Abstract:

Wisconsin Fast Plants, purple stem, non purple stem, variegated and wildtype were grown under LED and daylight to show the effects on plant growth and expression of phytochrome a and b. It is predicted that no change in growth or phytochrome expression is shown when comparing the two light sources. It was found that all plants had a larger step growth percentage under LED light. The hgihest growth rate was in variegated plants which ha a percent growth of 31.78%. This suggests that plants grow taller under LED light as supposed to daylight.

Introduction:

LED lights are used in a variety of places. For example, lamps, televisions, tarific signals and remote controls. LED lights used in this lab can have altered intensities of red, infrared, blue and green lights. When LED lights are turned on, energy is released in the form of photons. The color of the light depends on the energy gap of the semicondctor (2). Daylight on the other hand is the combination of light light inside and outside during the day. It has a larger spectrum of electromagnetic radiation but is not necessarily stronger. Daylight spectrum of light spans from 100 nm to 1mm. Here is a graph of the light spectrum for LED lights:

Wisconsin Fast Plants have a life cycle of 40 days and need little to no care when growing. This makes them perfect for lab work. The four types of Brassica rapa used in the following lab are: purple stem, non purple stem, variegated, and wild type.

Phytochromes are photoreceptors that detect light and the color of light. They exist in two forms phtochrome red (Pr) and phytochrome far-red (Pfr). Pr absorbs at a peak of 666nm and Pfr absorbs at 730nm. Photochrome a is responsible for early events in germination and seedling while phytochrome b is involved in shade detection and avoidance. Phytochrome b's response elongated the plants stem when it becomes shaded by competitors (1). It is known that the presence of protein effects the phenotype of a plant. It is predicted that there will be no noticeable difference in stem and leaf growth under LED and daylight and therefore will have no difference in the amount of phytochrome present.

Materials and Methods:

Week 1:

Four genotypes of Wisconsin fast plants were obtained: purple stemmed, variegated, non-purple stemmed (anthocyaninless), and standard.  Two of each strain of plant were individually planted in plastic cells grouped into two 4x1 cell complexes.  The arrangement of the plants in the cells was random.  The plants were watered and baseline measurements were obtained for stem length and leaf area.   A cardboard box with no holes or seams was fitted with an LED light source which allowed for precise manipulation of red, green, blue, and IR light sources.  Each light source was set to level 9.  One plant cell complex was placed in the LED box, under 24-hour light, and the other complex was placed in the University greenhouse under 24-hour fluorescent light.  The stem-length and leaf area were measured each day between noon and 2:00pm for one week (excluding the weekend) and watered as necessary. 

Week 2:

After the final measurements of the plants were made, the largest leaf from each plant was removed and crushed in a micropipette tube with a pestal.  1 mL of QB buffer (100mM potassium phosphate buffer pH= 7.8, 1mM EDTA, 1% Triton-X-100, and 10% glycerol) was added to the plant matter and this was centrifuged at top speed in the microfuge for 15-20 minutes.  When finished, the supernatant containing the plant protein was removed and placed in a new microcentrifuge tube.  Three dilutions for each of the 8 plants (24 total) were prepared.  The dilutions were 1:2, 1:20, 1:200; protein to buffer.  These dilutions were each prepared to 100 µL; therefore the first dilution contained 50 µL protein and 50 µL QB, the second contained 5 µL protein and 95 µL QB, and .5 protein and 99.5 µL QB.

A 1mg/mL stock solution of protein standard solution (Bovine Serum Albumin or BSA) in QB was prepared in order to prepare a standard curve.  Six microcentrifuge tubes were obtained and labeled 1-6.  Each tube was prepared as follows:

Each tube was vortexed upon preparation.  The protein determination reagent was prepared by adding 200 µL of Copper (II) Sulfate Pentahydrate (4%) solution into 10 mL of Bicinchoninic Acid (BCA) solution using a 15 mL falcon tube.  10 µL of standards and unknown proteins were loaded into a 96-well plate.  200 µL of protein determination reagent was added to each well with the protein.  The well was incubated at room temperature for 30 minutes.  The Molecular Devices UV Max Kinetic microplate reader was used to determine the concentration of unknown protein from microsomal preparation (λ= 560nm).  The plate was read and a standard curve was prepared by plotting the average protein standard concentration vs. the net absorbance values at 560 nm, and the line of best-fit was drawn.  Using Excel, a linear regression was run on this data and the concentration of unknown samples was determined based on this information. 

