Gene Expression and Metabolic Network Dynamics during Embryo Development in Arabidopsis

 

Arabidopsis_thaliana-zandraket02Introduction

Arabidopsis thaliana is a small flowering plant with a short life cycle. More information on the flowering plant is given on the angiosperm home page. It is widely used for studying plant sciences and it is useful for understanding the genetic, cellular, and molecular biology of flowering plants(Meinke et al., 1998). Arabidopsis thaliana is used in most experiments as a model organism because it is well studied and has several genetic resources and databases. From this organism, metabolic networks can be mapped, embryo development can be further analyzed, and maternal and paternal gamete contribution can be established. These will provide a comprehensive view  of gene expression patterns and can lay the foundation for further examination of embryogenesis (Xiang et al., 2011).

You might be asking yourself, “What is Embryogenesis?” Embryogenesis is the mechanism by which the embryo forms and develops. It is central to the life cycle of most plant species and consists of two cycles, morphogenesis (establishes the embryos body plan) and maturation (cell expansion and storage) (Park et al., 2011). The cycle begins with the zygote, which is the product of fertilization of female gamete (egg cell) with the male gamete (sperm cell). During embryogenesis, the single celled zygote follows a defined pattern of cell division to form a mature embryo. The zygote divides asymmetrically to produce an apical cell and a basal cell. The apical cell undergoes two divisions to form an eight-celled embryo, or the octant-stage. The embryos divide further and eventually become a mature embryo (Xiang et al., 2011). This is described in the figure below.

 

actual

Figure 1: Representation of the stages of Arabidopsis embryo development. This figure emphasizes the apical, central, and basal embryonic domains. (a) two cell embryo, (b) four celled embryo, (c) octant-stage embryo, (d) dermatogen-stage embryo, (e) globular-stage embryo, (f) heart-stage embryo, (g) torpedo-stage embryo. 

Isolation of Developing embryos from Arabidopsis seeds

To understand embryogenesis, embryos from seeds were isolated. Isolating zygotes in early stages has been quite difficult because of the small size of the ovules after fertilization and the small size of the embryos within. A method was developed to extract the embryo from the ovule. In this study, two incisions were made at the micropylar tube (MT) in the ovule and the embryos were carefully extracted. Using this procedure, embryos at various stages were removed and examined. The figure below shows the distinct stages where the embryo was removed. These stages include zygote stage (B1-B3), quadrant stage (B4), octant stage (B5), dermatogen (B6), globular (B7), heart (B8), torpedo (B9), bent (B10), and mature (B11). BA represents the ovule at fertilization and C1 and C2 are the two incisions made to remove the embryo (Xiang et al., 2011).

figure 1

Figure 2: C1 and C2 are incisions made to release the embryo from the ovule (A); elongated zygote (B1); one-cell (B2); two-cell (B3); quadrant (B4); octant (B5); dermatogen (B6); globular (B7) heart (B8); torpedo (B9); bent (B10); mature (B11) embryos.

Results

Majority of the nuclear-encoded genes are expressed during embryogenesis

To determine if certain genes are expressed during embryogenesis, the embryos were directly compared to the adjacent embryo stages as well as combinations of embryos that were not sequential. This means that the sequential embryos consisted of 10 combinations: Z vs Q, Q vs G, G vs H, H vs T, T vs B, B vs M, Z vs H, Z vs M, Q vs T, and G vs B.

  • zygote (Z)
  • quadrant (Q)
  • globular (G)
  • heart (H)
  • torpedo (T)
  • bent (B)
  • mature (M)

The results from the micro-array were used to analyze the global gene expression levels of the expressed genes during embryo development. An analysis of the results suggested that cumulatively, 78% of the genes in the Arabidopsis genome were expressed during embryogenesis (table 1). The earliest stage (Z) had 58% of genes expressed. When the embryo developed from the globular to the heart (G–> H), the percentage was 62%. However, when the embryos reached maturity (M), the percentage of genes decreased to 55%, despite the embryo containing more cells at the mature stage (Xiang et al., 2011).

table

Table 1: The table represents the percent of genes expressed at each stage.

