Arabidopsis thaliana and the GRF-Interacting Factor (GIF) gene family

By: Skyler Brennan

Background:

A. thaliana flowering plant. Image source: Wikipedia

A. thaliana flowering plant. Image source: Wikipedia

Arabidopsis thaliana is a flowering plant that originates from Eurasia, but has a very wide geographic range. This dicotyledonous angiosperm is very commonly used as a model organism for research on genetics, evolution, and general plant and flower development. A. thaliana is an ideal model organism due to its small genome and size, short generation time, and high number of offspring. The flowers found on A. thaliana are solely responsible for fertilization and reproduction, as they contain both female reproductive organs (gynoecium or pistil) and male reproductive organs (anther). For more general information on flowering plants, visit the angiosperm homepage.

Approximately 5% of the A. thaliana genome is devoted to coding transcription factors, with half of those transcription factors belonging to gene families that are only found in plants. One of these gene families is Growth-Regulating Factor (GRF) transcription factors. The nine members of the GRF transcription factor gene family primarily regulate leaf and cotyledon growth. GRF transcription factors form a complex with GRF-Interacting Factors (GIF), a group of transcriptional co-activators that control cell proliferation. Lee et al. (2013) shows that the three main GIF proteins (gif1, gif2, and gif3) in A. thaliana play a major role in cell specification maintenance during the development of reproductive organs.

Methods:

Growth conditions

The A. thaliana seeds were planted on wet soil at 4°C. After three days, they were moved to a growth room, which was maintained at 23°C. In the growth room, the seeds were exposed to light for 16 hours followed by darkness for 8 hours.

Formation of triple mutants

Triple mutants (loss of function of gif1, gif2, and gif3) were used extensively throughout the experiment. The F2 progeny of a gif1 gif2 (male) and gif2 gif3 (female) cross were collected and used as the triple mutants.

Scanning electron microscopy (SEM)

The A. thaliana flowers were placed in FAA solution and incubated at 4°C. The samples were then rinsed with a sodium phosphate buffer three times and stored in ethanol. After the samples dried, they were covered with gold particles for the SEM.

Results/Discussion:

Figure 1: Expression of gif1, gif2, and gif3 in developing flowers via in situ hybridization. A-H: gif1 expression; I-N: gif2 expression; O-Q: gif3 expression.

Figure 1: Expression of gif1, gif2, and gif3 in developing flowers via in situ hybridization. A-H: gif1 expression; I-N: gif2 expression; O-Q: gif3 expression. Source: Figure 6 from Lee et al. 2013

Expression of GIF genes in flowers

Lee et al. looked at the general expression of GIF genes in A. thaliana flowers through in situ hybridization of GIF mRNAs. The gif1 and gif2 transcript signals were detected in stages 1 and 2 of flower bud development, and then were expressed more strongly in stage 3 (Figs. 1A and I). Figures 1H and N show the expression of gif1 and gif2 in meiotic cells, tapetal cells, and microspores in the later stages of flower bud development. Additionally, gif1 and gif2 were expressed in the gynoecium starting in stage 7 (Figs. 1B, J, and K). The gif3 transcript signal, however, was not detected at all until stage 8 (Fig. 1O); eventually, the gif3 signal was observed in the embyro sac (Fig. 1P).

Figure 2: SEM analysis of ovule development. A-F: wild-type ovules; G-M: Triple mutant ovules.

Figure 2: SEM analysis of ovule development. A-F: wild-type ovules; G-M: Triple mutant ovules. Source: Figure 3 from Lee et al. 2013

Effects of gif triple mutant on gynoecium

Lee et al. studied gif1 gif2 gif3 triple loss-function mutants (triple mutants) and wild-type A. thaliana to determine the effects of the GIF proteins on flower bud development. Scanning electron microscopy (SEM) was used to distinguish the difference between development in wild-type ovules (Fig. 2A-F) and triple mutant ovules (Fig. 2G-M). The triple mutant ovules were found to be normal at bud initiation, but ended with spilt gynoecium (Fig. 2G). Also, integument parts of the wild-type ovules grew asymmetrically (Fig. 2F), but the triple mutant did not undergo this asymmetrical growth (Fig. 2L and M).

Other noticeable differences in ovule development include the fact that the triple mutant has no embryo sac. Triple mutants were also found to have defective carpel margin meristem (CMM) tissues, which is the likely cause of the split gynoecium and lack of an embryo sac. Triple mutants additionally experience slower sepal growth and reduced sporogenesis.

