1. DIGGING DEEPER INTO THE RELATIONSHIP BETWEEN SR45 AND
GPX7 IN ARABIDOPSIS THALIANA.
Sarah Metcalfe, Alicia Worthylake, and Xiao-Ning Zhang, PhD
Department of Biology, St. Bonaventure University, 3261 West State Rd, St. Bonaventure, N.Y 14778
ABSTRACT
GPX7 is an antioxidant enzyme that catalyzes the reduction of H2O2 that is produced.
Research has suggested that GPX7 helps protect against photooxidative stress and
limits PCD due to pathogen invasion. SR45 is a splicing factor involved with
alternative splicing and RNA metabolism. RNAseq analysis showed that GPX7 is
upregulated by SR45. The goal of this experiment is to further study the relationship
between GPX7 and SR45. We hypothesize that basal H2O2 levels will be higher in
gpx7 and sr45-1 mutants compared to WT seedling. Also, it is expected that GPX7
transcript levels will be lower in the sr45-1 mutant than in the WT and transgenic
lines. To begin this investigation, total RNAs were extracted, purified, and converted
to cDNA with RT-PCR from an SR45 null mutant (sr45-1), two SR45-GFP transgenic
lines (OX1-1 and OX1-9), along with the Col-0 wild-type. Primers were designed to
study GPX7 expression in each genotype using qPCR. The primers had an optimal
temperature at 57.3°C and an efficiency of 58%. To investigate the change of
expression levels in the different genotypes qPCR with GPX7 primers was compared
to qPCR with GAPDH primers, from this the relative fold increase in GPX7 was
calculated. The H2O2 levels of different genotypes in normal and stress conditions
were observed in seedlings from Col-0, sr45-1, and gpx7 mutants (SALK_072007 and
SALK_023283) using a DAB stain and compared to the WT coloration.
METHODS
RESULTS
Figure 2. Gel electrophoresis for a total RNA extraction and RT-PCR. A) Gel electrophoresis of the
total RNA extracts for Col-0, sr45-1, OX1-1, and OX1-9 to check the integrity of the RNA. The large
band as 28S shows that the RNA had not been degraded. The ladder was a low mass ladder (NEB
N0550). B) Gel electrophoresis of the RT-PCR products from the four genotypes. This was done to
detect RNA expression. The expected size using GAPDH primers was under 0.5 kb.
RESULTS
CONCLUSIONS & FUTURE DIRECTIONS
• Interestingly, the true leaves appeared darker (suggesting more H2O2) than the
cotyledons. There is no visible difference of staining in the different genotypes, except
that the sr45-1 (+) seedling has some dark staining around the wounds. (figure 1).
• The GPX7 primer was shown to work with an efficiency of 58% at 53.7°C (figure 3).
• qPCR did not show any significant fold change in GPX7 expression for Col-0, sr45-1,
OX1-1, and OX1-9, most likely due to a large deviation (figure 4).
• In the future we would like to look at GPX7 protein expression levels in gpx7 mutants
and the sr45-1 mutant.
• Using end point RT PCR we would like to look for a trend in GPX7 expression in the
sr45-1 and OX1 mutants.
ACKNOWLEDGMENTS & REFERENCES
• Chang, C. et al. (2009). Plant Phys, 105, 670-683. http://dx.doi.org/10.1104.pp 109.135566
• Passaia, G. et al. (2014). Journ Exp Bot, 65(5), 1403-1413. http://dx.doi.org/ 10.1093/jxb/ert486
• Zhang, X.N. & Mount, S. (2009). Plant Phys, 150, 1450-1458. http://dx.doi.org/
10.1104.pp.109.138180
Thank you to the rest of the BIO466 class. This work was funded by NSF.
Figure 3. The standard curve based on the serial dilution of Col-0. The serial
dilution began from the total cDNA and was diluted to 1/10, 1/100, 1/500 and
1/1000. The optimal concentration using GPX7 primers was determined to be
1/100. Calculated efficiency of GPX7 primers was calculated to be 58%.
A
B
Figure 1. H2O2 detection in leaves of Arabidopsis mutants. Leaves of 11 day old seedlings
were stained with DAB to detect presence of H2O2. (+) indicates physical damage inflicted
with a needle to induce stress, and (-) indicates no physical damage to plant. (bar = 1mm)
Figure 4. Fold change in GPX7 expression in different Arabidopsis mutants.
The fold change of GPX7 mutants compared to GAPDH expression found using
qPCR. Bars represent standard deviation.