Plant Biology 150L
Fall 2003
LIGHT-INDUCED PROTEIN EXPRESSION
Objectives of this laboratory exercise
I. To observe light-induced "greening" of maize seedlings
II. To demonstrate the biochemical basis for the greening process
Why are we doing this exercise?
Light is an important environmental factor that controls plant growth and development. A
principal reason is that light makes photosynthesis possible. Besides photosynthesis, light is also
involved in other plant processes, mostly affecting the appearance of a plant-- i.e.,, its structural
development or morphogenesis (origin of form). The control of morphogenesis by light is called
photomorphogenesis. In this lab we will observe how light changes the appearance of darkgrown maize seedlings (Zea maize) and demonstrate the molecular basis of that change.
Relevant background information
When a seed is sown in darkness (in the soil), it germinates and grows using mostly stored
nutrients. Once the seedling penetrates the upper layer of soil and is exposed to light, it begins to
alter its form. Perhaps the most striking alteration is the color of the cotyledons (or the leaves)
and the stem, from pale yellow to green. This color change is referred to as the "greening"
process which prepares the seedling to initiate photosynthesis.
Plants can absorb light because they have various photoreceptors, including phytochrome (red far red light), cryptochrome (blue - long wave ultraviolet), UV-B photoreceptor ( UV 280-320
nm), and protochlorophyllide a, which is a precursor of chlorophyll a (red and blue light). The
photoreceptor most familiar to us is phytochrome. When phytochrome is exposed to red light, it
becomes activated, inducing a series of biochemical changes in the plant cell.
One such phytochrome-induced change is the formation of chloroplasts from preplastids. The
preplastids already have trace amounts of chlorophyll and proteins required for photosynthesis.
Many of the proteins are present at very low levels in dark-grow plants, but upon illumination
their expression is dramatically increased. This induction mostly occurs at transcriptional level-i.e., the level of mRNA synthesis.
One of the best studied cases in light-induced gene expression is the accumulation of mRNA and
protein for ribulose-bisphosphate carboxylase and oxygenase (Rubisco). Studies have shown that
the genes coding for the small subunit protein (15 kD) are located in the nucleus while those for
the large subunit (50 kD) are in the plastid genome. The small subunit protein is synthesized in
the cytoplasm and transported into the plastid. The large subunit is synthesized in the plastid and
united with the small subunit, after the latter arrives, to form the holoenzyme. Each functional
Rubisco enzyme consists of eight small subunits and eight large subunits. As the enzyme
catalyzes the first step in CO2 fixation, Rubisco accumulation rapidly increases during the
greening process.
Laboratory Procedure
1 Cut 5-10 g (roughly) of etiolated leaves in the dark and store them in liquid N2.
2 The instructor will show you different plants. Compare the plants: dark-grown, greening
and green. Examine how significantly light can change the appearance of the plants.
Part 1. Light-induced protein synthesis.
3 The instructor will show students how to grind plant tissues in liquid nitrogen.
4 Students will be divided into groups to grind plants and extract proteins from different
categories of leaves (etiolated, greening and green). The etiolated leaves are already picked by
the instructor before bringing the pots to the lab to avoid illumination. One or two groups can do
the etiolated leaves; other groups will pick leaves (5-10 seedlings) from green and “greening”
plants and drop them in the mortar containing liquid nitrogen.
5 Grind the tissue into fine powder with a mortar and pestle.
6 Pour the tissue powder into a beaker with 5 ml extraction buffer and mix briefly.
7 Pour into a 15 ml tub, cap the tube and resuspend by inverting several times.
8 Leave the tube on ice for 5 minutes.
9 Take 1 ml of the supernatant and put into an eppendorf tube. Centrifuge for 5 minutes at
14000 rpm at 4°C. This is the crude protein extract stock for further analysis by all groups.
10 Label five eppendorf tubes as “blank,” “control,” “green,” “etiolated,” “greening” (or any
other labeling system that will work for you).
11 To each eppendorf tube, add the contents according to the protocol below. Distilled water
(DW) is the blank. Bovine Serum Albumin (BSA) is the control.
Blank
Mix well again by inverting tube.
Control
Assay Dye. Mix well again by inverting tube.
Etiolated
Protein Assay Dye. Mix well again by inverting tube.
Greening
Protein Assay Dye. Mix well again by inverting tube.
Green
Protein Assay Dye. Mix well again by inverting tube.
12 Measure the absorption at 595 nm in a spectrophotometer.
13 Calculate the protein concentration according to the concentration of the standard (BSA).
14 Compare the results obtained by different groups and take the average if they are close
enough (the instructor will make that call).
Tube
Blank
Control
Etiolated
Greening
Green
Contents
780
Absorption
Protein Concentration
Part 2. Protein SDS-PolyAcrylmide Gel Electrophoresis (SDS-PAGE)
1 The gels and running buffer will be made by the instructor during lab.
2
eppendorf tubes, and add an equivalent amount of protein loading buffer to each tube.
3 Heat the samples at 80-90°C for three minutes before loading the MW marker and samples
to the gel.
4 Run the gel for 45 minutes at 200 V (vary according to the size of the gel and the position of
the dye front).
5 Take off the gel and stain it with Comassie blue for 5-10 minutes.
6 Destain to observe the protein bands around 15 and 50 kD.
7 Compare the abundance of the proteins in different samples
Discuss the results according to the following questions:
Why are plants changing their color upon illumination?
What is the molecular basis for light-induced color change?
What other experiments can we do to study the mechanism involved in the light-regulated
expression of Rubisco? (e.g., To determine whether the regulation occurs at transcriptional
or post-transcriptional level; how the light signal is transmitted to the nucleus; etc.)