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This is to certify that the

thesis entitled
ISOLATION AND CHARACTERIZATION OF REVERTANTS
OF A PHOTOSYNTHETIC MUTANT OF
NIACOTIANA TABUCUM
presented by

Paul John Koivuniemi

has been accepted towards fulfillment
of the requirements for

Ph. D. degpein Genetics

M

V
Mm

 

Date May 15, 1980

0-7639

   
 

    

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OVERDUE FINES;
25¢ per day per item

RETURNING LIBRARY MATERIALS:

Place in book return to remove
charge from circulation records

 

ISOLATION AND CHARACTERIZATION
OF REVERTANTS OF A PHOTOSYNTHETIC
MUTANT OF NICOTIANA TABACUM

 

By

Paul John Koivuniemi

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Genetics Program

1980

ABSTRACT

ISOLATION AND CHARACTERIZATION OF REVERTANTS OF A PHOTOSYNTHETIC
MUTANT OF NICOTIANA TABACUM

 

BY

Paul John Koivuniemi

Increasing crop productivity is the long-term goal of most basic
plant research. The use of mutations for breeding is a very economic-
ally and ecologically sound method for doing this. This dissertation
introduces a method for isolating revertants of photosynthetic mutants
in plants which can be used for characterization of mutants and also as a
potential source of useful variation for future breeding purposes.
Additionally, a reductive approach is utilized to more accurately
describe the phenotype of the sulfur (§_) photosynthetic mutant of
tobacco.

Several green autotrophic revertants were recovered from haploid
tobacco plants containing the semidominant sulfur gene (S2) which
produces albinism and thus lethality in either the homozygous or haploid
configuration. The system used consisted of visual selection of 31
green somatic sectors on the yellow background of haploid §g_leaves,
their excision, and proliferation of the presumed revertant cells on a
callus inducing medium. Callus tissue was then placed on a regeneration
medium and l2 clones of phenotypically green revertant plants were
recovered. Of the l2 clones produced, 3 were selected for further
characterization based on the number of plants recovered from the clone,

their relative vigor as evidenced by growth rates, and their fertility.

Analyses of the revertants (_l, 3;, 3_) included classical Mendelian
genetics, cytogenetics, and observations of biological parameters such
as pollen fertility, somatic sectoring, and seed fertility. Revertant
31 is a second site genetic revertant which is closely linked to the
original §u_mutation and is probably intragenic. Revertant 3; is most
probably a monosomic for chromosome S which contains the sulfur mutation
and therefore is hemizygous for sulfur (Sg/O). Revertant Bg_is highly
aneuploid having a chromosome nunber of approximately double the normal
diploid number while still containing the sulfur mutation.

The enzymes ribulose-l, S-bisphosphate carboxylase/oxygenase and
polyphenol oxidase from the 3 revertants, the heterozygous mutant §gA§g,
and the wild-type plant sgfisg were examined. Kinetic parameters for
ribulose-PZ carboxylase/oxygenase were identical in all plants
examined except that the Vmax for the carboxylase from 3g was approxi-
mately half that of enzyme from the other plants. Specific activities
for ribulose-PZ carboxylase/oxygenase were also f0und to vary in §g[§u
and sstg over the course of a generation, but this variation correlated
with induction of polyphenol oxidase by either high light intensity,
growth conditions or old age of the plants.

A reductionistic approach was used to more clearly define the §u1§g
heterozygous mutant phenotype, which is enhanced by high light intensity,
and to correlate the observed phenotypic variation with the phenotypes
of the 3 revertants and the homozygous mutant Sg/Sg.

The phenotype of §gfl§g_heterozygous varied from yellow to light-
green when the plants were grown under high and low light intensity
conditions respectively. Bleaching of §EA§E is correlated with a

decreased content of chlorophyll per leaf area, agranal chloroplast

ultrastructure, changes in the number of chlorOphyll-protein complexes,
and absence of one or more of the light harvesting chlorophyll-
polypeptides of 25,000 to 29,000 daltons. The homozygous mutant grown
under low light intensity conditions was completely lacking in grana
stacks and deficient in chlorophyll-protein complexes.

Revertant Bl_was found to be identical to wild-type plants in all
parameters examined (visual phenotype, chloroplast ultrastructure,
chlorophyll-protein complexes, and chlorophyll-protein complex
polypeptides), with the exception of some small differences in pigment
complement under high light conditions.

The other 2 revertants, 32 and 3;, were similar to the heterozygous
mutant SEZEQ in all parameters examined with one exception. They
yellowed due to loss of chlorophyll and an increase in the amount of
carotenoids, had agranal chloroplasts, and had variant chlorophyll-
protein complexes when grown under high light intensities. However,
each appeared to contain some of the light harvesting pigment-protein
complex polypeptide(s) missing in Su/su when it is grown under high

light intensity conditions.

ACKNOWLEDGMENTS

To thank all those who contributed to the completion of this
dissertation is not possible. However, I would like to thank the mem-
bers of my committee Dr. Albert Ellingboe and Dr. Leonard Robbins,
especially for their support during the beginning and formative years of
my graduate career and also my co-mentors, Dr. N.E. Tolbert and Dr.
Peter Carlson for their helpful support, guidance and time during the
last 3 years.

Finally, I would like to acknowledge the encouragement given to me
by my parents and family and particularly by my wife Linda, who turned
what could have been a trying ordeal into a happy and balanced

experience.

TABLE OF CONTENTS

PAGE
LIST OF TABLES . . . . . . . . . . . . . . . . . . . ....... iv
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . vi
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . I

General Phenotypic Characterization . . . . . . . . . . . . . l
Chloroplast Ultrasturcture and Photosynthesis . . . . . . . . 5
Photorespiration and Ribulose-P Carboxylase/Oxygenase. . . . 8
Components of the ChlorOplast Membranes . . . . . . . . . . . 12
Somatic Cell Variegation . . . . . . . . . . . . . . . . . . l3
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . l5

CHAPTER
I. ISOLATION AND CHARACTERIZATION OF SOMATIC REVERTANTS OF
DOMINANT AUREA MUTATION IN TOABCCO . . . . . . . . . . . . l6
AbstraCt O O O O O O O O O O O O O O O O O O O O O O I O O 17
IntrOduction O O I O O O I O O O O O O O O O O O O O O O O 18

Materials and Methods . . . . . . . . . . . . . . . . . . 20

Plants . . . . . . . . . . . . . . . . . . . . . . . . . 20
Culture . . . . . . . . . . . . . . . . . . . . . . . . 20
Isolation of Revertants . . . . . . . . . . . . . . . . 20
Cytogenetics . . . . . . . . . . . . . . . . . . . . . . 22

Resu‘ ts O O O O O O O O O I O O O O O O O O O O O O O O O 23

Characterization of R1

Characterization of R3 . . . . . . . . . . . . . . . . 32
R3 Progeny . . . . . . . . . . . . . . . . . . . . . . . 4O
Characterization of R2 . . . . . . . . . . . . . . . . . 44
Other Clones . . . . . . . . . . . . . . . . . . . . . . 48

DiSCUSSiOn O O O O O O O O O O O O O O O O O O O O O O O O 52

CHAPTER

II.

III.

Literature Cited ............ . . . . . . .

RIBULOSE-l,S-BISPHOSPHATE CARBOXYLASE/OXYGENASE
AND POLYPHENOL OXIDASE IN THE TOBACCO MUTANT Suflgg

AND THREE GREEN REVERTANT PLANTS . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . ..................
Materials and Methods .................

Ribulose-P2 Carboxylase/oxygenase ..........

Results ............. . ..........

Effect of Age and Growth Condition on Total and
Specific Activity of Ribulose- -P2 Carboxylase/
Oxygenase . . . . . . . . . . . . . . . . .

Effect of Age and Growth Conditions on Polyphenol
Oxidase . . . . . . . . . . . . . . . . . . . . . .
Effects of Age and Growth Conditions on the Ratio
of Ribulose- P2 Carboxylase to Ribulose- P

Oxygenase from Su/su and su/su Plants . . . .
Kinetic Parameters of Ribulose- -P2 Carboxylase/
Oxygenase from SJ/Lu, Lu/SJ and three Revertant

Plants Recovered from Haploid SJ . . . . . . . . . .

Physical Paramters of Ribulose- -P2 Carboxylase/
Oxygenase from SJ/SJ, SJ/Lu, and Three Revertants
Of Hap] Oid §_Ll_ . O O O O O O O O O O O O O 0 O 0 0 0

DiSCUSSion O I O O O O O O O O O O O O O O O O O O O 0
Literature CitEd C O O O I O O O I O O O O O O O O O O

CHARACTERIZATION OF THE THYLAKOID MEMBRANES OF THE
TOBACCO AUREA MUTANT SJ/Lu AND OF THREE GREEN
REVERTANT PLANTS O O C O O O O O O O O O O O O O O O O
Abst ract O O O O O O O O O O O O O O O O O O O O O O 0

Introduction . . . . . . . . . . . . . . . . . . . . .

Materia15 and b1eth0ds O O O O O O O O I O O O O O O O 0

Plant Material . . . .
Light Conditions . . . . . .
Pigment Measurement .
Electron Microscopy .
Chlorophyll- -protein Complexes .

ChlorOphyll- -protein Complex Polyp

ii

(D o o o o o

ptides

Page
56

59
6T
63
65
65
7O

7O

77

8O

83

84

88
9O

CHAPTER PAGE

RESUItS O O O O O O O O O O O O O O O O O O O O O O O O O 102

Pigment Complement . . . . . . . . . . . . . . . . . . . l02
Chloroplast Ultrastructure . . . . . . . . . . . . . . . l07
Chlorophyll- ~protein Complexes . . . . . . . . . . . lll
Chlorophyll- protein Complex Polypeptides . . . . . . . . llS
DISCUSSIOn O O O O O O O O O O O O O O O O O O O O 0 O O O 119

References 0 O O O O O O O O O O O O O O O O O O O O O O O 122

SUr‘1MARY O O O O O O O O O O O O O O O O O O O O O O O O O 125
BIBLIOGRAPHY O O O O O O O O O O O O O O O O O O O O O O O 127

TABLE

LIST OF TABLES

INTRODUCTION

Summary and comparison of the phenotypic
characteristics of the semidominant aurea tobacco
mutant Sgfigg and its wild-type sibling 5g7§g . . . .
CHAPTER I

Description of plants used . . . . . . . . . . . . .
Seedling segregation ratios for revertant Bl . . . .
Biological characteristics of experimental plants .
Genetic model for revertant Bl_. . . . . . . . . . .

