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This is to certify that the

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A System to Study Plastid Development in Spruce

presented by

Chishih Chu

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-——.—-——— » -

A SYSTEM TO STUDY PLASTID DEVELOPHEIT IN SPRUCE

BY

Chishih Chu

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Plant Breeding and Genetics - Forestry
1992

ABSTRACT
A SYSTEM TO STUDY PLASTID DEVELOPMENT IR SPRUCE
BY
Chishih Chu

The objectives of this study were 1) to develop a callus system to
study plastid development associated with the change of pigments and
gene expression and 2) to map the spruce chNA genome by RPLP analysis.

Protocols were developed for the induction and maintenance of
white megagametophyte-derived callus and green callus derived from white
callus in Picea glauca.

Induction of pigmentation changes in callus, from white to green,
red, or brown, were observed using different levels of BAP or zeatin.
The effects of zeatin significantly differed from those of BAP causing
the white callus change to green, but not to red or brown. In contrast,
the addition of 2,4-D inhibited the color changes induced by BAP. Gluta-
mate caused browning of white calli, while ALA alone or glutamate and
ALA together maintained white color. Heat shock at 37°C for an hour
altered the responses of white calli to BAP and zeatin by significantly
increasing browning while decreasing greening.

Variations were found between green callus and white callus for
dry weight and protein concentration. A lack of chlorophyll in white
callus and a chlorophyll a/b ratio (1.339) for green callus were ob-
served. Neither white callus nor green callus showed photosynthetic
activity. In comparison with needles, green callus and white callus
contained an extra DNA band on CsCl density gradients. Differences in

polypeptides and differential expressions of five isozymes (IDH, GOT or

AAT, SKDB, GSPDH, and DIAor MNR) were found between green and non-green
callus.

The chNA genome size is 120.4, 120.2, and 120.2 kb for blue
spruce, Engelmann spruce, and white spruce respectively. Spruce chNA
lacks one of the inverted repeat sequences and has multiple rearrange-

ments when compared to that of radiata pine or Douglas-fir.

This dissertation is dedicated
to my parents,
I-Wan Chu and Chun-Ing Thom
and

to my brothers and sister

iv

AWS

I would like to thank my major professor Dr. Daniel E. Keathley,
and my committee members Drs. James Hancock, James Hanover, Amy Iezzoni,

and Kenneth Sink for guidance throughout this study.

Thanks to Dr. Michael Stine for the initial instruction in the
isolation of chNA, and to Dr. Robert Jensen for the gift of tobacco
chNA subclone library, my close friend Andrew David for the helpful

discussion and proofreading of manuscript.

Most of all I want to thank my parents, brothers, and sister for
their love, support, and understanding during the completion of this

dissertation and throughout my graduate studies.

TABLE OF CONTENTS

Page
LISTS OF FIGURES vii
LISTS OF TABLES viii
INTRODUCTION 1
CHAPTER
I. White spruce (Picea glauca (Moench) Voss)
megagametophyte culture 11
Introduction 12
Materials and Methods 13
Results 15
Discussion 19
Literature Cited 24

II. Factors that effect pigmentation change in megagametophyte

callus of Picea glauca 27

Introduction 28
Materials and Methods 29
Results 31
Discussion 38
Literature Cited 43

III. Variations in physical characteristics, proteins and DNA
DNA between green and nongreene callus culture

of Picea gIauca 45
Introduction 46
Materials and Methods 47
Results 51
Discussion 58
Literature Cited 62
IV. Physical mapping of Picea chloroplast DNA 65
Introduction 66
Materials and Methods 67
Results 69
Discussion 74
Literature Cited 77

vi

CHAPTER I

Figure

Figure

Figure

CHAPTER III
Figure

Figure

Figure
Figure
CHAPTER IV

Figure

Figure

Figure

Figure

LIST OF FIGURES

Page
Tissue culture of the megagametophytes of
white spruce. 16
The chromosome complements at mitotic metaphase. 18

Representative diagrams corresponding to 5 different
isozyme loci for different materials. 20

The absorbency spectrum of acetone-water extraction. 52

Silver staining SDS-PAGE gel of polypeptides from
extracts, which were separated by discontinuous
sucrose gradient, of needles, green callus and

white callus. 54
The megagametophyte callus DNA bending pattern. 55
Nuclear genomic DNA methylation pattern. 57

Southern blot analysis of DNA which was isolated at
different centrifuge forces and separated by TAFE. 70

Southern blot analysis of single and double digested
spruce chNA. 71

Physical and genetic map of blue spruce chNA. 73

Arrangement of homologous sequences in blue spruce
and radiata pine chloroplast DNA genome. 75

vii

CHAPTER II

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

CHAPTER III

Table

Table

CHAPTER IV

Table

Table

9.

10.

LIST OF TABLES

Page
The effects of cytokinin level and light intensity
on color change of white callus. 32
Average of green, red, and brown color in white
callus grown on medium containing zeatin and BAP. 33

The effects of different concentrations of 2,4-D and
BAP on the greening and browning of white callus. 33

The effects of GLU and ALA on the color change of
the white calli on different media. 34

Average of white and brown color in white callus
grown on media containing glutamate, ALA or a
combination of the two. 35

The effects of different concentrations of glutamate
and ALA on the color change of white callus in
liquid media. 36

The effects of heat shock, exposure time and
cytokinin concentration on color change of
white callus. 37

Average of white, green, red, and brown color in
white callus grown on medium containing zeatin and
BAP. 37

Average of white, green, red, and brown color in
white callus at different heat shock exposure time. 37

Summary of factors and their effects on color change
in white callus. 38

Relative moisture content, chlorophyll content and
protein concentration of white and green calli. 52

Methylation sensitive restriction enzymes and the

methylation sequences which will not be cleaved. 56
Spruce chNA restriction fragment size. 72
Similarity of chNA and mtDNA among spruces. 74

viii

Introduction

Tissue culture techniques have been used to propagate superior
genotypes, produce disease and herbicide-resistant plants by gene trans-
fer, generate biochemical mutants to study metabolic pathways, and
investigate the relationships between gene expression and plant develop-
ment.

In tissue culture, long-term cultures often result in tissue
culture-induced variability (D'Amato, 1977). This somaclonal variation
is thought to arise from a combination of different chromosomal abnor-
malities, such as aneuploidy (Murashige and Nakano, 1967, for tobacco),
polyploidy (Bayliss, 1973, 1975, for carrot), or chromosomal transloca-
tion (Jill Roth et al., 1989). Changes in DNA methylation have also been
associated with the level of auxin in the culture medium, especially
2,4-dichlorophenoxy acetic acid (2,4-D) (LoSchiavo et al., 1989). In
addition to nuclear genomic DNA changes in tissue culture, changes in
plastid number and their structure have been observed in cells undergo-
ing protoplast culture and plant regeneration (Thomas and Rose, 1983).

Plastids are characterized by their different morphology, pigmen-
tation, and metabolic activities and are typically grouped into func-
tional categories: proplastids, chloroplasts, chromoplasts, amyloplasts,
etioplasts, and elaioplasts. In addition, plastid type is closely corre-
lated to the cell type which contains them. Proplastids are the simplest
and least differentiated type of plastid and are found in the zygote,
root and shoot meristems, and reproductive tissue. Amyloplasts are non-
green plastids which accumulate starch and are typically found in stor-

age tissues such as cotyledons, endosperms and tubers. Elaioplasts

accumulate essential oils or fat. Chromoplasts are yellow, orange, or
red plastids and are found in petals, fruits, and roots. Chloroplasts,
which are the most functionally important and widespread member of the
plastid family, contain the site of photosynthesis and occur in all
green tissues. Finally, the etioplasts have a highly ordered paracrys-
talline region, called the prolamellar body, and protochlorophyllide,
but no chlorophyll.

In plants, the main pathway of plastid development and intercon-
version was identified by Whatley (1977, 1978). This interconversion
involves changes in proteins, size, internal structure, pigments, and
sometimes alternations in DNA methylation-demethylation. The physiology,
biochemistry, and genetics of amyloplasts, chromoplasts, etioplasts and

chloroplast have been broadly studied (Boyer et al., 1989).

DNA.methylation

It has been postulated that methylation is involved in the regula-
tion of plastid DNA transcription. This is supported by evidence of
different methylation sites in the various plastid types. In comparison
to chloroplast DNA, chromoplast DNA and amyloplast DNA are methylated in
regions of little or no transcriptional activity (Ngernprasirtsiri et
al., 1988a, 1988b; Macherel et al., 1986). In addition, some plastid-
specific gene expression has been reported. In the case of ripening
tomato fruit, chloroplast-specific mRNA levels continuously decrease
(Piechulla et al., 1985, 1986; Piechulla, 1988), while two chromOplast-
specific polypeptides are either not detectable or barely detectable in

chloroplasts (Hadjeb et al., 1988).

Pigmentation

In plants, pigments are usually found in the plastids and vac-
uoles. The green color of plants is due to chlorophyll, which is located
in the chloroplasts. Carotenoids, the yellow to red pigments, are also
found in chloroplasts but they are masked by chlorophylls. Flavonoids
(anthocyanins and flavones or flavonols) are generally present in vac-
uoles and contribute colors other than green to plants. These pigments
are functionally important for plant survival. Chlorophylls absorb light
energy and transfer it to other participants in the photosynthetic
process. Carotenoids are involved in trapping light for photosynthesis
and probably protect chlorophyll from photo-oxidation. Flavonoid pig-
ments are also important in attracting insects and birds during polli-
nation and seed dispersal. The biosynthesis of these pigments has been
well studied. The regulation, limiting factors, and major enzyme in-

volved in the biosynthesis of each pigment are described below.

A. Chlorophyll biosynthesis

5-aminolaevulinic acid (ALA) is the universal precursor for the
synthesis of chlorophyll and other tetrapyrroles. In higher plants, ALA
is synthesized from glutamate, and its formation is the rate-limiting
step for pigment accumulation during the greening of dark-grown cells
and tissues following exposure to light (Beale and Castelfranco, 1973,
1974). Most of the ALA synthesizing capacity assayed in organello is
dependent upon the light and dark treatments of the seedling prior to
chloroplast isolation (Haung and Castelfranco, 1989). This light regula-
tion was shown to be a low fluence phytochrome response (Haung et al.,
1989). After incubation with ALA and then exposure to light, dark-grown

cress seedlings (Lepidium sativum L.) showed a reduction in the rate of

greening and an inhibition of grana formation (Kittsteiner et al.,
1991); thus, indicating that excess ALA inhibits normal plastid develop-

ment.

B. Carotenoid synthesis and conversion of chloroplasts to chromoplasts
Carotenoids are members of the tetraterpenes. Their C4o carbon
skeletons are built by the successive addition of Cs units to form
geranylgeranyl pyrophosphate, a C20 intermediate, which then condenses
tail-to tail with another molecule of geranylgeranyl pyrophosphate to
form phytoene. Phytoene, the C40 condensation product, is dehydrated to
yield lycopene. Cyclization of both ends of lycopene gives b-carotene.
In the conversion of green fruit to red fruit in Capsicum annuum,
chlorophyll disappears and the amount of total carotenoids increases,
especially the carotenoid precursors phytoene and phytofluene. These
precursors were detected at the beginning stage of conversion and their
contents increased up to the red stage (Camera, 1978). Changes in other
carotenoids such as xanthophyll pigments were also observed. Lutein, the
main xanthophyll of green fruits, decreased gradually, while violaxan-

thin and neoxanthin levels persisted during the ripening period.

C. Flavonoid biosynthesis

Flavonoids consist of two aromatic rings joined to form a chromat-
ic structure by a three carbon unit (06-C3-C6). The phenylpropane resi-
due (ring B and 02'3'4) is derived from p-coumaric acid, itself formed
via the shikimate pathway. Ring A is basically formed from acetate and
is a rather special case of polyketide synthesis. Shikimate dehydroge-

nase (SKD) is the major enzyme of the shikimate pathway.

D. Plant growth regulators

It is known that cytokinins promote chloroplast development
(Parthier, 1979) and that in the absence of kinetin plastids do not
accumulate chlorophyll (Stetler and Laetsch, 1965). The application of
cytokinins restores chlorophyll accumulation as demonstrated in cucum-
ber, where the chlorophyll content of etiolated cotyledons increased 450
percent following cytokinin application (Fletcher and McCullagh, 1971).
There is no evidence of an auxin influence on chlorophyll accumulation,
chloroplast formation, or replication (Laetsch and Boasson, 1972;

Schmerder et al., 1978).

