Methods in plant biochemistry and molecular biology (gnv64)

PLANT BIOCHEMISTRY and MOLECULAR BIOLOGY... Library of Congress Cataloging-in-Publication Data Plant biochemistry and molecular biology / edited by William V.. Methods for Analysis of P

METHODS IN

PLANT BIOCHEMISTRY

and

MOLECULAR BIOLOGY

PLANT BIOCHEMISTRY

and

MOLECULAR BIOLOGY

PLANT BIOCHEMISTRY

and

MOLECULAR BIOLOGY

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Library of Congress Cataloging-in-Publication Data

Plant biochemistry and molecular biology / edited by William V Dashek

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the

CRC Press Web site at http://www.crcpress.com

Dr Dashek wishes to dedicate this manual to his children, Kristin Ann Simpson and Karin Ann Dashek, who patiently dealt with his need for years of scholarship He also thanks the following for his intellectual development: Dr W G Rosen, Dr Wm F Millington, Dr D T A Lamport,

Dr J G Varner, Dr L Margulis, and Dr M W Schein Dr Dashek extends his gratitude to Ms Kim Chancey of the University of Georgia for her patience and thoughtful clerical assistance Dr Dashek is grateful to Terry L Highley, Project Director, Biodeterioration of Wood, U.S Department

of Agriculture - Forest Service, Forest Products Laboratory, for the use of facilities and his encouragement He also expresses his appreciation to Ms K K Nelson of the Forest Products Laboratory for graphics Finally, this volume is dedicated to the late Dr J E Varner, who invented methods to advance our knowledge of plant biochemistry

The staff at CRC Press has been crucial in bringing this project to fruition My thanks go out

to Paul Petralia, Senior Acquisitions Editor, for signing this project; to Cindy Carelli, Editorial Assistant, for her excellent organizational and follow-up skills; and to Carrie Unger, Project Editor, for bringing together the final product in a well-edited, well-presented format

This laboratory manual is designed for the trained scientist performing research in a college, industrial, or federal laboratory The well-illustrated volume is a convenient assemblage of usable, contemporary research protocols and relevant literature citations covering the full range of plant biochemistry, including selected areas of plant molecular biology The detailed protocols are presented within the framework of a contributing author's research program, thereby offering insight into implementation of protocols to current plant biochemistry research Thus, the volume contains

a wide variety of contributing authors, reflecting the thinking and expertise of active investigators who generate advances in technology in order to answer significant research questions In this regard, contributing authors were selected for their ability to create and/or implement novel research methods While some of the investigators are well-known, established scientists who offer years

of valuable laboratory experience, others are at the "outset" of their careers, thus presenting new outlooks on some timely research

Neil R Bowlby

Department of Biochemistry

Michigan State University

East Lansing, Michigan

Mary V Duke

U.S Department of Agriculture Agricultural Research Service Southern Weed Science Laboratory Stoneville, Mississippi

Stephen 0 Duke

U.S Department of Agriculture Agricultural Research Service Natural Products Utilization Research Unit University of Mississippi

Julie K Ellis

Department of Life Sciences John A Logan College Carterville, Illinois

W Scott Grayburn

Northern Crop Science Laboratory U.S Department of Agriculture Agricultural Research Service Fargo, North Dakota