Within each stage, expression patterns that were less shared with adjacent stages were found

Expression patterns were found in the genes that function during cell cycle, metabolism, storage reserve synthesis, auxin and abscisic acid biosynthesis, and signaling. Within each stage, expression patterns that were less shared with adjacent stages were found. Additionally, overlapping functional gene groups were established within Z and Q stages (phase I), G and H stages (phase II), T and B stages (phase III), and M (phase IV). These expressions reflect the biologically specific programs that correlate to the phases. Auxin stimulus and signaling are more common in the first phase, while morphogenesis genes are more common in the second phase when the body is established and elongated. Phase three contains mostly storage proteins and fatty acids for storage reserves. Phase four includes abscisic acid and the embryo dessicates (Xiang et al., 2011).

Ribosomal proteins have a high presence during the earlier stages of embryogenesis during the Z, Q, and G stages. This is possible because the ribosomal proteins correlate with the embryo growth rate.

Maternal and paternal gene expression programs in zygote and quadrant stage embryos

Are parental gene expressions in fertilization important? The answer is yes! Chromatin activation, maternal transcripts, and maintenance of the zygote all play a crucial role. Gametes and zygote expression share an immense number of genes at the early stages Z and Q. Two samples were selected, one from pollen and the other from the embryo sac. The GUS reporter was used for paternal gene expression and GFP for maternal gene expression. Analysis of the results showed that pollen expression continued in the pollen tube after fertilization of the zygote, but was absent in the ovule. After fertilization, there was presence of the paternal gene in the zygote. For the maternal gene, there was no detectable expression in the pollen or the pollen tube, but there was GFP expression in the unfertilized ovule. This is represented by the figure below (Xiang et al., 2011).

Panels A-E includes the GUS reporter for paternal genes. Panel A is the pollen and panel B is the ovule. Panel C is the zygote after fertilization. As you can see in C, the red circle indicates expression of the genes. When a female GUS reporter was crossed crossed with a male wild type (WT), there was no expression seen (D). When a male GUS reporter was crossed with a female WT, there was expression as seen by the red outline in panel E.

Panels F-J includes the GFP reporter for maternal genes. Panel F and G show no expression in the pollen or in the pollen tube, respectively. Panel G1 shows a staining for sperm, and G2 shows no expression. Panel H displays GFP expression in an unfertilized ovule (yellow outline). In Panel I, pollen GFP reproter was crossed with WT female, and the results showed a GFP signal in the zygote (yellow outline). When the pollen from WT was crossed with female reporter line, GFP expression was observed (panel J).

parental

Figure 3: Paternal and maternal gene expression in early Arabidopsis embryogenesis. A through E, expression of paternal GUS reporter, F-J expression of maternal GFP reporter. pollen expression shown in blue (A); no paternal expression in ovule (B); expression in fertilized zygote (C); female GUS with WT male, no expression (D); male GUS with WT female, expression (E); no expression of maternal gene, GFP reporter in pollen (F); no expression of maternal gene in pollen tube (G); showed expression in ovule (H-J).

Conclusion 

In this study, live zygote to late stage embryos of Arabidopsos were successfully isolated. This is a huge success since it is very difficult to extract because of the small size. The first genome-wide gene expression was found using these embryos, and the data found will serve as a basis for future studies that address molecular and developmental mechanisms in embryogenesis. The key notes in this study include the dynamics of embryo-specific gene expression as the embryo matures. The findings of Arabidopsis embryogenesis will contribute to future plant embryo research.

Most of the parts of this experiment were easy to understand and it helped grasp concepts of why it was important. One of the weaknesses in this paper was that they did not go in-depth on the genomic or metabolic dynamics. They included one figure on metabolic networks for torpedo-bent stages, but it was very complicated. They did not explain the figure as well as needed. One of the strengths in this experiment was that it was easy to understand. The first half of the figures included descriptions which related to why the authors did the experiment. Not only will this study contribute to future studies using Arabidopsis thaliana,but the gene expression set can also be used to compare this plant to other species.

References

1. Xiang, D., et al. 2011. “Genome-Wide Analysis Reveals Gene Expression and Metabolic Network Dynamics during Embryo Development in Arabidopsis.” Plant Physiology. 156 (346-356).

2. Park, S., et al. 2011. “Arabidopsis Embryogenesis.” Plant Embryogenesis. 427.

3. Meinke, D.W., et al. 1998. “Arabidopsis thaliana: A Model Plant for Genome Analysis. ” Science. 282 (662).

4. https://www.kuleuvenkulak.be/kulakbiocampus/lage%20planten/Arabidopsis%20thaliana%20-%20Zandraket/Arabidopsis_thaliana-zandraket02.jpg (Arabidopsis image)

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