Physical and genetic mapping of chloroplast DNA

The chloroplast genome is small relative to plant nuclear genomes
and is encoded on a circular molecule. In angiosperms, the chloroplast
genome contains two inverted repeats and ranges in size from 120 to 217
kb. Exceptions to this, species lacking one repeat region, are known
only in the subfamily Papilonoideae of the legume family (Palmer and
Thompson, 1982; Palmer et a1. 1987). In gymnosperms, chNA maps have
been developed for three species Gingko, Douglas-fir, and radiata pine
(Palmer and stein, 1986; Strauss et a1. 1988). In Douglas-fir and radia-
ta pine, chloroplast genome lacks one inverted repeat region and is
approximately 120 kb in size, Gingko differs from these species in
having two inverted repeats and genome size 158 kb.

Changes in genome size accure from two processes: changes in the
amount of repeated DNA and changes in sequence complexity. Both process-
es are important in chNA evolution (Palmer, 1990). Chloroplast DNA
(chNA) RFLPs provide a tool for ascertaining amounts and patterns of

species introgression (Govindaraju et al., 1988, Szmidt et al., 1988).

The comparison of chNA maps between different species is probably the
best current procedure for developing phylogenies of plant species.
Generally, chloroplast genes are either single gene or organized
and cotranscribed as multigene transcriptional units. Recombination or
inversion of chNA results in chNA variation and typically takes place
at the ends of the multigene units. Examples are rps2-ath—atpH-ath-
atpA and rpoA-rpoB-rpoCl-rpoC2 (Herrmann et al., 1986; Hudson et al.,

1987, 1988; Hudson and Mason, 1988).

Objectives of this study

The interconversion of plastids involves changes in gene expres-
sion for nuclear and chNA. Megagametophyte tissue of Picea glauca was
used as explant to develop a tissue culture system that would allow the
study of both these processes and the relationship between plant devel-
opment and plastid differentiation. Megagametophpyte tissue was chosen
because callus derived from megagametophyte tissue lacks pigmentation. A
shoot regeneration protocol using megagametophyte-derived callus was
developed (Chapter 1).

Factors that induced color change in megagametophyte callus from
white to green, red, or brown were studied. These factors included plant
growth regulators (cytokinins and auxin), chlorophyll precursors (gluta-
mate and ALA), and heat shock (Chapter 2).

The differences between green and non-green tissue were also
studied. These differences included expression of five different
isozymes - aspartate aminotransferase (AAT), shikimate dehydrogense
(SKDH), isocitate dehydrogenase (IDH), glucose-6-phosphate dehydrogenase

(G6PDH), and menadione reductase (MNR), as well as physical

characteristics - dry weight, chlorophyll concentration, chlorophyll a/b
ratio, photosynthetic activity, protein amount and genomic DNA (Chapters
1 and 3). In addition, the physical map of chNA for Picea engelmannii,

P. glauca, and P.pungens was determined (Chapter 4).

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10

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plast genomes of two conifers lack a large inverted repeat and are
extensively rearranged. Proc. Natl. Acad. Sci. USA 86:6030-6034.

Szmidt, A.E., T.A. El-Kassaby, A. Sigurgeirsson, T. Alden, D. Lindgren
and J. E. Hallgren, 1988. Classifying seed lots of Picea
sitchensis and P. glauca in zones of introgression using restric-
tion analysis of chloroplast DNA. Theor. Appl. Genet. 76:841-845.

Thomas, M. R. and R.J. Rose, 1983. Plastid number and plastid structural
changes associated with tobacco mesophyll protoplast culture and
plant regeneration. Planta 158:329-338.

Whatley, J.M. 1977. Variations in the basic pathway of chloroplast
development. New Phytologist 78:407-420.

---- 1978. A suggested cycle of plastid developmental interelations. New
phytologist 80:489-502.

Chapter 1
White Spruce (Picea glance (Moench) voss) Megagametophyte Culture

Abstract

Shoot regeneration of white spruce (Picea glauca) mature megagame-
tophyte derived callus was obtained using seeds that had been stored for
six years. Megagametophyte calli were induced on MS medium (pH 5.8)
supplemented with 2% sucrose, 1.0% agar, 10 uM 6-benzyl-aminopurine
(BAP), and 10 uM 2,4-dichlorophenoxyacetic acid (2,4-D) for 3-4 weeks in
the dark. Calli were subcultured on the same medium but in either low
light (8 uE/mz/sec) or high light (50 uE/mz/sec). Megagametophyte calli
remained white when grown under either light intensity.

When transferred to MS medium containing either 1 uM zeatin or the
combination of 0.5 uM zeatin and 0.05 uM naphthaleneacetic acid (NAA),
white calli produced organized green or green and red regions. Continued
subculture and maintenance on the same medium resulted in shoot regener-
ation from the green regions. A chromosome number of 2n=24 was deter-
mined for all tissue types. Differential expression of isocitrate dehy-
drogenase (IDH), glutamate oxaloacetic acid transaminase (GOT)(also
called aspartate aminotransferase; AAT), shikimate dehydrogenase (SXDH),
glucose-6-phosphate dehydrogenase (G6PDH), and diaphrase (DIA) (also

called menadione reductase; MNR) was observed in different tissues.

11

12

Introduction

Constant progress has been made in the development of micropropa-
gation and tissue culture systems for conifers during the past three
decades. La Rue (1950), using Cycas revoluta was the first to obtain in
vitro cell masses from mature gymnosperm megagametophyte tissue. Since
that time, tissue culture systems have been developed for many conifer
species. The successful in vitro propagation of conifers through organo-
genesis and embryogenesis by the use of cotyledon, hypocotyl, embryo,
needle, pollen, and megagametophyte explants, and from cell suspension,
and from protoplast cultures has been reviewed by Durzan and Campbell
(1974), David (1982), Berlyn et al. (1986), Rohr (1987), Bonga et al.
(1988), and Dunstan (1988).

There has been extensive work on the development of tissue culture
systems for members of the genus Picea. Somatic embryogenesis from
immature embryos and cotyledons has been reported in Picea abies,
(Hakman et al., 1985; Hakman and von Arnold, 1985; Gupta and Durzan,
1986; von Arnold and Hakman, 1986; von Arnold, 1987; Krogstrup, 1986;
Lelu et a1. 1987), P. sitchensis (von Arnold and Woodward 1988), and P.
mariana (Attree et a1. 1990).

In white spruce, plantlet regeneration from different explants has
been accomplished. Plantlets were first regenerated from hypocotyl and
cotyledon segments (Campbell and Durzan, 1975, 1976). Currently, the
successful development of somatic embryogenesis using immature embryos
(Hakman and Fowke, 1987; Lu and Thorpe, 1987), mature embryos (Trem-
blay, 1990) or cultured shoots and cotyledons from stored seeds (Attree
et al., 1989) as explants has made mass propagation of particular geno-

types possible.

13

Isozymes have been used as markers to indentify the specific
genotypes or hybrids and to study the variation in physiological condi-
tion during plant differntiation. Ernst et a1. (1987) studied the iso-
zyme pattern of Engelman spruce (Picea engelmannii) and blue spruce (P.
pungens) and Gruber (1990) used phosphoglucoisomerase (PGI-2), which
gave different banding patterns, to identify hybrids of Engelmann and
blue spruce. In additon, variations in isozyme patterns demonstrate the
various physiological and developmental states in intact plants
(Scandalios 1974, 1977).

This study reports the development of a tissue culture system for
mature megagametophytes of white spruce. Differential expression of
isozymes among different explants and change in ploidy were also inves-

tigated.

Materials and Methods
Tissue culture

White spruce seeds that had been stored at 4°C since 1984 were
surface-sterilized for 12 minutes in 25% Clorox with 5-6 drops of Tween
20 (Sigma) and washed 3-4 times with sterilized, double distilled water.
They were then placed on agar (0.4% or 0.6%) containing 3% sucrose and
allowed to germinate in a growth chamber with an 18 hr light/6 hr dark
photoperiod at 26°C. The megagametophytes used as explants were removed
from the seeds 1 to 2 weeks following germination.

Megagametophyte calli were induced on MS medium (pH 5.8) supple-
mented with 2% sucrose, 10 uM BAP, 10 uM 2,4-D, and either 0.8% or 1.0%
agar. Cultures were stored in the dark at 26°C. After 3-4 weeks, callus
was subcultured on the same medium and stored in either low light (8

uE/mZ/sec) or high light (50 uE/m2/sec) with an 18 hr light/6 hr dark

14

photoperiod at 26°C. After 6-8 months of subculture on the callus induc

tion medium, white calli were transferred to Ms medium (pH 5.8) with 2%
sucrose, 0.6% agar, and either 1 uM zeatin or 0.5 uM zeatin and 0.05
uM NAA and then transferred to high light to stimulate shoot regenera-
tion. When green spots appeared in the otherwise white callus, they were
subcultured on white callus induction medium to establish green callus.
Chromosome Analysis

Chromosome number was examined in shoots, white calli, and green
calli derived from white callus using the method of Dyer (1979).

Isozyme studies

Isozyme polymorphisms were analyzed in tissues from seeds, seed-
lings and calli. Tissue samples were homogenized in sample vials con-
taining 200 ul of extraction buffer (100 mM Tris-HCl pH7.5, 5% w/v
sucrose, 5% w/v PVP-40, 0.1% v/v 2-mercaptoethanol, 50 mM ascorbic acid,
10 mM diethiothreitol, 10 mM sodium metabisulfate and 0.1% w/v bovine
serum albumin). All explants were derived from seeds collected from
white spruce accession number 6719-0916. Seedlings and calli were germi-
nated or induced from those seeds. The sample size and types are listed
in Figure 3. Filter paper wicks 2 x 20 mm (Whatmann No.3) were dipped in
the homogenate and inserted into a vertical slit at the gel origin. Two
different samples of each tissue were loaded in each gel.

The composition of electrophoresis buffers and power requirements
followed Cheliak and Pitel (1984) and Scandalios (1969). The buffer
system for each enzyme: shikimate dehydrogenase (SKDH) EC 1.1.1.25,
isocitrate dehydrogenase (IDH) EC 1.1.1.42, glucose-6-phosphate dehy-
drogenase (G6PDH) EC 1.1.1.49, aspartate aminotransferase (AAT) (also

called glutamate aminotransferase; GOT) EC 2.6.1.1, and diaphrase (DIA)

15

(also called menadione reductase; MNR) and the staining method followed
those described by O'Malley et a1. (1980) and Conkle et al. (1982).
Connaught starch lot 400-1 (Connaught Laboratories, Willowdale, Ontario,
Canada) was used to prepare the 20 x 15 x 1.6 cm, 12.5% w/v starch

gels.

Results
A. Callus induction

Seed germination averaged 82.0%. Initial experiments using combi-
nations of BAP (1 uM, 10 uM and 25 uM) and 2,4-D (1 uM and 10 uM) and 20
explants of each treatment on 0.6% agar solidified medium yielded a low
callus induction rate (8.6%). Medium containing 10 uM BAP and 10 uM
2,4-D gave the highest callus induction rate (16.7%). Therefore this
combination was selected for the subsequent experiments. Increasing the
agar concentration to 0.8% resulted in increased rate of callus induc-
tion to 28.2% (22/78) for megagametophytes 1 week after seed germination
and 10.5% (4/38) for megagametophytes 2 weeks after seed germination.
Megagametophyte callus grown on 0.8% agar stopped growing after two or
three subcultures. Increasing the agar concentration to 1% and using
megagametophytes 2 weeks after seed germination on either 0.6% or 0.4%
agar resulted in callus induction rates of 40.5% (17/42) and 35.7%
(15/42), respectively. The megagametophyte calli grew well but slowly
after several subcultures. When callus size reached 1 cm in diameter,
the calli were maintained in the callus induction medium with the agar

concentration changed to 0.6 %.

16

 

Figure 1. Tissue culture of the megagametophytes of white spruce. A) 1-
year-old white callus. B) green regions on white callus after transfer-
ring the callus on the shoot regeneration media. C) green callus derived
from white callus. D) shoot regenerated from green regions of white
callus.

