryo sac of some species such as maize, barley, tobacco, wheat and rice [11, 26–29]. This technique combined with the transcriptome assay broadens our knowledge of gene expression in egg cell, central cell, zygote and proembryo [30–34], and these valuable information help us to understand certain critical questions such us zygote gene activation in higher plants.
Some genes up- or down-regulated in the two daughter cells from in vitro fertilized maize zygote were identified by Okamoto et al. . However, besides the difference of embryogenesis in vitro and in vivo, there are even greater differences between embryo development in monocotyledon and dicotyledon plants. In contrast with the fixed and traceable division pattern during early embryogenesis in classic dicotyledon plants, variant cell division occurs during the proembryo development of monocotyledon plants . Up to now, the analysis of transcriptional profiles in the in vivo apical and basal cells from the two-celled proembryo in dicotyledon plants is not reported. Therefore, in the present study, we focused on the transcriptome differences between the two cells in dicotyledonous plant tobacco. We originally established a procedure for isolating the live apical and basal cells of the in vivo two-celled proembryo just after the first cell division of zygote, and then the SMART PCR synthesized cDNAs from these two cells were used for SSH analysis. After macroarray screening and sequencing of the candidate clones, we successfully identified 797 ESTs that were specifically or predominantly expressed in the apical or basal cells. The ESTs were further analyzed, including comparative studies on the different transcript composition, functional classification, and validation by real-time PCR and in situ hybridization in the zygote and its apical and basal daughter cells. Also, the expression patterns of some identified ESTs were analyzed in different organs and tissues. The transcriptional composition differences in the apical and basal cells and possible function of some candidate differential transcripts are further discussed.
Establishment of isolation procedures for the apical and basal cells from the two-celled proembryo
After the first zygotic division, tobacco zygote produces two asymmetric daughter cells just like most classic dicotyledonous plants. Morphologic observation of the isolated two-celled proembryoes revealed that the shape and size of apical cell is distinct from those of the basal cell. As shown in Figure 1h, the apical cell is small and nearly spherical, whereas the basal cell is relatively large and elongated. To epitomize the cell size differences between the two cells, we measured cell length in vertical axis, width in transverse axis as well as the diameters of the two protoplasts (Figure 1k). The results show that the ratios of the basal cell to the apical cell are 2.28 in length and 0.80 in width, and the diameter of basal cell protoplast is larger than that of apical cell, with 18.37 ± 2.05 μm versus 15.41 ± 1.54 μm. To display the distinct difference in size, several couples of the isolated apical and basal cells were shown in the same bright field (Figure 1b).
cDNA synthesis and identification of differentially expressed genes between the apical and basal cells
To reveal the transcriptome differences of the apical and basal cells, suppression subtractive hybridization (SSH) was applied to identify the differentially expressed genes. Both forward (apical cell/basal cell, apical cell cDNA as tester) and reverse (basal cell/apical cell, basal cell cDNA as tester) subtracted cDNA libraries were constructed to enrich the genes specifically or predominantly expressed in the apical or basal cells. Pools of putative differentially expressed cDNA were obtained after two rounds of subtraction. Compared with their respective unsubtracted control, both the subtracted DNA samples displayed a quite different distribution with a number of distinct bands. The forward subtracted cDNA ranged mainly from ~300 bp to ~1 kb and the reverse subtracted cDNA from ~250 bp to ~750 bp (Figure 2b).
