Comparative Transcriptomics of Defense Hormones & Transcription Factors in Selenium Hyper-Accumulator

Stanleya pinnata & Non-Accumulator Stanleya elata: A Novel Inter-Specific Approach

Michael Adair, Dr. Jennifer Cappa, Jiameng Wang, Dr. Mark Simmons, Dr. Elizabeth Pilon-Smits
Oklahoma State University, Stillwater, OK; Colorado State University, Ft. Collins, CO

Introduction

(Table 1)

Transcript Expressions

Results:

Quantification of ICS2

Some plants accumulate extraordinary levels of toxic

elements. Stanleya pinnata, a native of the Western U.S.,
hyperaccumulates selenium (Se) up to 0.5% of its dry
weight. Selenium is similar to sulfur, and accumulated via
the same mechanisms. With the goal to discover genes
instrumental for Se hyperaccumulation in S. pinnata, I
quantified differences in gene expression between this
hyperaccumulator and related non-accumulator Stanleya
elata, using RNA sequencing. I focused on variation in
expression of genes involved in synthesis and signaling of
salicylic acid (SA), a hormone involved in biotic and abiotic
stress resistance, as well as some other stress response
genes and transcription factors.

(Table 2)

(Fig. 1) Expression quantification of isochorismate synthase 2 (ICS2). Bioreplicate contigs were summed,
sums were averaged for total gene expression, standard error was calculated and a t-test was performed
between S. pinnata & S.elata, 0/20 M Se growing conditions, in each location, root/shoot.
(Table 1-4) S. pinnata and S. elata transcript levels of SA biosynthesis pathway genes from root and shoot
in 0M or 20M Se growing conditions. Each read is the averaged RPKM of 3 bioreplicates with standard
error of mean (SEM, n=3). Star (*) denotes significant difference between species (t-test = p<0.05). Red
RPKMs = highest transcription rate / condition / local. Red name = highest transcription rates solely in
S. pinnata.

www.ncbi.nlm.nih.gov

http://www.plantphysiol.org

Methods & Materials

Root and shoot transcript levels in S. pinnata and S. elata

treated +/- Se were statistically compared, using three
bioreplicates. Contig sequences were aligned with
Arabidopsis, and homology confirmed using a series of
bioinformatic tools: TAIR, MAFFT, BLASTn, and Mesquite.
Up- and down regulated contigs were segregated and
contigs with the same expression pattern summed. T-tests
were performed to identify significant differences in gene
expression between species and treatments.
Computer Programs & Hardware used:
TAIR- The Arabidopsis Information Resource. Used to
retrieve Arabidopsis sequence for alignment to
transcriptome contigs.
MAFFT- A multiple alignment program
General alignment of contig sequences.
Mesquite- A modular system for evolutionary analysis.
Quality control check for gene homology.
BLASTn- Basic Local Alignment Search Tool.
Gives confidence level of gene homology if
alignment fails in Mesquite.
RESEARCH POSTER PRESENTATION DESIGN 2012

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Transcript Expressions

(Table 3)

Transcript Expressions

(Fig. 4) Bar graph visualizing increase or decrease, RPKM & approximate fold
change in expression of each enzyme involved in SA biosynthesis in 0 & 20M
Se concentrations.

Conclusions
Several genes involved in the synthesis and signaling of
salicylic acid (SA) were more highly expressed in the
hyperaccumulator. Many of them were also affected by Se
treatment. Furthermore, a transcription factor associated
with sulfur deficiency (SLIM) was upregulated in the
hyperaccumulator. These findings suggest that the
hyperaccumulator senses it is being attacked by
pathogens, and is sulfur-deficient. This fits well with our
earlier work that showed Se hyperaccumulators have
upregulated levels of sulfate transporters and sulfur
assimilation enzymes.
These results provide better insight into genetic
mechanisms of Se hyperaccumulation. The key genes
identified in this project may be used to create plants with
better ability to accumulation and tolerate Se. This can
advance phytoremediation (environmental cleanup using
plants) and Se biofortification.
Further research is needed to determine the role of other
transcription factors and other enzymes involved in
hyperaccumulation. Future expression comparisons of key
enzymes could lead to finding a master switch that
allows toxins to hyperaccumulate.

Acknowledgements
(Fig. 2) Transcript levels of MYB transcription factor
pathway genes in 0M Se treated roots of S.
pinnata and S. elata. MYB= Myeloblastosis. The
values are mean RPKM and SEM (n = 3). Stars
denote significant differences between species (ttest, p<0.05).

(Fig. 3) Transcript levels of MYB transcription factor

pathway genes in 20M Se treated roots of S.
pinnata and S. elata. MYB= Myeloblastosis. The
values are mean RPKM and SEM (n = 3). Stars
denote significant differences between species (ttest, p<0.05).

Thank you:
Dr. Elizabeth Pilon-Smits, Dr. Jennifer Cappa, Dr. Mark
Simmons and Jiameng Wang for the support,
encouragement and direction, REU coordinator Dr. Paul
Laybourn, Colorado State University & Dr. Diana Spencer
Funded by: National Science Foundation.