17

B. Shoot regeneration

One to two months following transfer onto MS medium with either 1
uM zeatin or a combination of 0.5 uM zeatin and 0.05 uM NAA, green
colored regions developed on the surface of most calli. The surface of
these green regions was either smooth or rough. After subculturing for
2-3 months, shoots regenerated from the smooth surface of the green
regions (Figure 1). More than forty shoots were regenerated. Actual
shoot length was not measured, however, it was noted that shoot elonga-
tion ceased when shoots reached approximately 3 mm in length. Root
regeneration was attempted using 1/10 strength MS without sucrose and

lacking plant growth regulators, but none occurred.

C. Chromosome Studies

Despite the fragility of callus tissues and the low frequency of
white and green callus cells undergoing cell division, approximately 20-
30 callus cells could be found for each tissue type that were suitable
for use in counting chromosome numbers. The chromosome number of
shoots, white calli, and green calli derived from white callus; was
2n=24 for all cells (Figure 2). However some cells showed chromosome
overlapping occurred. Despite chromosome overlap, chromosome number of
callus cells were clearly higher than the haploid number of 12, but

chromosomal abnormalities such as aneuploidy can not be excluded.

18

,‘_
2?

Figure 2. The chromosome complements at mitotic metaphase. Magnifica-
tion of each picture was 800 folds. (a) shoot cell of seedlings. (b)
white callus cell. (e) green callus derived from embryo.

 

D. Isozyme studies

Both the staining intensity of individual bands and the presence
or absence of bands for specific isozymes varied among the different
tissue types that were used for sample extraction. A schematic represen-
tation of the isozyme banding patterns is shown in Figure 3.

The IDH isozyme banding pattern showed that variation among tis-
sues was mainly observed in the faster zone (Figure 3A). The band at
Rf=0.20 appeared in all samples and was highly expressed in both green
callus types. The band at Rf=0.33 did not appear in white callus and was
expressed at a lower level in root, stem, and needle tissues than that
observed in green callus. One extra band at Rf=0.23 was present only in

samples from green calli derived from embryo.

19

The AAT isozyme banding pattern showed that two zones of activity
were evident on gels (Figure 38). White calli did not have the band
at Rf=0.29. White calli and green calli derived from white calli showed
less intensity at band Rf80.47 than the other tissues. Root, stem, and
needle tissues produced low intensity bands at Rf30.23 and 0.29. Inter-
estingly, an extra band was observed in samples from embryos (Rf=0.53)
and both green callus types (Rf80.25).

Two zones of activity of DIA (MNR) isozyme were observed (Figure
3C). Bands Rf=0.67 and 0.71 did not appear in white callus, embryo, and
megagametophyte samples. The band at Rf80.46 was only observed in seed-
ling samples.

Only one band (Rf=0.17) was observed for SKDH. It was present in
all samples except those from white calli (Figure 3D).

Three GSPDH isozyme bands were observed (Figure 3E). In white
callus, the band at Rf=0.31 was barely visible, and band at Rf=0.39 was
very weakly expressed. Both green calli showed high intensity at band
with Rf=0.24. The band shown at Rf=0.39 had the highest staining inten-

sity in megagametophyte samples.

Discussion

Immature megagametophyte tissue has typically been the explant of
choice in studying organogenesis or embryogenesis in vitro. The problem
with using immature seeds for megagametophyte culture is timing the
collection period to coincide with the desired developmental stage.
Using mature seeds to develop the megagametophyte culture circumvents

that problem and has the advantage of long term storage.

20

#2 Eb m 5: Ne Gc Wc Gw Rf
‘—‘ 0

 

A _ _ _ ._ _. a, — _. . 0.20

__. ._. -—— -—— -—— -—- -—- 4 Q33

F! Eh I S! No 0: We Gw Rf

i O

1 — — — — — - — - ‘ 0-23

| B — - -— --- — - : ‘ 029

‘ — - — - — - — - "'0.42

x —:---"'— "‘ 323'33
.

F9 Eb Rt 59 Ne Gc Wc Gw R
=

 

— - —I- — _ — —— —— v 0.4!
— _ " "'O.45
—- — -— -— _ — —- ‘ ¢~- 0.54
: : : —‘ — “W
'_ _ *”0.71
Fg Eb Rt 5: Ne Gc Wc Gw Rf
0

‘ 0.17

 

 

 

E — _ _ — — _ ‘ 0.24
— — _ — — . 0.3‘
— _ — — -— _ . 039

 

Figure 3. Representative diagrams corresponding to 5 different isozyme
loci for different materials. A. IDH isozyme pattern. 8. AAT (GOT)
isozyme pattern. C. DIA (MNR) isozyme pattern. D. SKDH isozyme pattern.
E. GGPDH isozyme patterns. ngmegagametophytes of 7 seeds. Ebzembryos of
7 seeds. thnoots of S seedling. St:shoots of 5 seedlings. Nezneedles of
5 seedlings. Gc:green calli derived from embryo. We: white megagameto-
phyte callus. Gw:green calli derived from white callus.

21

When the agar concentration in the callus induction medium was
increased from 0.6 t to It, the callus induction rate increased. This
suggests that moisture content in the medium may play an important role
in developing the success and rate of callus induction, especially
during the first few subcultures. The opposite relationship appears to
exist during seedling germination where megagametopyte tissues from
seedlings germinated on 0.4% agar, overlaid with water on seeds, had a
higher callus induction rate than those germinated on 0.6% agar.

The morphogenetic capacity of megagametophyte tissues in vitro is
often determined by the developmental stage. Immature seeds are often
used to initiate megagametophyte cultures. In general megagametophyte
tissue of gymnosperms has been far more responsive in vitro than the
microspore tissue. In this study, shoot regeneration was obtained from
callus derived from mature megagametophyte tissue, but shoot growth
arrested at about 3 mm in length and the shoots failed to regenerate
roots. The attenuated shoot growth may have resulted from many factors,
including media that were not suitable for continued shoot development.

The ploidy level of mature megagametophyte tissue is often deter-
mined from the callus derived from the megagametophyte tissue. In previ-
ous studies, callus from megagametophyte tissue remained haploid after
five months in culture (Bonga, 1974, 1981), became dihaploid for almost
all of the cells after several months in culture (Berlyn, 1968), or had
mixed ploidy levels (70% haploid, 25% dihaploid, and small number of
tetraploid) after six months in callus culture (Renfroe and Berlyn,
1986). The diploid chromosome number of white spruce is 2n-24 (Sax and
Sax 1933). In the case of mature megagametophyte calli of white spruce,

the chromosome number (Zn-24) indicated the calli may have originated

22

from haploid tissues through the nuclei fusion of the multinucleate
cells, endomitosis, or chromosome non-disjunction. In this study, it may
be that the long subculture period resulted in chromosome doubling, but
for whatever reason, all countable samples showed a diploid chromosome
number, consequently no haploid white callus cell lines were identified.
Isozyme patterns frenguently vary in intact plants as a function
of various physiological and developmental stages (Scandalios, 1974) and
similar responses have been demonstrated in cultured cells of tobacco
(Lee, 1972a,b). In tobacco, kinetin and zeatin cause the appearance of
new isozyme forms, as does gibberellin GA3in the presence of IAA.
During shoot regeneration from callus, changes in the expression of the
isozymes peroxidase (Thorpe and Gaspar, 1978) or esterase (Kosinski et
al., 1988) were observed. This differential expression of isozymes from
shoots regenerated from the same genotype explant indicates that culture
conditions can cause epigenetic effects. Moreover, it has been shown
that enzymes or isozymes for carbohydrate metabolism associated with
plastid type differ between green tissue and etiolated tissues (ap Rees
and Entwitle, 1989; Keeling, 1989; Madore, 1990; Frehner et al., 1990).
The data from this study showed differential expression of isozymes in
different tissues. In white callus, isozyme IDH (band Rf=0.33) and AAT
(band Rf=0.29) were absent (Figure 3) in comparison with green calli
derived from white calli and other materials. This could be explained by
either differential expression of both isozymes or the existence of null
alleles in the genotypes used in this study. Given that the green calli
were established using white callus explants, it most likely indicates

differential gene expression between these two callus types. In addi-

23

tion, differential expression of isozymes IDH, AAT, DIA (MNR), SKDH, and
SKDH was observed in various tissues.

White megagametophyte callus and green callus derived from white
callus responded differently when placed on shoot regeneration media.
Although shoots arose from small isolated green regions in white calli,
no shoot regeneration from green calli derived from white calli has been
established. However, white calli and green calli maintain their origi-
nal color white or green under both high and low light regimes. Fur-
thermore, green calli is inducible from white calli and cytokinins can
stimulate chloroplast development (Partier, 1979).

The prospects for mature megagametophyte culture of Picea glauca
are promising. The success of using mature megagametophyte tissue to
produce megagametophyte derived callus and plantlets would be an advan-
tage to tree breeding programs in terms of a long-term supply of haploid
derived sources. The lack of root regeneration and failure to obtain
continued shoot elongation currently pose significant barrier to the
commercial application of this process. Future research should concen-
trate on the regeneration of plantlets and the mechanisms underlying
embryogenesis.

The differential expression of isozymes between white callus and
green callus derived from white callus along with the inducible change
of white callus to green callus provides an interesting mechanism for
studying the genetic regulation of this essential metabolic process in

plants.

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Attree, S.H., S. Budimir, and L.C. Fowke, 1989. Somatic embryogenesis
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Cheliak, W.M. and J.A. Pitel, 1984. Genetic control of allozyme variants
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---- 1977. Regulation of isozyme patterns in plant cell differentiation.
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Chapter 2

Factors that effect pigmentation change in white megagametophyte
callus of Picea glance
Abstract

When grown on MS medium containing 10 uM 6-benzylaminopurine (BAP)
and 10 uM 2,4-dichlorophenoxyacetic acid (2,4-D) spruce megagametophyte
tissue produced white callus. Transfer of white calli to MS medium
containing BAP or zeatin resulted in a change of color to green, red, or
brown. The effects of zeatin differed from BAP, causing the color
change to green, but not to red or brown. The addition of 2,4-D to the
MS medium inhibited the color change induced by the cytokinins, espe-
cially the development of green and red regions. White callus also
showed different responses to the addition of glutamate and S-aminolevu-
linic acid (ALA) to the medium. Glutamate caused browning of white
calli, while ALA alone or glutamate and ALA together caused no change in
color. Exposure to 37°C for an hour also changed the response of white
calli to BAP and zeatin by significantly increasing browning while

decreasing the development of green regions.

27

28

Introduction

Plastids are characterized by differences in their morphology,
pigmentation and metabolic activities. Their differentiation is governed
by developmental and environmental factors which alter the gene expres-
sion of the plastid and nuclear genomes. Changes in both nuclear and
plastid gene expression are involved in plant developmental processes
(Piechulla et al., 1986; Piechulla, 1988; Kuntz et al., 1989), and
changes in plastid polypeptide composition have been observed during
the greening process of etiolated peas (Dietz and Bogorad, 1987).

In plants, the main pathway of plastid development has been iden-
tified by Whatley (1977, 1978). The interconversion of plastids includes
changes in protein compliments, plastid size, internal structure, and
pigments. Cytokinins have been linked to the promotion of chloro-
plast development (Parthier , 1979), and it has been shown that in the
absence of kinetin plastids do not accumulate chlorophyll (Stetler and
Laetsch, 1965). This was also convincingly shown in etiolated cucumber
cotyledons that the chlorophyll content increased 450 percent after a 14
hour cytokinin pretreatment (Fletcher and McCullagh, 1971). No evidence
for an auxin influence on chlorophyll accumulation, chloroplast forma-
tion, or replication has been found (Laetsch and Boasson, 1972;
Schmerder et al., 1978).

The chlorophyll molecule has two major parts, porphyrin and phytol
and is a member of the metallotetrapyroles, which have been referred to
as ”pigments of life” because of their importance in living systems. ALA
is the universal precursor for the synthesis of chlorophyll and other
tetrapyrroles, and in higher plants ALA is synthesized from glutamate

(Beale and Castelfranco, 1973, 1974). ALA formation is the rate-limiting

29

step for pigment accumulation during the greening of dark-grown cells
and tissues when exposed to light (Beale and Castelfranco, 1974). This
light regulation was shown to be a low fluence phytochrome response
(Huang et al. 1989)

This paper reports inducible changes in the color of white mega-
gametophyte callus of Picea glauca. The role of different cytokinins,
chlorophyll precursors (glutamate and ALA), and heat shock in this
process were investigated using callus cultures grown in a controlled

environment.

