The effect of low phosphate and no sulfur nutrient deficiencies on microRNA

expression in Arabidopsis thaliana demonstrating the up regulation of miR395 in low

sulfate soil and up regulation of miR399 in low phosphate soil
Danling Ye
TA: Hongchen Cai
Biology 240M Section Number: 001
March 7, 2014









Introduction
Around the world, many people rely on wheat and other grains as a staple food
crop.
1
As the world population increases and climate change occurs, it is becoming more
and more important to find ways to feed the world population and to be able to grow
crops such as wheat on nutrient deficient soil. In the study, scientists used Arabidopsis
thaliana as a model organism to study the effect of nutrient deficient soil, temperature
extremes, heavy rainfall, and drought on the expression of different microRNAs
controlling flowering and response to environmental stress with the goal to gain more
knowledge about the levels of expression of microRNAs that lead to optimal crop yield in
wheat grown under the different conditions.
1
Arabidopsis thaliana is used as a model
plant organism because it has a variety of characteristics that make it easy to study in the
laboratory setting. It is small, completes its life cycle in a short span of time, 6 weeks,
self-fertilizing, and easily cultured in the lab.
2
In addition, its genome is known and
genetic and physical maps of the chromosomes are available.
2

The work was divided up, with individual groups of scientists focusing on one of
the four factors affecting growth. In the study done in this lab, the scientists focused on
the impact of nutrient stress on the expression of microRNAs controlling nutrient uptake
and flowering in Arabidopsis thaliana.
1
The scientists also had to decide how to modify
standard microRNA purification and qRT-PCR procedures for the research study.
1
Arabidopsis thaliana was grown on plates deficient in phosphate, having no sulfur, or in
a full medium to determine the impact of nutrient deficiencies on microRNA expression.
1



MicroRNAs (miRNA) are important because they control the expression of certain genes
after transcription. They regulate messenger RNA (mRNA) by increasing the rate of
target mRNA degradation and decreasing the rate of target mRNA translation; thus
functioning as negative, specific regulators of gene expression.
1
Thus microRNA controls
the plants physiological response to environmental stresses and signals for the transition
from vegetative to flowering phrase.
1
In addition, they control nutrient uptake. In the
study, the nutrients phosphate and sulfur were studied because they are both important
macromolecules in plants. Phosphate is a component of nucleic acids, phospholipids,
ATP, and coenzymes, making it important in cell division and in energy
transformations.
1,3
Phosphate deficiency causes stunted plant growth or causes plants to
have an abnormal dark-green color.
3
Sulfur is an important component of proteins and
coenzymes.
1
Sulfur deficiency will cause plants to be smaller, have more slender stalks,
grow slower, have delayed maturity, and have yellowish to light green young leaves.
4
In
the environment, a dearth of phosphate or sulfur limits plant growth, making them
important macromolecules for agriculture and plant growth in general.
1
They only exist
in limited quantities in soils, and in agriculture, the same soil is used year after year to
plant crops, depleting the soil of nutrients and making fertilizers an useful product for the
agricultural industry.
The four microRNAs studied were miR156, miR395, miR398, and miR399. The
different microRNAs serve different purposes. MiR156 is responsible for the regulation
of the emergence of vegetative leaves and for controlling the transition from juvenile to
the adult stage.
5
MiR395 targets two components of sulfate uptake and assimilation.
6
MiR398 targets the messenger RNA of two copper/zinc superoxide dismutases and it can
trigger their cleavage or repress their translation.
7
MiR399 targets the gene PHOSPHATE
2 and regulates phosphate homeostasis, and it also regulates flowering at different
temperatures.
8