U.S Department of Agriculture - Forest

Michigan State University

East Lansing, Michigan

Alan M Jones

Department of Biology

University of North Carolina

Chapel Hill, North Carolina

University of British Columbia

Vancouver, British Columbia, Canada

Department of Plant and Soil Science Southern Illinois University

John E Mayfield

Department of Biology North Carolina Central University Durham, North Carolina

Kerrie L McDaniel

Department of Biology Western Kentucky University Bowling Green, Kentucky

Lee Mcintosh

Department of Biochemistry Michigan State University East Lansing, Michigan

University of California/Davis Davis California

Part 1: Structure

Chapter 1 Methods for Analysis of Plant Cell and Tissue Ultrastructure 3

Chapter 2 Carpogenesis and Basidiosporogenesis 13

Suki C Croan

Chapter 3 Decolorization of Wood Sapstain 23

Suki C Croan

Part II: Chemistry

Chapter 4 Isolation, Assay, and Characterization of Plant Carbohydrates 29

Chapter 5 Assay and Purification of Enzymes-Oxalate Decarboxylase 49

Chapter 6 Antibody-Mediated Immunochemistry and Immunoassay

in Plant-Related Disease 73

Carol A Clausen and Frederick Green Ill

Chapter 7 Extraction and Assay of Plant Lipids: Phospholipids 89

Chapter 8 Isolation and Analysis of Plant Nucleic Acids 97

Chapter 9 Isolation, Separation, and Characterization of Organic Acids 107

Chapter 10 Photoaffinity Labeling with 5-Azidoindole-3-Acetic Acid 115

Chapter 11 Methods for the Analysis of Cytokinin Content,

Metabolism, and Response l33

David A Lightfoot, Kerrie L McDaniel, Julie K Ellis,

in Plant Tissue by GC/FID 153

Chapter 13 The Role of Plant Growth Regulators During

Filament and Floral Development in Ipomoea nil Flowers 165

Helen G Kiss

Chapter 14 Extraction and Assay of

Terpenoids-Including Certain Plant Hormones 177

Chapter 15 Analytical Methods for the Analysis of Alkaloids 185

Chapter 16 Phenolics and Compartmentalization

in the Sapwood of Broad-Leaved Trees 189

Chapter 1 7 Lignin Analysis 199

Jeffrey F D Dean

Chapter 18 Flavonoid Applications in Research 217

W Dennis Clark and Gregory P Titus

Chapter 19 Analysis and Manipulation of the Chorophyll Pathway

in Higher Plants 229

Chapter 20 Isolation and Characterization of Plant

and Algal Pigment-Protein Complexes 243

Chapter 21 The Isolation and Assay of Elicitins 265

Chapter 22 Chemistry, Extraction, and Assay of Plant Vitamins 281

Part III: Metabolism

Chapter 23 Simultaneous Measurement of Oxygen Uptake

and Quinone Pool Reduction in Potato Tuber Mitochondria 289

Chapter 24 Biosynthesis of Plant Cell Wall Polysaccharides 305

Chapter 28 eDNA Library Construction 349

Part IV: Plant Molecular Biology

Chapter 31 Plant Transformation Techniques and Vectors 361

Chapter 34 Molecular Analysis of cis-Acting Transciptional Regulatory

Elements and Transcriptional Factors

in the Bean Storage Protein Phaseolin Gene 397

Norimoto Murai

Chapter 35 Manipulation of Plant Gene Expression

Using Antisense RNA 423

lndex 443

Structure

Methods for Analysis of Plant Cell

and Tissue Ultrastructure

John E May field and William V Dashek

Contents

1.1 Introduction 3 1.2 Protocols 4 1.2.1 Collection and Fixation of Buds 4 1.2.2 Postfixation in Osmium Tetroxide and Dehydration 4 1.2.3 Infiltration and Embedding 5 1.2.4 Block Trimming and Sectioning 5 1.3 Expected Results 6 References 1 0

1 1 Introduction

The balsam fir (Abies balsamea), a transcontinental gymnosperm, is economically important

because of its value as a source for lumber and wood pulp Besides its economic value, balsam fir, like many other conifers, contains the embryonic tissue that will produce the mature structures during the next growing season within relatively dormant vegetative buds These buds have several advantages that make them very attractive for the preparation and study of plant ultrastructure Thus, transmission electron spectroscopy (TEM) of buds is presented as an example of plant electron microscopy Methods for the preparation of a wide variety of plant tissues can be found in the supplementary references to this chapter (Table 1.1) For a discussion of plant cell fractionation and the assay of organelle fractions for purity, the reader is referred to the review by Quail.1

One advantage is the ease of handling because of the ideal size of the buds They are large enough to treat as macroscopic structures, but small enough to allow good TEM fixation without

a labor-intense effort Terminal buds have a mean height of 2.60 mm Because they have relatively well-described developmental cycles, gymnosperm buds can be collected and prepared at intervals that coincide with temporal-related cytological information Since the preburst bud has a localized region (the apical dome) which contains an abundance of undifferentiated cells, many difficulties associated with the fixation and sectioning of differentiated plant tissues are avoided Also, the