Materials and Methods
Subculture Media

All white megagametophyte callus of Picea glauca used for the
following experiments was maintained on the MS medium (pH 5.8) supple-
mented with 10 uM 2,4-D and BAP, 2% sucrose, and 0.6% agar (see chapter
1). Cultures were subcultured at 4 week intervals. All experiments were
initiated with callus that had been maintained in culture for at least
8 months.
Standard Media

White calli were transferred onto MS medium (pH 5.8) containing
different sucrose concentrations (0.5%, 1%, 2%, 3%, 4%), and either 0.6%
agar or without agar. Changes in coloration were scored after 3 and 7
weeks in culture. A 2% sucrose concentration was chosen as a component
of the standard medium (MS medium with 2% sucrose, either with 0.6% agar
or lacking agar). All cultures were grown in a controlled environment

chamber under an 18 hr light / 6 hr dark photoperiod at 26°C. Two

30

light intensities, high (50 uE/m2/sec) and low (8 uE/m2/sec)
were used.
Scoring Coloration

Callus coloration was scored as: Red: regions of white callus
developing red color; Green: regions of white callus developing green
color: Brown: regions of white callus developing brown color; White: no
regions developing any other color. An individual callus could be scored
for more than one color.
Plant growth regulators (PGR)
Cytokinins: Eighteen explants of each treatment (six calli per
perti-dish) were grown on 20 ml of the standard solid medium with dif-
ferent levels of either zeatin (0.4 uM, 1 uM, and 2.5 uM) or BAP (5 uM,
10 uM, and 15 uM) and either high light or low light intensity.
Auxin with cytokinin: A different concentration of 2,4-D (1 uM,
2 uM, 5 uM, and 10 uM) and BAP (4 uM, 5 uM, and 10 uM) and thirty ex-
plants of each treatment under high light regimes were used to test the
effects of the interaction of BAP and 2,4-D on the color change. After 4
weeks, color change was recorded as green, red or brown color.
Glutamate and ALA

Treatments containing liquid standard medium with no glutamate,
20 mM glutamate, 3 mM ALA, and 20 mM glutamate with 3 mM ALA were
tested using twenty explants for each treatment (one callus in each 35 x
10 mm petri dish). These calli were grown in 5 ml of liquid medium in
the dark. Coloration of calli was scored at 2 and 4 weeks.

The long-term effects of glutamate and ALA were studied by main-
taining calli in the same petri dish without subculture for up to nine

weeks. Individual white calli were placed in the dark in either 5 ml of

31

liquid standard medium (sixteen calli per treatment and each in one
petri dish) or 20 ml of solid standard medium (twenty-five calli per
treatment and five calli per petri dish 100 x 15 mm) with the same
treatments. The color was scored at 3, 7, and 9 weeks.
Heat shock

White calli were exposed to 37°C for 0 min, 30 min, or 60 min.
Fifteen white calli of each treatment (five per petri-dish) were trans-
ferred onto standard solid medium with zeatin (0.4 uM, 1 uM, and 2.5 uM)
or BAP (5 uM, 10 uM, and 15 uM) under the high light regime. Changes in
callus color were scored after 4 weeks.
Statistical analysis

The data for color change were analyzed for each experiment using
the Multiple General Linear Hypothesis Analysis Program in Systat
(version 4.0). The data for each treatment were separated by Duncan's
New Multiple-Range Test (Steele and Torrie, 1980) if the MGLH-ANOVA

indicated significance.

Results

White callus underwent color changes in the absence of plant
growth regulators in the MS culture medium supplemented with different
sucrose concentration. After three weeks in culture no change in color
was observed in calli grown on media containing 1% or 2% sucrose. De-
creasing the sucrose concentration to 0.5% resulted in 8% browning.
Increasing the sucrose concentration to 3% or 4% resulted in an even
more pronounced change, with 24% and 27% browning, respectively. Follow-
ing seven weeks of growth without subculture, brown color was observed

at the rate of 24%, 64%, 67%, 61%, and 52% for 0.5%, 1%, 2%, 3%, and 4%

32

sucrose concentrations, respectively. No other color (green or red) was

observed in this experiment.

A. The effects of plant growth regulators
cytokinins (zeatin and BAP):

The addition of cytokinins to the standard medium caused a color
change from white to green, red and brown (Table l and 2). Differences
in color change between zeatin and BAP were statistically analyzed.
Zeatin and BAP have significantly different effects on the induction of
green color, but not on the induction of red and brown color (Table 2).
Analysis of the data in Table 1 (ANOVA not shown) indicated there was
also a significant interaction between light and cytokinins on color
change to red and brown coloration. The BAP treatment averages for red
color in high light and low light were 33% and 5%, respectively. The
comparable values for the zeatin treatments were 3% and 8%. Calli which

did not change color were not scored in this experiment.

Table 1. The effects of cytokinin level and light intensity on color
change of white callus.

 

 

 

Light Explants Zeatin(uM) BAP(uM) Green Red Brown

high 18 0 5 0.78(14/18) 0.28(5/18) 0.56(10/18)
low 18 0 5 0.61(11/18) 0.17(3/18) 0.44(8/18)

high 18 0 10 0.61(11/18) 0.61(11/18) 0.17(3/18)

low 18 0 10 0.72(13/18) 0(0/18) 0.44(8/18)

high 18 0 15 0.83(15/18) 0.11(2/18) 0.33(6/18)

low 18 0 15 0.61(11/18) 0(0/18) 0.44(8/18)

high 18 0.4 0 0.50(9/18) 0(0/18) 0.17(3/18)

low 18 0.4 0 0.50(9/18) 0.06(1/18) 0.50(9/18)

high 18 1 0 0.39(7/18) 0.06(1/18) 0.28(5/18)

low 18 1 0 0.22(4/18) 0.06(1/18) 0.61(11/18)
high 18 2.5 0 0.17(3/18) 0(0/18) 0.89(16/18)
low 18 2.5 0 0.28(5/18) 0.11(2/18) 0.33(6/18)

 

33

Table 2. Average of green, red, and brown color in white callus grown
on solid standard medium containing zeatin or BAP.

 

 

Cytokinin Green Red Brown
Zeatin 0.3410.14a 0.0510.04a 0.4610.26a
BAP 0.69:0.10b 0.2010.23a 0.40:0.l3a

 

1) Mean + SE of color induced from white calli.

2) Values followed by different letters are significantly different by
Duncan's NMR test (alpha - 0.05).

The effect of 2,4-D with BAP:

The addition of both 2,4-D and BAP on the color change of white
callus is shown in Table 3. The addition of both growth regulators
resulted in a reduction in the amount of color change when compared to
calli grown on standard medium containing only cytokinins. Only one
treatment (5 uM BAP and 2,4-D) resulted in calli containing any green
regions and no red coloration was observed. High levels of 2,4-D were

also associated with a reduction of brown color in the calli.

Table 3. The effects of different concentrations of 2,4-D and BAP on the
greening and browning of white callus.

 

 

2,4-D BAP 2,4-D Explant White Green Brown
(uM) (uM) BAP number
1 10 0.10 30 0.83(25/30) 0(0/30) 0.17(5/30)
1 4 0.25 30 0.97(29/30) 0(0/30) 0.03(1/30)
2 10 0.20 30 1(30/30) 0(0/30) 0(0/30)
5 10 0.50 30 1(30/30) 0(0/30) 0(0/30)
5 5 1.00 30 0.97(29/30) 0.03(1/30) 0(0/30)
10 10 1.00 30 1(30/30) 0(0/30) 0(0/30)

 

34

Table 4. The effects of the Glu and ALA on the color change of the
white calli on different media.

 

 

Treatments Explants Weeks Media White Brown

Control 25 3 Solid 1(25/25) 0(0/25)

20 mM Glu 25 3 Solid 0.40(10/25) 0.60(15/25)

3 mM ALA 25 3 Solid 0.92(23/25) 0.08(2/25)

20 mM Glu + 25 3 Solid 0.96(24/25) 0.08(2/25)
3mMALA

Control 25 7 Solid 0.40(10/25) 0.60(15/25)

20 mM Glu 25 7 Solid 0(0/25) l.00(25/25)

3 mM ALA 25 7 Solid 0.92(23/25) 0.08(2/25)

20 mM Glu + 25 7 Solid 0.84(21/25) 0.16(4/25)
3 mM ALA

Control 25 9 Solid 0.36(9/25) 0.64(16/25)

20 mM Glu 25 9 Solid 0(0/25) 1(25/25)

3 mM ALA 25 9 Solid 0.64(16/25) 0.36(9/25)

20 mM Glu + 25 9 Solid 0.80(20/25) 0.20(5/25)
3mMALA

Control 16 3 Liquid l.00(16/16) 0(0/16)

20 mM Glu 16 3 Liquid 0.50(8/16) 0.50(8/16)

3 mM ALA 16 3 Liquid l.00(16/16) 0(0/16)

20 mM Glu + 16 3 Liquid l.00(16/16) 0(0/16)
3 mM ALA

Control 16 7 Liquid 0.25(4/25) 0.63(10/16)*

20 mM Glu 16 7 Liquid 0(0/16) 1(16/16)

3 mM ALA 16 7 Liquid 0.88(14/16) 0.06(1/16)*

20 mM Glu + 16 7 Liquid 0.94(15/16) 0.06(1/16)
3 mM ALA

Control 16 9 Liquid 0.25(4/16) 0.56(9/16)*

20 mM Glu 16 9 Liquid 0(0/16) 1(16/16)

3 mM ALA 16 9 Liquid 0.63(10/16) 0.25(4/16)*

20 mM Glu + 16 9 Liquid 0.80(12/16) 0.13(2/16)*
3 mM ALA

 

Controlzstandard solid or liquid medium.

‘*' indicates that remaining calli developed yellow

color.

35

B. The effects of Glutamate and ALA

No green regions developed on calli when either glutamate or ALA
were added to the standard medium. Red and brown colored regions ap-
peared on the some of the white calli (Tables 4 and 5). However, the
glutamate and ALA treatments had different effects on callus color
change. Following the addition of glutamate and ALA (Tables 4, 5, and
6); the calli either showed no color change or developed brown regions.
No differences in color change were observed among cultures on solid or
liquid standard medium. Media augmented with ALA alone or ALA in combi-
nation with glutamate resulted in a high percentage of calli that re-
mained white and which rarely became brown (average 14% or 10% respec-
tively). For media augmented with glutamate alone, the duration of the
subculture period greatly altered this response. After seven weeks
without subculture, 100% of the white calli grown in media containing

only glutamate turned brown.

Table 5. Average of white or brown color in white callus grown on media
containing glutamate, ALA, or a combination of the two.

 

 

Treatment Number of Qallug Cglgr
Calli White Brown
Control 41 0.5410.36a 0.4010.32a
20 mM Glutamate 41 0.15:0.23b 0.85:0.23b
3 mM ALA 41 0.8310.16c 0.1410.l4c
20 mM Glutamate
+ 3 mM ALA 41 0.89:0.09c 0.1010.08c

 

1) Mean 1 SE color induced on white calli.
2) Values followed by different letters are significantly different by
Duncan's NMR test (alpha 8 0.05).

36

Browning of calli greatly increased as the number of weeks since
the previous subculture increased, but red, brown, or white color of
callus was observed in the four week treatment (Table 6). Red color
appeared in calli grown on ALA alone or glutamate with ALA within 2
weeks, but disappeared completely after 4 weeks. Also brown and white

color increased at same period as red color disappeared.

Table 6. The effects of different concentrations of glutamate and ALA on
the color change of white callus in liquid media.

 

 

Medium Explants Weeks White Green Red Brown
Control 20 2 1(20/20) 0(0/20) 0(0/20) 0(0/20)

20 mM Glu 20 2 1(20/20) 0(0/20) 0(0/20) 0(0/20)

3 mM ALA 20 2 0.40(8/20) 0(0/20) 0.60(12/20) 0(0/20)

20 mM Glu 20 2 0(0/20) 0(0/20) 1(20/20) 0(0/20)

+ 3 mM ALA

Control 20 4 0.10(2/20) 0(0/20) 0(0/20) 0.90(18/20)
20 mM Glu 20 4 0(0/20) 0(0/20) 0.10(2/20) 0.90(18/20)
3 mM ALA 20 4 0.70(14/20) 0(0/20) 0(0/20) 0.30(6/20)
20 mM Glu 20 4 0.75(15/20) 0(0/20) 0(0/20) 0.25(5/20)

+ 3 mM ALA

 

C. The effect of Heat Shock

A combined heat shock and cytokinin treatment also resulted in
calli of all four color types (Table 7). In the heat shock treatments
zeatin and BAP had significantly different effects on the formation of
green color and red color, but not brown color. In addition, white
calli grown on zeatin remained white more frequently than white calli
grown on BAP (36.8% versus 3%) (Table 8). The 30 minute heat shock
treatment did not significantly effect callus color (Table 9). However,
in calli exposed to 37°C for 60 minutes, the percentage of green and red
regions decreased while the percentage of brown regions increased signi-

ficantly.