It is hypothesized that miR156 and miR399 will be induced in phosphate deficient
soils and that miR395 and miR398 will be repressed in phosphate deficient soils based on
previous data about microRNA expression in phosphate deficient soils.
9
It is expected
that miR395 will be induced by sulfate deficiency because miR395 increases the
movement of sulfate to the shoots during periods of sulfate starvation.
10
It is also
hypothesized that miR398 will be repressed in no sulfate soil and miR156 and miR399
will be induced in no sulfur soil.
Materials and Methods
1
(adapted from Axtell et. al., 2014)
The prep crew grew seedlings of Arabidopsis thaliana in three kinds of medium:
full medium, low phosphate, and no sulfur. Approximately 20-25 seedlings were placed
on each plate, except for the low phosphate plate, where the number was doubled. The
plate was sealed with surgical tape and allowed to grow for three weeks. Many scientists
worked together on the experiment. Each pair was assigned a specific plate; the assigned
plate was the full medium plate. 40 seedlings were placed in a 1.5 mL microcentrifuge
tube. 750 ul of Lysis buffer was added to the tube, and the seedlings were ground with a
blue pestle. The samples were centrifuged at maximum speed for 5 minutes. A filtration
column was inserted into a 2 ml collection tube and the lib was closed; a binding column
was inserted into another collection tube and the lid was closed. The lysate supernatant
was pipetted into the filtration column and centrifuged at maximum speed for 1 minute.
550 ul of the clarified lysate was transferred into a clean 1.5 mL microcentrifuge tube.
The microRNA was extracted according to the following the Sigma mirPremier
microRNA isolation kit protocol, using with minor modifications.
11
For the elute RNA
step, 30 ul of elution solution was used. 9.5 ul of primer/dNTP/water master-mix,
prepared by a prep crew in advance, was dispensed into a PCR tube. The step was
repeated because there were two RNA samples for each pair. 4 ul of the RNA was added
to one tube, and the process was repeated for the other sample. The samples were heated
at 65C for 5 minutes on a thermal cycler, then placed directly on ice. 6.35 ul of Reverse
Transcriptase Master Mix, prepared by a prep crew in advance, was added to each tube
and the solution was pipetted up and down a couple of times to thoroughly mix. The tube
was capped, making sure the lid was on firmly. Pulsed reverse transcriptase was
performed using the thermal cycler; step 1 was at 16C for 30 minutes, step 2 at 30C for
30 seconds, step 3 at 42C for 30 seconds, step 4 at 50C for 1 second, step 5 was a
repetition of step 2 50 more times, step 6 at 70C for 15 minutes, and step 7 at 4C until
the samples are removed, preserving the cDNA for as long as needed.
Each pair made a U6 master mix by adding 35 ul of 2X SYBR Green Master Mix,
3.5 ul of U6 Primer F, 3.5 ul of U6 Primer R, and 31.5 ul of RNase-free water. It was
placed in the centrifuge for 30 seconds. Each pair also made a miRNA master mix using
an assigned primer; the scientists doing the experiment were assigned miRNA primer
395. The mix was made with 35 ul of 2X SYBR Green Master Mix, 3.5 ul of miRNA
primer 395, 3.5 ul of Universal SL-Primer R, and 31.5 ul of RNase-free water. It was
placed in the centrifuge for 30 seconds. Since there were many scientists working on the
project, different combinations of cDNA from plants grown on different soil mediums
and with different microRNA primers were tested. The possible combinations were the
U6 control and cDNA from plants grown on full medium plates, U6 control and cDNA
from plants grown on low phosphate plates, U6 control and cDNA from plants grown on
no sulfur plates, master mix with miR156 primer and cDNA from plants grown on full
medium plates, master mix with miR156 primer and cDNA from plants grown on low
phosphate plates, master mix with miR156 primer and cDNA from plants grown no
sulfur plates, master mix with miR395 primer and cDNA from plants grown on full
medium plates, master mix with miR395 primer and cDNA from plants grown on low
phosphate plates, master mix with miR395 primer and cDNA from plants grown on no
sulfur plates, master mix with miR398 primer and cDNA from plants grown on full
medium plates, master mix with miR398 primer and cDNA from plants grown on low
phosphate plates, master mix with miR398 primer and cDNA from plants grown on no
sulfur plates, master mix with miR399 primer and cDNA from plants grown on full
medium plates, master mix with miR389 primer and cDNA from plants grown on low
phosphate plates, and master mix with miR399 primer and cDNA from plants grown on
no sulfur plates.
For each pair, 21 ul of U6 master mix was mixed with 1 ul of cDNA 1, cDNA
from one person in the pair, in a small tube. In another tube, 21 ul of U6 master mix was
mixed with 1 ul cDNA 2, cDNA from the other person in the pair. 21 ul of U6 master
mix was mixed with 1 ul of water to serve as a negative control. 21 ul of miRNA master
mix was mixed with 1 ul cDNA 1. 21 ul of miRNA master mix was mixed with 1 ul
cDNA 2. 21 ul of miRNA master mix was mixed with 1 ul water, to serve as a negative
control. The qPCR reactions were loaded into precise assigned well locations in a 96 well
plate, 24 ul of the appropriate mix into each well. The contents of each location were
recorded. Thus each type of microRNA being studied miR156, miR395, miR398, and
miR399 was used in the qPCR setup. In qPCR specific fragments of DNA are
amplified. The primer used binds to a target DNA, serving as the start for the synthesis of
DNA by a DNA polymerase enzyme. At high temperature, the two strands of DNA
separates; thus by alternating between high and low temperatures, qPCR allows the
cyclical process of denaturation, annealing, and extension to occur over and over again;
doubling the target DNA amount each cycle. A special dye in the qPCR wells bind only
to double-stranded DNA and will only be fluorescent when bound to the double-stranded
DNA, thus collecting data on the amount of fluorescence over time and graphing
fluorescence, in relative fluorescence units vs. time, in number of cycles shows the
amplification of the target DNA during the course of the PCR reaction.
For the PCR reaction, there is a threshold, or minimal level of fluorescence that is
set to mean detected. The cycle threshold, or C
t
, is the x-axis value where the
fluorescence value crosses the threshold. When multiple samples are compared,
differences in C
t
values imply different initial concentrations of the target DNA in the
reaction: higher C
t
values indicate lower amounts of target DNA concentration. The
difference between two cycle thresholds (!C
t
) can be found using the equation: !C
t
=C
t
control
C
t
experimental
. The efficiency of the PCR reaction, represented by E, is the
increase in target DNA per PCR cycle. The relative abundance, represented by RA, is
E
!Ct
. Though the ideal efficiency for a reaction is 2, however, that is rarely the actual
efficiency value because it is influenced by a variety of factors. Thus E must be
determined experimentally for each target reaction. To do so, one cDNA sample that
contains the target cDNA can be prepared and analyzed in a series of ten-fold dilutions.
The C
t
values can be plotted as a function of log
10
dilutions, creating a linear equation.
Thus the efficiency of the reaction can be calculated as E = 10
(-1/S)
where S is the slope of
the line where C
t
values are plotted as a function of log
10
dilutions. A cDNA reference is
known to accumulate at equal levels in all samples being used. The C
t
value for the
reference cDNA should be measured to normalize the relative accumulation data to
account for unequal unloading, and the normalized relative accumulations, abbreviated
RA
n
, is equal to [(E
target
)
!Ct target
]/[(E
reference
)
!Ct reference
].
