Bioinformatic analysis of ESTs
Functional annotation of the differentially expressed contigs with two or more ESTs between the apical cells (a) and basal cells (b)
BLASTX sequence similarity (aBlastN)
(a) Apical cell
cytochrome P450 like protein
40s ribosomal protein s23
lipid transfer proteins related
40s ribosomal protein
60s ribosomal protein l23
cytochrome P450 protein
hypothetical protein isoform 2
ribosomal protein s27
tabacum cDNA clone mRNAa
tabacum cDNA clone mRNAa
tabacum cDNA clone mRNAa
ubiquitin extension protein
Petunia × hybrida
(b) Basal cell
NADH dehydrogenase subunit 7
tabacum cDNA clone mRNAa
pathogenesis-related protein 10
cacao cDNA clone mRNAa
tabacum cDNA clone mRNAa
embryo abundant methyltransferase
sugar transport protein 8
tomato cDNA clone mRNAa
tabacum cDNA clone mRNAa
tabacum cDNA clone mRNAa
tabacum cDNA clone mRNAa
nucleoside diphosphate kinase
defender against cell death 1
N.suaveolens × N.tabacum
histone h3.3B isoform 2
60s ribosomal protein l35a
tabacum cDNA clone mRNAa
tabacum cDNA clone mRNAa
60s ribosomal protein l29
cell growth defect factor 2
tabacum cDNA clone mRNAa
40s ribosomal protein s19
DNA-directed RNA polymerase II
nucleoside diphosphate kinase
tabacum cDNA clone mRNAa
tabacum cDNA clone mRNAa
Putative tobacco homologies of Arabidopsis genes involved in normal embryo development
BLAST e-Value/% Identity/%% Similarity
1e-80 96% 100%
Ribosomal Protein L17/L23
1e-41 98% 100%
Ubiquitin Fused to Ribosomal Protein L40
4e-16 35% 46%
WUSCHEL RELATED HOMEOBOX 2
2e-07 35% 40%
Adherin sister-chromatid cohesion 2
1e-15 33% 61%
Chloroplast RNA Binding Protein
2e-10 36% 52%
Spliceosome Associated Protein
4e-49 55% 75%
Heat Shock Protein (Hsp90)
4e-10 50% 70%
Digalactosyl Diacylglycerol Synthase
1e-12 39% 59%
Ubiquitin Fused to Ribosomal Protein L40
2e-87 88% 95%
Ribosomal Protein S5
7e-31 75% 87%
Protein Tyrosine Phosphatase Like
2e-08 33% 47%
Immunophilin-like FK506 Binding Protein
Validation of the differential expression in zygote and its two daughter cells
Combining the zygotic expression of transcripts with the expression in apical and basal cells, two different expression patterns were observed in the examined transcripts: (i) specifically expressed only in the apical cell (Figure 4d, e, l, m, x) or in basal cell (Figure 5a-c, h, j); (ii) expressed in zygote, and subsequently predominantly in the apical cell (Figure 4k, n, p) or in the basal cell (Figure 5e, f, l, m). It's very interesting that about one third of the tested transcripts showed only negligible signals in the zygote, while greatly enhanced the expression in one of the daughter cells after zygotic division, suggesting an mechanism controlling specific gene activation in the apical and basal cells for early embryogenesis.
Whole mount in situ hybridization of the isolated zygote and two-celled proembryos
Expression analysis of candidate genes in different organs and tissues
The apical and basal cells of tobacco two-celled proembryo possess distinct transcriptional profiles
Zygotic gene activation (ZGA) is a critical event during early embryogenesis, which means the transfer of development control from parents to zygote and embryo. Some evidences substantiate the assumption that ZGA in higher plants occurs shortly after fertilization [6, 33, 34, 41]. In our study, some differential transcripts were expressed in zygote and then only in one of the daughter cells, suggesting the possibility that these transcripts were specifically inherited by the apical or basal daughter cell (Figure 8). The other tested transcripts just displayed weak or negligible expression in zygote, but strong in the apical or basal daughter cell. The results indicate that apart from ZGA, further gene activation in early embryogenesis may also happen in the apical and basal cells, respectively (Figure 8). These cell specific transcript inheritation and activation may lead to the transcriptome differences in the apical and basal cells.