Seedling segregation numbers for revertant R§_ . . .

Seedling segregation numbers for the variegated
plant Y; I O O O O O O O O O O O O 0 O O O O O O O O

Seedling segregation numbers for revertant fig, . . .

CHAPTER II

Properties of crystalline ribulose-l,S-bisphosphate
carboxylase/oxygenase from various tobacco mutants .

Protein ratio of large to small subunits of
ribulose-P2 carboxylase/oxygenase . . . . . . . . .
CHAPTER III

Description of plants, growth conditions and summary
of the phenotypic analysis of the sulfur mutant, its

wild-type sibling and three green revertants grown
under high and low light conditions . . . . . . . .

iv

PAGE

2l
25
30
33
35

43
47

85

87

lO3

TABLE PAGE

2. Pigment content of leaves from wild-type fl/fl,
heterozygous mutant iu/fl and 3 green revertant
plants (BL, 32, 33) when grown under high and
low light intensity conditions . . . . . . . . . . . . . . .l04

LIST OF FIGURES

FIGURE PAGE
CHAPTER I
l. Microsporocyte from the diploid revertant 3L . . . . . . . . 29

2. Microsporocyte from revertant R3 showing 23
pairs of chromosomes . . . . . . . . . . . . . . . . . . . . 29

3. Leaf from the variegated plant 11, an F1 progeny
Of the cross B§_X éflléfl' O O O O O O O I O O O O O O O O O O 42

4. Leaf from the variegated plant 12, an F1 progeny
Of the cross‘B§_x éflléfl. O O O O I O O O O O O O O O O O O O 46

5. Root tip chromosomes from the highly aneuploid
revertant 32 containing 93 chromosomes . . . . . . . . . . . 50

CHAPTER 11

l. Changes in ribulose-Pz Carboxylase activity wnth
change in age and growth conditions for tobacco
p1ants O O O O O O O O O O O O O O O O O O O O O O O O 0 O O 72

2. Changes in ribulose-Pz oxygenase activity as a
function of plant age and growth conditions. . . . . . . . . 74

3. Induction of polyphenol oxidase activity in tobacco
plants grown in the growth chamber . . . . . . . . . . . . . 79

4. The ratio of ribulose-P2 carboxylase to ribulose-PZ
oxygenase activity as a function of plant age and
growth conditions . . . . . . . . . . . . . . . . . . . . . 82

CHAPTER III

1. Chloroplast from revertant_RL grown autotrophically
under greenhouse conditions . . . . . . . . . . . . . . . . l09

2. Chloroplast from revertant 32_grown autotrophically
under greenhouse conditions . . . . . . . . . . . . . . . . l09
vi

FIGURE
3.

4.

Chloroplast from revertant 3; grown autotrOphically
under greenhouse conditions . . . . . . . . . . . . . . . .

Chloroplast from the homozygous sulfur mutant SgfiSg
grown hetertrOphically on a sucrose supplemented

mEdi Um O O O O O O O O O O O O O O O O O O O O O O O O O O O

ChlorOphyll-protein complexes isolated from (a)

wild-type (§g[§g), (b) heterozygous mutant (Sufigg),

and (c) homozygous mutant (§2/§E) plants grown
heterotrOphically on a sucrose supplemented medium .....

Chlorophyll-protein complexes isolated from

(a) revertant 33, (b) revertant 32, (c) hetero-

zygous mutant Sg/gg, and (d) wild-type (sgysg)

plants grown in the greenhouse . . . . . . . . . . . . . . .

ChlorOphyll-protein complexes isolated from

wild-type plants grown under high (b) and ow

(d) light intensity conditions - 330 uE m'

sec' and 32 uE m’ sec‘ respectively - and

heterozygous mutant plants grown under the same

conditions (c and a respectively). . . . . . . . . . . . . .

Polypeptides solubilized from the chlorophyll-

protein complexes as described in Materials and
MethOdS O O O O I O O O O O O O O C O O O O 0 O O 0 O O O 0

vii

PAGE

l09

l09

ll3

ll3

ll7

ll7

INTRODUCTION

The sulfur mutation Ii!) is a semidominant aurea mutation in

tobacco (Nicotiana tabacum L.) and was the first dominant aurea (yellow

 

leaf phenotype) mutant found in plants. The sulfur mutant was discover-
ed by Burk and Menser in l964 (l), and its name was derived from the
color of its yellow leaves. It occurred spontaneously in heterozygous
condition (Sg7§_) in an aged seed lot. Burk and Menser demonstrated
through selfings and reciprocal back crosses that the mutation occurred
at a single nuclear locus. In addition to these crosses done to esta-
blish the mode of inheritance, Sg/sg plants were crossed with several
monosomics of N. tabacum (3) to map the S3 mutation to a particular
chromosome. Evidence from these crosses indicated that §g_was located
on chromosome 5. A summary of the phenotypic comparisons of the hetero-

zygous mutant §EA§E and its wild-type sibling sgfigg is presented in

Table l.

General Phenotypic Characterization

 

Seeds from the self-fertilized heterozygous mutant Sngg segregated
plants of 3 types in a ratio of l dark-green wild-type plant (22(52):?
yellow-green heterozygous mutant plants (Sg/sg):l yellow to albino homo-
zygous mutant plant (Sg/Sg) (l). The latter are lethal when grown
autotrophically.

Burk and Menser (l) noted that green and yellow sectors were
present on the yellow-green'§g[§g leaves in several cases. These sec-

tors included twin Spots in which the two homozygous phenotypes (green

and yellow) appear in the heterozygous yellow4green background of the

TABLE I

Summary and comparison of the phenotypic characteristics of the
semidominant aurea tobacco mutant Sgfigg and its wild-type sibling gufigg

 

Parameter Su/su* Reference
ChlorOplast Size 2/3 X l
Chloroplast Ultrastructure
-High Light no grana 3
-Low Light rudimentary grana 3
Total Chlorophyll
~Leaf Area 1/8 X 3,4,5,6,9
Total Carotenoids
-Leaf Area l/3 X 3,4,5,6,9
Chlorophyll a/b l.3 X 3
l.8 X 9
Growth Rate
-Leaf Area
High Light 3/4 X 7
Low Light l/3 X 7
-Dry Weight
High Light X 7
Low Light 2/3 X 7
Alkaloids
-Fresh Weight 0.7 X 2
Total Sugar
-Fresh Weight l/2 X 2
Photosynthetic Unit Size l/2 X ll,l3
Plastocyanin
-mg Chlorophyll 2.5 X 27
Cytochrome f
-mg Chlorophyll 2.5 X 27
Light Harvesting
Pigment-Protein Complex l/4 X 23,28
Photosynthesis
-mg Chlorophyll 3-5 X 4,7
-Leaf Area X 7

Hill Reaction 2X 9

Table l (cont'd)

 

Parameter Su/su* References
Photorespiration
-Leaf Area 2X 20
RuPZ Carboxylase/Oxgyenase
~Km( CO l.l X l9,20
-Km( (5)3 0.7 x 19,20
Dark Respiration
-Leaf Area X 14
0.1 X 6
-mg Chlorophyll 2-3 X 14
X 6

 

*The values are given relative to wild-type'_g[§_ where X denotes the
wild-type value.

leaf. It was noted that chloroplasts from §g7§g plants were only 2/3 as
large as those found in the wild—type leaves and that they were abnonnal
in structural detail, although the abnormalities were not Specified

(l).

Menser e; 31., (12) continued the phenotypic description of the
heterozygous mutant Sngg and wild-type.§g[§g plants. I§g7§g had a lower
carotenoid and chlorophyll content than did £3752 when the plants were
grown in the greenhouse in winter. '§g[§_ had only 25% as much total
chlorOphyll and 55% as much carotenoid as the wild-type on a leaf area
basis in young plants. In older plants, gufigg had 2.2 times more total
chlorophyll per leaf area than did the mutant Sg/gg. The chlorophyll
a/b ratio was identical in plants of all ages. Varying chlorophyll and
carotenoid complements have been found in these plants by other investi-
gators. Schmid and coworkers (29) reported that.§fl(§! had only 12% of
the total chlorOphyll and approximately 33% of the total carotenoid per
unit leaf area as did §_[§g. The chlorOphyll a/b ratio was 2.9 and 2.2
for égZEQ and §27§g respectively: the total chlor0phyll/total carote-
noid ratio was 1.9 f0r.§fl(§! and 4 for ggfigg. This is in contrast to
values of l and 2.5 found for Sngg and ggfigg respectively by Menser et
a1. (12). Homann and Schmid (8) reported chlorophyll a/b ratios of 2.9
15. 5.1 and chlorophyll/carotenoid ratios of 3.8 gs 2.7 for ggfigg and
.§g[§g respectively. The wildtype 5275! had 5 times as much total
chlorOphyll as did the mutant Sgflsg (8). These plants were grown in
Florida under high summer light intensities.

§gf§g has been reported to grow much more slowly than does §g[§g.
Su/su has a 20-fold lower growth rate than wild-type controls as mea-

sured in either fresh weight or dry weight accumulation under low light

intensity conditions in the greenhouse in winter (12). High light
intensities have been reported to overcome this disadvantage. .Under
high light conditions,_§u[§g grew at nearly the same rate as did the

wild-type su/s (21).

Sg/gu has been shown to differ from sgfigg in its alkaloid content,
free amino acid content and in its total sugar content (12). The wild-
type has approximately 1.6 times more total alkaloids than does Sgfisg.
This is reflected primarily in a 50% higher content of nicotine in
.§_A§!- The increase in free amino acids was attributable largely to an
increase of 2 to 3 times in aspartic acid, alanine, asparagine, gluta-
mine, serine, and threonine. The sugar content in heterozygous plants
was not only quantitatively, but also qualitatively different than in
the wild-type plants. Sg/sg contained a large amount of sucrose while
.§g[§g contained only glucose and fructose and no sucrose (12).

The number of stomata and the degree of stomatal opening has been
reported to be the same in both plant types by some investigators (12),
while others have observed that stomata in §EK§E plants open more
rapidly than in wild-type plants (36). .§g[§_ is reported to withstand
high temperatures and high illumination better than does_§g[§g (21).