0-8493-9480-5/97/$0.00+$ 50

TABLE 1.1 Summary of Ancillary Electron Microscopic Techniques

Scannmg electron mtcroscopy

High-resolutton autoradtography

Immunoelectron microscopy

Confocal laser microscopy

Scanned probe microscopy

Scanning tunneling microscopy

Electron energy system imaging and X-ray microanalysis

An examination of the cells within these zones will yield a variety of cytological forms at the electron microscopic level

1.2 Protocols

1.2.1 Collection and Fixation of Buds

In this laboratory, vegetative balsam fir buds were collected in Vermont during April Other related species such as Fraser fir from other locations can be used with an equal degree of success if the collection times are changed to account for species- or locality-related differences in temporal sequence

As in most biological materials, it is important to keep the interval between its natural environment and the TEM-fixed state as short as possible Bud-containing terminal branch sections were removed from trees and stored in plastic bags for transportation to the laboratory In the laboratory, the buds were dissected from the enclosing scales and placed into 3% glutaraldehyde

in 0.05 M sodium cacodylate buffer, pH 7.4 While covered with buffered glutaraldehyde, the apical

portion of the buds was cut into l-mm3 portions and fixation continued in fresh buffered dehyde for 2 to 24 h After 2 h, we were unable to notice any differences in ultrastructural appearance

glutaral-1.2.2 Postfixation in Osmium Tetroxide and Dehydration

Following glutaraldehyde fixation, the bud sections are washed in three changes of buffer during

a l-h period While within fresh buffer, the tissue-containing vials should be placed into an ice bucket in a fume hood All preparations of osmium tetroxide solutions and subsequent fixation must be conducted in a chemical fume hood The buds are now postfixed in cold 2% osmium tetroxide (Os04) in the same buffer for 2 h The postfixation in Os04 is followed by ten changes

of cold distilled water'during a 1-h period The final water wash is replaced by 0.5% uranyl acetate

To enhance the electron-dense staining contrast, the tissue should remain in uranyl acetate overnight After uranyl acetate, tissue is placed into water for several minutes before the next step (dehydration) Dehydration is necessary for the tissue to become permeated by the embedding medium The tissue is dehydrated in a graded series of ethyl alcohol that includes 50, 70, and 95% Tissue is

acetone with 10 min in each change From acetone, the tissue is introduced to the embedding medium as a I: 1 mixture of acetone and Spurr's3 low-viscosity embedding medium Since acetone

is highly volatile, it is necessary to make certain that the tissue is always completely covered with the liquid

1.2.3 Infiltration and Embedding

The next steps, infiltration and embedding, are started when the tissue is placed in a 1: 1 mixture

of acetone and Spurr's and immersed from 6 h to overnight Infiltration is followed by embedding

We obtain best results when most of the infiltrating mixture is removed from tissue before placing

into 100% Spurr's A plastic Petri dish is lined with a piece of filter paper and about two to three

drops of embedding medium are placed on the paper Using a toothpick, individual sections are removed from the infiltration mixture and placed onto Spurr's-laden filter paper and rolled around

on the filter paper to be certain that the tissue section remains covered with Spurr's Several drops

of embedding medium may be added before the tissue is removed from the filter paper and placed into a BEEM capsule

For observations in which orientation is not a concern, embedding may be carried out using BEEM capsules The capsules should be placed into a suitable holder Place a label (5 x 15 mm) that contains enough information to identify the specimen Please use a pencil to record information

on the label Place about three drops (enough to fill the conical tip of the capsule) of Spurr's embedding medium into the BEEM capsule Using a wooden toothpick, place the small piece of tissue into the capsule and gently tease it to the bottom Completely fill the remainder of the capsule with Spurr's

If one is interested in embedding the tissue sections with a specific orientation, it will be necessary to use a fiat-embedding procedure A simple way of accomplishing this is by using the lid of the BEEM capsule as the support surface

