Effects on microRNA in Arabidopsis Thaliana Grown with Nutrient Deficiencies

Alexis Moyer

Ioanna Georgalakis

Bio240, Section 902

Introduction

MicroRNA’s are small RNA known to adapt and regulate gene expression within

organisms (Zhang, 2015). These small RNA components can influence growth, metabolism, and

most importantly, adaptive responses by targeting the mRNA of enzymes (Djami-Tchatchou,

2017). Stress that builds up on the organism, can cause the miRNAs to change expression, in

order to produce optimal conditions for the organism to grow and produce properly. Some of

these stresses, include drought, salinity, amount of nutrients, and temperature extremes (Zhang,

2015). This experiment looks at the effect on microRNA’s in Arabidopsis thaliana, when grown

in environments with nutrient deficiencies.

The nutrients in the soil that plants are grown in, have a lot to do with how well the plants

grow. Phosphorus and Sulfur are just two of the macronutrients that are essential to plant growth.

However, these two nutrients are not needed in big amounts, like carbon and oxygen. The two

experimental groups in this experiment use a medium with low phosphate and a medium with no

sulfur and the control group uses a full medium, made up of all the essential nutrients. Phosphate

is used for nucleic acids, phospholipids, ATP, and coenzymes, while sulfur is used in proteins

and coenzymes (Ward 2016).

Arabidopsis Thaliana was used as the experimental plant to analyze miR156, miR395,

miR398, and miR399. These microRNAs were known to target mRNA and decrease the amount

of protein in the plant, by acting the same a mutation in the genes for the enzymes. The decrease

in protein of the plant is determined by the rate of transcription, mRNA degradation, translation,

and protein degradation. For example, miR156 is known to interfere with proteins, called

Squamosal Promoter Binding Proteins. When this microRNA increases in expression, less of that
protein is produced within the plant and when the expression decreases, more of those proteins

are produced (Xu, 2016).

In order to analyze the microRNA in the Arabidopsis, the central dogma of biology must

be understood. Genes are copied through the process of transcription in the nucleus, which forms

messenger RNA. Then this mRNA leaves the nucleus and is translated into a protein. The RNA

can be reverse transcribed into cDNA, which is what is done in this experiment. Translation of

the mRNA is where the miRNA interferes and influences the protein making process (Ward,

2016). qRT-PCR is used to amplify the cDNA of the Arabidopsis, which is then used to analyze

the expression of miRNA’s by measuring the RNA levels.

By doing this experiment, the impact of nutrient stress on expression of miRNAs should

be found and the aspects of purification and qRT-PCR that need to be modified for research

study can be found. The expression of each miRNA is dependent on the function of the protein it

effects. It is expected that growth miRNAs would go down in expression, since there is more

protein needed towards growth, since there are nutrient deficiencies in the mediums used to grow

the plants.

Materials and Methods (Ward 2016)

Arabidopsis seeds were prepared with three different variables, full media, No sulfur, and

Low Phosphate. These seeds were grown for two weeks before the experiment actually begun.

On week 4 of the experiment seeds were collected from each of the three different plates. Then

the seed’s RNA is extracted. First, the lysis mix is added and the seeds were grounded up, then

put into a filtration column. With this supernatant, ethanol was used to get the nucleic acids out

of the solution then a binding solution was used on the liquid, in order to bind the miRNA’s. A

wash solution is then used to get rid of impurities within the solution. With the remaining
flowthrough, a master mix is mixed with the miRNA that was extracted and put into a PCR tube.

This master mix will allow reverse transcription to occur. The next week, qRT-PCR was set up,

using the cDNA of the miRNA’s. This was done by setting up 3 control tubes of U6, two with

the cDNA and one with water, and 2 tubes of the miR399, with the cDNA, and one with miR399

and water. When this is set up, the qRT-PCR is run and the results are analyzed on a computer.

To analyze the results, you must first find the slope of the graphs given, in order to

calculate the efficiency. To calculate the efficiency, the equation is E=10 (-1/slope). These

efficiencies that are calculated will be used later on in another equation. The next step to

analyzing the results is calculating the Ct values by using the equation Ct = Ctcontrol -

Ctexperimental. This uses the Ct values of the full media plants and subtracts the Low P and No S C t

values. This is then used to find the RA values, using the equation, RA = ECt. This is where

efficiency comes back into play. RAn is the next thing that is calculated, using the equation RA n

= RAtarget/RAreference, where the target is the miRNA’s being analyzed, and the reference is the U6

that was put into the qRT-PCR. From these numbers, the medians are found and used to see

whether the specific miRNA in Low P and No S was effected.

Results

With this experiment the primary goal was to see the effect that the amount of sulfur and

phosphate used to grow the plants, had on the miRNAs within the plants. After running the qRT-

PCR, an excel document was used to find the median RA n’s for each miRNA that was analyzed.

The sample data gives a basis for what the results should look like.
Table 1: Sample Data of the medians for miRNA

Median RAn's
LowP NoS
miR156 1.862930111 1.227683226
miR395 0.046397251 1158.413858
miR398 0.035445039 0.150537893
miR399 20.76552394 1.198111257

Figure 1: Sample Data of the median RAn’s for miRNA

Changes in microRNA accumulation in

Arabidopsis seedlings grown on nutrient-
deficient media
10000
Normalized Relative
Abundance (log10)

1000
100
10 LowP
1 NoS
0.1
0.01
miR156 miR395 miR398 miR399

This data was used as a control to compare the medians that were calculated for the

section and class microRNA’s and to see what the expression of each should turn out to be.