Finally, the Sgfisg mutant has a more pronounced short-day response than

does the wild-type (21).

Chloroplast Ultrastructure and Photosynthesis

Examination of the chloroplasts of the sulfur mutation has been
done principally by Schmid, Gaffron and coworkers (7-8, 13-15, 18-31).
The first electron microscope study of the chloroplast ultrastructure of

the Sgfisg mutant demonstrated that Sg/gg plants grown under the high

light intensity of the Florida summer had essentially agranal chloro-
plasts, containing at most 3 appressed membranes, whereas the wild-type
.§u[§g plants were routinely seen to have 20 or more lamellae stacked
into grana (8,23,24,28,29). It was also incidentally observed that
there were many mitochondria in close proximitiy to chloroplasts in
.§g[§g plants grown in high light.

As mentioned above, Sg/sg grew more slowly under low light inten-
sity conditions than did ggfigg (12). .Sgfigg also had a lower photosyn-
thetic rate than did sy/sg at low light intensities (less than 3000 ergs
sec'1 cm'z) (24). However, at high light intensities (greater
than 5000 ergs sec"1 cm'z) §g7§g showed no saturation of photo-
synthesis while the wild-type ggfigg was saturated at approximately 3000
ergs sec"1 cm'z. These experiments were done wfith red light -
greater than 600 nm - to obviate problems with the possibility of
increased efficiency of Sngg plants due to the relatively higher
carotenoid content (24).

Saturation rates for photosynthesis were shown to be 3 times as
great in the heterozygous mutant as in the wild-type (25) on a leaf area
basis. Saturation for the green control was fbund at 10,000 ergs
sec"1 cm'2 and at approximately 50,000 ergs sec'1 cm"2
fori§gA§g.

In a more detailed analysis, S3753 plants were grown in sterile
flasks on an artificial medium under high (12,750 ergs sec"1
cm‘z) and low (1400 ergs sec"1 cm'z) light intensities. In
subsequent measurements of photosynthetic rates of these plants as
determined by 02 gas exchange, it was shown that Sngg plants,

previously grown in high light rapidly evolved oxygen and were never

saturated at high light intensity. 0n the other hand, Sg/ég plants,
previously grown in low light, showed a net consumption of 02 until
light intensities reached 12,000 ergs sec"1 cm'z. The wild-type

.§g[§g plants were intermediate in response and generally were saturated
at 12,000 ergs sec'1 cm‘2 evolving approximately 33% the amount

of oxygen as the.§EA§E plants grown in high light (29).

In older mutant plants the chlorOphyll content increases. However,
photosynthesis still remains 3 times as high in Sgfiég as compared to
§_[§_ on a per mg chlorOphyll basis (21).

Photosynthesis in §gX§g (measured as 02 Evolution) has been shown
to be independent of temperature from 15 to 35°C (24,29). Dark C02
evolution (respiration) was much lower in §EA§E plants than it was in
'ggfigg when these plants were grown in high light. The wild-type evolved
approximately 10 times as much C02 as did Sgfisg on a leaf area basis.
If measured on a per mg chlorophyll basis, the dark evolution of C02
was shown to be identical for both S3733 and §g7§g (24).

Chloroplasts isolated from Sg/sg showed greater instability than
chloroplasts isolated from gngg as measured in terms of loss of the
Hill reaction over time (8). However, chloroplasts from Sg/gg plants
showed a 2-fold higher Hill reaction immediately after isolation than
did ggfisg chloroplasts, which was interpreted as indicating a more
efficient PS 11 (8).

As mentioned previously, Sg/sg has been shown to grow as well as
.§_L§g under high light intensity conditions (21). An increase in leaf

area per plant (12-fold increase) and also in dry weight per leaf area

has been correlated with growth of Sgfigg at high light intensities as

compared with low light conditions. However, a concomitant decrease in
the amount of chlorOphyll per leaf area has also been observed (21).
The wild-type.§g[§g grows 3 times faster in low light intensity condi-
tions than Sngg even though in high light conditions it grows only
slightly faster (21).

Incidental observations which relate to photosynthesis have also
been made on Sgfigg plants. The photosynthetic unit size is smaller in
the heterozygous mutant Sgfigg than it is in the wild-type_§g[§g (26,27).
Fluorescence emission spectra done on extracts from Sngg plants have
shown a peak at 635 nm which was not present in wild-type égflgg plants
(7). Homann (7) interprets this as being protochlorophyll. Young Sngg
plants have 2 to 3 times the wild-type amount of primary electron
acceptor for PS II. Older Sngg plants have an amount equal that found
in wild-type plants of any age (7). However, this is given in terms of
molecules of chlorophyll per electron acceptor and therefore may only
reflect the fact that young Sg/gg plants have less chlorOphyll than do

older Sg/sg plants or wild-type plants of all ages (21).

Photorespiration and Ribulose-P2_Carboxylase/Oxygenase
Photorespiration is defined as the uptake of oxygen and evolution
of C02 by plants in the light (for review see Refs. 5 and 33). This
is an apparently unnecessary and wasteful process in plants.
Photorespiration as measured by the ratio of C02 released in the
light to C02 evolved in the dark was found to be 1.64 1 0.69 for gy/gg
based on 5 trials and 4.73 i 1.62 for Sgfigg based on 3 trials (36).

These values were cited as indicative of more photorespiration in Su/su

mutants. Specific light intensities used were not reported, with only a
range of 1000 to 2000 ft candles being given. Another tobacco variety,
Havana Seed, had a light/dark ratio of C02 release of 3.05 based on 2
experiments, indicating that the exceptional plant might not be the
mutant Sg/su but its wild-type sibling sg/gg. The heterozygous mutant
has been cited as the paradigm for a plant with a genetically altered
rate of photoreSpiration (34,35), when indeed this may not be the case.
As mentioned above, only a small number of actual measurements were done
with the light/dark C02 release assay, and the original data show

large variations between the individual experiments, yet no analysis of
variance was presented.

Recently, doubt has been cast upon the validity of the light/dark
ratio of C02 release as a true measure of photorespiration and it was
suggested that determinations done in this manner should be correlated
with other measures of photorespiration (4). Most importantly however,
in the study cited above (36), the light intensities used in growth of
plants and in doing the photorespiration assays were not presented.
Schmid has demonstrated (21,22,24), and I have confinned that light
intensity has a profound affect on the phenotype of the heterozygous
mutant and therefore this environmental parameter must be rigorously
controlled and specified. At any rate, Zelitch and Day's original
report (36) has provided the impetus for extensive research on this
mutant and its photorespiratory pathway.

Some of the reported findings of Zelitch and Day (36) are contrary
to those of previous workers. Zelitch and Day feund that the Sg/sg
plants had rates of photosynthesis 25% lower than the wild-type plants

even at increased concentrations of C02 (500 ppm). However,

10
this was done at 2000 ft-candles of light intensity which may not have
been high enough to demonstrate Sgfigg's true ability to photosynthesize
(23). They also reported that Sngg had a more rapid rate of stomatal
opening than did the wild-type plants, which is contrary to earlier
reports (12), and that it grew more slowly than_§g[§g under the high
light intensity conditions in the growth chamber (no specific data were
given).

Salin and Homann (19) included the heterozygous mutant Sngg and
wild- type ggfigg plants in a study examining changes in photorespiration
with leaf age. Their photorespiration assay was based on the inhibition
of photosynthesis by 21% 02 as compared to 1% 02. They found young
‘Sgfigg and §g7§g leaves had identical rates of photosynthesis on a per mg
chlorOphyll basis and that these rates were unaffected in both genotypes
by changes in 02 concentration, thus indicating that photoreSpiration
was not measurable in either young Sgfisg or young_§gL§g plants. The
rate of photosynthesis in ngég was fOund to be approximately 3 times
that in wild-type plants at both oxygen concentrations with the relative
rates of photosynthesis found to be 1.6 for Sngg and 1.4 for ggfigg in
1% ys 21% 02 respectively in older leaves. This number was 1 in young
plants of both genotypes. Therefore, an increase in photorespiration
with age and a possible difference between the two genotypes, with the
mutant Sngg having a slightly higher rate, were indicated. Plants were
greenhouse grown in Florida and assay light intensities were 5 x 104
ergs sec“1 cm'z.

Phosphoenolpyruvate carboxylase was found to be approximately 2

times higher on a per mg chlorOphyll basis in S375! than in sgfisg in

11

both young and old plants (19). Dark respiration was identical on a
leaf area basis for both genotypes but 2 to 4 times higher in Sngg on a
per mg chlorophyll basis than for §g7§g in both young and old plants
(19). This is somewhat contradictory to results presented earlier
(24).

The photorespiratory pathway originates with the splitting of
ribulose-PZ by 02 and the subsequent production of phosphoglycolate.
The reaction is catalyzed by the enzyme ribulose-Pz carboxylase/
oxygenase. Therefore, several investigators have attempted to demon-
strate an altered ribulose-P2 carboxylase/oxygenase enzyme in Sg/gg
heterozygotes to account for the reported higher rates of photorespira-
tion. Kung and Marsho (11) reported that the carboxylase and oxygenase
reactions were 40% and 60% lower respectively for enzyme crystallized
from the heterozygous mutant Sg/gu than for wild—type crystalline
enzyme. Isoelectric focusing showed no differences in the small or
large subunits of the enzyme. The small subunit of the enzyme is
nuclear encoded and the large subunit gene is located in the chloroplast
(10).

Schmid's group has also examined ribulose-Pz carboxylase/
oxygenase from the Sngg mutant (13,14). Although they did not use
crystalline enzyme, they found that the Km(C02) was approximately 10%
lower for enzyme from wnld-type plants as was the Ki(C02) for the
oxygenase reaction. The Km(02) was roughly 30% lower for enzyme from
the heterozygous mutant as compared to enzyme from the wild-type plants.
These results could be predicted based on the measured differences in
photorespiration (36). Photorespiration as measured with the light/dark

14C02 - evolution technique was again shown to be 2 times higher

12

irISE/sg than in the wild-type (l4).