37

Table 7. The effect of heat shock, exposure time and cytokinin concen-
tration on color change of white callus.

 

 

 

 

Time Explant Zeatin BAP White Green Red Brown

(min) # (0M) (“14)

0 15 0 5 0(0/15) 0.80(12/15) 0.27(4/15) 0.07(1/15)
30 15 0 5 0.07(1/15) 0.93(14/15) 0.27(4/15) 0.07(1/15)
60 15 0 5 0.07(1/15) 0.33(5/15) 0(0/15) 0.67(10/15)

0 15 0 10 0(0/15) 0.93(14/15) 0.40(6/15) 0(0/15)

30 15 0 10 0(0/15) 0.93(14/15) 0.40(6/15) 0.20(3/15)
60 15 0 10 0.07(1/15) 0.60(9/15) 0.20(3/15) 0.60(9/15)

0 15 0 15 0(0/15) 0.93(14/15) 0.53(8/15) 0.27(4/15)
30 15 0 15 0(0/15) l.00(15/15) 0.80(12/15) 0(0/15)

60 15 0 15 0.07(1/15) 0.73(11/15) 0.40(6/15) 0.73(11/15)

0 15 0.4 0 0.33(5/15) 0.60(9/15) 0.l3(2/15) 0.l3(2/15)
30 15 0.4 0 0.20(3/15) 0.60(9/15) 0(0/15) 0.27(4/15)
60 15 0.4 0 0.60(9/15) 0.27(4/15) 0(0/15) 0.27(4/15)

0 15 1 0 0.27(4/15) 0.47(7/15) 0(0/15) 0.27(4/15)
30 15 1 0 0.73(11/15) 0.13(2/15) 0.07(1/15) 0.07(1/15)
60 15 1 0 0.47(7/15) 0.27(4/15) 0.07(1/15) 0.40(6/15)

0 15 2.5 0 0.33(5/15) 0.53(8/15) 0(0/15) 0.13(2/15)
30 15 2.5 0 0.l3(2/15) 0.87(13/15) 0.20(3/15) 0.07(1/15)
60 15 2.5 0 0.27(4/15) 0.20(3/15) 0(0/15) 0.60(9/15)
Table 8. Average of white, green, red, and brown color in white callus

grown on medium containing zeatin and BAP.

 

 

Cytokinin White Green Red Brown
Zeatin 0.3710.20a 0.4410.24a 0.0510.07a 0.2410.17a
BAP 0.03:0.04b 0.8810.22b 0.3610.22b 0.2910.29a

 

1) Mean + SE color induced on white calli.

2) Values followed by different letters are significantly different by
Duncan's NMR test (alpha s 0.05).

Table 9.

at different heat shock exposure time.

Average of white, green, red, and brown color in white callus

 

 

 

Time White Green Red Brown

0 min 0.1510.17a 0.7210.02a 0.2210.22a 0.1410.12a
30 min 0.1910.28a 0.7410.33a 0.29:0.28a 0.1110.10a
60 min 0.2610.23a 0.4010.22b 0.1110.16a 0.5410.18b
1) Mean percent 16E color induced on white calli.

2)

Values followed by different letters are significantly different by
Duncan's NMR test (alpha 8 0.05).

38

Discussion
In order to give an overview of the factors and their effects on
color change, the results of the preceding experiments have been summa-

rized in Table 10.

Table 10. Summary of factors and their effects on color change in white
callus.

 

Color

Factors Green Brown Red

 

BAP and Yes, but different Yes, but same effect Yes, same as for

 

 

 

Zeatin effects between BAP between BAP and brown color
(Table l) and zeatin zeatin
2,4-D No, inhibition of Yes, but only in No
(Table 3) BAP effects on high BAP:2,4-D ratio

greening
Glutamate No Yes (about 100%) No
ALA or No Yes (ls-36%) Yes, it showed within
Glu+ALA two weeks, but
(Tables 4 changed to either
and 6) brown or white later
Time at reduced greening increased browning no difference among
37°C at 60 min at 60 min exposed periods
(Table 7)

 

It has been reported that increasing the sucrose concentration in
the nutrient medium alters the production of phenolics (Knobloch and
Berlin, 1980; Suzuki et al., 1981; Khouri et al., 1986). In this study,
a high sucrose concentration and a long culture period resulted in an
increase in brown colored regions in the calli. Sucrose concentrations
at 3% and 4%, but not 1% or 2%, increased browning of white callus after
a 3-week period. The percentage of white calli turning brown also in-
creased for all sucrose concentrations when cultures were allowed to

grow for seven weeks without subculture. Therefore, a 2% sucrose con-

39

centration and a 4-week subculture were selected to minimize the effects
of browning due to these factors.

Parthier (1979) suggested that cytokinins alter gene expression
for plastogenesis. In one study, thirty-nine polypeptides decreased and
twenty-three polypeptides increased in concentration during the greening
process (Dietz and Bogorad, 1987). In this work, the greening of white
megagametophyte-derived callus was inducible by the addition of either
BAP or zeatin. However, the response differed significantly for the two
plant growth regulators. BAP was more efficient at inducing the forma-
tion of green regions (Table 1). This appears to indicate the involve-
ment of cytokinins in the induction of chlorophyll biosynthesis and
probably chloroplast development in white callus of white spruce.

Previous work has shown that both the quality and intensity of
light, such as blue light and UV-B light (280-320 nm), influence the
production of phenolics in cultured tissue (Suzuki et al. 1981; Duell-
Pfaff and wellmann, 1982; Knobloch et al., 1982). In gymnosperms. chlo-
roplast development can occur in either the light or dark. Our results
demonstrate that white callus grown at either high or low light intensi-
ties had a similar response to the BAP and zeatin treatments when scored
for the induction of green regions. This is consistent with evidence
from other studies indicating that chloroplast development may be
trigged either by light through photoreceptors, such as phytochrome, or
by plant growth regulators, such as cytokinins (Lerbs et al., 1981:
Mohr, 1984).

The type and concentration of auxins and cytokinins, as well as
their relative ratios in the culture medium, play a role in the biosyn-

thesis and accumulation of secondary metabolites. An increase in the

40

concentration of phenolics has been reported in the presence of low
levels of auxins, especially 2,4-D (Kadkade, 1982). In carrot cells,
pigment accumulation was inhibited by low levels of 2,4-D, but was
stimulated by 0.01-l.0 uM of kinetin, BAP, or zeatin (Ozeki and Romaine,
1981). In our work the presence of auxin (2,4-D ) seems to counteract
the effects of the cytokinin for color induction (Table 3). When the
control medium (MS medium with 10 uM BAP and 10 uM 2,4-D) was used to
culture white calli and green calli, neither white calli nor green calli
changed color. This indicates that cytokinins tend to initiate and
auxins inhibit the greening process. Once the greening process begins,
the addition of auxins does not reverse it.

The browning of white callus in media containing either glutamate
or ALA or both glutamate and ALA differed (Tables 4, 5 and 6). This
indicates that there may be a different pathway for glutamate and ALA
metabolism in white callus. The interaction of the metabolites of ALA
and glutamate increased the red colored regions in the white calli but
these then changed to brown or back to white after a short period (Table
6). This reddening process appeared to be independent from the other
color changes since it was not observed in any brown or green colored
regions of the calli. The turnover rate for red color to white or brown
was fast and generally required only one to two weeks. Whether the brown
calli found in the different treatments are derived from red calli or
directly induced from white calli is still unknown.

In glutamate and/or ALA treatments, no green coloration has ob-
served in these treatments (Tables 4 and 6). Apparently, white calli did
not synthesize any chlorophyll, even after being treated with chloro-

phyll precursors glutamate and ALA. This indicates that the greening

41

process induced by the cytokinins is blocked after the step(s) of ALA
biosynthesis in the chlorophyll biosynthesis pathway in white callus.
Alternatively it is possible that the genes for chlorophyll synthesis
are regulated by cytokinins through major regulatory protein(s), al-
though these experiments do not allow this question to be addressed .
Furthermore, green and brown color existed alone or together when white
calli were grown in the presence of cytokinins and glutamate and ALA
(Tables 1, 4, 6, and 7). It is possible that different pathways exist in
white callus for the color change to either green or brown, yet chloro-
phyll biosynthesis either promoting or inhibiting brown coloration is
still unknown.

In plants, heat hardening and heat shock protein (HSP) synthesis
are concomitant phenomena, and a good correlation has been found between
HSP synthesis and thermotolerance (Lin et al., 1984; Sachs and Ho,,
1986). Shifts from the optimal growth temperature result in a stress for
plants and, even if not lethal, affect their development (Christianson,
1982). Plastid HSP synthesis fluctuates through the day and is con-
trolled in a circadian fashion (Otto et al., 1988). However, the effect
of plastid HSPs is still not understood. In this case, heat shock
changed the response of the white callus to BAP and zeatin. A signifi-
cant increase in browning, and decrease in greening, took place when
white calli were exposed to 37°C for 60 min. It is possible that changes
in the response of white callus to cytokinins resulted from the break-
down of the cytokinins, or the expression of HSPs.

The objective of this paper was to understand the factors that
effect the color change of white megagametophyte callus of P. glauca.

This research showed that cytokinins can induce greening, and that

42

auxins inhibit the greening process. Furthermore, we have shown that
color change in white callus is inducible, but how that change occurs is
still a question. Because green callus can be induced from white callus,
this provides a system for studying chloroplast development. More

research is needed to evaluate the differences between white and green
calli at the DNA, RNA, and protein levels before this will lend any real
understanding the regulation of chloroplast development and pigmentation

in plants.

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variations in physical characteristics, proteins, and DNA between green
and non-green callus cultures of Picea glance

Abstract

Variations in callus dry weight and protein concentration were
found between white calli and green calli derived from white calli. The
chlorophyll a/b ratio for green calli was 1.339. White calli lacked
chlorophyll. Both green calli and white calli showed absorbency profiles
from 200 to 300 nm and a strong peak at 320 nm, but green calli had addi
tional absorbancy peaks at 410 nm, 430 nm, 470 nm, and 670 nm. No meas-
urable photosynthesis activity occurred in either white or green calli.
In comparison with needles, green calli and white calli contained an
extra DNA band on CsCl density gradients. There was also differential
expression of polypetides among white calli, green calli derived from
white calli, and needles. Comparison of HpaII and MspI restriction

patterns detected differences in genomic DNA methylation among tissue

types.

45

46

Introduction

Chloroplasts and mitochondria are theorized to have originated
endosymbiotically with subsequent transfer of genes from these orga-
nelles to the nucleus resulting in the dependence of both organelles on
the nuclear genes for the synthesis of essential polypeptides. Chloro-
plast DNA encodes approximately 60-100 polypeptides and the remaining
80-90% of plastid proteins are encoded by the nuclear genome (Sugiura,
1989: Taylor, 1989). Organelle development and genetic regulation are
also largely under nuclear control (Attardi and Schatz, 1988; Taylor,
1989).

Plants have five major types of plastids: proplastids, chloro-
plasts, chromoplasts, amyloplasts, and etioplasts. All plastids can
develop directly from proplastids. The reverse process, that is, the
simplification of plastid structure back from a specialized form to the
proplastid form, is conditional upon cell division and the stage of
plant development or differentiation (Whatley, 1977, 1978).

The regulation of plant development and plastid interconversion
is accomplished at several levels. Changes in both nuclear and plastid
gene expression are involved in this developmental process (Piechulla
et al., 1986; Piechulla,1988: Kuntz et al., 1989). Transcriptional
and post-transcriptional regulation may play a major role in the inter-
conversion of plastids. Light also has a profound influence on morpho-
genesis and biochemical differentiation of plastids and regulates
the expression at both transcriptional and post-transcriptional
levels through light-photoreceptor interactions (Thompson, 1988).

Furthermore, some plastid and nuclear genes, such as psbA and rch

47

are temporally and spatially expressed during plant development (Fiebig
et al., 1990).

