Plant Physiology

Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu

Pigments in Rapid-Cycling Brassica rapa (RCBr)

Learning Objectives: Upon completion of this lab you should be able to:

grow an RCBr ("Fast Plant") from seed to seed
describe the life history of Brassica rapa using terms such as radicle, hypocotyl, cotyledon, epicotyl, node, internode, pedicel, receptacle, sepals, petal, androecium (or stamen); filament, anther, gynoecium (or pistil), stigma, style, ovary, nectary, pollination, silique (pod), raceme, inflorescence, fertilization
cross pollinate a flower
compare and contrast anl (anthocyaninless and ygr (yellow-green) mutants
measure anthocyanin and chlorophyll concentrations of leaves

Introduction:
The purpose of this lab is two-fold. First, this lab will provide the opportunity for you to study the growth of a plant, rapid-cycling Brassica rapa (RCBr) throughout its entire life cycle. To learn more about the history and biology of RCBr visit the Wisconsin Fast Plants^� web site. This site provides a wealth of information, including the on-line version of Dr. Williams article and slides/images of the plants. The second major purpose of this lab is to use Fast Plants^� as a model experimental organism to study pigment composition and quantification.

Wild type RCBr plants have purple stems and dark green leaves. The purple coloration is due to anthocyanin pigments. One RCBr mutant has non-purple stems (anl) while another has yellow-green leaves (ygr). These observations lead to a variety of questions:

Do non-purple (anl) mutants have lower concentrations of anthocyanins than wild-type purple plants (ANL)?
Do yellow-green mutants (ygr) have lower concentrations of chlorophyll than wild-type plants (YGR)?
Do yellow-greem mutants (ygr) have higher concentrations of yellow pigments (carotenoids) than wild-type plants (YGR)?
Do hybrid plants have intermediate amount of pigments?

The purpose of this lab will be to answer these questions.

Pre-Lab Assignment:
Before coming to lab, print a copies of this exercise and RCBr Growth Techniques.

EXERCISE 1: Growing Rapid-Cycling Brassica rapa

Obtain 3 seeds each of the wild type (purple stem, dark green leaves; ANL/YGR) and non-purple stem/yellow-green leaves (anl/ygr) mutant. Plant each in one cell of a quad according to the instructions in the RCBr growth techniques handout. Thin to one plant per cell which will yield one plant of each type in each quad. When it is time to pollinate, cross-pollinate the two individuals to produce hybrid seeds (ANL anl/YGR ygr). Allow these plants to produce seed. Collect the F1 hybrid seeds.

EXERCISE 2: Growing Plants for Pigment Analysis

Obtain a quad and in two cells plant hybrid seeds. In the other cells, plant seeds from the original parent stocks. Be sure to label the cells appropriate using toothpick flags. Harvest the seedlings after about 10 days and analyze for chlorophyll, anthocyanin and carotenoids.

EXERCISE 3: Measuring Chlorophyll & Carotenoid Concentrations

Use a cork borer to cut a disk from a leaf. Measure the diameter and then calculate the area of the disk. Place the disk in a mortar and add a small amount of acetone/Tris buffer (80:20 volume; pH = 7.8). Grind the disk using the pestle. Transfer the homogenized mixture into a 10 mL graduate cylinder. Rinse the mortar/pestle with some fresh extraction mixture and pour into the graduate cylinder. Make up to a final volume of 6 mL. Transfer this to a centrifuge tube and spin at the highest setting for 3 minutes. Measure the absorbance of the supernatant at wavelengths of 470, 537, 647, and 663 nm. Then using the equations of Sims and Gamon (2002) calculate the total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid concentration. Click here for details.

Table 1. Chlorophyll pigment data for RCBr plants
		ANL/YGR					Hybrid					anl/ygr
Wavelength (nm)		1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
	470
	537
	647
	663

Table 2. Chlorophyll pigment concentrations for RCbr. These values are calculated using the equations developed by Sims and Gamon (2002). Final calculations are corrected for solution volume (6 mL) and leaf disk area (dia = mm; area = m2)
	ANL/YGR					Hybrid					anl/ygr
	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
chl a (�mol ml^-1)
chl b (�mol ml^-1)
carotenoids (�mol ml^-1)
Anthocyanin (�mol ml^-1)
chl a (�mol m^-2)
chl b (�mol m^-2)
carotenoids (�mol m^-2)
Anthocyanin (�mol m^-2)
ttl Chl (�mol m^-2)
chl a/b ratio

Table 3. Summary chlorophyll pigment concentrations for RCBr
	ANL/YGR	Hybrid	anl/ygr
chl a (�mol m^-2)
chl b (�mol m^-2)
carotenoids (�mol m^-2)
ttl Chl (�mol m^-2)
chl a/b ratio

EXERCISE 4: Measuring Anthocyanin Concentrations

Because the equations used for calculating anthocyanin described above are not particularly accurate, we will use a better technique for measuring anthocyanin concentrations. Use the cork borer to cut a disk from a leaf (or obtain the stem/hypocotyl and record the fresh weight). Grind the tissue in a small amount of acidified methanol ( methanol / HCL / water; 90:1:1) using the mortar and pestle. Transfer to a graduate cylinder, rinse with mortar/pestle with fresh acidified methanol and then pour into a graduate cylinder. Make the final volume up to 6 mL with fresh acidified methanol. Then transfer to a centrifuge tube and spin at the highest setting for 2 minutes. Record the absorbance of the solutions at 529 and 650 nm. Then using the equations of Sims and Gamon (2002) calculate the total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid concentration. Click here for details.

Table 4. Anthocyanin pigment data for RCBr plants
		ANL/YGR					Hybrid					anl/ygr
Wavelength (nm)		1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
	weight tissue (g)
	529
	650

Table 5. Anthocyanin pigment concentrations for RCbr. These values are calculated using the equations developed by Sims and Gamon (2002). Final calculations are corrected for solution volume (6 mL) and leaf disk area (dia = mm; area = m2) or tissue weight (g)
	ANL/YGR					Hybrid					anl/ygr
	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
Anthocyanin absorbance corrected
[Anthocyanin] (mmol/ml)
[Anthocyanin] (mol; correct for solution volume)
[Anthocyanin] (mmol)
[Anthocyanin] (mmol/m2 or g)
Mean [Anthocyanin] (mmol/m2 or g)

Post-Lab Results: At the conclusion of the experiment, write an abstract summarizing your results. Among the questions to consider in your abstract: Why are ygr mutants yellow-green? How is pigment production affected in teh hybrids? Append to your abstract:

A completed version of Tables 1-6.
A histogram (bar graph) summarizing the results for chlorophyll, carotenoid and anthocyanin concentration for the three groups.
Suitable statistical test(s) to determine if there is a meaningful difference in pigment concentration. Show your data/work.

References:

Williams, P. B. and C.B. Hill. 1986. Rapid-cycling populations of Brassica. Science 232: 1385-1389.
Williams, P.B. 1989. Rapid-cycling Brassicas. Carolina Tips 52: 5-7.
Wisconsin Fast Plants Web Site - there is a wealth of information available at this site.