Glycolate oxidase, another enzyme involved in photorespiration, has
also been studied in the heterozygous mutant (20). Glycolate oxidase
was shown to be less sensitive to blue light inhibition in §g[§g than in
EEAEE- The plants used were probably grown under low light intensity
since they were grown in the greenhouse in Germany during the winter.
The plants were described as growing more slowly than in Florida and the
chlorOplasts were reported to have higher grana stacks. On a per mg
protein basis, crude extracts from Sgޤg had 2 times as much activity

for glycolate oxidase as did the wild-type gg/sg. Therefore, the SQ

mutation may have a pleitropic effect on glycolate oxidase.

Components of the ChlorOplast Membranes

 

Since the chloroplasts of the heterozygous mutant Sg/gg had been
shown to have an altered fine structure (8,21,22,23,24,25,29) much
research has been directed toward determining whether or not components
of the chlorOplast thylakoid membranes are altered in the mutant.

§_/_g_has been shown to have approximately 2.5 times as much
plastocyanin and cytochrome f as does the wild-type on a per mg chloro-
phyll basis (16). A comparable figure is not given in terms of these
components per leaf area. The wild-type had a larger amount of total
copper per mg chlorophyll than did the heterozygous mutant (l6).

Remy and coworkers (17,32) have begun an analysis of the pigment-

protein complexes found in the thylakoid membranes. They have

demonstrated that the Sngg mutant contains only small amounts (4 times

13

less than wild-type) of the light harvesting pigment-protein complex
which is thought to be associated with photosystem II. A deficiency of
one of the polypeptides associated wnth this complex was also reported
(32). Plants were grown in the greenhouse with supplemental light, but

precise intensities were not given.

Somatic Cell Variegation

 

The mechanisms underlying somatic variegation in plants have long
been of interest to plant geneticists. There are many types of variega-
tion and some have been shown to be genetic in origin while others are
due to physiological or morphological causes (for a review see Ref. 9).
One occasionally observes the spontaneous appearance of twin spots on
the yellow-green leaves of Sgޤg heterozygotes. They consist of an area
of dark green tissue adjacent to an area of yellow tissue of a similar
pattern (1). Mitotic crossing-over was proposed as a mechanism to
account for these twin spots. Carlson (2) used the Sg/ég mutation to
demonstrate that mitotic crossing-over occurs in the somatic tissue of
these plants. Twelve plants were regenerated from the green portion of
the twin spots and were shown to be identical to wild-type plants, both
cytogenetically and through classical breeding experiments (2). This is
the result predicted by the mitotic crossing-over model.

Sg/sg has also been used to characterize the frequency of occurance
of somatic sectors (6) and the affect of various physical and chemical

agents on these frequencies (2). The distribution of spots (both twin

l4

and single) has been shown to be random on Su/ u leaves (6). Several
agents, including gamma rays, UV light, psoralen, mitomycin C and
caffeine, have been shown to cause an increase in the frequency of twin

spots observed on leaves with mitomycin C being the most effective.

15

Discussion

 

My thesis work has touched on aspects presented in all of the
preceeding work. An initial interest in possible selection systems for
photorespiratory mutants - and in particular, those having an altered
ribulose-PZ Carboxylase/oxygenase enzyme such that the oxygenase no
longer functions, or functions at a low level - lead to the use of
haploid §g_plants as a starting material from which to isolate somatic
revertants of the §g_mutation. After these had been isolated, work
proceeded in 3 major areas, the first being the characterization of
ribulose-PZ carboxylase/oxygenase from the heterozygous mutant Sngg
and recovered revertant plants. This is treated as a separate chapter
since it is a most controversial and important aspect of the sulfur
phenotype. Secondly, but closely related to the first area, I attempted
to determine the primary genetic lesion caused by the §g_mutation. This
analysis was based on much of the early work described above; however, I
attempted to pay particular attention to the affects of the environment
on the heterozygous mutant Sgfigg and to use a reductionistic approach to
better distinguish the high and low light intensity phenotypes.
Finally, while these biochemical analyses were being done, I proceeded
with a genetical analysis of the nature and origin of the revertant
plants which were isolated, mainly through classical Mendelian

techniques.

CHAPTER I

Isolation and Characterization of Somatic Revertants of a Dominant Aurea

Mutation in Tobacco.

Paul J. Koivuniemi, Peter S. Carlson, and N.E. Tolbert

Program in Genetics, Department of Crop and Soil Sciences and

Department of Biochemistry

Michigan State University, East Lansing, Michigan 48824

16

17

Abstract

Tobacco plants which are genetic revertants of the semidominant
aurea sulfur (§_) mutation have been isolated with 13 gigu techniques,
and regenerated from somatic leaf tissue of haploid §E plants. The in
§i§g rescue technique requires visual selection of green sectors on the
yellow background of a leaf. Tissue from green cellular sectors was
transferred to a callus inducing medium and subsequently to a regenera-
tion medium. Twelve of the 31 initial green explants produced clones of
green plants. Plants from three of these clones were genetically char-
acterized. One clone (3;) contained a second site suppressor mutation
which mapped very near to the original §2 mutation. Two others were
found to result from aneuploidy; R;_was monosomic for chromosome S,
which contained the original Sp mutation, while 32 had a highly aneu-

ploid chromosome number. This study is the first extensive use of

revertants in the genetic and biochemical analysis of a plant mutant.

18

INTRODUCTION

Genetic variegation in somatic leaf tissue is found in many species
of plants and has been extensively studied to determine its underlying
causes (for a review see Kirk and Tilney-Bassett 1978). Elucidation of
the genetic mechanisms underlying variegation has relied on the produc-
tion of gametes from variegating cells. However, genetic analysis is
not always possible since variegated plants are often deficient in
chlorophyll and do not reach maturity. In addition, variegated tissues
often occur at a low rate in a differentiated vegetative structure (e.g.
a leaf) so there is little possibility that cells in the variegated
region will be included in the germline.

Some investigators have begun to use the techniques of plant tissue
culture to characterize these otherwise inaccessible variant somatic
cells (i.e. those which are not ordinarily fOund in germinal tissue).
These techniques involve recovery, regeneration and genetic characteri-
zation of plants derived from leaf tissues which display a variegating
phenotype (Carlson 1974; Dulieu 1974, 1975). We have utilized these
procedures to determine what genetic events (if any) underlie the
appearance in haploid plants of spontaneous green revertant spots in a
yellow (SQ) mutant of tobacco (Burk and Menser 1964).

Regeneration of plants from phenotypically distinct somatic sectors
is useful not only for genetic characterization of somatic variegation
but also for the isolation of variants which modify the expression of a
specific mutation. Genetic revertants can be used in biochemical and
physiological studies to more fully analyze the primary genetic lesion

in an attempt to make the difficult connection between a gene and its

19

phenotype. A problem encountered wnth in vitro selection utilizing
plant cell cultures is the lack of expression of whole plant character-
istics. This problem can be circumvented by visually selecting cells_1g
.Eigg with subsequent use of the techniques for plant tissue culture to
derive entire plants from these genetically variant cells. Hence,
selection of altered traits can be accomplished where they are being
normally expressed (e.g. in the leaf). Radin and Carlson (1978) have
successfully used this technique for selecting herbicide-tolerant cells
in plant leaves.

The semidominant sulfur mutation is reported to produce altered
photosynthetic pr0perties in the heterozygous (Sgigg) condition when
compared to its wild-type sibling §g£§g (Zelitch and Day 1968). These
altered properties are attributed to a modified ribulose-1,5-
bisphosphate carboxylase/oxygenase in the mutant (Kung and Marsho 1976;

Okabe and Schmid 1978). We have used an i situ selection technique to

 

isolate revertants of the sulfur mutation, in order to investigate these
claims (Koivuniemi, Tolbert and Carlson 1980 a,b). Our 13 sigg tech-
nique depended upon heterotrOphic growth of haploid yellow plants (§2)~
The yellow color of the §g_haploid is a clear visual phenotype associ-
ated with the mutant. Therefore, by simply recovering green spots
spontaneously produced in the pale yellow leaves it was possible to
isolate apparent revertants. The jg sjgg selective system used to
isolate revertant plants is described, and an analysis of the genetic

basis of the observed revertant phenotypes is presented in this paper.

20

Materials and Methods

Plants: A summary description of the plants used is presented in Table
1. These included the heterozygote of the semidominant aurea mutant of

Nicotiana Tabaccum L., sulfur, Su/su. Upon selfing, the heterozygote

 

segregates 1:2:1, greenzyellow-greenzyellow plants (yellow plants die as
seedlings), demonstrating a single gene two allele inheritance pattern
(Burk and Menser 1964). For details of plant growth conditions see
Koivuniemi, Tolbert and Carlson (1980a). Haploid §E plants were
obtained by anther culture of the heterozygous mutant Sglsg. Both
haploid mutant I§EI and wild-type plants (gg) are recovered (Burk
1970).

Culture: Linsnaier and Skoog (1965) minimal salts were used throughout.
Haploid yellow §g_plants were grown on a medium containing 1.0 mg/l
thiamine-HCl, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, and 1 mg/l
indoleacetic acid (IAA) in petri dishes (80 x 100 mm) to a maximum
height of 7 cm and an approximate diameter of 7 cm with 8 to 24 leaves
ranging in size from 3 to 7 cm2. Seedlings used in determining segre-
gation ratios were germinated aseptically on unsupplemented Linsmaier
and Skoog minimal salts medium.

Isolation of revertants: Green spots appeared spontaneously on the

 

yellow leaf background of the haploid §g_plants with a frequency of 0.4
to 1.5 spots per plant. Spot size ranged from 2 to 3 mm in diameter.
Spots were excised and placed on a callus inducing medium containing 1.0
mg/l thiamine-HCl, 3 mg/l IAA and 0.3 mg/l kinetin for 2.5 to 4.0
months. Callus was transferred to a regeneration medium containing 1.0

mg/l thiamine-HCl, 0.3 mg/I IAA, and 10 mg/1 6-(x,X-dimethylallylamino)-

21

TABLE 1

Description of Plants Used

 

 

Genotype Origin Phenotype*
(Growth Chamber) (Greenhouse)

su/su wild-type green green

Su/su semidominant mutant yellow-green light-green
Su/Su mutant (lethal) yellow yellow

Su mutant (lethal) yellow yellow

R1 revertant green green

R2 revertant green/yellow-green green

R3 revertant yellow-green light-green
V1 revertant F1 Variegated -

V2 revertant F1 Variegated -

 

* There is a large environmental influence on the phenotype of these
plants. Plants in the growth chamber at temperatures of 25°C day and
20°C night, received a relatively high and constant light intensity of
330 u E x m"2 x sec'1 for 16 hr per day. The light intensity

was generally much lower in the greenhouse in Michigan during the winter.
In general, mutant and revertant plants bleach under the high light
intensities of the growth chamber or full sunlight and their chloroplast
ultrastructure degenerates (i.e. under high light intensities the grana
lamellae become unstacked). There is also a development effect on the

phenotype: younger plants are not as green as are older plants.

