The Expression of Chlorophyll a/b

Binding Protein in Glycine Max

Alexander WardCMB 426

What is Chl-A?

The primary photosynthetic pigment (core pigment).

Interacts directly with the light requiring reactions of photosynthesis.

Exists in all phototrophs, save bacteria.

Primarily absorb red and blue light.

3x more abundant than Chl-B.

What is Chl-B?

An accessory photosynthetic pigment (antenna pigment).

Exists in all phototrophs save diatoms, cyanobacteria, and algae.

Primarily absorb red and blue light.

What is Chlorophyll a/b Binding Protein? Binds both Chl-A and Chl-B. Forms a photoactive protein-

chlorophyll or protein-pigment complex.

A few different forms of chl a/b binding protein (3 in this figure).

Forms the light harvesting complex (LHC) for photosynthesis.

In changing light conditions, represents a system for balancing the two photosystems.

λ-Phage

Lane 1 and 1’ – MJ-LF Lane 2 and 2’ – MJ-ST Lane 3 and 3’ – DEPOD Lane 4 – Positive Control Lane 5 – MW Ladder Lane 7 – Negative Control

Restriction Digest

Genomic DNA present without enzyme.

XhoI and EcoRI show single bands.

The double digest indicates no internal cut sites.

PCR Product is from the λ-Phage, DEPOD.

Treatments

Three different filtered light conditions consisting of 18 hours light and 8 hours dark at room temperature.

The filters were red, green, blue, and gray for the control. One week old Glyine max seedlings were grown in vermiculite for one

week. The trifoliate leaves were harvested for RNA extraction.

Primer Test

Lane 1 – Primer Test Lane 2 – MW Ladder Lane 4 – Negative Control

The test shows a single band around the expected size.

RNA

Mass of RNA used = 1μg. Normalization based on the pixel count from the control sample. Gel consists of 1.5% agarose in 1X glyoxal running buffer. Red, green and blue are filtered light experimental conditions.

RT-PCR

From right to left, 100, 10-1 and 102 dilutions for the 3 lanes of each experimental condition.

MW

La

dder

Control Red

+ Co

ntrol

Green Blue

- Con

trol

Condition Net IntensityControl 84420Red 67789Green 49589Blue 21910

Net Intensity based on 10-2 dilution.

Results

Downregulation of mRNA for Chlorophyll a/b Binding Protein in each treatment.

Different results than expected, expected upregulation for Red and Green light, due to upregulation of light harvesting complexes.

Expression could be upregulated through alternative splicing and adding or removing functional groups from the DNA expression level.

It could be the protein being examined is coded by the plastid, chloroplast DNA (ctDNA).

Conclusion/Future Work

Repeat the experiment with variation in incubation time. Use different amounts of transmitted light, based on the filter used. Vary the distance from the light source. Test ctDNA and chloroplast RNA expression.

References

"Botany Online: Photosynthesis - Structure - Thylacoid Membrane." Botany Online: Photosynthesis - Structure - Thylacoid Membrane. N.p., n.d. Web. 24 Apr. 2016. <http://www1.biologie.uni-hamburg.de/b-online/e24/16.htm>. (Slide 4)

"The Difference between Chlorophyll A & B and Photosynthesis Overview « Dyna-gro Blog." Dynagro Blog RSS. N.p., n.d. Web. 24 Apr. 2016. <http://dyna-gro-blog.com/the-difference-between-chlorophyll-a-b-and-photosynthesis-overview/>. (Slide 2-3)

Questions

Figure Caption

A model of the four complexes (PS II: photosystem II, cyt b6/f: part of the electron transport chain between PS II and PS I, PS I: photosystem I, CF1: ATP synthase, CF0: proton channel). The topology of some peptide subunits has been marked. Non-marked polypeptides are in literature characterized solely by their molecular weights: chl a/ b: chlorophyll a/b binding protein, chl a: chlorophyll a binding protein, 559: cytochrome 559, b6: cytochrome b6, f: cytochrome f, 700: reaction centre of PS I, 680: reaction centre of PS II, PQ: plastoquinone, PC: plastocyanin, FeS: iron-sulphur protein, R: Rieske-protein, N: NADP-binding protein. The subunits of CF0 have Greek letters as names, K: proton channel. Part A (at the top) of the picture: proteins encoded by the nucleus are blue, that of plastids have different colours.

LHC

The light-harvesting complex (or antenna complex) is an array of protein and chlorophyll molecules embedded in the thylakoid membrane of plants, which transfer light energy to one chlorophyll a molecule at the reaction center of a photosystem.