sr45-1, sr45-1; oif2-1, and KIN11GFP; sr45-1; oif2-1 mutants and their affect on Arabidopsis thaliana reproduction

Alexandra Lyndsley ’15, Christa Wentworth ‘15, Alicia Worthylake ’16 and Dr. Xiao-Ning ZhangDepartment of Biology, Saint Bonaventure University, Saint Bonaventure, NY

Abstract:

SR45 activates pre-mRNA splicing and is involved in recruiting spliceosome proteins (Zhang, 2014). A loss of function gene will lead to developmental defects in developing plants (Zhang, 2009). KIN11 is a protein that is thought to play a role in gene regulation through signal transduction (UniProt, 2015). The four genotypes, Col-0, sr45-1, sr45-1; oif2-1, and KIN11GFP; sr45-1; oif2-1 were used to study flowering, reproduction and embryogenesis. FLC inhibits flowering in wild-type plants. FLC expression was measured using RT-PCR. We found that all four genotypes expressed FLC, showing that they are all capable of inhibiting flowering. The ABCE model determines which reproductive organs a flower will have and how many of each organ. A null mutation will lead to defects in one of the four whorls. We were able to show that neither the mutants nor the transgenic plant significantly differed from the wild-type in organ number. Therefore we were able to determine that none of the genotypes we studied had an effect on the ABCE model. The four stages of Arabidopsis embryogenesis are globular, heart, torpedo and mature. By using microscopy and ovules from siliques of varying ages, we were able to identify all four stages of embryo development in all genotypes except for sr45-1; oif2-1 and KIN11GFP; sr45-1; oif2-1. In conclusion, our study suggests that FLC expression is higher in sr45-1, organ number does not differ in the studied mutants, and there was a prominent delay in embryogenesis in sr45-1; oif2-1.

Results:

Figure 1. Expression of FLC, KIN11, and GAPDH in Arabidopsis Thaliana. ImageJ software was used to determine the area of the bands. GAPDH was used as a standard.

Figure 3. Phenotypic affects on Arabidopsis thaliana flowers. A) Col-0 B) sr45-1 C) sr45-1;oif2-1 D) KIN11GFP;sr45-1;oif2-1 E) cs25 F) cs77 G) fl2. (E – G) were used as negative controls.

Figure 5. Visualization of stages of embryogenesis in Arabidopsis thaliana. (A – D) Col-0 globular, heart, torpedo and mature stages. (E - H) sr45-1 all stages were found as in Col-0. (I) sr45-1; oif2-1 globular stage – the heart, torpedo and mature stages were not found. (J – L) KIN11GFP; sr45-1; oif2-1 globular, heart and torpedo stages – the mature stage was not found. The scale bar is 0.20mm.

Conclusion:

• It was found that FLC was expressed in all of the studied genotypes. Preliminary results show that FLC expression was higher in sr45-1. Seeing FLC expression in all studied genotypes was expected, however the higher level of expression in sr45-1 was not expected.

• It was found that the ABCE model, that governs development of flower whorls, is not effected by the mutations studied, except in the negative controls. This was expected due to the fact that when the E gene is present there will be whorl development.

• It was found that embryogenesis occurs normally, stage wise, in the mutants, though with a delay in sr45-1; oif2-1 and KIN11GFP; sr45-1; oif2-1. It was expected that embryogenesis would occur in all genotypes, however, the delays found in sr45-1; oif2-1 and KIN11GFP; sr45-1; oif2-1 were not expected.

Figure 2. Ratios of KIN11 and FLC to GAPDH in Arabidopsis thaliana genotypes. The ratio of FLC and KIN11 compared to GAPDH through the use of gel electrophoresis band areas.

References:

1. SNF1-related protein kinase catalytic subunit alpha KIN11. March 31, 2015. Retrieved April 17, 2015, from http://www.uniprot.org/uniprot/P92958

2. Zhang, X.-N., & Mount, S. M. (2009). Two Alternatively Spliced Isoforms of the Arabidopsis SR45 Protein Have Distinct Roles during Normal Plant Development. Plant Physiology, 150(3), 1450–1458. doi:10.1104/pp.109.138180

3. Zhang, X., Mo, C. et al. (2014). Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis. Plant Signaling and Behavior, 9(7). Retrieved April 14, 2015.

Figure 4. The average organ number in Arabidopsis thaliana genotypes. The only statistically significant difference in organ number observed among the plants was among the negative controls cs25, cs77 and fl2 when compared to the wild type Arabidopsis. A t-test was performed (p-value> 0.01). Error bars indicate a standard deviation (unique to each genotype). A indicates a significant difference.

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