22

purine. Visual slection was made for green regenerating tissue. Shoots
containing leaves were placed on a medium without hormones for root
induction and were subsequently transferred to soil.

Cytogenetics: Chromosome counts were done on pollen mother cells by

 

squashing freshly picked anthers in a propiocarmine staining solution.
Root tips were soaked for 50 min in a 50% saturated solution of ortho-
dichlorobenzene, perfused by evacuation in 3:1 ethanolzacetic acid

fixative and then stained by squashing in pr0piocarmine.

23

RESULTS

Of 31 distinct green sectors excised from yellow haploid S3 leaf
tissue, 12 individual isolates yielded entire green plants. In a
control experiment, 1 green callus clone was recovered from 8 isolates
of yellow haploid §g_1eaf tissue. Of the approximately 50 plants
regenerated from this clone, all bleached out to the yellow phenotype
characteristic of haploid §g_plants. Hence, this variation can be
attributed to effects of cell culture.

Much variation is found in plant cell tissue culture, both in terms
of genetic stability (e.g. variation in chromosome number) as well as in
the visual phenotype of the cells in culture. For instance, the high
cytokinin used in regeneration media sometimes causes greening of
genetically albino cells. However, regenerated plants show the typical
albino phenotype (Parke and Carlson, 1979).

There was much variation in gross characteristics such as leaf and
flower morphology, chlorophyll content, and fertility amongst the clones
of plants regenerated. The 3 most vigorous isolates (3;,‘32, 53) were
selected for biochemical (Koivuniemi, Tolbert and Carlson 1980 a,b) and
genetic characterization.

All three clones were somewhat fertilie indicating that they were
no longer haploid. We have no way of knowing whether the increase in
chromosome number occurred in the leaves, or during in 31559 culture of
the leaf cells. However, the spots were originally selected as green
revertants on a haploid leaf, and changes in chromosome dosage

relationships caused by increase in numbers of homeologous chromosomes

24

is a frequent basis for these events (Dulieu 1974). Therefore, the
former possibility seems to be the most likely.

Characterization 9f.RL: .EL has been most completely analyzed.

 

When grown under both greenhouse and growth chamber conditions the 3;
clone of plants was virtually indistinguishable from wild-type plants.
In an effort to determine whether 3; resulted from a genetic or epigene-
tic event an initial selfing was done. In this selfing, fl proved to be
fertile and only green seedlings were found in the offspring of the
cross (Table 2, cross 1). The production of a diploid plant was not
sufficient to explain this phenotype since diploid EEAEE plants are
yellow and die as seedlings. Therefore, to further characterize the
genetic origin of El, backcrosses were done (Table 2, crosses 2 to 5).
In reciprocal backcrosses to wild-type.§u[§g plants, all progeny
produced were green (Table 2, crosses 2 to 3). However, when Rl_was
reciprocally backcrossed to the heterozygous mutant Sglsg, an entirely
new phenotype was produced which we named yellow-green/yellow to indi-
cate that it was intermediate between the heterozygous mutant Sgigg
(yellow-green) and the homozygous mutant Sngg (yellow) (Table 2,
crosses 4 to 5). In a cross between the wild-type_sg£§g and the hetero-
zygous mutant Sgigg a 1:1, green:yellow-green segregation ratio is
nonnally produced. The yellow-green/yellow plants were like the homozy-
gous mutant Sngg in that they too did not survive past the seedling
stage. However, when grown aseptically these plants visually showed a
faster growth rate and had a higher final chlorophyll content than did
Sgigg plants.

With this information it was possible to predict that green F1

plants (i.e. glfigg) resulting from crosses between R; and either §3y§g.

25

 

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26

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27

or §gfigg should show segregation patterns different from green plants
derived from selfings of 31 or green wild-type plants (§_/§_). There-
fore, several further crosses were done. To establish that green plants
derived from Rl_selfings were genetically identical to the R1 parents
they were self-fertilized once again and also backcrossed to wild-type
.EEAEE and heterozygous mutant plants (Sgfigg). All these control crosses
had segregation patterns identical to the equivalent crosses done whth
the original 3; revertants (compare Table 2, crosses 1 to 5 and 6 to 8).

Selfing of green {1 hybrids resulting from the cross Rl_x §g7§g
(i.e. RL[§_) yielded all green plants (Table 2, cross 9). Crosses
between green F1 backcross progeny and wild-type.§g[§g also yielded all
green plants (Table 2, crosses 10,13,15,17). However, when these green
F1 progeny were crossed to Sglsg a new segregation ratio of 2:1:1,
green:yellow-green:yellow-green/yellow, was produced (Table 3, crosses
11,12,14,16). No maternal inheritance was observed in any of the
crosses.

There are several possible genetic mechanisns which can explain
these data. First, the revertant could be due to a nullisomic for
chromosome S which carries the S2 gene. This hypothesis requires that
the §g_mutant polypeptide (or RNA) would be toxic to the cell so that
the revertant plants would be more normal because they lack the mutant
gene product. Revertants of this type would be classically termed
amorphs. There are precedents fbr this model (Hartman and Roth 1973).
One prediction of this hypothesis is that the original mutation is a
partial dominant; this is the case fOr SQ. However, the chromosome

number (Figure 1) and other parameters contained in Table 3 which

indicate little or no pollen and seed abortion, suggest that the plant

28

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29

 

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sum:m_ _m5co= xm a_muaewxocaqm mam; mmnzp cm_Poa u

2 3:3 m .82: 5:8 +

:m\:m op vmmmoeu mm: “cm—a «

 

 

+ e. a}: 2 o; H mam 2
+ + mam a: 1: m 3m 2
.. - 33 +3 o.m H 0.2 a
+ - .. a we :33
u 1 N.@@ mm we :m\=m
E E
m:_copumm xu___cmum :o_pm:wewa «cowumc_Eme amass: ucm_a
u_uceom ope: ummm cm~_oa meomoeoczu

 

mpcmpq _mu:ms_cmaxm we mu_um_cmuumem;u _cuwmo—o_m

m m4m<h

31

is not aneuploid. The data in Table 2 also indicate that there is no
selection against any class of gametes or progeny which would be
predicted to be nullisomic or monosomic. Additional, circumstantial
evidence comes from work of Gerstel and Parry (1973) who have produced
nullo-S tobacco plants which are very different from wild-type (and thus
_L also) in many characteristics including morphology and fertility.

A second possibility is that the revertant is due to a deletion of
all or part of the S3 gene. The same biological data (Table 3) mitigate
against this mechanism, but not as forcefully. The observance of no
selection against any of the classes that would carry the deletion
provides strong circumstantial evidence against a deletion hypothesis.
One might, however, expect that an organism would be able to tolerate
loss of part of a chromosome better than loss of an entire chromosome.

Action of a second site suppressor mutation is a third possible
mechanism to explain the BL revertant. This mode of reversion is more
compatible with the evidence previously discussed (Tables 2 and 3;
Figure 1) than is either preceeding possibility. The Rl_revertant grows
and reproduces indentically to wild-type plants and it is only through
measurement of pigment content under high light intensity conditions
(Table 1; Koivuniemi, Tolbert and Carlson 1980b) that the 2 phenotypes
can be distinguished. Under high light intensity 3; behaved differently
from both the heterozygous mutant Sg/gg and the wild-typeisgigg
(Koivuniemi, Tolbert, and Carlson 1980b). Genetic analyses also
demonstrate that R; is not wild-type and therefore is not a first site
revertant. If it were a first site revertant, it would segregate

identically to the wild-type ggfisg. However, we have shown that when 31_

32

is in trans to SE, it does not produce the yellow-green phenotype as
would §g, but rather the new phenotypic class, yellow-green/yellow.

A summary of the second site mutation model for R; is presented in
Table 4. Of the 9 possible genotypes predicted by this model, many are,
and others probably would be, indistinguishable by visual inspection;
however, they might be detected by careful measurement of chlorophyll
content under different light regimes. Several of the possible geno-
types have not been recovered because of the close linkage of §g_and its
suppressor. No recombinants have been recovered among 3322 back-cross
progeny. Thus, with 95% confidence, §2 and its suppressor are within
0.15 map units of each other. In summary, the evidence indicates that
R1 is a second site intragenic suppressor mutation.

Characterization 91.33: R§_was characterized following Rl_and

 

therefore we had reason to expect that this clone of plants would also
have a genetic origin. Three initial crosses were done. These included
selfing R§_and backcrossing it to both wild-type'§g[§g and the heterozy-
gous mutant §_7§g (Table 5, crosses 1 to 5). None of the crosses demon-
strated a segregation pattern indicating the presence of a first or
second site revertant. However, the presence of the SQ allele was indi-
cated (Table 5, crosses 1,4,5) along with erratic segregation patterns.
Based on these facts, hypotheses invoking chromosomal abnormalities were
proposed and the subsequent crosses presented in Table 5 (crosses 6 to
13) were done to test for predicted segregation ratios based on these
hypotheses.

The data regarding R§_conform best to the hypothesis that it
resulted from a monosomic condition for chromosome S which carries the

.Sg gene. In these same plants the homeologous chromosome is assumed to

33

TABLE 4

Genetic model for revertant Bl

 

Genotype*

Phenotype

 

su+ SQ+
W
Su- sp_+
W
Su- 59+
W
Su- sp+
Su- sp—
W
Su- sp-
W
Su- sp-
W
su+ sp-
W
Su- sp-
W
su+ SQ-

su+ sp-

wild type homozygote, green (recovered)

Sulfur heterozygote, yellow-green (recovered)

Sulfur homozygote, yellow-white (recovered)

Sulfur haploid, Yellow (recovered)

Rl_second site revertant homozygote, green (recovered)+
Double heterozygote, green (recovered)

Suppressor heterozygote in Sulfur, yellow-green/yellow
(recovered)

Predicted green

Predicted green

Predicted green

 

* g2: stands for the second site suppressor gene in its original

wild-type fbrm, g2; is the newly recovered revertant fOrm, wfild-type

sulfur is sgi_and the sulfur mutant is S31.