Some candidate genes from the apical and basal cells play potential roles in embryo and post-embryo development
It seems that some of the 299 transcripts encode proteins required for gamete and early embryo development based on the homology search against Arabidopsis and rice. As shown in Table 2, 12 transcripts encode homologies of the genes involved in Arabidopsis embryo and seed development, such as PASTICCINO1/2 (PAS1/2) and WOX2. In Arabidopsis, WOX2 and WOX8 genes are expressed complementarily in the apical and basal cells in a lineage-specific manner and regulate respective cell fate decision during early embryogenesis [39, 42]. Our results show that the tobacco homology of WOX2 (ACC43) is also predominantly expressed in the apical cell of two-celled proembryo. Another apical cell transcript (ACC12), which encodes a tobacco lipid transfer protein, showed specific expression activation in the apical cells. In Arabidopsis embryogenesis, lipid transfer protein gene (AtLTP1) showed a specific position expression in the embryo, with transcript accumulation exclusively in the protodermal cells of the globular embryos and in the cotyledons and the upper end of hypocotyl in late stage of embryos .
In the basal cell transcripts, BCC12 transcript, a putative embryo abundant methyltransferase, displayed predominant expression in zygote and its basal daughter cell (Figure 5). In mouse embryogenesis, histone arginine methylation mediated by arginine methyltransferase 1 (CARM1) contributes to cell fate decision in the four-cell-stage of embryo . Moreover, the mutant analysis for Arabidopsis METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE3 (CMT3) gene revels that DNA methylation is critical for the regulation of cell fate decision during early embryogenesis . Besides, the basal cell transcript BCC39 encodes a tobacco homology of the cell growth defect factor 2 (Cdf2) in Arabidopsis , and the overexpression of Cdf2 caused Bax-like lethality in yeast . Bax is a mammalian proapoptotic member of the Bcl-2 family, and the overexpression of Bax in Arabidopsis mesophyll protoplasts resulted in cytological apoptosis characteristics . Therefore, such gene may involve in the programmed cell death (PCD) mediated degeneration of the future suspensor (Figure 8).
In our study, tissue expression analyses also show that several transcripts are abundant in the different stages of ovules, but barely detectable in vegetative tissues, indicating their possible functions in embryo and ovule development as well as seed formation, such as transcripts ACC34, AC190C, AC373C and BC388C (Figure 7). Besides expression in ovules, five differential transcripts (AC334C, AC338C, AC356C, BCC13 and BCC45) in the apical and basal cells displayed predominant expression in mature anthers. In Arabidopsis, the mitogen-activated protein kinase gene YDA functions in the process of zygote elongation and subsequent cell division, and regulates the first cell fate decision of the basal lineage . Recently, the study reveals that the SHORT SUSPENSOR (SSP) transcripts accumulate in mature pollens, and then are delivered via the sperm cells to zygote, where SSP protein is produced to activate YDA-dependent signalling . On one hand, our anther expressed transcripts may play roles in anther development, and on the other hand, it is reasonable to speculation that these transcripts are transferred to zygote via sperm cells and regulate the subsequent embryo development. Furthermore, another three transcripts (ACC13, BCC05, BCC18) show preponderant expression in tobacco roots (Figure 7). It's well known that auxin is important for pattern formation in embryo and root development [49, 50]. In our study, one root expressed transcript BCC18 encodes an auxin induced parA protein in tobacco , suggesting that this protein may involve in auxin regulated embryo differentiation and subsequent root formation. Further research of these candidate genes in this study will contribute to elucidate the regulation mechanism of early embryo polarity establishment and pattern formation as well as succedent organ development in higher plants.
Here we first established a procedure for isolating the live apical and basal cells of tobacco two-celled proembryo just after the first zygotic cell division in vivo. In dicotyledon plant, for the first time, we carried out a global investigation to the transcription profiles of the apical and basal cells in vivo by applying SSH technique coupled with macroarray hybridization. Further validation by quantitative RT-PCR and ISH technique showed that some differential and specific transcripts in the apical and basal cells of two-celled proembryos were successfully isolated, and the differential and specific expression of these transcripts revealed that the transcription compositions in the apical and basal cells are significantly distinct. Transcripts with specific expression in the apical and basal cells provide useful markers for research on the early embryogenesis. Some identified genes specifically expressed in the ovules, suggesting close relation to specific events of the embryo and seed development. Therefore, functional analysis of these genes will promote promising research on molecular mechanism of embryogenesis and seed development.
All 797 EST sequences in the study (library ID AC001C- 385C and BC001C-412C) were deposited in GenBank with accession numbers from GT270790 to GT271586.
