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

Introduction to the Protoplast Lab

Objectives: Upon completion of this set of labs you should be able to:

use a centrifuge and understand its principle of operation.
describe the action of pectinase, hemicellulase and cellulase on plant tissues.
isolate and observe protoplasts from plants
identify the major structures in plant cells and protoplasts that are visible with light microscopy.
describe some of the properties of protoplasts.
determine the viability of a plant protoplast preparation.
perform simple statistical analyses
use an ocular micrometer to measure cell size.
use a hemocytometer to count cell populations.

A Protoplast Primer:
Protoplasts, plant cells without walls, are important tools in the study of plant physiological activities. They have been used to: (1) separate cell types; (2) study the intracellular location of particular metabolites; (3) gently isolate organelles from plant cells; (4) study cell wall and plasma membrane biosynthesis; (5) produce somatic cell hybrids (fusion of protoplasts); (6) introduce foreign genetic material and organelles into recipient plants; and (7) achieve highly efficient virus infections (Wagner et. al., 1978).

Protoplasts are most easily isolated by enzymatically digesting the cell wall. The general procedure is: (1) surface sterilize the leaf; (2) rinse the tissue in the proper osmotic agent such as sorbitol or mannitol. Protoplasts must be maintained in an isotonic medium so they don't burst; (3) cut the leaf into strips or peel the epidermis to expose the tissue for enzymatic digestion; (4) treat with enzymes (sequentially or mixed). Typically, a combination of cellulase, pectinase and hemicellulase, is used. The specific amount and type of enzyme used is generally a result of trial and error. Cellulysin and Meicelase are trade names for cellulase and pectinase, respectively; (5) rinse the preparation to remove the enzymes; and (6) isolate and purify the protoplasts. An alternative method to isolate protoplasts is to mechanically chop up the plant tissue in isotonic medium (McLaughlin, Giannini, Fishbeck, 1993).

Protoplasts can be ruptured in a variety of ways to release the cell contents. Three commonly used methods include: (1) mechanical shear; (2) osmotic shock; and (3) mild detergent. Each method has specific advantages and disadvantages and the method selected depends upon the application. For example, forcing protoplasts through a syringe or fine nylon mesh (~ 20 μm) will rupture protoplasts including the vacuoles. Osmotic shock will gently disrupt the protoplast releasing intact vacuoles. Treating protoplasts with K₂HPO₄ causes the cytoplasm to congeal and release vacuoles.

An Anthocyanin Primer:
Anthocyanins, which are a type of flavanoid pigment, are responsible for the coloration of red cabbage leaves. These pigments are water soluble and are dissolved in the cell sap in the vacuole. Thus, they are a good marker to observe the size of the vacuole. In cabbage, anthocyanins may be synthesized in anthocyanoplasts which are tiny, transient, organelle-like structures found in the vacuole (look for discrete red spots or speckles). The concentration of anthocyanin in the vacuole of grape skin cells is 97 mM (Moskowitz and Hrazdina, 1981).

Anthocyanins are polyphenolic (see structure), water-soluble molecules responsible for the color of many flowers and fruits. There are 15 different anthocyanins in red cabbage. Most have the basic cyanidin skeleton to which are attached various groups including glucose, coumarin, ferulic acid, and sinapic acids. The basic anthocyanin skeleton is drawn below:

Anthocyanins are pH sensitive. The color of an individual anthocyanin will vary from red (acidic conditions) to blue to yellow (alkaline conditions). The final tissue color is ultimately dependent upon several factors including the type and concentration of anthocyanin pigments present, pH, and the presence of other metabolites (such as ions, sugars, hormones).

Anthocyanin synthesis requires light. Low levels of anthocyanin are present in dark grown plants (0.35 nmol/g) but the amount increases rapidly after exposure to light (5 nmol/g after 6 days). IAA treatment decreases anthocyanin synthesis and K⁺ stimulates it (Mazza & Minati, 1993).

Overview:
In this lab, we will isolate and purify protoplasts from red cabbage and examine them in the microscope. If time we will examine the properties and the viability of the protoplasts. We will spectrophotometrically determine the anthocyanin content of the red cabbage protoplasts. To accomplish this task we will also need to determine the protoplast volume with an ocular micrometer and quantify protoplast numbers in our preparation using a hemocytometer.

Pre-Lab: Print and complete the pre-lab questions.

Post-Lab: At the completion of the lab experience you are required to complete:

An abstract summarizing the main activities of the lab. The abstract should be typed and be approximately one page in length and should include information about the plants used, techniques, results and conclusions. The questions scattered throughout the exercises provide a guide to the types of items to include in the abstract. I recommend starting by saying; "The purpose of this lab was to....." Then, describe briefly each major thing we did and the outcome. These could be mini-paragraphs (two or so sentences each) or part of one larger single paragraph.
Append to your abstract the following:

Sketch or print of digital imags of the red cabbage and green leaf (species to be determined) protoplasts we observed including an appropriate caption. Label the visible structures. Include plate magnification. Label your images (e.g., Figure 1, 2) giving each an appropriate caption.

Tables 1 & 2 summarizing protoplast yield data. Are the data significant at the 0.05 confidence level?

Table 1 for the frequency of pigmented and unpigmented protoplasts

Table 2 & 3 summarizing volume data for pigmented and unpigmented protoplasts

On a separate sheet of paper show a statistical analysis to answer the question, "Are pigmentd protoplasts larger than unpigmented protoplasts?" Record H_o, t value from t-test, p value and your conclusion about the hypothesis/question.

Tables 1 & 2 summarizing anthocyanin data

References:

Curtright, Robert, Randy Emry, and John Markwell. 1996. Plant Pigments Course. University of Nebraska, Lincoln. (source of anthocyanin diagram). From web site.
Galun, E. 1981. Plant Protoplasts as Physiological Tools. Ann. Rev. of Plant Physiol. 32:237.
Hrazdina, G.G. Marx and H.C. Hoch. 1982. Distribution of secondary plant metabolites and their biosynthetic enzymes in pea (Pisum sativum L.) leaves. Plant Physiol. 70: 745-748.
Hrazdina, G., H. Iredale, and L. R. Mattick. 1977. Anthocyanin composition of Brassica oleracea cv. Red Danish. Phytochemistry 16:278.
Hrazdina, G., G. J. Wagner, and H. W. Siegelman. 1978. Subcellular localization of enzymes of anthocyanin-biosynthesis in protoplasts. Phytochemistry 17:53.
Lin, W. and V. Wittenbach 1981. Subcellular localization of proteases in wheat and corn mesophyll protoplasts. Plant Physiol 67: 969-972.
Mazza, G & E Miniati. 1993. Anthocyanin in Fruits, Vegetables, and Grains. CRC Press, Ann Arbor, MI
McLaughlin, E, Giannini, J., Fishbeck, K. 1993. Color Coded Organelles. American Biology Teacher.
Moshkowitz, and G. Hrazdina. 1981. Vacuolar contents of fruit subepidermal cells from Vitis species. Plant Physiology 68: 686-692.
Wagner, G.J., W.C. Butcher IV and N.W. Siegelman. 1978. The plant protoplast: A useful tool for plant research and student instruction. Bioscience 28: 95-101.