The functions of plastids include the biosynthesis of amino acids
and fatty acids, nitrate assimilation and photosynthesis. Photosynthetic
reactions, which provide metabolic energy, are not present in non-green
plastids. Enzymes for carbohydrate metabolism have differential expres-
sion between green and non-green tissues (ap Rees and Entwistle, 1989;
Reeling, 1989; Madore, 1990; Frehner et al., 1990). In ripening tomato
fruit, the chloroplast-specific mRNA levels continuously decrease
(Piechulla et al., 1985; Piechulla et al., 1986; Piechulla, 1988), while
two chromoplast-specific polypeptides (Mr 35 and 58 kd) are either not
detectable or barely detectable in chloroplasts (Hadjeb et al., 1988).
Obviously, different plastid types have various mechanisms that regulate
their metabolism.

DNA methylation has been postulated as a mechanism that controls
the transcription of genes thus regulating gene expression. In studies
of plastid DNA, chromoplast DNA and amyloplast DNA, but not chloroplast
DNA, are methylated in regions containing genes that are not expressed
or are expressed at low frequency (Ngernprasirtsiri et al., 1988a,
1988b; Macherel et al., 1986).

This study is a attempt to better understand plastid development
through the characterization of differences among green and non-green
tissues of white spruce. Differences in physical characteristics, pro-

teins, and genomic DNA were studied and discussed.

Materials and Methods
Culture conditions for white calli and green calli were described

previously in Chapter 1.

48

Relative moisture content

The relative moisture content of white calli and green calli was
measured by weighing calli before and after placement in an oven at 80°C
for 24 hours. Six samples of each tissue type were measured.
Chlorophyll measurement and total protein content

Photosynthesic activity of white calli and green calli (ca. 59
each) was measured using a Port Photosynthesis System (LI-COR, INC).
Approximately 10 g of callus tissue was homogenized at speed 7-8 for 20
seconds with a Teckmar Tissuemizer in 150 ml of grinding medium (Stine
et al. 1988). The homogenate was centrifuged at 12,000 rpm for 15 min in
a Sorvall GS-3 rotor. Distilled water (40 ml) was added to suspend the
pellet and blended for 4 seconds with the Teckmar Tissuemizer. Acetone
(160 ml) was then added and blended for 10-20 seconds at speed 7-8,
after which the homogenate was centrifuged at 10,000 rpm for 5 min in a
Sorvall GSA rotor. The supernatant was used to measure chlorophyll
concentration and chlorophyll a/b ratio. The pellet was used to measure
the total protein content.
A. Chlorophyll measurement Chlorophyll content and the chlorophyll
a/b ratio were determined by measuring the A663 and A645 of the superna-
tant respectively. Three samples of each supernatant material were
measured. Wavelengths from 200 nm to 800 nm were used to measure the
absorbency spectra using a Perkin-Elmer photospectrometer and a P-E
Lamda 4B program.
B. Protein determination Approximately 1-2 mg of each air-dried
pellet was used to estimate the protein concentration by Lowry assay

kit (Sigma) and A750. Three samples of each tissue were measured.

49

Protoplast isolation

Protoplasts were isolated from white and green calli. Calli (5-10
9) were slowly ground through a 100 um nylon or iron mesh in 12 ml of
protoplast isolation medium (5% mannitol, 2% sucrose, 5% sorbitol, 18 mM
CaClz, 15 mM CaSO4, 0.4% cellulase (Rarlan Chemical), 0.2% macerase
(Calibiochem), and 0.3% hemicellulase (Sigma)). After incubation for 5-6
hours at 70 rpm in the dark, protoplasts were concentrated at 1,000 rpm
for 5 minutes in a Sorvall HB-4 rotor. The pellet was resuspended in a
8% mannitol solution.
Sucrose gradient separation of needle and protoplast solutions

The protoplast solution was passed through an 18 gauge needle 3-4
times. Needle tissue (10 g) was ground for 20-30 seconds at speed 7-8 in
grinding buffer (Stine et al. 1988) with a Techmar Tissuemizer. The
homogenate was filtered through 1 layer of 100 um mesh nylon and 2
layers of Mirocloth (Calibiochem.) and centrifuged at 12,000 rpm for 15
minutes in a Sorvall GS-3 rotor. Grinding buffer (10-15 ml) was added to
resuspend the pellet. Protoplast and needle solutions were loaded on a
discontinuous sucrose gradient, (0.7M, 1.1M, 1.4M, and 1.6M) with a
sucrose gradient buffer of 50 mM Tris, 6% sorbitol, and 25mM EDTA at pH
7.5. Organelles and cell particles were separated at 12,000 rpm for 1
hour in a Sorvall HB-4 rotor. Each interface was collected and an equal
amount of T50320 (50 mM Tris pH8.0 and 20 mM EDTA pH 8.0) was added. The
solutions were centrifuged at 11,000 rpm for 10 minutes in a Sorvall HB-
4 rotor and the pellets were used for SDS-PAGE electrophoresis.
SDS-PAGE Electrophoresis

Distilled water (0.4 ml) was added to dissolve each pellet and

then equal amounts of 2x sample load buffer (0.12 M Tris, 6%(w/v) SDS,

50

10% 2-mercaptoethanol, 20%(v/v) glycerol, and 2 mg/ml bromophenol) was
added. The solution was boiled for 2 min before loading on the gel.
Sample volumes ranged from 1-10 ul. A 15% acrylamide separating gel and
6% stacking gel (Laemmli 1970)were used to separate proteins. The gel
size was 15cm x 15cm x 0.15 cm. Constant current (3 mA) was applied
until the blue dye front reached the bottom of the gel (approximately 5
hours). Bio-Rad silver stain plus was used to visualize the proteins
under conditions described by the manufacturer (Bio-Rad laboratory).
Total DNA isolation

Total DNA isolation from needles followed the protocol described
by Neale et al. (1986). The method used to isolate the total DNA of
green calli and white calli was modified from Ausubal et al. (1987).
Callus tissue (12 g) was ground to fine powder in liquid N2 with a
prechilled mortar and pestle. The powder was transferred into 250 ml
plastic bottles and 67 ml of extraction buffer (50 mM Tris pH 8.0, 10 mM
EDTA pH8.0, 1M NaCl, 3 mM cysteine, 1 mM ascorbic acid, and 50 ug/ml
proteinase K (Boehringer Mannheim)) was added to dissolve the powder.
The homogenate was then filtered through a 100 um nylon mesh. Sarkosyl
(20 ml of 5% sarkosyl) was added and the solution was incubated for 3 hr
at 55°C, and then centrifuged for 10 min at 4,000 x g, 4°C in a Sorvall
GSA rotor. Isopropanol (0.6 volume) was added to the supernatant and
gently mixed. Total DNA was precipitated for 15 min at 8,000 x g, 4°C in
a Sorvall GSA rotor. DNA purification and precipitation followed methods
described by Stine et al. (1988).
Southern analysis

DNA was digested with restriction enzymes under the conditions

specified by the manufacturer (Boehinger Mannheim). A 0.6 % (w/v)

51

agarose gel (Sigma) in TBE buffer and l-l.2 V/cm constant power were
used to separate DNA fragments. Zeta-probe blotting membrane (Bio-Rad)
was used for DNA alkaline blotting (Bio-Rad Laboratory). Petunia chNA
88 and maize mtDNA coxII clones were used as probes. Probes were labeled
using random primer labeling according to the manufacturer's (Boehringer
Mannheim Biochemicals) directions. Prehybridization, hybridization and
post hybridization washeswere performed according to Bio-Rad instruc-
tions for Zeta-Probe membrane. Autoradiography was carried out at -70°C
with DuPont Cronex Lightning plus intensifyingscreens and Kodak x-Omat

XARS x-ray film.

Results
A. Physical characteristics

The relative moisture content, chlorophyll content and protein
concentration of green calli and white calli are summarized in Table 1.
Green calli differed significantly from white calli not only in chloro-
phyll content but also in dry weight and protein concentration. Green
calli on average weighed 1.4 times more than white calli. The ratio
of total protein amount between green and white callus was 4:1 in fresh
weight and 2:1 in dry weight. In white calli extraction, absorbency
occurred in the A663 and A645 regions, but no definite peak was observed
and no chlorophyll was detected (Table 1 and Figure 1). The chlorophyll

a/b ratio of green calli was 1.339. Neither callus types showed the

evidence of photosynthetic activity. The absorbency peaks of green
callus were at 680nm, 470nm, 430nm, and 410nm. Both callus types shared

an the absorbency peak at 320nm and from 200 nm to 320 nm (Figure 1).

52

Table 1. Relative moisture content, chlorophyll content, and protein
concentration of white and green calli

 

 

 

Green calli White calli
Relative moisture 0.9384 0.9651
Dry weight 0.0616 0.0349
(g/g of fresh weight)
Photosynthetic rate 0 0
Chlorophyll conc. 47.4650 0.0315a
(ug/g of fresh weight)
Chlorophyll a/b 1.3390 2.3360a
Total protein 6.0850 1.4736

(mg/g of fresh weight)

 

a:the value of white callus was calculated as formulas for the measure-
ment of chlorophyll and chlorophyll a/b ratio.

075'

 

 

Abserbance

 

 

 

A
0.50 "
0.25 '
200 300 400 500 600 750' :00
050? B
azs'
200 . ‘ :00 400 so? :00 700 :0:

Wavelength nm

Figure l. The absorbency spectrum of acetone-water extraction. A. The
spectrum of green callus. B. The spectrum of white callus.

53

B. Protein analysis

Differences in polypeptides were found among needles, green cal-
lus, and white callus (Figure 2). There was little polypeptides varia-
tion among samples from the different interfaces of the sucrose gradi-
ents. In samples from white callus, no polypeptides (Figure 2. a and b
regions) were observed in samples from the extraction solution-0.7 M and
0.7-1.1 M interfaces, but a number of bands were found in samples from
the 1.1-1.4 M and 1.4-1.6 M interfaces. In green callus, the polypep-
tides migrating to the a and b regions of gel were present in samples
from all interfaces of the sucrose gradients. In the sucrose gradient
the extraction solution-0.7 M interface, white calli did not contain
polypeptides of 45, 38, and 32 kd that were observed in samples from
needles. Samples from green calli and needles were more similar. The
only difference between needles and green callus was that needle samples
produced a band at 38 kd and green callus samples showed a similar band

at 39 kd.

C. DNA Studies

After ultracentrifugation of total DNA, two strong DNA bands were
detected in samples from needles (a third band was either faint or
undetectable) and three DNA bands were visible in samples from green
calli and white calli. When white spruce chNA and the bottom DNA band
(band 3) of white calli were digested with restriction enzymes PstI or
SacI, the restriction band patterns differed, especially in the 16-24 kb
range (Figure 3A). DNA from the third band did not hybridize to the

maize mitochondrial CoxII DNA used as a probe (Figure 3B).

54

 

 

Figure 2. silver staining SDS-PAGE gel of polypeptides from extracts,
which were separated by discontinuous sucrose, of needles, green callus
and white callus. 1:interface between loading solution and 0.7 M. 2:
interface between 0.7 M and 1.1 M. 3: interface between 1.1 M and 1.4 M.
4; interface between 1.4 M and 1.6 M. N: needles. szhite callus.
G:green callus.

55

Pet! Sacl P3“ 53°. ‘ -2 3 t
F—fl F—_1 r'1 r—1
Vk:G‘Wc G I“ B “I B “I II.-

 

A B

Figure 3. The megagametophyte callus DNA banding pattern. A. PstI or
SacI-cut DNA was fractionated by electrophoresis in 0.6% agarose gel
with THE buffer.B. Band 1, 2, and 3 was digested with PstI and hybri-

T:Total DNA. W: White

dized with maize mitochondrial CoxII probe.
spruce. B: blue spruce. G:Green callus. Wc:White callus.

56

0. DNA methylation

Four different methylation sensitive restriction enzymes: HaeIII,
HpaII, MspI, and NciI, were used to study the methylation pattern of
genomic DNA. These restriction enzymes and the methylation patterns they
fail to cleave are listed in Table 2.

Table 2. Methylation sensitive restriction enzymes and the methylation
sequences which will not be cleaved.