34

TABLE 4 (cont'd)

+ The R; revertant plants resulted from two events (a) induction of the
suppressor mutation (sp-) and (b) diploidization of the resulting
plant which occurred either in the leaf or during subsequent tissue

culture.

35

m o mm Ne o _w Aux x =m\=mv
x cmmemlzo_—mxfiam\:m x mmv .__

 

\cmmem
o o N_ cm 0 m“ =m\=m x camemfizm\=m x may .o_
+N¢ o o o o m¢_ :m\:m x cmmemfizm\:m x mmv .m
o _ mm NR N mm A=m\=m x Hay x mm .m
_ N ¢o_ mm a N mm x _m .N
o P mm_ __ o m Hz x mm .w
o mm m_ mm o «N. mm x :m\:m .m
o QFP N_ o__ N m :m\:m x mm .¢
o _ o No_ 0 *mN mm x :m\:m .m
_ m c NNF o «me :m\:m x mm .N
0 mm_ No a o c mm x mm .—
mu:m_m mo Loganz
umummmwcm> soPPm> zop_w> cmmcmuzo_—m> :mmcmnzo_—m> :mmcw mmogu

\cmwcmuzo__m> \cmmco

 

mm ucmpcw>mm com mcmnE:z cowpmmmcmmm mcw_ummm

m m4m<~

36

ummm we mc__—maw_mm_s a 0» one me has pan .czocx yo: mw amass: cow; a—umpumaxmcs mPgu cow :omomc use +

mco_pwucou u_mww ewes: :mmcmuzoppm>\cmmeu mm vm_w_mmmpuwc cmump mew: mucmpa weaves meow s

 

 

o o o Nw m— on_ =m\:m
x :mmcmuzo__mafimm x =m\:mv .mp
\cmmcm
o o o o o mom =m\=m x emaemfiam\=m x may .N.
35$ 08 .5952
emucmmwcm> Zo_—m> zo__m> :mmcmuzop_m> :mmcmlzop_m> :mmcw mmoeu

\cmmemuzo__m>

\cwwco

 

“v.3couv m mum<e

37

be disomic. Chromosome dosage would then affect the reversion of 33_to
a green autotrophic plant. The production of a monosomic most likely
occurred at a stage when most of the genome underwent diploidization.
R; is aneuploid (Table 3, Figure 2) however, it cannot be aneuploid for
much of the genome since it is fertile both as a male and as a female
parent. The monosomic hypothesis is consistent with the seedling segre-
gation pattern presented in Table 5. Data from an 33_self-fertilization
are presented in cross 1 of Table 5. Three classes of progeny are seen
to be segregating: yellow-green, yellow-green/yellow, and yellow.
According to our model, (i.e. 33 is monosomic for chromosome 5) there
are 3 major phenotypic classes expected which can be denoted symboli-
cally as 979. §g/Q, and Sgigg respectively. Theoretically, these 3
classes are expected to be present in a ratio of 1:2:1; however, when
one considers that there is almost certainly selection at both the
gametic and zygotic levels against both the nullisomic gametes and
zygotes, and also against the monosomic zygote, a deviation in these
ratios is not unexpected. (A coefficient of selection of 0.44 for the
nullisomic gamete and coefficients of selection of 0.64 and 0.82 for the
heterozygous and homozygous nullisomic zygotes respectively are
sufficient to account for the departure from the expected ratio.)

All of the other crosses presented in Table 5 (crosses 2 to 13) can
also be explained with this model. As an example, consider the
segregation ratios presented in cross 8. This is a cross between R§_and
a hybrid produced by crossing Rl_and‘§g7§g. From this cross, two
diploid classes are predicted to occur. They can be symbolized as
§£_§E:, which is yellow-green/yellow based on our model for 3;, and

Su sp+
su s + which is the heterozygous sulfur mutant. These plants were
Su spT

38

predicted to be, and were present in the greatest number since they are
normal disomics for chromosome 5. Two other classes are predicted to be

present at a lower frequency since they result from fertilization events

Su sp-

involving nullisomic gametes. These are , which would to be

 

intermediate in phenotype to yellow-green and yellow-green/yellow and
sp+

therefore subject to classification as either, and which would be

 

green since it has no copies of the §g_gene. This latter class was
found in a relatively high frequency; however, only 30% as many of these
aneuploid progeny were present as were found in either of the 2 euploid
classes. The classes green/yellow-green and yellow can be accounted for
in two ways. (a) Misclassification of seedling genotype: this is a
possibility since seedlings were observed only as they first germinated.
All of the plants, other than the wild-type, varied in their chlorophyll
concentrations depending upon their age and particularly upon environ-
mental conditions (light intensity). (b) Production of variant pheno-
types through pairing of chromosome S with its homeologous chromosomes
and subsequent aberrant segregation is also a possibility since tobacco
is an amphidiploid. The homeologous chromosomes from the two progenitor
species of N. tabaccum are available for pairing with the monosomic
(Smith 1979). Pairing of homeologous chromosomes would produce
subsequent aberrant disjunctions and thus the anomalous classes of
progeny .

Additional data to support the monosomic hypothesis is found in
Table 3. R; had a chromosome number lower than the diploid number of
48 (see also Figure 2), high pollen abortion rates and low seedling
germination rates. The lower germination is demonstrated clearly by the

data in Table 5 on seedling germination rates in reciprocal crosses.

39

Through pooling of the data from lines 2 to 7, the mean seedling germi-
nation rate with 33 as a female parent is 0.6 :_0.1 as compared to 0.9 :_
0.1 when 33 is the male parent in the cross. These observations support
the monosomic hypothesis, particularly since pollen is more susceptible
to chromosomal number abnormality than is the egg (Smith 1979). One
would expect greater zygotic lethality when a presumed aneuploid is the
female parent since aneuploids would not be lost at the gametic level.
Additional circumstantial support comes from the work of Burk and Menser
(1964) who attempted to map the Su mutation with monosomics. They
expected that in the cross Sgfigg x monosomic S, half of the progeny
would be lethal since the homozygote, §EK§EA was lethal. However, they
found only some "atypical lethals“, that is, plants which fonned one or
two true leaves, but not 50% lethality. Carlson (1974) has subsequently
mapped §g_to chromosome S. Therefore, §g7Q_is apparently not lethal in
all cases and could account for the R3 revertant.

The most serious criticism of this model is that the original 33
revertant is predicted to have the genotype §g7g_while being essentially
diploid for the remainder of the genome. The original R§_plants were
green under low light intensity conditions, though not as green as wild-
type, and they grew vigorously. The yellow-green/yellow plants, on the
contrary, were very obviously lacking in chlorophyll and we were
unable to propagate any of these plants to a flowering stage. The
environmental influences on phenotypic variation (Table 1; Koivuniemi,
Tolbert and Carlson 1980b) discussed previously might explain this
discrepancy. Additionally, the 33_plants are evidently highly aneuploid,
as evidenced by chromosome counts and zygotic mortality in reciprocal

crosses. Therefore, the genetic composition and hence the phenotype of

4O

33_may be impossible to duplicate exactly after stabilization of the
genome.

Other models invoking deletions, translocations and other chromoso-
mal rearrangements could possibly explain the results with 33. However,
the limiting fact is that the 3g allele segregated in 33_in a fashion
somewhat analogous to a heterozygote. Since our selection process was
begun with haploid material, it is reasonable to postulate that monosomy
for chromosome S which could generate the observed patterns of genetic
segregation in the offSpring of an aneuploid plant.

R3 Progeny: An interesting plant, 3;, (Figure 3) was recovered
from the cross 33 x wild-type. It was profusely variegated, had good
growth characteristics and was entirely fertile. This was true in spite
of the fact that approximately 50% of its leaf area showed the yellow-
green phenotype of the heterozygous mutant §gfi§g which was itself sub-
ject to extreme bleaching and would not flower under field conditions.

This variegation was transmitted at a rate of about 14% upon
selfing or in a backcross (Table 6), regardless of whether the
variegating plant was used as a male or female in the cross. Such data
indicate that whatever was inducing the variegation was nuclear encoded.
Additionally, there was no "sorting out" of the various tissues into
more phenotypically stable patterns (i.e. leaves and shoots which are
yellow-green and green only), which would have been indicative of a
mutant plastid (Kirk and Tilney-Bassett 1978).

The F1 progeny showed a segregation pattern which indicated that y;_
was heterozygous at the sulfur locus (337gg) (Table 6). Apparently,
whatever was causing the variegation was producing its affect by inter-

acting with the sulfur locus since the change in color was the only

41

..:Im.\mw x Mm 398 93
Lo xcmooca Ha an «N» ucmpg umpmmmwcm> mg» soc» emmu

.m mesmvu

42

 

43

.Locem cw on has omega mo beam mcemuuma cowpmmmwem> cw wwwucpmucmucs op man Lm>mzoz

.mucw_a mcwumamwcm> we mmcpo an» 0» mm __m3 we mgauocmsa m op mu=m_a :mwmmm op meme mm: uqewuaw c<

 

 

 

 

i.
0 N_ o_ we No_ we H> x :m\:m .m
o o e. —m «N Na :m\:m x H> .q
o Nm ON 0 PNN va H> x :m\:m .m
o __ vN o mNF mmp :m\:m x H> .N
N c_ o. Nm mm Fe H> x H> ._

zoppw> :wmemnzo__m> cube: zo_—m> :mmcmuzop_m> cameo mmocu
«umummwwcm> cmummmwcm>ucoz

 

.ww uce_a umummwwcm> ms“ Low mcmn522 cowummmemmm mew—ummm

o m4m<p

44

phenotypic difference apparent in the variegating tissue. No twin Spots
have been observed on this plant. Currently we are regenerating plants
from variegated tissues in order to determine their precise genotypes.
Preliminary evidence indicates that yellow-green plants regenerated from
variegating sectors are stable and do not variegate themselves.
However, at least some of the green plants have variegation patterns
similar to 3;. At present we do not postulate a specific mechanisn to
explain the observed variegation, however, it appears that the sulfur
locus has become a mutable gene. That is,the mutant allele §g_is
reverting back to an apparently wild-type phenotype at a high frequency.
Examples of similar situations are cited by Kirk and Tilney-Bassett
(1978). I

A second interesting variegated plant, 33, has been recovered from
the cross 33 x 3;. It appeared to have some chromosomal or allelic
instability but not of the same nature as that found for 3;. Its leaves
had the appearance of a normal diploid §37§g plant which had been
irradiated with Gamma rays (Figure 4). There was a very high frequency
of green spots and twin spots (green and yellow) on a yellow-green
background. !3_has not yet been characterized genetically.