Results
Figure 1: Photos of Arabidopsis thaliana grown on different mediums over the course of
3 weeks







Figure 1: Key
A: Arabidopsis thaliana grown on full medium plates after one week of growth
B: Arabidopsis thaliana grown on full medium plates after two weeks of growth
C: Arabidopsis thaliana grown on full medium plates after three weeks of growth
D: Arabidopsis thaliana grown on low phosphate plates after one week of growth
E: Arabidopsis thaliana grown on low phosphate plates after two weeks of growth
F: Arabidopsis thaliana grown on low phosphate plates after three weeks of growth
G: Arabidopsis thaliana grown on no sulfur plates after one week of growth
H: Arabidopsis thaliana grown on no sulfur plates after two weeks of growth
I: Arabidopsis thaliana grown on no sulfur plates after three weeks of growth

A B C
D E F
G H I
After one weeks of growth, there was minimal growth of Arabidopsis thaliana on
all three types of plates. During the second week of growth, Arabidopsis thaliana grown
under full medium conditions were more clustered and a lighter green color than the
Arabidopsis thaliana grown under low phosphate or no sulfur conditions. During the
third week of growth, Arabidopsis thaliana grown on the full medium plate had healthy
leaves and stems and were a green color. Arabidopsis thaliana grown on low phosphate
plates were more scattered and had smaller shoots and leaves than the ones grown on the
full medium plate; in addition, there were many plants that had a darker green-black
color. The plants grown on the no sulfur plate were a bit bigger than low phosphate
plants, but the number of plants on the plate was fewer. Compared to the full medium
plate, the plants had smaller shoots and leaves, were less clustered, and were darker
green-black or yellowish-green.
Most of the qPCR reactions worked well, however for the section, the negative
control for miR156 and low phosphate had a C
t
value of 9.6253987, which is
significantly smaller than 36 and thus should be treated with suspicion. Thus the section
results about miR156 grown on low phosphate plates should be treated with suspicion.