 

Restriction enzymes

 

HaeIII HpaII MspI NciI
Sequences Gemscc cmsccc m5cccc cmscccc
not cleaved mSCCGG cmscccc

 

The nuclear genomic DNA pattern for all tissue and callus types
was constant for each restriction enzyme, but differed among the re-
striction enzymes (Figure 4). In contrast to the restriction patterns
of HpaII, MspI, and NciI, nuclear genomic DNA was obviously digested by
restriction enzyme HaeIII, even though HaeIII is also a methylation
sensitive enzyme. Digestion of total DNA by restriction enzymes HpaII
and MspI, (which are isoschizomers and differ only in the methylation
position of sequences they will not cleave) resulted in different dis-
persion patterns among samples from needles, green callus and suspension
cells. The nuclear genomic DNA patterns of green callus and suspension
cells were much more widely dispersed in MspI-cut DNA when compared to
HpaII-cut DNA. The nuclear genomic DNA patterns of needles were the same

for both restriction enzymes (Figure 4, a region).

57

HaeIII HpaII MspI Nci I

 

NWGSNWGS NWGSNWGSM

III I II

 

 

Figure 4. Genomic DNA methylation pattern. Total DNA was digested by
methylation-sensitive restriction enzymes: HaeIII, HspII, MspI, and
NciI. Ethidium bromide was used to stain DNA. N:Needles. s:suspension
cells. G:green calli derived from white callus. W. White calli. M:1 kb
ladder. Medium for suspension cell culture was same as callus induction
medium without agar. DNA concentration in each tissue type is same for
all restriction enzymes.

58

Discussion

Plastid development require the regulation and expression of both
genomic and plastid genes. These studies showed that white calli and
green calli differ not only in chlorophyll biosynthesis but also in
protein concentration (Table 1). Total protein concentration of green
calli was four times more than that in white calfi and there was a 50%
increase in dry weight. The difference in total protein amount may
contribute to the difference in dry weight. These differences are also
apparent in the lower enzyme concentration and shorter time to isolate
protopasts from white calli. It is probable that this indicates a dif-
ference in cell wall components for white versus green callus cells.

The most striking difference between green callus and white callus
was the absence of detectable chlorophyll in white callus (Figure 2).
The presence of the absorbency peak around 320 nm may indicate that some
water or acetone soluble polypeptides exist in both green calli and
white calli. Similarly some secondary metabolites may exist in the
acetone-water extraction solution and cause the absorbency peak from 200
nm to 300 nm (Figure 1). An analysis of this acetone-water extraction
solution may provide a clue to understanding the variations of second-
ary metabolites between green calli and white calli.

It is interesting that no photosynthesis was observed in green
callus. The reason for this may be 1) green callus does not photosynthe-
size and fix carbohydrate because the sucrose in the culture medium
provided a carbon source (feed-back effect): 2) a low chlorophyll a/b
ratio (1.3990) and different absorbency spectra from chlorophyll a and b
(Figure 1) indicates that the photosynthetic apparatus may not be well

developed; 3) light intensity (20 uE/mZ/sec) may not be high enough to

59

stimulate photosynthesis in callus tissue; or 4) polypeptides necessary
for photosynthesis may not be completely expressed in green callus
(Figure 2). In one dimensional SDS PAGE gels, polypeptide differences
occurred among needles, green calli, and white calli (Figure 2). These
variations in polypeptides clearly indicate that the expression of genes
needed to maintain the cell functions differs according to plant tissue
type, developmental stage, and environmental conditions.

Qualitative variations in nuclear DNA result from the changes in
the amount of repetitive DNA together with changes in cell developmental
patterns (Buitti, 1977; Bassi et al., 1984). A heavier satellite DNA
band in CsCl density gradients has been observed in dedifferentiating
tissues, but not in differentiating tissues, of Vicia faba. This
suggests the amplification of G+C rich nuclear DNA sequences during the
cell dedifferentiation process (Natali et al., 1986). In the present
study, three DNA bands appeared in samples from both callus types after
CsCl density centrifugation. This indicates the possible amplification
of nuclear DNA sequences occurs in callus, because band 3 DNA neither
hybridized to mitochondrial DNA nor showed the same banding pattern as
chloroplast DNA (Figure 23 and 20).

Despite the variation between green callus and white callus,
plant growth regulators play an important role in the regulation of the
greening process from white to green calli. Green calli can be induced
from white calli by the addition of cytokinins (either zeatin or BAP) or
inhibited by the addition of auxin (2,4-D) (Chapter 2). In addition,
auxins are known to increase genomic DNA methylation. Cytokinins have
no known effect on DNA methylation (LoSchiavo et al. 1989). There-

fore, the greening process observed in the present study results from

60

the activation or inhibition of specific genes that regulate the expres-
sion of nuclear DNA through the methylation or demethylation of nuclear
or plastid DNA in response to the plant growth regulators .

In green plant, up to 30% of total cytosine residues of genomic

DNA are comprised of 5-methylcytosines (mSC). The m5

Cs occur predomi-
nantly in CpG dinucleotides, but also in CprG trinucleotides (Shapiro,
1968, Gruenbaum et al., 1981), and the mSC content is both species- and
tissue-specific (Sharpiro, 1968; Vanyushin, 1984). To detect differ-
ences in methylation we used the isoschizomer restriction enzymes MspI
and HpaII, which recognize a difference in methylation position within
restriction sequences. No methylation differences in nuclear genomic DNA
were observed within tissue types for each restriction enzyme. However,
variation of nuclear genomic DNA methylation was observed between tissue
types. More of the spruce nuclear genomic DNA sequences from green
callus and cell suspension cultures than those from needles are methy-
lated at the 3' cytosine of sequences (CmSCGG) cut by MspI, as was
evidenced in the broader dispersion of DNA from those tissues when
digested with MspI as opposed to HpaII, the most complete digestion was
by HaeIII, suggesting that nuclear genomic DNA may be less methylated or
not methylated at the 5' end cytosine (GGmSCC) (Figure 4). It has been
reported that DNA methylation is considered to play an important role in
the epigenetic control of gene transcription (Holliday, 1987). It is
possible that the differences in genomic DNA methylation patterns be-
tween needles and green callus may be a result of the addition of 2,4-D
to the culture medium, given that 2,4-D is known to increase DNA methy-

lation levels (LoSchiavo et al. 1989). It is unknown whether this in-

61

creased methylation level is responsible for the lack of photosynthetic
activity in green calli.

In this report, differences in dry weight, protein quality, pro-
tein quantity were observed between green and white calli. Variations in
polypeptides, total genomic DNA and nuclear genomic DNA methylation
pattern was observed between needles and green and white calli. These
are a clear indication of differential gene expression among those
tissues. Although these differences were documented, additional research
will be needed to establish the mechanism governing the greening of

white calli.

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Benth. Plant Physiol. 93:617-622.

Natali. L., A. Cavallini, R. Cremonini, P. Bassi and P.G. Cioini, 1986.
Amplification of nuclear DNA sequences during induced plant cell
dedifferentiation. Cell Differentiation 18:151-161.

Neale, D.B., N.C. Wheeler, and R.W. Allard, 1986. Paternal inheritance
of chloroplast DNA in Douglas-fir. Can. J. For. Res. 16:1152-1154.

Ngernprasirtsiri, J., H. Nobayashi, and T. Akazawa, 1988a. DNA methyla-
tion occured around lowly expressed genes of plastid DNA during
tomato fruit development. Plant Physiol. 88:16-20.

--- 1988b. DNA methylation as a mechanism of transcriptional regulation
in nonphotosynthetic plastids in plant cells. Proc. Natl. Acad.
Sci. USA 85:4750-4754.

Piechulla, B. 1988. Differential expression of nuclear- and organelle
encoded genes during tomato fruit development. Planta 174:505-512.

Piechulla, B., E. Pichershy, A.R. Cashmore, and W. Gruissem, 1986.
Expression of nuclear and plastid genes for photosynthesis-
specific proteins during tomato fruit development and ripening.
Plant Mol. Biol. 7:367-376.

Shapiro, H.S. 1968. In. Sober, H.A. (ed.) Handbook of Biochemistry. pp.
H39-48. CRC Press, Cleveland.

Stine, M., 8.8. Sears and D.B. Keathley, 1989. Inheritance of plastids
in interspecific hybrids of blue spruce and white spruce. Theor.
Appl. Genet. 78:768-774.

Sugiura, M. 1989. The chloroplast chromosomes in land plants. Annual

64

Taylor, W.C. 1989. Regulatory interactions between nuclear and plastid
genomes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:211-233.

Thompson, W.F. 1988. Photoregulation: diverse gene responses in greening
seedlings. Plant, Cell and Enviroment 11:319-328.

Vanyushin, B.F. 1984. In. Trauntner, T.A. (ed.) Methylation of DNA. pp.
99-114. Springer-Verlag, New York.

Whatley, J.M. 1977. Variation in the basic pathway of chloroplast
development. New Phytologist 78:407-502.

---- 1978. A suggested cycle of plastid developmental interrelations.
New Phytologist 80:489-502.

Chapter 4

Physical Mapping of Picea chloroplast DNA

Abstract
The chNA genome of Picea engelmannii, P. glauca, and P. pungens

are 120.2, 120.2, and 120.4 kb respectively. No difference in chNA
size chNA was observed between white spruce and Engelmann spruce. Small
size difference in the blue spruce chloroplast genome versus that found
in white and Engelmann spruce may be the result of a deletion, insertion
or duplication. A comparison of spruce chNA with known maps of radiata
pine and Douglas-fir indicated the loss of an inverted repeat region in
spruce chNA. Sequence homology was observed between chloroplast and

mitochondrial DNA fragments in spruce.

65

66

Introduction

Paternal inheritance of chNA has been demonstrated in the genera
Pinus (Neale and Sederoff 1989, Wagner et al. 1987, 1989), Picea (Stine
and Keathley, 1990: Stine et al., 1989; Sutton et al.,1991; Szmidt et
al., 1988), Pseusotsuga (Neale and Sederoff, 1988: Neale et al., 1986),
and Larix (Szmidt et al., 1987). Occasionally, biparental inheritance of
chNA occurs, as in Pinus monticola (White, 1990).

In general, the organization of chNA in gymnosperms differs from
that in angiosperms in that the former only has one copy of the inverted
repeat region (Lindolm et al., 1988: Strauss et al., 1988) and has a
genome size of approximately 120 kb. The only known exception to this is
Ginkgo biloba, which has two inverted repeats and a genome size of 157
kb (Palmer and Stein, 1986).

RFLPs of chNA provide a tool for ascertaining the amount and
pattern of introgression between species. Govindaraju et al. (1988)
found variation in chNA within trees of a Pinus banksiana - P. contorta
sympatric region and suggested that there is increased variability in
the organelle genome in regions of sympatry. Szmidt et al. (1988) used
this technique to classify seedlots of Picea sitchensis and P. glauca in
zones of introgression.

The phylogenetic diversity of the gymnosperms and the ecological
and economic importance of the Pinaceae justify a closer analysis of the
variation in the spruce chloroplast DNA genome. Currently, the best
procedure for developing phylogenies is to map and compare the chNA
of related species. The physical and genetic map of chloroplast DNA has
been determined for Ginkgo biloba (Palmer and Stein, 1986) and both

Douglas-fir and radiata pine (Strauss 1988).

67

This chloroplast DNA mapping study increases our understanding of
chloroplast DNA variation among members of the genus Picea. Physical
maps of Picea pungens, P. glauca, and P. engelmanii were constructed by
analyzing RFLP's of spruce chNA probed with Petunia and tobacco chNA

clones.