Figure 3: Analysis of gynoecium and anther development via SEM. A-G: Wild-type phenotypes; G-Y: Triple mutant phenotypes.

Figure 3: Analysis of gynoecium and anther development via SEM. A-G: Wild-type phenotypes; G-Y: Triple mutant phenotypes.  Source: Figure 1 from Lee et al. 2013

Effects of gif triple mutant on anther

Triple mutants and wild-type were also examined via SEM in order to determine the impact of the GIF genes on the anthers of A. thaliana. Wild-type anthers have four distinct lobes that are bilaterally symmetrical (Figs. 3D, E, and G), but triple mutants have misshapen lobes (Figs. 3Q, T, and X). It was also observed that triple mutant anthers are typically bound to petals or carpels (Fig. 3W and Y).

Interestingly, the triple mutants didn’t have any viable pollen grains or microsporangium, and over time the anther only contained connective cells; the triple mutants are clearly not capable of reproduction. This result implies that GIF proteins are necessary for the production of pollen grains.

Further Investigations: 

While Lee et al. thoroughly examined the function of GIF proteins in A. thaliana, there are still unanswered questions that should be addressed in future studies. For example, very little research has been done on the molecular mechanisms of GIF proteins. Studying these molecular mechanisms could answer how GIF proteins maintain cell specification, as well as provide more insight on the interactions between GIF proteins and GRF transcription factors in the GRF-GIF complex.

Broader Implications:

Arabidopsis thaliana im Labor

A. thaliana is one of the most common model organisms in botanical research. Image source: Max-Planck Institute for Developmental Biology 

Lee et al. states that the GIF gene family is unique to A. thaliana, so similar plants (specifically dicots) may contain a gene family that functions like the GIF gene family. Also, as A. thaliana is one of the most commonly used model organisms in botanical research, knowledge of GRF transcription factors and GIF proteins can be useful to scientists that want to use A. thaliana in a study. A. thaliana is so important to plant research, and therefore it is imperative to understand its developmental process as completely as possible.

Critique: 

I thought the main strength in this study was the investigation of the function of GIF proteins using SEM, in situ hybridization, and histological analysis. By using multiple methods, the analysis of the GIF proteins was more comprehensive and detailed. As for weaknesses, the study only looked at the GIF proteins and their effects on reproductive development. In reality, the GIF proteins are very closely associated with the GRF transcription factors, and the GRF-GIF complex has more influence on the formation of reproductive organs than the GIF proteins alone.

Conclusions:

Overall, the A. thaliana gif1 gif2 gif3 triple mutants develop an abnormal split gynoecium as well as abnormal anthers that contain neither microsporangium nor pollen grains. The experiments conducted by Lee et al. showed that a lack of GIF proteins severely impacts the CMM tissues, which consequently impacts ovule development. GIF proteins maintain cell specification, but the reason why has not been extensively researched. This research has a major application to botanical science, due to the fact that A. thaliana is often used as a model organism.

Resources: 

Brennan, A., Mendez-Vigo, B., Haddioui, A., Martinez-Zapater, J., Pico, F., & Alonso-Blanco, C. (2014). The genetic structure of Arabidopsis thaliana in the south-western Mediterranean range reveals a shared history between North Africa and southern Europe. [Article]. Bmc Plant Biology, 14.

Kaul, S., Koo, H., Jenkins, J., Rizzo, M., Rooney, T., Tallon, L., et al. (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. [Review]. Nature, 408(6814), 796-815.

Kim, J., Choi, D., & Kende, H. (2003). The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis. [Article]. Plant Journal, 36(1), 94-104.

Lee, B., Ko, J., Lee, S., Lee, Y., Pak, J., & Kim, J. (2009). The Arabidopsis GRF-INTERACTING FACTOR Gene Family Performs an Overlapping Function in Determining Organ Size as Well as Multiple Developmental Properties. [Article]. Plant Physiology, 151(2), 655-668.

Lee, B., Wynn, A., Franks, R., Hwang, Y., Lim, J., & Kim, J. (2013). The Arabidopsis thaliana GRF-INTERACTING FACTOR gene family plays an essential role in control of male and female reproductive development. [Article]. Developmental Biology, 386(1), 12-24.

Riechmann, J., Heard, J., Martin, G., Reuber, L., Jiang, C., Keddie, J., et al. (2000). Arabidopsis transcription factors: Genome-wide comparative analysis among eukaryotes. [Article]. Science, 290(5499), 2105-2110.

 

 

 

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