Characterization of R2: 33 has proven more resistant to genetic
analysis than either 3; or 33, Segregation data (Table 7) were incon-
clusive in demonstrating the reversion mechanism but were consistent
with the hypothesis that the plants were highly aneuploid. There were 2
major problems with evaluating the segregation ratios from crosses with
33, First, it had a germination rate indicating that approximately 50
to 75% of all the progeny were not viable. Second, it was difficult to

distinguish many of the phenotypes which appeared in the offspring.

45

1| .mélm. x Mm 398
mgu +0 xcwmocq Pm cm .N> ucmpa umpmmmwgm> mg“ seem womb

.v mcamwm

46

 

47

.meummou umucaou mew; zo—Fma\:mmemuzo_pm> use cmmcmuzo_pm> «

 

 

o 0 am o ON :m\=m
x :mmcmuzo—Pmaazm\:m x va .N
\cmmgw
m o 2 o we 3&3 x 522323 x E .c
o o we 0 amp :m\=m
x :mmcmuzo__mxasm\=m x va .m
\cmmco
o o N o o__ =m\:m x :mmLmA=m\=m x Nay .q
o 1 «m¢_ o w Hm x Na .m
mm c O Na N =m\=m x N: .N
o o m ow me =m\:m x Nm ._
muse—a mo amassz
zo_—m> zo_~m> :mmcmuzoppm> :mmcmuzoP—o> :mmcw mmocu
\cmmcmuzo__m> \cwmcw

 

mm ucmuem>wz com mcmnE:z cowummmcmmm m:_—ummm

m m4m<h

48

There were no clear cut segregations such as the green, yellow-green and
yellow derived from a §gf§g_self-fertilization. However in a cross
involving an apparently green F1, which was isolated from the cross 33 x
EEA§2,.§E alleles still segregated (Table 6, line 4).

These data are consistent with the hypothesis that the original 33,
revertant resulted from an increase in chromosome number, particularly
of the chromosome homeologous to chromosome S. The homeolegous chromo-
somes presumably carry a gene homeologous to sulfur, which if normal,
could mask the affects of 33. This plant is itself evidence for the
existence of such a gene, since it is green and yet still maintains the
__3 allele.

Chromosome counts indicated that the 3g_plants were near the
tetraploid level (Table 3, Figure 5). The fertility data reinforced
this conclusion, indicating that pollen and seed germination rates were
much decreased in 33_when compared to controls (Table 2). Additionally,
these plants were completely male sterile. Apparently the high
chromosome number results in genetic instability since plants from this
clone segregated somatic sectors (which appeared yellow in the greenish
background) at a very high frequency. These sectors followed cell

lineage patterns.

Other Clones: The remaining 9 clones recovered from green sectors

 

have not been analyzed genetically. They were quite variable in pheno-
type. Under greenhouse conditions they ranged in color from some which
were much yellower than the yellow-green heterozygote 337§g, to others
which were apparently as green as wild-type plants. Morphologically
there was also great variation, with leaves from some plants having a

long slender lancelot shape, and others being rounder than normal

49

.meeemeeecee mm me_eweueee mm
ueeecm>ec ewe—esmee »_;mw: ecu Eeee mmeemeseccu a?» poem

.m me=m_u

50

 

51

tobacco leaves (this is common in plants regenerated from tissue
culture). Only 4 other plants have flowered to date. Of these, 2 were
completely sterile, while 2 others had apparently good seed set, but
seed germination was below the 5% level. Based on these observations we
have concluded that aneuploidy is potentially responsible for at least
some of these 9 clones.

One plant which has been recovered is a periclinal chimera. It did
flower but was completely infertile. It then reverted to a completely
vegetative state and has remained so for approximately 1 year. A green
plant showing some variegation was also isolated from the same callus
tissue. It has apparently stabilized, and is almost completely green at
present. This plant also flowered, but unfortunately, it too was

completely infertile.

52

DISCUSSION

Our data indicate that it is possible to isolate spontaneously
occurring revertants of a mutation in plants. The technique involves
visual selection of apparent revertant events occurring in the somatic
tissue of plant leaves, propagation of such variants through techniques
of 13 11339 tissue culture, and regeneration of whole plants from these
tissues. In particular, this study indicates that it is possible to use
these techniques on haploid tissue, where one can recover recessive
events which would not be recognized in diploid plants.

The use of second site revertants has proven invaluable in genetic
studies with viruses, bacteria and fungi both for the isolation of muta-
tions in specific genes as well as for illucidation of interactions
between various genes and their products (Hartman and Roth 1973). Me
have demonstrated that these potentials exist for higher plants as well.
Our revertants (particularly the second site suppressor mutation 3;)
have been used in attempts to delineate the cause of the reported
increase in photorespiration in the heterozygous mutant §g7§g and also
to determine the primary genetic lesion caused by the §g_mutation
(Koivuniemi, Tolbert and Carlson 1980 a, b). Evidence obtained from
analysis of these revertants has reinforced our conclusion that the 33
mutation does not primarily affect the small subunit of ribulose-1,5-
bisphosphate carboxylase/ oxygenase. Instead, the data indicate that
there may be some changes in thylakoid manbrane polypeptides associated
with the 33 mutation. The usefulness of revertants in studies to
confirm or deny the association of a gene and a fuctional polypeptide is

self-evident. Undoubtedly production of revertants could be enhanced

53

through the use of mutagens. These could also be used to produce more
specific lesions (e.g. deletions or point mutations) through the choice
of pr0per mutagenic agents.

Visual selection techniques could be used not only for selection of
nuclear mutants, but they might be useful for selection of chloroplast
mutants as well. The possibility of isolating chloroplast mutants could
be greatly enhanced through the use of a known plastome mutant as the
progenitor plant. It has been difficult in the past to recover chloro-
plast mutants due to the great reiteration of DNA within the chloroplast
and the large number of chloroplasts per cell. This technique enchances
the probability of recovery of a mutant since it isn't necessary for the -
mutant to occur in germline tissue.

A major advantage of selecting mutants in this manner is that
problems with lack of expression in whole plants of traits selected in
culture (Parke and Carlson 1979) are avoided since the cells expressing
the characteristics of interest are in their natural physiological and
developmental state. Murakishi and Carlson (1976) and Radin and Carlson
(1978) have used similar techniques for isolation and regeneration of
virus-free and herbicide-tolerant plants respectively. The sole limit-
ing factor is the ability to discern the cells of interest. However,
in most selection schemes, this is not a problem with leaf tissue since
the revertant cells appear green and/or viable against a bleached or
necrotic background.

This study has also demonstrated that there is a great deal of
change in chromosome number in plants derived from variant somatic leaf
tissue. This is in accord with the findings of Dulieu (1974). Of 12

regenerated clones, at least 3 and possibly 4 or 5, had a chromosome

54

number greater than or equal to the diploid number of 48. It may be
speculated that this is a normal condition in some somatic tissues.
Since somatic tissue is not committed to the production of gametes, it
may not be so stringent in preserving its genetic integrity. Conse-
quently there may be much more genetic variation available in somatic
tissue than there is in the germline of an individual plant. If this is
the case, the 13.3133 rescue technique provides a method for
capitalizing on this variation.

The variegated plants which we recovered from the crosses, 33_x
3_733_and 33 x 33 are potentially very interesting. First, the mecha-
nisms causing variegation may give more information on the interaction
of genes. The 33 allele apparently demonstrates nuclear instability.
Perhaps the mechanism is similar to that of controlling elements in
maize (McClintock 1956); or the controlling element system which Sand
(1976) has indentified as being associated with a mutable flower color
locus in tobacco.

33 may also be useful in studying physiological processes in the
plant. For instance, the heterozygous plant 33733 has been reported to
have higher photorespiration than its wild-type sibling 33733 (Zelitch
and Day 1968). It would be of interest to know if this were true also
for 33thich is composed of approximately 50% 33733 and 50% 33733
tissue. Cell-cell interactions in differentiated plant tissue may thus
be amenable to study.

Finally, our data indicate that the 33 mutation is not caused by a
gross chromosomal aberration such as a deletion, since we have isolated

an apparently intragenic, second site suppressor mutation. As discussed

in the text, the data also suggest that the product of 33 could be a

55

toxic polypeptide which is actively harmful to plant cells. The mutant
is semidominant and apparently reverts at a high frequency through
several different mechanisms, two criteria which are predicted by the

toxic polypeptide model.

56

LITERATURE CITED

 

Burk, L.G., 1970 Green and light-yellow haploid seedlings from anthers
of sulfur tobacco. J. Heredity 61:279.

Burk, L.G. and H.A. Menser, 1964 A dominant aurea mutation in tobacco.
Tobacco Sci. 8:101-104.

Carlson, P.S., 1974 Mitotic crossing-over in a higher plant. Genet.
Res. 24:109-112.

Dulieu, H.L., 1974 Somatic variations on a yellow mutant in Nicotiana
tabacum L. (aIT/al aZT/azl I. Non-reciprocal genetic
events occurring in leaf cells. Mutation Res. 25:289-304.

Dulieu, H.L. 1975 Somatic variations on a yellow mutant in Nicotiana
tabacum L. (aIT/al) (aZT/az) II. Reciprocal genetic
events occuring in leaf cells. Mutation Res. 28: 69-77.

Hartman, P.E. and J.R. Roth, 1973 Mechanisms of suppression. Advan.
Genet. 17:1-105.

Gerstel, D.U. and D.C. Parry, 1973 Production and behavior of nullisomic
S in Nicotiana tabacum. Tobacco Sci. 17: 78-79.

Kirk, J.T.0. and R.A.E. Tilney-Bassett, 1978 The Plastids. Elsevier

 

North-Holland, New York.