III. Materials and Methods
DNA preparation

Needles from blue spruce, Engelmann spruce, and white spruce were
collected and washed thoroughly with distilled water. The procedure and
media for chloroplast DNA isolation was the same as described by Stine
et al. (1989), except sucrose gradients, concentrations were changed to
80%, 66%, 52%, 38%, and 10%. Chloroplasts were collected from the 52%-
38% interface. Digestion of chNA samples with restriction endonu-
cleases was carried out according to the directions supplied by the
manufacturer of each enzyme. Chloroplast DNA was subjected to either
single restriction endonuclease digestion with PstI, SacI, or SmaI, or
double restriction endonuclease digestion with PstI and SacI or SacI and
SmaI.
Electrophoresis

DNA fragments were separated by standard agarose gel electropho-
resis techniques as described by Maniatis et al. (1982). The DNA samples
were loaded to give approximately equal concentrations of chNA. Aga-
rose gels were 0.6% (w/v) and ethidium bromide was incorporated into
both the gel and the TBE buffer (0.089 M Tris, 0.089 M boric acid, 2.0
mM EDTA, pH 8.0). High molecular weight markers ranging from 8 to 50 kb

(Sigma) and a 1 kb ladder ranging from 0.1 to 12 kb (Boehringer Mannheim

68

Biochemicals) provided molecular markers for each gel. Electrophoresis
conditions were 1 V/cm for 40 hr for the single digested DNA and 1.5
V/cm for 16 hr for the double digested DNA. Following electrophoresis,
the DNA in the agarose gels was transferred to Zeta-Probe blotting
membranes (Bio-Rad) using the alkaline blotting procedures as described
by the manufacturer.
Southern analysis

Probes were labeled using random primer labeling according to the
manufacturer's (Boehringer Mannheim Biochemicals) directions. A bank of
cloned chNA of petunia and tobacco was obtained from Dr. J. Palmer and
Dr. R. Jansen. Prehybridization and hybridization steps were performed
according to Bio-Rad instructions for Zeta-Probe membranes. Post hybri-
dization washes were as follows: the hybridization filter was washed
once for a 10-20 seconds at room temperature with 1 mM EDTA, 40 mM
NaHPO4, pH 7.2 and 0.5% 808. This was followed by two washes with 1 mM
EDTA, 40 mM NaHPO4, pH 7.2, 1% SDS and 1 mM EDTA, 40 mM NaHPO4, pH 7.2,
0.2 SDS at 65°C for 30 min each. Autoradiography was carried out at
-70°C with DuPont Cronex Lightning plus intensifying screens and Kodak
x-Omat XARS x-ray film. The exposure time ranged from 2 days to 1 week
depending on the radiation intensity on the Zeta-Probe membranes. 0.1%
SSC and 0.05% 808 were used to strip membranes of labeled DNA.
Construction of chNA physical map

Following hybridization of single and double digested chNA with
total tobacco subclones, homologous sequences between tobacco chNA
clones and spruce chNA were compared and analyzed. By arranging the
overlapping spruce chNA restriction fragments with the labeled tobacco

chNA clones, a spruce chNA physical map was constructed.

69

Transverse alternative field electrophoresis (TAPE)

The procedure and media used to prepare cell homogenate from
needles followed the methods of Stine et al. (1989). The homogenate was
centrifuged at 2,000 rpm for 3 minutes in a Sorvall GS-3 rotor. The
supernatant was then centrifuged at 4,250 rpm for 10 minutes in a Sor-
vall GS-3 rotor, and that supernatant was centrifuged at 10,000 rpm for
10 minutes in a Sorvall GS-3 rotor. The pellets from the 2,000 rpm,
4,250 rpm, and 10,000 rpm centrifugations were used to make agarose
plugs as described by the manufacturer (Beckman). 0.8% LE agarose and
running conditions of stage I: 30 minutes, 250 volts, and a field alter-
nation period of 90 seconds; Stage II 10 hours, 250 volts, and a field
alternative period of 45 seconds were applied to separate the DNA. The
gel was soaked in 0.2 N HCl and slowly shaken at 70 rpm for 12 minutes.

Southern blotting was performed as previously described.

Results

Petunia chNA clones and maize mtDNA clone pZmEl (Fox and Leaver,
1981), which contains the CoxII gene, were used to probe the DNA sepa-
rated by the TAFE method. Two DNA bands of approximately 60 kb and 120
kb hybridized to the probes (Figure 13 and 1C). The hybridizing bands
were identical in size regardless of whether the chloroplast or mito-
chondrial probes were used. When DNA separated at the same centrifuga-
tion forces, but subjected to CsCl density gradients, was probed with
the chNA probe, a smeared region from 50 kb to 200 kb was observed
(Figure 10).

The chNA restriction fragment sizes of spruce were determined by

electrophoresis followed by comparison to DNA molecular size markers

70

 

 

Figure 1. Southern blot analysis of DNA which was isolated at different
centrifuge forces and separated by TAFE electrophoresis. 1A. Blue spruce
and white spruce DNA was separated by TAFE. 18. DNA of 1A was hybridized
to mtDNA clone CoxII. 10. After stripping radioactive labeled DNA of 13,
DNA was rehybridized to petunia chNA clone p8. 1D. DNA after CsCl
density gradient was hybridized to chNA clone P3. Bs:blue spruce.
WS:white spruce. 1:2,000 rpm. 2:4,250 rpm. 3:10,000 rpm.

71

Pet I See I Sma I P+Sa Sa+Sm.
t—I l—_l r—I r—fi l——1
BEWMB EWBEWMB EWB EW

 

Figure 2. Southern analysis of single and double digested spruce chNA.
A. Restriction patterns of single digested spruce chNA. B. Restriction
patterns of double digested spruce chNA. C. Hybridization of cloned,

-P labeled Petunia chNA P10 to gel indicated in Fig. 1A. D. Hybridi-
zation of cloned, 2-P labeled Petunia chNA P1 to the gel indicated in

Fig. 13. szlue spruce. E:Engelmann spruce. W: white spruce. M:molecular
size marker.

 

72

(Figure 1 and Table 1). However, repetitive sequences within the spruce
chNA and homologous sequences between spruce chNA and mtDNA (Figure 1)
caused problems in constructing the physical map. Forty-two tobacco
chNA subclones ranging from 1.0 to 6 kb were used to finally create
the chNA physical map by comaparing the hybridization of single and
double digested chNA and homolougous sequences of chNA fragments.

The chNA size of P. pungens, P. engelmannii, and P. glauca is
about 120.4, 120.2, and 120.2 kb respectively (Table 1). The variation
of chNA among the different spruce species could be visualized in SacI-
and SmaI-cut fragments, but not in PstI-cut fragments. No chNA varia-
tion in size was detected between white spruce and Engelmann spruce in
this study. Values for those two species are reported jointly in Table

1. Variation in total chNA between blue spruce and white and Engelmann

Table 1. Spruce chNA restriction fragment size.

 

 

P. pungens P. engelmanni and P. glauca
PstI SacI SmaI PstI SacI SmaI
23.7 21.4 32.3 23.7 21.4 32.3
21.5 17.2 21.6e* 21.5 17.2 21.4e*

18.7 10.6 13.7 18.7 10.6 13.7

16.4a* 10.6 11.6 16.2a* 10.6 11.6

11.4 9.6 11.3 11.4 9.6 11.3

8.1 9.6 9.9 8.1 9.6 9.9

6.5 9.4d* 5.8 6.5 9.2d* 5.8

5.8 9.2 3.1 5.8 9.2 3.1
4.8 9.2 3.1b 4.8 9.2 2.3, 0.8b
2.0 4.4 2.5c 2.0 4.4 2.1, 0.4c

1.5 4.2 1.9 1.5 4.2 1.9

2.7 1.2 2.7 1.2

1.1 1.2 1.1 1.2

0.6 0.8 0.6 0.8

0.6 0.4 0.6 0.4

 

120.4(kb) 120.4(kb) 120.4(kb) 120.2(kb) 120.2(kb) 120.2(kb)

 

Note: same letter indicates the RFLP polymorphism of chNA among
speices. '*' indicates same chNA fragments which showed variation among
species.

PS"

SmaI

73

spruce was 0.2 kb. Following hybridization of single and double digested

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DNA with chNA probes, a physical and genetic map of spruce chNA was
constructed (Figure 3).
BLUE SPRUCE
ll.
'5 0 J 3 q, j Y i V V“,
5'33 1’! 3’ a”? 3 3"?“ :3: 8°? 5; {I
5.: 10.: u 10.: u u 23.: m I as
1.5 20
.01 :4 0: 41 at: +.\ I) 101 ll! 9: 96 2 106
11 0.6 0.6
s
dI\ns :u .J.1u anFu n: u as In
1.8 0.4 1.2 1.2

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3. Physical and genetic map of blue spruce chNA.

Discussion

Identical banding patterns were found for DNA separated by differ-
ent centrifugation forces and TAFE when the DNA was probed with petunia
chNA or maize mitochondrial CoxII clones (Figure 1). Both hybridiza-
tions showed strong signal for DNA in the pellet resulting from centrif-
ugation forces between 4,250 rpm and 10,000 rpm. It has been found that
maize mtDNA has regions that are homologous to the chNA inverted repeat
(Stern and Palmer, 1984). In this study, spruce chNA and maize CoxII
DNA appeared to share some sequences.

The TAFE method is designed to separate large DNA fragments and

whole chromosomes. The 120 kb band from southern analysis of the TAFE

gel may be chNA because the estimation of spruce chNA size is 120.4 kb

74

for blue spruce and 120.2 kb for white spruce separately (Table 1).
However the size of the lower band is estimated at 60 kb band. This band
could be supercoiled chNA or mtDNA, or plasmids which share some
sequence homology with chNA and mtDNA. Circular dimers and concatenat-
ed dimers of chNA have been found in the higher plants (Tewari and
Meeker 1979). However they do not appear to be present in the DNA analy-
sis in this study since no hands were located in the 240 kb region of
the TAFE gel.

The variation of chNA among these spruce species is very low,
(Table 1 and Figure 3) and may be only the result of few deletions or
duplications or insertions. This may suggest that their divergence
is recent event. Morphological similarity between blue spruce and
Engelmann spruce, coupled with a smaller geographic range and a reduced
variability in the reproductive structure of blue spruce has been taken
as an indication that blue spruce is a relatively recent derivation of
Engelmann spruce (Daubenmire, 1972). However, paternal inheritance of
chNA and maternal inheritance of mt DNA in spruce (Stine et al. 1991),
the variation of chNA and mtDNA between blue spruce and Engelmann
spruce (Table 2), and unidirectional interspecific hybridization of blue
spruce and Engelmann spruce (only when Engelmann spruce is used as

female parent) (Ernst et al. 1990, Fechner and Clark 1969), indicate

Table 2. Similarity of chNA and mtDNA among spruces.

 

 

 

DNA Blue spruce Engelmann spruce White spruce
chNA different same same
mtDNA same different same*

 

*data from probing with maize CoxII clone (personal communication with
Andrew David).

75

that blue spruce and Engelmann spruce may not be as closely related to
each other as suggested. Gene sequencing of conserved regions such as
168 and 23S rDNA will help resolve this issue.

It is well documented that the inverted repeat typically found in
angiosperms is absent in conifers (Lidholm et al., 1988; Strauss et al.,
1988). Our data also showed the absence of the large inverted repeat in
spruce chNA, resulting in the small size (120 kb) of the chloroplast
genome when compared to most other land plants. It has been proposed
that the loss of a large repeat sequence increases the frequency of
chNA rearrangements (Palmer and Thompson 1982). A chNA rearrangement
of nearly 50 kb in size between Douglas-fir and radiata pine has been
documented. This variation may have resulted from the recombination of
the repetitive sequences within the chNA (Tsai and Strauss 1989). A

comparison of chNA maps for radiata pine and blue spruce indicates

 

 

 

 

BLUSSPRUCE
o e ° 9
v ‘y 9'
5L! 1 ,2. «3‘ use e .e :23"
N_ L A . % 7 LI

 

 

 

‘ I 3 W 3 4 S X 7 #1
: g* 3 :1— 3 1% 4 #5 i I V“ 7 fi
.26 3.3." a a.” a. awe 3., we. '9.” we.

RADIAI’A PINE

Figure 4. Arrangement of homologous sequences in blue spruce and radiata
pine chloroplast genome. chNA map of radiata pine is from Strauss et
al. 1988. Number above or below maps indicate fragment blocks. Block
points are approximate. Number on the left side indicate the steps of
inversions between radiata pine and blue spruce.

76

that several rearrangements (at least 5 inversions) of the chNA have
occurred (Figure 4).

In the Pinoid group: Picea, Pinus, and Pseudotsuga, a study of
seed protein immunology indicated that Picea is distant from the other
two genera due to a greater time of divergence (Price and Olson-Stojko-
vich, 1987). Douglas-fir may have arisen from a pine-like ancestor 50
million years ago (Herman, 1985) and the first evidence of spruce in the
fossil record dates back 65-135 millions years ago (Florin, 1963). The
estimated divergence of the chNA sequence between Douglas-fir and
radiata pine is 3.8 percent (Strauss et al., 1988). The chNA map of
spruce is dramatically different from the chNA map of Douglas-fir and
radiata pine. The chNA map analysis among Douglas-fir, radiata pine and
spruces supports the seed protein study that HPicea has diverged from

other two genera and evolved for a considerable time.

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