For this, the capsule is prepared by removing the conical portion with a razor blade The identification label is then inserted from the lid end of the capsule and pushed toward the cut end

so that it will be removed from the vicinity of the tissue Holding the capsule so that the lid is in

a horizontal position, a drop of Spurr's is placed on the center of the surface of the lid The desired tissue section is removed from the Spurr's-soaked filter paper, as previously described, and placed within the drop of Spurr's with the desired surface against the surface of the lid The capsule is now closed in the inverted orientation by inserting the bottom portion into the lid The closed capsule (with the cut end facing up) is placed into the capsule holder The capsule is now filled with Spurr's from the cut end

The BEEM capsule lids fit snugly into the openings of the holders If it is necessary to remove the capsule before curing of the Spurr's is completed, this can be accomplished by pushing the capsule in the direction away from the hinge of the lid This will allow the removal of the capsule without leaking the embedding medium

For curing or polymerization of the Spurr's, all capsules are placed into a 60 to 70°C oven After 30 min in the oven check to see if the tissue sections are remaining within the capsule tips

or against the bottom of the lid If they have moved away from their original position, a gentle push or relocation with a toothpick should be adequate After overnight polymerization, the blocks are ready for trimming and sectioning

1.2.4 Block Trimming and Sectioning

Remove the cured block from the BEEM capsule by removing the snap lid and make a longitudinal cut with a single-edge razor blade and remove the block Securely place the block into the chuck

Figure 1.1

Trimmmg the block of flat-embedded l!ssue The final tnm

is made With an unused section of a smgle-edge razor blade

The side (b) which approaches the kmfe should not be

greater than 0.5 mm

of a specimen holder and place the assembly on the stage of a dissecting microscope With a edge razor blade, trim the end of the block to produce a trapezoid surface Trim so that the surface

single-of the block includes, as far as possible, only tissue (Figure 1.1) The width of the block face to

be sectioned should be no greater than 0.5 mm Edges (a) and (b) should be parallel It is better to use a fresh double-edge razor blade for the final trim It is extremely important that surface (b) has

a very lustrous appearance

The trimmed blocks may be sectioned on an available ultramicrotome with either a glass or diamond knife Although reasonably good sections may be obtained with glass knives, sectioning with a diamond will consistently yield thin sections The use of glass or diamond knives will depend

on both the resources and skill of the electron microscopist We will not attempt to provide instructions for ultramicrotomy, since specific instructions must be provided for each type of ultramicrotome Pale gold or silver sections are adequate for examination with the transmission electron microscope These sections are floated onto 200-mesh copper grids and stained with lead citrate stain.4 The samples in this article were viewed with a Philips® 300 electron microscope with

an accelerating voltage of 80 kV

1.3 Expected Results

Examples of cell ultrastructure are provided from apical dome cells of buds collected in April.5

Some cells contained electron-dense inclusions and nuclei with highly condensed chromatin (Figure 1.2) Also, one may observe dictyosomes with associated highly distended vesicles (Figure 1.3) Often the vesicles were confluent with the plasma membrane A dense population of distended vesicles is observed in association with a high density of endoplasmic reticulum (ER) (Figure 1.4 ) Besides dictyosome-vesicle complexes and ER, the cytoplasm also contains a rela-tively high density of free ribosomes and mitochondria with a matrix of reduced electron density (Figure 1.5)

Many cells are characterized by an abundance of thin primary cell walls and nuclei with varying degrees of condensed chromatin (Figure 1.6) Also, microtubules are shown extending through developing cell plates (phragmoplasts) (Figure 1.7) This stage of cell plate formation coincides with both nuclear membrane reconstitution in daughter nuclei and the occurrence of ribonucleopro-tein granules within the nuclei

Figure 1.6

Pnmary cell wall (W) clo;e to nucleus with h1ghly conden<;ed chromatm Bar= 0.5 11m

Figure 1.7

Cytoskeletal elements extendmg through developing cell plates Bar= 1.0 Jlm

References

I Quail, P H., Plant cell fractionation, Annu Rev Plant Physiol., 30, 425, 1979

2 Owens, J N and Molder, M., A study of DNA and mitotic activity in the vegetative apex of Douglas fir during the annual growth cycle, Can J Bot., 51, 1395, 1973

