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, 144 (1), 155-72

Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated With the Establishment of Rapeseed Microspore-Derived Embryo Development

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Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated With the Establishment of Rapeseed Microspore-Derived Embryo Development

Ronny Joosen et al. Plant Physiol.

Abstract

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.

Figures

Figure 1.
Figure 1.
Developmental fate of isolated rapeseed microspores. The culture temperature controls the developmental fate of isolated microspores (A). Conventional MDEs are obtained by continuous culture at 32°C (B–D). Suspensor-bearing MDEs are obtained by culturing for 24 h at 32°C followed by transfer to 25°C (E–H). Developing pollen are obtained by continuous culture at 18°C (I–K). The timeline at the bottom of the figure indicates the day of culture. Illustrated developmental stages are representative of the samples analyzed in this study; however, the timing of their appearance varies depending on the individual culture. The solid and dashed lines under each developmental pathway represent, respectively, the period of viable pollen formation and the period of pollen collapse. A, Freshly isolated late unicellular microspore. B, Four-celled embryo after 2 d of culture. C, Randomly divided multicellular embryo bursting out of the exine wall (white arrow) after 5 d of culture. Note the absence of a defined protoderm layer. D, Globular to early heart-stage embryos formed after 10 d of culture. E, Two-celled embryo at 4 to 5 d of culture. F, Uniseriate suspensor formed at day 6 to day 7 of culture. G, Longitudinal divisions in the distal tip cell of the filament leading to a four-cell embryo proper after 8 d of culture. Two additional nuclei are present behind the two nuclei in focus. The original microspore wall (white arrow) is visible at the opposite end of the cell file. H, Preglobular-stage embryo after 9 to 10 d of culture showing differentiation of the protoderm (white arrow) and hypophysal cell (asterisk). I, Bicellular pollen with a large vegetative nucleus and a smaller generative nucleus. J, Tricellular pollen with a large vegetative nucleus and two smaller sperm nuclei. K, Collapsed pollen-like structures. The developmental stages of the samples used for microarray analysis are as follows: 0d, starting microspore culture; 5p, 5-d pollen culture; 2e, 2-d conventional MDE culture; 5e, 5-d conventional MDE culture; 10e, 10-d conventional MDE culture; 8se, 8-d suspensor-bearing MDE culture; 10se, 10-d suspensor-bearing MDE culture. Photographs A to J are digital images from 4′,6-diamidino-2-phenylindole-stained material observed using a Zeiss Axioskop microscope or with an IM Zeiss microscope. Bars = 10 μm for A, B, E, F, I, and J; 30 μm for C, D, G, H, and K.
Figure 2.
Figure 2.
Effect of different periods of heat-stress treatment on suspensor formation in rapeseed microspore culture. A, The frequencies of embryos with long, normal-looking suspensors ≥30 μm (dark gray bars), embryos with short suspensor-like structures (irregular-shaped appendant structures or uniseriate structures <30 μm; light gray bars), or embryos without a suspensor (white bars), B, Total embryo yield. All cultures were started with a density of 40,000 microspores per milliliter. After the heat-stress treatment, cultures were kept at 25°C. For the determination of total embryo yield (B), embryos larger than 0.4 mm were counted after 16 d of culture when the largest embryos had already reached 3 mm. Data are the means of five replicate experiments, each with three 6-cm petri dishes per treatment and 3 mL of original microspore suspension per dish. Bar = ±se.
Figure 3.
Figure 3.
PCA of gene and protein expression profiles detected in rapeseed pollen and MDE cultures. PCA plots of gene (A) and protein (B) expression profiles are shown. For both plots, the replicated culture samples/pools are indicated. For the PCA plot of the gene expression profiles, culture 1 and culture 2 are indicated as 1 and 2, respectively. For each plot, the corresponding distribution of genes or proteins is shown in the inset. The composition of the analyzed samples in terms of the percentage of gametophytic and embryogenic structures was incorporated into the plot of the gene expression profiles (vectors). 0d, Starting microspore culture; 5p, 5-d pollen culture; 2e, 2-d conventional MDE culture; 5e, 5-d conventional MDE culture; 10e, 10-d conventional MDE culture; 8se, 8-d suspensor-bearing MDE culture; 10se, 10-d suspensor-bearing MDE culture.
Figure 4.
Figure 4.