Table 1: Medium Relative Abundances of Sample Data for Four MicroRNAs studied
grown under Low Phosphate and Low Sulfur Conditions
Median RAn's
LowP LowS
miR156 1.862930111 1.227683226
miR395 0.046397251 1158.413858
miR398 0.035445039 0.150537893
miR399 20.76552394 1.198111257

Figure 1: Graph of Medium Relative Abundances of Sample Data for Four MicroRNAs
studied grown under Low Phosphate and Low Sulfur Conditions as determined by data
on fluorescence collected after qRT-PCR












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Table 2: Medium Relative Abundances of Section Data for Four MicroRNAs studied
grown under Low Phosphate and Low Sulfur Conditions
Median RAn's
LowP LowS
miR156 0.111820765 7.234385601
miR395 0.508479963 969.3807593
miR398 17.20884977 6.59568177
miR399 151.660697 7.92026409






















Figure 2: Graph of Medium Relative Abundances of Section Data for Four MicroRNAs
studied grown under Low Phosphate and Low Sulfur Conditions as determined by data
on fluorescence collected after qRT-PCR












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Table 3: Medium Relative Abundances of Whole Class Data for Four MicroRNAs
studied grown under Low Phosphate and Low Sulfur Conditions
Median RAn's
LowP LowS
miR156 0.7733 1.0354
miR395 1.3411 751.4459
miR398 17.2088 2.1197
miR399 25.2056 0.7390

Figure 3: Graph of Medium Relative Abundances of Whole Class Data for Four
MicroRNAs studied grown under Low Phosphate and Low Sulfur Conditions as
determined by data on fluorescence collected after qRT-PCR

For all of the samples, Arabidopsis seedlings after four weeks of growth were
used. For the sample and whole class data, there was a no significant increase in miR156
relative accumulation under low phosphate or low sulfur conditions. However, for the
section data, there was a more significant decrease in miR156 expression under low
phosphate conditions, with a relative accumulation value of 0.111820765. In addition, for
the section data under the low sulfur conditions, there was a more significant increase in
miR156 expression, with an increase of over 7-fold. For plants grown on low sulfur
plates, the expression of miR395 increased significantly across the sample, section, and
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Changes in microRNA accumulation in
Arabidopsis seedlings grown on nutrient-deficient
media (whole class data)
LowP
LowS
class data; there was a 1158-fold increase in the sample data, a 969 fold-increase in the
section data, and 751-fold increase in the class data. For plants grown on low phosphate
plates, the expression of miR395 did not have a significant change for the section or
whole class data; however there was a relatively significant decrease for the sample data.
For plants grown on both the low sulfur and low phosphate plates, there was an increase
in miR398 expression in the whole class data and section data and a decrease in its
expression in the sample data. For plants grown under low phosphate plates, the
expression of miR399 was induced across the sample, section, and whole class data.
However, under low sulfur conditions, there was no significant change in miR399
expression in the class or sample data, while the section data showed a significant
increase in miR399 increase under low sulfur conditions.
The class data was somewhat consistent with published results. The most
significant change in relative abundance was the change in miR395 expression under low
sulfate conditions, with an over 751-fold increase. This change is consistent with
predictions in published literature.
10
The second greatest change in relative abundance
was the change in miR399 expression in low phosphate conditions, with an over 25-fold
increase. The increase is consistent with predictions in published literature.
9
However, for
the other microRNAs tested, there was either no significant change or the change was
inconsistent to that predicted through looking at published research.
9


Discussion
The data collected was mostly inconclusive and thus does not conclusively
answer the initial questions. As seen by Figures 1 and 3, for the sample and whole class
data, there was a no significant increase in miR156 relative accumulation in Arabidopsis
under low phosphate or low sulfur conditions. However, as seen by Figure 2, for the
section data, there was a more significant decrease in miR156 expression under low
phosphate conditions and a more significant increase in miR156 expression under low
sulfur conditions. A study using deep-sequencing techniques showed a significant
increase in miR156 expression in low phosphate conditions and cross talk by miR156 in
other nutrient deficient conditions, demonstrating that the data collected in the study for
miR156 was inconclusive when compare to published literature.
9

The expression of miR395 in Arabidopsis thaliana grown under low sulfate
conditions showed the greatest change out of all the microRNA and nutrient deficiency
combinations. As seen by Figure 1, 2, and 3, miR395 increased significantly across the
sample, section, and class data when Arabidopsis was grown under low sulfate
conditions; there was a 1158-fold increase in the sample data as seen by Table 1, a 969
fold-increase in the section data as seen by Table 2, and 751-fold increase in the class
data as seen by Table 3. The results are consistent with those present in literature that
predict that miR395 is strongly induced by sulfate deficiencies.
6
MiR395 targets three of
the four isoforms of ATP sulfurylase, the first enzyme involved in the pathway of sulfate
assimilation.
10
It also increases translocation of sulfate to the shoots, thus it is logical that
a decrease in sulfate in the soil would cause miR395 induction.
10
For plants grown on low
phosphate plates, the expression of miR395 did not have a significant change for the
section or whole class data as seen by Figure 2 and 3; however there was a relatively
significant decrease for the sample data as seen by Figure 1. The results for the sample
data are consistent with published results that show miR395 repression in low phosphate
conditions.
9