3 Spurr, A R., A low viscosity epoxy resin embedding medium for electron microscopy, J Ultrastruct Res., 26, 31, 1969

4 Reynolds, E S., The use of lead citrate at high pH as an electron opaque stain in electron microscopy,

J Cell Bioi., 17, 208, 1963

5 Oyofo, B A., Schmitt, D., Llewellyn, G C., Dashek, W V., and Mayfield, J E., An ultrastructural analysis of spruce budworm-promoted defoliation in Abies balsamea, Biodeterioration Research, Ple- num Press, New York, 2, 571, 1989

6 Lawes, G., Scanning Electron Microscopy and X-ray Microanalysis, Chichester, West Sussex, New York, 1987

7 Hall, J L and Haves, C., Electron Microscopy of Plant Cells, Academic Press, New York, 1991

8 Knox, R B and Clarke, A., Localization of proteins and glycoproteins by bindmg to labeled antibodies and lectins, in Cytochemistry of Plant Cells, Hall, J L., Ed., Elsevier, Amsterdam, 1978, 150

9 Pawley, B., Handbook of Biological Confocal Microscopy, Plenum Press, New York, 1990

10 Wickramasinghe, H K., Scanned Probe Microscopy, American Institute of Physics, New York, 1992

Publishers, New York, 1993

12 Chen, C., Introduction to Scanning Tunneling Microscopy, University Press, New York, 1993

13 Neddermeyer, H., Scanning Tunneling Microscopy, Kluwer Academic Publishers, Boston, 1993

14 Egerton, R F., Electron Energy Loss Spectroscopy in the Electron Microscope, Plenum Press, New

York, 1986

15 Morgan, J A., X-ray Microanalysis in Electron Microscopy for Biologists, Oxford University Press,

New York, 1985

16 Red, S J B., Electron Microprobe Analysis, Cambridge University Press, Cambridge, 1993

17 Scott, V D and Love, G., Electronprobe Microanalysis, E Horwood, Chichester, 1985

18 Sigree, D C., X-ray Microanalysis in Biology: Experimental Techniques and Applications, Cambridge

University Press, Cambridge, 1993

19 Zierold, K and Hagler, H K., Eds., Electron Probe Microanalysis: Applications in Biology and

Medicine, Springer-Verlag, New York, 1989

and White-Rot Basidiomycetes 14 References 20

2.1 Overview- Carpogenesis and Basidiosporogenesis

by Wood-Deteriorating Basidiomycetes

Wood-deteriorating basidiomycetes produce fruiting bodies that generate enormous quantities of basidiospores in nature They are the primary source of infection of wood and wood products in above-ground use It is very difficult to induce cultures to produce fruiting bodies and basidiospores

in vitro Therefore, most investigators have studied mycelial growth of wood-deteriorating

basidi-omycetes instead of basidiospore germination in order to evaluate potential wood preservatives Basidiospores can be produced in the laboratory by artificial means involving dikaryotic isolates

of brown-rot (Figures 2.1, 2.2, and 2.5) and white-rot basidiomycetes (Figures 2.3, 2.4, and 2.6) and complex or chemically defined media

*The use of trade or firm names m this publicatiOn is for reader mformatwn and does not 1mply endorsement by the U.S Department of Agriculture of any product or service The Forest Products Laboratory is mamtamed in cooperation with the Umversity of Wisconsin This article was wntten and prepared by U.S Government employees, and 1t 1s therefore in the public domain and not subject to copyright

13

2.2 Protocols

2.2.1 Fruiting Body Formation and Basidiospore Production

by Brown-Rot and White-Rot Basidiomycetes

Dikaryotic isolates of the wood-deteriorating basidiomycetes are maintained on 2% malt extract agar medium (MEA) or potato-dextrose agar (PDA) in the dark at 27°C and 70% relative humidity The following media can be used for fruiting body formation and basidiospore production: complete plus yeast extract medium (CYM),11 sawdust medium,5 PDA medium (Difco), MEA medium, and a chemically defined medium2 containing glucose or Walseth cellulose12 and ammo-nium tartrate An additional25 mM KH2P041 is added to the basal medium, BIII6 to the chemically defined medium Pyrex® storage dishes (Coming No 3250) containing 50 mL of the complex medium or chemically defined medium are inoculated at the center agar of plates with a 6-mm mycelial disc obtained from a fresh culture Plastic petri dishes containing 20 mL of the same medium can be used in place of the storage dishes