Hierarchical cluster analysis of the expression profiles of rapeseed probes expressed in freshly isolated microspores, pollen cultures, or MDE cultures. The columns represent the samples and the rows the individual probes. Probes that are up- or down-regulated compared to the common reference are indicated in red and green, respectively. The intensity of the colors increases with increasing expression differences, as shown in the bar at the bottom. The pollen and embryo up-regulated clusters are indicated with arrows. Only probes for which an expression ratio for both culture samples could be calculated were included in the analysis (1,059 probes). 0d, Starting microspore culture; 5p, 5-d pollen culture; 2e, 2-d conventional MDE culture; 5e, 5-d conventional MDE culture; 10e, 10-d conventional MDE culture; 8se, 8-d suspensor-bearing MDE culture; 10se, 10-d suspensor-bearing MDE culture. LF, Mature leaves; FB, flower buds (3–4 mm).
Figure 5.
Figure 5.
Robust markers for MDE development. The number listed beside each marker corresponds to the assigned probe number. The expression profile of a representative probe is presented when a marker is represented by multiple probes (number of multiple probes indicated in parentheses). The expression ratio (log2 scale) of each probe or probe set relative to the common reference is indicated. Color coding of the expression ratios is as in Supplemental Figure S4. S, Probes that are expressed at significantly higher levels in preglobular suspensor-bearing MDEs as compared to preglobular conventional MDEs. 0d, Starting microspore culture; 5p, 5-d pollen culture; 2e, 2-d conventional MDE culture; 5e, 5-d conventional MDE culture; 10e, 10-d conventional MDE culture; 8se, 8-d suspensor-bearing MDE culture; 10se, 10-d suspensor-bearing MDE culture; LF, mature leaves; FB, flower buds (3–4 mm).
Figure 6.
Figure 6.
BNM2 BURP domain expression analysis. A, RNA gel-blot analysis of BNM2 gene expression in rapeseed pollen and MDE cultures. Total RNA was isolated from microspores at the start of culture (0); from embryo cultures after 4 d in culture at 32°C (E); from nonembryogenic heat-stressed microspore cultures (NE) obtained by culturing microspores for 1 d at 25°C, followed by 3 d at 32°C; from pollen cultures after 4 d at 25°C (P); and from purified MDEs at the globular (G), heart (H), torpedo (T), 21-d cotyledon (C1), 28-d cotyledon (C2), and 28-d cotyledon stage (C3). B, RNA gel-blot analysis of BNM2 transcripts in developing seeds. RNA samples were isolated from whole seeds collected at specific days after pollination (DAP). These developmental time points correspond approximately to the globular (7 DAP), heart/torpedo (14 DAP), early cotyledon (21 DAP), midcotyledon (28 DAP), and mid-to-late cotyledon (35 and 42 DAP) stages of development. C, RNA gel-blot analysis of BNM2 transcripts in nonseed tissues. RNA samples were isolated from mature leaves (L), roots (R), and stem (S), flowers at anthesis (F), small flower buds (SB, <3 mm), large flower buds (LB, 5–7 mm), and anthers (A) and pistils (P) and from flowers at anthesis. For A, B, and C, 10 μg of total RNA was loaded per sample. Ethidium bromide staining of RNA was used to compare sample loading. D to G, mRNA in situ hybridization analysis of BNM2 expression in MDEs (D and E) and seeds (F and G). The probe used for RNA gel-blot analysis and mRNA in situ hybridization detects at least two duplicated copies of the BNM2 gene in the amphidiploid rapeseed genome.
Figure 7.
Figure 7.
Heat map of the relative signal intensities of proteins expressed in rapeseed pollen and MDE cultures. Signal intensities for each protein spot were normalized to the highest signal intensity for that protein spot (100%) in the culture series. Values shown are relative (i.e. they do not reflect the absolute expression levels of the proteins in the samples). The heat map was created in GeneMaths, version 2.01 (Applied Maths). The color-code bar is shown on the right. Sample legend as in Figure 3.
Figure 8.
Figure 8.
Integration of sequence and expression data obtained from transcriptome and proteome analyses of rapeseed pollen and MDE development. In the network, the nodes represent the cDNA probes (circles) and protein spots (squares), whereas the line connecting the nodes represents the sequence similarity between the protein and cDNA sequences. The sequence similarity is indicated by the thickness of the lines (see inset). The similarity in expression profiles is represented by the color of the connecting lines (see inset). The assigned probe and protein numbers are shown inside the nodes. The cytoscape output is shown in Supplemental Table S5.

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