Isolation of the zygote and the apical and basal cells from the two-celled proembryo
Tobacco (Nicotiana tabacum cv. SR1) plants were grown in a greenhouse with a photoperiod of 16 h light/8 h dark at 25-27°C. The elongated zygotes and the two-celled proembryos were isolated respectively from ovules at 84 and 108 h after pollination (HAP) according to the method of Qin et al. . The isolated two-celled proembryos were collected into a droplet of 13% (w/v) sterile mannitol solution (pH 5.7) with a micropipette. To avoid the confusion of apical cells and basal cells from different two-celled proembryos, each proembryo was then transferred into an individual droplet of mannitol solution containing 1% cellulase onozuka-R10 (Yakult), 0.5% pectinase (Sigma), 1% hemicellulase (Sigma) and 0.5% snailase (Sigma) for enzymolysis. The two-celled proembryos were incubated in the enzyme solution for 10-15 min at 25°C. By gently sucking and spitting with a micropipette, a pair of protoplasts with a small apical cell and a large basal cell was separated. The two kinds of protoplasts were respectively collected into fresh 13% (w/v) mannitol droplets and washed twice, then transferred into the lysis/binding buffer and immediately frozen in liquid nitrogen. The viability of isolated protoplasts was detected using 50 mg/L fluorescein diacetate (FDA; Sigma) staining. Two transcription inhibitors, actinomycin D (50 mg/L, Sigma) and cordycepin (100 mg/L, Sigma), proven effective in suppressing the expression of stress-inducible genes , were added to all solutions in the process of cell isolation.
RNA isolation of the zygote, apical and basal cells and cDNA synthesis
For each independent cDNA synthesis, RNA from about two hundred zygotes, three hundred apical cells or basal cells were respectively extracted using the Absolutely RNA Nanoprep Kit (Stratagene) according to the manufacturer's instructions. Then cDNA was synthesized and amplified using a Super SMART PCR cDNA Synthesis Kit (Clontech). The optimal LD-PCR cycle number was determined empirically to ensure the cDNA remained in the exponential phase of amplification. Approximately 100 ng synthesized cDNA was analyzed on a 1.2% agarose gel alongside 100 ng 1 kb DNA ladder. Then, the amplified cDNAs of the apical and basal cells were used for SSH and templates for gene-specific expression analysis.
Suppression subtractive hybridization
The generation of forward- and reverse-subtracted cDNA and unsubtracted control cDNA from the apical and basal cells was performed using the PCR-Select cDNA Subtraction Kit (Clontech) following the manufacturer's instructions. Two rounds of hybridization and PCR amplification were performed to enrich the differentially expressed sequences, with 30 fold excess of the driver cDNA to select against for the first round subtraction and 2.5 fold for the second round subtraction. The subtracted apical and basal cell cDNAs were purified using QIAquick PCR Purification Kit (Qiagen), cloned with the pGEM-T Easy Vector System (Promega) and then transformed into Escherichia coli DH5α cell. The transformed bacteria were plated onto LB agar plates containing ampicillin, X-gal and IPTG. For constructing the subtracted apical and basal cell libraries, 4032 and 3300 recombinant white colonies were picked respectively, and cultured in 80 μl LB freezing medium with ampicillin in 384-well microtitre plates. After overnight culture, the plates were stored at -80°C for membrane printing.
Colony and cDNA macroarray preparation
Colony and cDNA macroarrays were used respectively for the first and second round screenings. For the colony macroarray, all selected clones from subtracted libraries were printed onto Hybond-N+ nylon membranes (Amersham Biosciences) using the Genetix QPix 2 Colony Picker Systems (Genetix Ltd). The nylon membranes were then placed onto LB agar plates with ampicillin and incubated at 37°C overnight. For the succedent cDNA macroarray, the first-round validated colonies were picked out for the second round screening with plasmids. The plasmids were isolated from overnight-grown bacterial cultures using a standard alkaline lysis protocol with SDS in 96-well format and then printed onto Hybond-N+ nylon membranes. All the macroarray membranes were treated following the user manuals, with DNA crosslinked to membranes by baking at 80°C for 2 h, and then were stored at -20°C for differential screenings.