Table 2: Section Data of the Median RAn’s for miRNA

Median RAn's
LowP NoS
miR156 0.099124183 0.040243599
miR395 298.5153959 796.1195861
miR398 556.6205377 37.78514417
miR399 13.94110787 0.30910186
Figure 2: Section Data of the Median RAn’s for miRNA

Changes in microRNA accumulation in Arabidopsis

seedlings grown on nutrient-deficient media
1000
Normalized Relative
Abundance (log10)

100

10

1
miR156 miR395 miR398 miR399

0.1

0.01

Median RAn's LowP Median RAn's NoS

The results for the section data was not very similar to the sample data, that was used as a

control to compare. The only microRNA that was similar between the two was miR399 in LowP

media. The numbers for the sample data is higher in all median RAn’s except for the medians of

miR398.

Table 3: Class Data of the Median RAn’s for miRNA

Median RAn's
LowP NoS
miR156 7.031565025 1.15489044
miR395 35.4242195 246.9614569
miR398 2.38634451 1.912012802
miR399 1.055767549 0.285372207
Figure 3: Class Data of the Median RAn’s for miRNA

Changes in microRNA accumulation in

Arabidopsis seedlings grown on nutrient-deficient
media
1000
Normalized Relative
Abundance (log10)

100

10

1
miR156 miR395 miR398 miR399

0.1

Median RAn's LowP Median RAn's NoS

For the class results, as seen in the table and graph above, for the most part all of the

microRNA that was analyzed was expressed more in both Low P and NoS medias, except for

miR399, which decreased in expression. The results for the miR395 were similar to the section

data and miR156 was similar to the sample data. For the most part, the section data has higher

medians for all of the RAn’s, except the miR156. When compared to the numbers of the sample

data, the medians are higher for the class data, except for the median Rans of miR399.

Discussion

For the control data, the results should have looked like Figure 1, which had miR156

showing a bit more expression with both Low Phosphate and No sulfur, MiR395 showing much

more expression in No sulfur groups and less expression in Low phosphate groups, miR398

showing lower expressions in both experimental groups, and higher expression in both groups

for miR399. However, the results for the section and class data were different. For the section
data, as seen in figure 2, miR156 showed less expression in both Low phosphate and No sulfur

groups, higher expression of miR395 and miR398 for both groups and higher expression of

miR399 for the low phosphate groups and lower expression in the no sulfur groups. For the class

data as seen in figure 3, there was a higher expression of miR156, miR395, and miR398, and

around the same expression of miR399 for the low phosphate group and lower expression for the

no sulfur group.

The background knowledge of these four microRNAs are needed to understand why the

results are the way they are. It must be known what kind of proteins are effected and how that

effects the plants life cycle. It is known that a higher expression of miR156 results in a longer

juvenile state for the plant, which makes them flower later and increases the branching. This is a

reason for the increase of this microRNA in the sample data. The increased branching leads to a

larger surface that can lead to more sun reaching the plants for photosynthesis. This is why more

of this miRNA is needed when there are nutrient deficiencies because this way the plant can

continue to grow without the specific nutrients (Week 9 powerpoint).

There could be many reasons for these differences in results between all three of the data

collections of median RAn’s. One of which could be because each plant was done by different

people and required intricate work. I know with my group, there was trouble counting out 150

leaves because there were not that many in the plates that were given. This could have messed up

the results for the section, which also went into the results for the class. Also, mistakes with the

pipets could have swayed the results as well.

It is known that the stage that must be looked at for the Arabidopsis is an early stage in

the life cycle and further investigation can be done to find out more about more specific

microRNA’s. This further research can lead to new understandings of gene expression when it
comes to post-transcriptional RNA and more crops, such as flax can also be used to understand

how each microRNA effects the growth and production of the plants (Melnikova, 2016). Also,

more microRNAs that are to be discovered could lead to ways to cure infections and diseases

found in plants (Soto-Suarez, 2017).

References

Djami-Tchatchou, A. (2017). Functional Roles of microRNAs in Agronomically Important

Plants - Potential as Targets for Crop Improvement and Protection. Frontiers in Plant

Science, 8, 378.

Melnikova, N. (2016). Identification, Expression Analysis, and Target Prediction of Flax

Genotroph MicroRNAs Under Normal and Nutrient stress Conditions. Frontiers in Plant

Science, 7, 399.

Soto-Suarez, M. (2017). The Arabidopsis miR396 mediates pathogen-associated molecular

pattern-triggered immune responses against fungal pathogens. Scientific Reports, 7,

44898.

Xu, M. (2016). Developmental Functions of miR156-regulated Squamosa Promoter Binding

Protein-like Genes in Arabidopsis thaliana. PLoS Genetics, 12, e1006263.

Zhang, B. (2015). MicroRNA: a now target for improving tolerance to abiotic stress. Journal of

Experimental Botany, 7, 1749-1761.

Ward, A., Axtell, M. Burpee, D., and Nelson, K. (2016). Using microRNA to enhance wheat

crop yields in nutrient poor conditions. Department of Biology, The Pennsylvania State

University, University Park, PA.