For plants grown on both the low sulfur and low phosphate plates, there was an
increase in miR398 expression in the section data and whole class data as seen by Figure
2 and 3. However, the sample data showed a decrease in miR398 for plants grown under
low sulfur and low phosphate conditions. The sample data is consistent to research
published that used deep-sequencing techniques to show a repression in miR398
expression under nutrient deficient conditions.
9

For plants grown under low phosphate plates, the expression of miR399 was
induced across the sample, section, and whole class data as seen by Figures 1, 2, and 3.
According to a research study, an overexpression of miR399 leads to an over
accumulation of phosphate, thus logically, miR399 should be induced under low
phosphate conditions.
8
However, under low sulfur conditions, there was no significant
change in miR399 expression in the sample or class data as seen by Figures 1 and 3,
while the section data, as seen by Figure 2, showed a significant increase in miR399
increase under low sulfur conditions. MiR399 targets a sulfate transporter called AST68,
and thus the section data is consistent with other research.
12
There are many possible sources of error in the experiment. First and foremost,
the experimenters were inexperienced and new to most of the techniques in the lab,
which meant that a lot experimental error was probably introduced in the section and
class data. Pipetting errors and other technical errors could have caused much of the error
in the experiment. In addition, the section data and class data was a compilation of results
from many different experimenters, all who probably had minor differences in
techniques. Different groups used different pipets, instruments, and samples of chemicals.
Small samples were used for the experiment, which may have led to inconsistent results
because minor differences in the instruments and chemical sample composition might
have had drastic consequences on the data. The sample data was obtained from one
scientist, Dr. Axtel, who was experienced in the techniques used in the lab, making it
more reliable than the class data. Thus experimental errors caused by inexperience
probably caused most of the inconsistency between the class data and the sample data and
between the class data and published results.
In addition, increasing a sample size usually increases the accuracy of the data by
decreasing the influence of outliers. The section data had a small sample size compared
to the whole class data or data used in published results, which might be one factor
accounting for the differences in results.
Also, the relative accumulation of the reference is used to correct for unequal
loading. A no template control with a C
t
value of less that 36 is regarded as suspicious,
and is probably caused by pipetting error or contamination by residual DNA in the
original RNA preparation.
1
For the section, the negative control for miR156 and low
phosphate had a C
t
value of 9.6253987, which is significantly smaller than 36, indicating
possible pipetting error or contamination. It is also possible that other samples had
contamination or major pipetting errors, leading to incorrect results.
Additional experiments can be done to better understand the results. First, one
scientist, instead of many, can do the same experiment. The instruments used should be
consistent for all samples; for example, instead of using many micropipettes, one of each
kind of micropipette necessary for the experiment can be used. The experiment should be
modified so that steps are put in place to check for contamination, including DNA
contamination on the instruments used. Care should be given not to place anything that
will come into contact with the sample onto the lab bench. In addition, researchers can
ask questions about the quantity of up or down regulation of different microRNA
necessary to cause a significant increase in accumulation of different minerals in the
plant. Also, Arabidopsis thaliana was used as a model organism for the experiment and
the experiment should be done on wheat and other crops to confirm the results and for the
results to have practical implications in agriculture. The overarching research question is
concerned with microRNA expression in four environmental conditions: drought, heavy
rainfall, temperature extremes, and soil nutrient deficiencies. The experiment was only
concerned with soil deficiencies, and the results should be put in conjunction with results
from experiments dealing with the other environmental conditions.
The experiment helps somewhat in answering the original research questions. One
can conclude that under sulfate deficient conditions, the expression of miR395 increases
in Arabidopsis thaliana. Thus for crops, such as wheat grown on soils with sulfur
deficiencies, an up regulation of miR395 might increase crop yields. One can also
conclude that under phosphate deficient conditions, an up regulation of miR399 occurs in
Arabidopsis thaliana. Thus an up regulation of miR399 in low phosphate conditions
might increase crop yields. Only minor modifications of standard microRNA purification
and qRT-PCR were made in the experiment.




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