Arabidopsis Proposal: February 10, 2012

Thomas Tarcha, Cheyenne Chadwick, Monica Patten; Stefan Kowynia

A.  Results from prior NSF support: N/A

B.  Specific Goals:

1.  Determine the effects of acid pH on the growth of Arabidopsis plants including:

  1. Width of largest leaf of each plant
  2. Length of largest leaf of each plant

2.  Determine if acid will affect growth of reproductive features and fertility of seeds.

3.  Determine if gene silencing of RDR and DCL affects plants ability to grow large leaves and reproduce, in acid rain or not.

C.  Background

  1. Arabidopsis is an ideal experimental plant due to its small manipulative genome, its known growth pattern of leaves (in doubles), and its ability to reproduce quickly. Due to quick reproduction (a month to maturity), we can see phenotypic changes emerge quicker, and stress would play its role more rapidly.

b. RNA interference, or RNAi, is involved in gene silencing in living organisms. It is a biotechnology technique used to silence the expression of specific genes. RNA molecules with sequences that correspond to particular genes trigger the breakdown of the genes mRNA. DCL or Dicer,

is a protein complex that attaches to a specific microRNA (miRNA). MiRNA tends to be about 20 nucleotides long and is single stranded. Once the DCL-miRNA complex is together, it binds to the mRNA strand with a complementary strand of nucleotides. This binding either cuts the RNA (Dicer) or blocks translation. This is an example of how we got 1 of our 2 mutants.

c. RNAi is necessary in gene expression. Without it organisms would not show any specific trait. Expressing specific traits comes from an “evo-devo” relationship as a result of natural selection. It is the way generations of plants and animals can respond to stimulus. When stress is applied to plants, it should change to ensure its survival and ability to reproduce.

D.  We can see how acid affects the Arabidopsis plants by observing how the plants react when they are exposed to acid. For our experiment, we are observing how a 4.4 acid affects the surface area of the leaves. If the acid causes the leaves to shrivel up, become flaccid, or change color, there would be implications/indications of a reduced surface area. This would cause the plant to absorb less sunlight, reducing its ability to perform photosynthesis, which may eventually lead to the plants death. If acid were to cause the plant leaves to increase in surface area, it would be an indication of a need for more sunlight, thus more photosynthesis. This increase in need for photosynthesis could be due to a need of more glucose to help grow, while helping combat the effects of the acid.

E. E. Experimental Methods and Analysis

Control Plants:

·  Two of each of the three types of plants are kept as controls for the

Variable, wild type, DCL, and RDR.

·  Wild Type= Water + Fertilizer / ·  Wild Type= Water + Fertilizer
·  RDR= Water + Fertilizer / ·  RDR= Water + Fertilizer
·  DCL= Water + Fertilizer / ·  DCL= Water + Fertilizer

·  The control plants are watered every Tuesday and Friday with a mixture of ½ tablespoon of fertilizer per gallon of tap water.

Variable Plants:

·  Two of each of the three types of plants are used as the variable for testing phenotypic effects of pH, Wild Type, DCL, and RDR.

·  Wild Type= Water+ Fertilizer+ Sulfuric Acid / ·  Wild Type= Water+ Fertilizer+ Sulfuric Acid
·  RDR= Water + Fertilizer + Sulfuric Acid / ·  RDR= Water + Fertilizer + Sulfuric Acid
·  DCL= Water + Fertilizer + Sulfuric Acid / ·  DCL= Water + Fertilizer + Sulfuric Acid

·  Sulfuric acid was tested for a pH of 4, and then a disposable transfer pipette was used to add two drops of sulfuric acid to the gallon mixture of water and fertilizer.

·  This solution of Sulfuric acid, water, and fertilizer was used to water the variable plants in a separate plastic container from the control plants so that cross contamination did not occur.

·  The Control and variable plants were stored separately with the variable plants in a plastic container inside of the large black plant container provided, and the control plants were kept loose in the large container.

·  All of the plants were kept under the same light source.

Wild Type= Water + Fertilizer / ·  Wild Type= Water + Fertilizer
·  RDR= Water + Fertilizer / ·  RDR= Water + Fertilizer
·  DCL= Water + Fertilizer / ·  DCL= Water + Fertilizer
·  Wild Type= Water+ Fertilizer+ Sulfuric Acid / ·  Wild Type= Water+ Fertilizer+ Sulfuric Acid
·  RDR= Water + Fertilizer + Sulfuric Acid / ·  RDR= Water + Fertilizer + Sulfuric Acid
·  DCL= Water + Fertilizer + Sulfuric Acid / ·  DCL= Water + Fertilizer + Sulfuric Acid

Analysis:

-Length and Width of the biggest leaf on each plant=quantitative

-General Leaf Shape= qualitative

-General Leaf color= qualitative

Part F: Significance

1.  This experiment shows the effects of acid precipitation (with a PH below 5.6) and its influence on the environment/plants.

2.  Acid rain has significantly been increasing since the 1950’s due to the demand for vehicles and electricity. Pollution from fossil fuels such as: gas, oil, and coal cause an increase of sulfur dioxide and nitrogen oxides into the atmosphere, which in turn causes precipitation to be more acidic. Acid rain will be a continuum of environmental stress as the demand for fossil fuels stays the same or increases.

3.  This experiment shows how plants will grow and reproduce with the stress of acid rain while genes RDR and DCL are turned off.