Homologs of the floral regulator FLO/LFY control the first zygotic cell division in the moss Physcomitrella patens

Takako Tanahashi, Naomi Sumikawa, Masahiro Kato, and Mitsuyasu Hasebe (2005)

Introduction

Like all multicellular organisms, Physcomitrella patens begins its diploid development from the single cell stage called the zygote.   However, the methods by which this zygote undergoes mitotic division and the genes involved are mostly unknown.  Physcomitrella patens has been found to be a useful model organism for study of these processes for several reasons.  These reasons include a high efficiency of homologous recombination, easily accessible and observable zygotes and sporophytes, and multicellular haploid generations that allow for maintenance of mutants with severe diploid defects.

Prior research indicated that homologs of the transcription factor FLO/LFY are required for the first zygotic division.  This report focuses on two FLO/LFY genes in P. patens and shows that FLO/LFY is essential to progression of the first cell division in the zygote as well as sporophyte development.

Methods

Gametangia, sporophytes, and protonema were cultured  on 9cm Petri dishes with BCDAT medium and inoculated onto sterile 42mm diameter peat pellets.  Sporophytes were then collected after a month and germination rate was estimated by spreading spores on solidified BCDAT medium and germinated protonema were counted after 1 week.  DNA was then extracted from protonema and gametophores and a southern analysis was performed.  Reverse transcription-PCR was performed on extracted RNA and the products were sequences to confirm the amplification of the expected fragments. Following this, targeting plasmids and generation of transformants in P. patens.  PpLFY1 fragments were then amplified via PCR and transferred into Agrobacterium tumefaciens and used for transformation of A. thaliana. A transformation of P. patens was then performed and candidates were analyzed by Southern analyses.  Wild-type and double disruptants were cultured separately and then transferred into the same box and submerged in distilled water and cultured for 5 weeks and harvested to create genetic crosses used in the experiment.

 

Results/Discussion

Figure 1 below shows GUS activity in PpLFY1-GUS lines (A-L) and in PpLFY2-Gus lines (M-AB).

This figure provides evidence that PpLFY1 and PpLFY2 genes function in similar ways and show expression patterns very similar to one another.

Figure 2 below displays the phenotypes of single and double mutant strains.  Sporangia are the small brown spheres and are significantly reduced in the double mutant strain (E) when compared to the single mutants(C-D) and the wild type(B).

This figure gives a qualitative and quantitative representation of the amount of sporangia formed from each different strain of P. patens.  It provides evidence that the double mutant produces almost no sporophytes and that PpLFY genes are essential to not only zygotic division but sporophyte development as well.

Figure 3 below shows the development of the egg cell, zygote and embryo.  A-G represents wild type and H-K represents PpLFY double mutants.

This figure proves that PpLFY double mutants do not progress to the first cell division.

Figure 4 below shows the development of sporophytes in the wild type (A-D) and in the PpLFY double mutant strains (E-K). Image E shows and undifferentiated archegonium venter, image F shows a double sporangia, and image G shows a withered sporophyte.

This figure similar to figure 2 shows that PpLFY disruptants have impaired sporophyte development and provides further evidence that PpLFY is essential for sporophyte development.

Conclusions

The results from this research shows that PpLFY1 and PpLFY2 show similar expression patterns and play roles in controlling the first zygotic division of P. patens. Mutants show mostly normal organogenesis but abnormalities in their pattern of cell division, They also have a substantially lower number of gametophores that form a sporophyte.  Zygotes in double mutants do not progress to the first cell division.

This paper has many strengths, including the number of assays and analyses it performs.  The researchers do a very good job of analyzing each genetic cross both qualitatively and quantitatively.  I do not see any need for improvement on this paper, however further research could be done to determine what may have caused the unexpected diversion of the two lineages of land plants in regard to body plans mentioned in the paper.

References

Tanahashi, Takako, et al.. “Diversification of gene function: homologs of the floral regulator FLO/LFY control the first zygotic cell division in the moss Physcomitrella patens.” Development. (2005): 1727-1736. Web. 3 Apr. 2014. <http://dev.biologists.org/content/132/7/1727.short>.

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