Previous Next

Plant Detectives Manual: a research-led approach for teaching plant science

Activity 3: Extraction and quantification of photosynthetic pigments

3.1) Introduction and objectives

Although most plant leaves appear to be green, several different colour pigments are usually present in the chloroplasts of green leaves (Fig. 5). Colours are a result of light absorption by pigments at specific wavelengths. These pigments are not simply decoration, but play critical roles in photochemistry — photosynthesis and photoprotective mechanisms. Mutations that affect pigment composition of leaves can have substantial functional impact, and can be used to explore the genetic control of pigment biochemical pathways and physiological processes.

 

The chlorophylls a (Chla) and b (Chlb) provide the green colour and absorb the light energy that is needed for photosynthesis. Chla has a methyl group in the position where Chlb has a formyl group, which gives Chla slightly more affinity for non-polar solvents relative to Chlb. Pheophytin plays a vital role in electron transport, but also may occur as an acid-induced breakdown product of chlorophyll. This other form of chlorophyll lacks the central Mg2+ that Chla and Chlb have; this results in a higher affinity for non-polar, hydrophobic solvents.

 

Closely associated with the chlorophylls in the chloroplast is another group of pigments, the carotenoids. They are yellow to red in colour and likely play a role in the gathering of light energy for photosynthesis, as well as helping to protect the chlorophylls against photo oxidation. The carotenoids are divided into two groups: the carotenes, a pure hydrocarbon group; and the xanthophylls, which are characterised by two additional oxygen atoms. The additional oxygen, which is present as hydroxyl, groups at the ends of the molecule, making the xanthophylls more polar. In other words, the xanthophylls have less affinity for non-polar solvents than the carotenes.

 

Differences in the chemical structure of pigments not only determine their absorbance spectrum and, thus, their function, but they also change the pigment affinity for different solvents. This difference in solubility allows the separation of pigments by chromatographic techniques and the specific absorbance spectrum can be utilised to identify individual pigments (Fig. 6C).

 

The main objectives of Activity 3 are to:

  1. isolate total pigments from both wild type and mutant plants grown in the light using a simple organic solvent extraction procedure
  2. quantify pigments based on their spectral properties.

 

Image

Figure 5. Structure of plant pigments

3.2) Materials

Numbers in parenthesis indicate number of specific items per group.

  1. 1.5 millilitre (ml) Eppendorf tubes and rack
  2. 80% (v/v) acetone in capped bottle
  3. block heater (optional)
  4. gloves
  5. microcentrifuge (one per class or one per group) (swing out bucket is best, but not indispensable)
  6. micropipettes of 200–1000 microlitres (µl) and tips
  7. safety goggles (one per person)
  8. vortex (one per class or one per group if available)
  9. plate reader BIO-TEK uQuant and 96 well plate or spectrophotometer and cuvette
  10. waste container for acetone in the chemical hood
  11. liquid nitrogen (–197 °C!!!)
  12. plastic pistils for grinding tissue or ball bearings and a TissueLyser

Image

3.3) Procedures

3.3.1) Pigment extraction for spectrophotometric analyses

 

Plant pigments can be extracted from plant tissues by organic solvents. The tissue is first frozen in liquid nitrogen and ground, the ground tissue is then treated with organic solvents to extract the plant pigments. Using the protocol below you will extract Chla and Chlb and quantify their content in your leaf tissues. Depending on who performs the harvest and the type of grinding method, you may be asked to continue from Step 3 or Step 4.

Image

 

Materials

Method

Eppendorf tubes

1. Harvest approximately 30 milligrams (mg) of leaf tissue from FOUR wild type and mutant plants and place each into a 1.5 ml Eppendorff tube. If leaves are small you may need to bulk samples. Keep design principles in mind. Make sure tubes are properly labelled beforehand and contain a bearing ball for the grinding if using the TissueLyser. Alternatively, you can grind tissue using plastic pistils (Step 3). See labelling tips in Appendix B.

Goggles, gloves, liquid nitrogen

2. Freeze the material immediately by dipping the sample in liquid nitrogen. IMPORTANT: wear goggles and gloves to avoid burning your skin. Liquid nitrogen boils at −196 °C (very cold!) and causes rapid freezing when in contact with living tissue. TIP: it is best to use a floating styrofoam rack to keep samples upright and avoid liquid nitrogen leaking into the tube.

Pistil for grinding

3. Grind the frozen tissue, taking care not to let the samples thaw. If thaw starts, dip the tubes in the liquid nitrogen again.

Acetone, pipettes, tips, vortex, gloves

4. Add 1000 µl of 80% (v/v) acetone to the sample. Wear goggles at all times while working with solvents!

Vortex

5. Vortex for ten seconds. TIP: if it is necessary to increase extraction efficiency, incubate under light (if available) or at 37 ºC to 40 ºC for ten minutes, vortexing periodically.

Microcentrifuge

6. Centrifuge the sample for two minutes at 7000 g in a microcentrifuge.

 

7. Carefully, transfer as much supernatant as possible to a new tubes. Be careful not to remove pellet. Usually, 700–900 µl can be recovered depending on how packed the pellet is at the bottom. Remember to label the new tubes accordingly!

 

8. For one of the four tubes per genotype, transfer 500 µl to another tube labelled ‘group#–WT’ or ‘group#–Mutant’. These two samples per group will be used in Activity 4. If they are to be used at another time they can be stored at –20 ºC.

3.3.2) Spectrophometric quantification

Dissolved pigments absorb light of specific wavelengths directly proportional to their concentration. This relationship is expressed quantitatively by the Beer-Lambert law:

Image

where Abs is the absorbance at wavelength , l is the light path length (centimetre (cm)), the millimolar extinction coefficient, or a molecule–specific constant in a given solvent (L g–1 cm–1), and C is the concentration (g L–1). We can calculate the concentration of any absorbing molecule if we know its absorbance at specific , l and . In this case, we need to use the for 80% (v/v) acetone.

Materials

Method

96 well plate

1. Add 200 µl of 80% (v/v) acetone in two wells as blanks.

96 well plate

2. Transfer 200 µl aliquots of your samples into two or three wells.

Plate reader

3. Select the wavelength of 664 nanometres (nm).

 

4. Read the absorbance of your sample at 664 nm and record the number.

 

5. Repeat reading for the wavelength of 647 nm.

 

6. Recalculate the absorbance of your samples by subtracting that of the blank.

 

7. Calculate chlorophyll concentration as:

 

Image

Image

Note that the ε values are those for chlorophyll in aqueous solution of 80% (v/v) acetone.

Waste container, chemical hood

8. Discard the extract in the proper waste container in the hood.

 

Alternative procedure for pigment characterisation using a regular spectrophotometer

The use of a plate reader gives more flexibility and higher replication power as many samples can be read and a whole spectrum (i.e., absorbance readings at different wavelengths) can be obtained in a matter of minutes. If a plate reader is not available, however, the same measurements can be performed with a regular single cuvette spectrophotometer. Make sure to run at least three replicate readings at 647 nm and 664 nm for each of the three biological replicates per genotype for the quantification of chlorophylls; the same equations can be used provided the solvent is 80% (v/v) acetone.

3.4) Expected outcomes

  1. calculate the amount of Chla and Chlb in a microgram (µg) of chlorophyll per milligram (mg) of fresh weight
  2. calculate the Chla/b ratio for all the samples. Graph and analyse the data and indicate if there is a significant difference between genotypes (See Appendix B).

 


Previous Next