Preparation of probes and cDNA differential screening
The probe labeling and macroarray hybridization were carried out using the PCR-select Differential Screening Kit (Clontech). The membranes were prehybridized for 40-60 min at 72°C, and then hybridized with the radioactive probes at 72°C overnight. The hybridized membranes were washed in 2 × SSC and 0.5% SDS for 4 × 20 min, in 0.2 × SSC and 0.5% SDS for 2 × 20 min at 68°C, and then exposed to PhosphorImager screens (Amersham Biosciences) for 24 hours. Images were acquired by scanning the membranes with a Typhoon 9210 scanner (Amersham Biosciences), and data analysis was performed using ArrayVision 8.0 software (Amersham Biosciences). The clones showing the most marked differential expression were selected for sequencing.
Sequence and bioinformatics analysis
The differentially expressed clones identified by screening were picked for sequencing with ABI3730 machines (Applied Biosystems). The vector and adaptor sequences were trimmed using Vector NTI Advance 9 software (Informax). After pre-processing, the expressed sequence tags (ESTs) were clustered and assembled into contigs using online tool EGassembler (http://egassembler.hgc.jp/; ). The assembled consensus sequences of contigs and valid ESTs were used as a query for BLASTN and BLASTX searches http://blast.ncbi.nlm.nih.gov/Blast.cgi, with significance threshold score >115, expected value <e-25 for BLASTN, and an e-value of <e-5, score > 50 for BLASTX. Transcripts encoding proteins of known functions were manually categorized into the functional classification described by Bevan et al. , with reference to the scarlet runner bean (SRB) embryonic EST project (http://www.mcdb.ucla.edu/Research/ Goldberg).
Gene expression analysis by quantitative real-time PCR
For expression analysis in the zygote and its two daughter cells, pre-amplified double-stranded cDNAs (ds cDNAs) using the Super SMART PCR cDNA synthesis kit were used. After purification and measurement, 20 ng of ds cDNA from each sample was used as template for real-time PCR analysis by SYBR-green fluorescence using the Rotor-Gene Q6000 system (Corbett Life Science). Cycling parameters were as follows: 94°C for 10 sec, 56°C for 20 sec, and 72°C for 30 sec. The cDNA samples used were independent from those of the SSH analysis and all expression patterns were confirmed by using two independent cDNA samples. For every examined gene, the expression levels in each sample relative to zygote were calculated.
For expression pattern analysis among different organs, the materials were taken as follows: root, stem and leaf from the one-month-old plants, anther and stigma/style from anthesis-stage flowers, 1 DAP (day after pollination ) ovules at the egg-celled stage, 3 DAP at the zygote, 5 DAP at early globular embryo, 7 DAP at late globular embryo, 8 DAP at heart-shaped embryo, 9 DAP at torpedo-shaped embryo and 12 DAP at cotyledon-staged embryo. Each reaction contained equal amount of sample cDNA, and the reaction was repeated at least twice. The constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPD) gene (Accession number AJ133422) was used as an internal standard. Primer pairs were all designed with Primer Premier Software (Premier Biosoft International) and listed in the Additional file 3.
Whole mount in situ hybridization
Digoxigeninlabeled RNA probes were generated with the DIG RNA labeling kit (Roche) according to the manufacturer's instructions, and in situ hybridization was performed as described by Hejátko et al. , with modification of embedding the isolated zygotes and the two-celled proembryos in 12% polyacrylamide. The elongated zygotes and the two-celled proembryos were isolated respectively from ovules at 84 and 108 h after pollination (HAP). Gel pieces containing the zygotes and proembryos after hybridization were incubated in a 1:2000 Anti-DIG-Antibody (Roche), and transcripts were detected colorimetrically by the DIG nucleic acid detection kit (Roche). The images were digitally recorded with a BH2 microscope (Olympus) and the digital sight DS-U2 camera system (Nikon).
This study was supported by National Natural Science Foundation of China (30970277, 30821064) and the Major State Basic Research Program of China (2007CB108704).
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