Preincubation is carried out in the dark at 27°C and 70% relative humidity After the colonies reach a diameter of 25 to 35 mm, the plates are inverted and incubated at 12 to l5°C under either black light or fluorescent light or cool white fluorescent light 3.4.9•10 A 12-h light and a 12-h dark cycle is used for the entire incubation All plates are aerated once a week by opening the lids of the inverted plates.7.8

The structures that produce basidiospores are regarded as the basidiomata, although their structures sometimes differ from those found in nature All the lids of the storage dishes or petri dishes are layered with precut colored weighing paper The basidiospores deposited on the weighing paper are collected from the inverted plates of the fruiting cultures by transferring the basidiospore prints onto a sterile plastic petri dish The basidiospores are usually produced continuously for a period of 2 to 5 months.2-5 The collected basidiospores are immediately lyophilized and then stored

at -20°C in the dark The basidiospore production is initially determined by means of microscopic examination of the basidiospore prints deposited under the fruiting cultures The production of basidiospores is usually visible to the naked eye because the prints are creamy white against the colored weighing paper The basidiospore prints on the weighing paper are suspended in a sterile 0.005% Tween® 20 solution and tested for viability on MEA plates after being counted with a hemocytometer (Protocol 2.1 )

Maintam the d1karyot1c 1solates of the brown-rot and wh1te-rot bas1d10mycetes on MEA or PDA medium

J._

Inoculate the center of agar storage d1shes or plastic petn plates contammg the complex medium

or chem1cally defined medium wlth a 6-mm mycelial disc obtained from a fresh culture

J._

Premcubate in the dark at 27°C and 70% relauve humidity for 3 to 10 d

or unul the colomes reach a d1ameter of 25 to 35 mm

J._

Invert and incubate at 12 to I5°C under a 12-h hght and a 12-h dark cycle for I to 4 months

Aerate once a week by opening the lids of the inverted plates

J._

Collect the bas1d1ospore pnnts on the surface of we1ghing paper placed on the hds of the petri plates

or storage dishes under bas1diomata (frmting bod1es) by transferrmg the layered weighmg paper

onto a stenle plast1c petn d1sh

J._

Lyoph1hze Immediately and then store at -20°C in the dark

J._

Suspend the basidw;pore pnnts on the we1ghmg paper in a sterile 0.005% Tween® 20 solutiOn and test

for vmb1hty on MEA plates after countmg w1th a hemocytometer

Protocol 2 1

Figure 2.1

Por01d rc,upmate krtile ba,tlhoma produced on 2'/r MEA by ;\nrrodw r arbontca

A

Figure 2.2

Porotd rc,urmalL' let tile ba"rhomata ptoduleu on the 'urtaLL' of the PDA :-.upp l emc ntcd wtth gluco:-.e !A) anrl chem t c<t ll)

Jefined agar rncdtlllll (B) hy Glo eopln /111111 trahcum

Figure 2.4

Fertile ba'>!diOmata of Pleurotus ostreatu.1 on PDA

A

B

Figure 2 5

(A) Po i o id re'>Uplnate fertile ha'idJOma IO!Ill<Illon on PDA plate only under cool\\ h1te tlume-,cent l1g:ht b) l'oll w ploc I'll/{!

Figure 2 6

Fertile bas1 d1omata on a chemically defined medmm w1ht Walseth cellulo se by Scluzophv llum commun e

References

ba s 1diomycete Gloeophy/lum trab eu m , Mater Org , 27 , , 1992

Preservation, IRG/WP/94- I 0081, 1994

4 Croan , S C., Basidiosporogenesis by brown-rot basidiomycete in vitro, Inter Res Group on Wood Preservation, IRG/WP/95-10126, 1995

5 Croan, S C., Carpogenesis a nd b as idiosporogenesis by Flammulina velutipes, Schizophyllum commune,

6 Kirk, T K., Croan, S C Ming Tien, Murtag, K E., and Farrell, R L., Production of multiple ligninases

Enzyme Microb Techno/ , 8, 27, 1986

7 Leatham , G F and Stahmann , M A , Effect of light and aeration on fruiting of Lentinula edodes , Trans