An SDS PAGE Gel was prepared by removing the comb from the gel and clamping it to the electrophoretic apparatus.  The apparatus was then filled with TBE buffer.  Each well was rinsed with running buffer and a pipette.  Using QB buffer, each sample was diluted to the same concentration as the most concentrated sample, which was the LED-grown standard plant (sample 5).  Each sample was then placed in boiling water to denature it for 10 minutes.  30 µL of diluted protein was then mixed with 15 µL of loading buffer.  15 µL of these mixtures were then loaded into wells 2-9 with the protein standard in well 1.  The gel was then run at 100v for one hour.

Western Blot:

6 pieces of Whatman paper (3mm) and 1 piece of nitrocellulose paper were cut to the exact size of the SDS gel.  One corner of the NC paper was marked with soft lead pencil. The NC paper was floated on transfer buffer and submerged. The Whatman paper was soaked in transfer buffer. The gel was rinsed in dH2O. A gel sandwich for gel transfer was constructed in the following order: positive terminal (+), western apparatus grid, 3 sheets of filter paper, NC paper (protein blot), SDS page gel, 3 sheets of filter paper, western apparatus grid, negative terminal (-).  This was run for one hour. The transfer unit was disassembled and NC was blocked in 50ml of Blotto overnight. The next day, NC was washed with 20ml of TBS-T one time for 15 minutes and then two times for 5 minutes. NC was then incubated in primary antibody solution (TBS-T) at room temperature, for 6 days.

Antibody Wash and Protein Determination:

NC was washed with 20ml TBS-T one time for 15 minutes and twice for 5 minutes.  NC was then incubated with secondary antibody solution (1:5000 dilution) for 90 minutes. NC was again washed one time with 20ml TBS-T for 30 minutes and then two times for 15 minutes. CD1 solution (50ml TBS and 20µL hydrogen peroxide) and CD2 solution (0.03g 4-chlor-1-naptol and 10ml MeOH) were prepared. After the membrane had been washed, the CD1 and CD2 solutions were mixed while stirring. CD1 and CD2 mixture was added to NC and developed until the bands appeared. The reaction was terminated by rinsing the NC with dH2O. The development did not take longer than 2 hours.

Results

Figure 1.docx  

Figure 1: Standard curve of protein STD concentration (µL) vs. absorption with linear regression (y=7.3617x-2.6766. R2 = 0.8908).

  Figure 2.docx

Figure 2: Percent change in plant stem length. A comparison of plants under daylight vs. plants under LED.

 Figure 3.docx 

Figure 3: Percent change in plants’ biggest leaf size. A comparison of the biggest leaf of each plant under daylight vs. LED.

Figure 2 shows a variation in the stem length between plants under daylight vs. LED. Each plant under LED shows a slightly higher percent change in stem length than plants under daylight. Figure 3 shows the percent change in biggest leaf size. There is no significant trend in percent change between the plants under daylight vs. LED.

Discussion:

It was predicted that there would be no change in stem length, leaf size and amount of phytochroms present when comparing plant growth under LED and daylight. This hypothesis cannot be accepted due to the information gained from this study. It was measured that all plants grew taller when grown under LED lights. The greatest stem length percent change was measured in the variegated plants 31.78% difference. This may be because LED lights have high intensity at specific nm wavelengths as supposed to daylight which covers all wavelengths.

There was no notable difference when looking at leaf size. Purple stem and non purple stems had a greater percent growth under daylight while variegated and wild type plants had a greater percent growth under LED lights. These results suggest that leaf growth is different for all types of plants. It also might mean that leaf growth does not respond to LED light, further experimentation may be need to compare leaf growth with plants under light with day/night changes.

Phytochrome western blots were hard to read. It is predicted that there would be no difference in phytochrome levels under LED and daylight. This may not hold true because of the difference in stem length. Recall that photochrome a is involved with stem elongation. This phytochrome may have been at higher levels in the plants grown under LED lights because of their higher percent growth of stem length. The variegated plants should have had the highest levels of this phytochrome.

1. Reed, J. W., A. Nagatani, T. D. Elich, M. Fagan, and J. Chory. "Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development." Plant Physiology 104.4 (1994): 1139-149. Print.

2. Zheludev, N. (2007). "The life and times of the LED – a 100-year history.” Nature Photonics 1 (4): 189–192. <http://www.nanophotonics.org.uk/niz/publications/zheludev-2007-ltl.pdf>.