Koivuniemi, P.J. , N.E. Tolbert and P.S. Carlson, 1980a
Ribulose-l,5-bisphosphate carboxylase/oxygenase and polyphenol
oxidase in the tobacco mutant 33733 and three green revertant
plants. Plant Physiol. (in press)

Koivuniemi, P.J., N.E. Tolbert and P.S. Carlson, 1980b Characterization
of the thylakoid membranes of the tobacco aurea mutant 33733 and

three green revertant plants. (In preparation)

57

Linsmaier, E.M. and F. Skoog, 1965 Organic growth factor requirements
of tobacco tissue cultures. Physiol. Plantarum 18:100-127.

McClintock, B., 1956 Controlling elements and the gene. Cold Spring
Harbor Symp. Quanti. Biol. 21:197-216.

Murakishi, H.H. and P.S. Carlson, 1976 Regeneration of virus-free
plants from dark-green islands of tobacco mosaic virus-infected
tobacco leaves. Phytopath. 66:931-9032.

Parke, D. and P.S. Carlson, 1979 Somatic cell genetics of higher
plants: Appraising the application of bacterial systems to higher

plant cells cultured 13 Vitro. In, J. Scandalios, ed. Physiological

 

Genetics Academic Press, New York.

Radin, D.N. and P.S. Carlson, 1978 Herbicide-tolerant tobacco mutants
selected in situ and recovered via regeneration from cell-culture.
Genet. Res. 32:85-89.

Sand, S.A., 1976 Genetic control of gene expression: Independent
location of FlT (3) and its interactions with the mutable 3 locus in
Nicotiana. Gentics 83:719-736.

Smith, H.H. 1979 The Genus as a genetic resource. In, R.D. Durbin, ed.
Nicotiana: Procedure for Experimental Use, U.S. Dpartment of
Agriculture Technical Bulletin 1586.

Zelitch, I. and P.R. Day, 1968 Variation in photorespiration. The
effect of genetic differences in photorespiration on net

photosynthesis in tobacco. Plant Phyiol. 43:1838-1844.

58

This research was supported by National Science Foundation Grants
PCM 78-15891 to N.E. Tolbert and AER 75-20882 to P.S. Carlson and is
part of a Ph.D. thesis by P.J. Koivuniemi in the Genetics Program at

Michigan State University.

CHAPTER II

Ribulose-l,5-bi5phosphate Carboxylase/Oxygenase and Polyphenol
Oxidase in the Tobacco Mutant Su/su and Three Green Revertant

Plants1

Paul J. Koivuniemiz, N.E. Tolbert and Peter S. Carlson,
Program in Genetics,
Department of Biochemistry and Department of Crop and Soil Sciences,

Michigan State University, East Lansing, Michigan 48824

Manuscript - received: October 16, 1979

Revised manuscript - received:

59

6O

1 This research was supported by National Science Foundation Grants
PCM 78-15891 t0 NeEoTo and AoEoRo 75-20882 t0 P.SoCo and pUbIIShEd
as Journal Article No 9170 of the Michigan Agricultural Experiment

Station.

2 This paper is part of a Ph.D. thesis by P.J.K. in the Program in

Genetics.

3 Abbreviations: ribulose-Pz, ribulose-I,5-bisphosphate; L-DOPA,
L-Dihydroxyphenylalanine; PVPP, insoluble polyvinylpolypyrroli-

done.

61

Abstract

Ribulose-l,5-bisphosphate carboxylase/oxygenase (E.C.4.1.1.39) was

crystallized from a heterozygous tobacco (Nicotiana tabaccum L.) aurea

 

mutant (33733), its wild-type sibling (33733), and green revertant
plants regenerated from green spots found on leaves of haploid 33
plants. No differences were found in the specific activity or kinetic
parameters of this enzyme, when comparing 33733 and 33733 plants of the
same age, which had been grown under identical conditions. The enzyme
crystallized from revertant plants was also identical to the enzyme from
wild-type plants with the exception of one clone, designated 33. 33 has
a chromosome number approximately double that of the wild-type (87.0 1
11.1 33 48). The enzyme from 33_had a lower Vmax for C02.
although the Km values were identical to those for the enzyme from the
wild-type plant. The enzyme from all mutant plants had identical
isoelectric points, identical molecular weight as demonstrated by
migration on native and SDS polyacrylamide gels, and the same ratio of
large to small subunits as the enzyme from the wild-type. The large
subunit of the enzyme from tobacco leaves exhibited a different electro-
phoretic pattern than did the large subunit from spinach; there were 2
to 3 bands on SOS-polyacrylamide gels fbr the tobacco enzyme whereas the
enzyme from spinach had only one species of large subunit.

Total polyphenol oxidase activity was the same in leaves from the
heterozygous mutant (33733) and wild-type (33733) plants when corre-
lated with developmental age as represented by morphology rather than by

the chronological age of the plants. There was a marked increase in the

62

soluble activity of this enzyme with increasing age of both plant types
and also as a result of varying environmental conditions.
Ribulose-l,5-bisphosphate carboxylase/oxygenase activity correlated
inversely with increases in the soluble activity of polyphenol oxidase
in crude homogenates from which the carboxylase/oxygenase was crystal-
lized over a generation of 33733 and 33733 plants. Criteria are out-
lined for determining if differences in activity of ribulose-1,5-bis-
phosphate carboxylase/oxygenase are caused by an effect of polyphenol

oxidase activity and/or by some other extrinsic parameter.

63

Introduction

 

The heterozygous tobacco aurea mutant 33733 (2), which resulted
fran a nuclear mutation, has been reported to have higher rates of
photoreSpiration than its wild-type sibling, John Williams Broadleaf,
33733 (19,26). In an effort to explain this observation, Kung and
Marsho (12) examined the activity of ribulose-Pz Carboxylase/
oxygenase (E.C.4.1.1.39) and by isoelectric focusing looked for struc-
tural changes in the small subunit of the enzyme. Their results indica-
ted lower activity for both reactions in the crystalline enzyme from the
mutant plant 33733; ribulose-Pz carboxylase had approximately 57% and
ribulose-Pz oxygenase 40% of the activity f0und in the wild-type plant
33733. By isoelectric focusing, no differences were seen in the poly-
peptides of the large or small subunit of the enzyme from both plant
types. Okabe (18,19) has reported that ribulose-PZ carboxylase/
oxygenase from the mutant 33733 has a higher Km(C02) and a lower
Km(02) in a crude desalted extract than does the enzyme from 33733.
Because these results would be significant in understanding the ratio of
photorespiration to photosynthesis, we sought to reproduce them with the
crystalline enzyme prepared from these plants. Because of large fluctu-
ations in the activity of the ribulose-Pz carboxylase/oxygenase it was
necessary to compare its total and specific activity from 33733 and
.33733 plants of the same age. This was done weekly, from the seedling
stage of development through the beginnings of senesence, for a single
group of plants in an attempt to correlate changes in enzyme activity

with plant age, growth conditions, and polyphenol oxidase activity.

64

Several kinetic properties of the ribulose-Pz carboxylase/
oxygenase from the wild-type 33733, the heterozygous mutant 33733, and
three different revertants isolated from haploid 33 plants were
examined. While our work was in progress Garrett (6) reported that in
ryegrass (331133) changes occur in the isoelectric point of the
holoenzyme and in the Km(C02) for ribulose-Pz Carboxylase/oxygenase
due to a change in ploidy level in the plant from diploid to tetraploid.
One of our tobacco revertants, 33, also has a high ploidy level
(chromosome number of 87.0 1 11.1). We therefore examined several
properties of the enzyme from this clone of plants.

Polyphenol oxidase is a chloroplast enzyme which is associated
primarily with the thylakoids (16,23). In the presence of soluble poly-
phenol oxidase in homogenates from leaves, other enzymes are inactivated
(14,16). Ribulose-Pz carboxylase/oxygenase has been shown to be
structurally altered in extracts, prepared for enzyme crystallization,
in which there is high polyphenol oxidase activity (7). Hence we have
compared the develOpment of soluble polyphenol oxidase in the heterozy-
gous mutant 33733 and the wild-type plant 33733 in an attempt to corre-
late its activity with changes in ribulose-P2 carboxylase/oxygenase

activity.

65

Materials and Methods

 

Plants used included the semidominant aurea mutant of Nicotiana
tabacum L., 33733, first described by Burk and Menser (2), and its wild-
type sibling, John Williams Broadleaf, 33733. Seeds were obtained from
selfing of 33733 plants. Yellow haploid 33 plants were derived from
another cultures of 33733 plants and were maintained by heterotrophic
growth on a Linsmaier a Skoog medium (13) supplemented with 20% sucrose
and 1 mg/l indole-3-acetic acid. Revertant plants were regenerated from
leaf tissue of haploid 33 plants (10). These revertants were phenotypi-
cally distinct from both 33733 and 33733 plants. A more complete
description of these plants will follow in a subsequent publication.
Seeds from the heterozygous mutant 33733 were germinated and the segre—
gating heterozygous mutant and wdld-type plants were grown in Bacto
potting soil in a growth chamber with a light intensity of 600 uE x
m'2 x 5']. Between 60 to 70 days post-genuination, plants were
transplanted to pots containing a 1:1 mixture of Bacto potting soil with
vermiculite and placed in a greenhouse. Regenerated revertant plants
were grown under the same conditions. Leaves used for comparing enzyme
activity with plant age were taken from similar nodes on plants of
different genetic character, but of the same age, grown under identical
conditions.

Ribulose-P2__carboxylase/oxygenase. This enzyme was crystallized

 

as previously described (3,21) except that 2% insoluble polyvinylpoly-
pyrrolidone (PVPP) was added to the grinding buffer to partially
protect against polyphenol oxidase activity released during grinding

of the tobacco leaves. This procedure involved homogenization,

66

filtration through G-25 Sephadex, and protein concentration by vacuum
dialysis. The enzyme was crystallized by dialysis in collodion bags
against a NaCl-free buffer. Crystallization was done only once, as it
was found that recrystallization had no effect on enzyme specific
activity or purity as shown by native and SDS polyacrylamide gel
electrOphoresis. The crystals were dissolved in a buffer containing 100
mM N,N-bis(2 hydroxyethyl)glycine (Bicine) at pH 8.3, 10 mM MgC12,
0.25 nM EDTA and 200 nM NaCl, and a stock solution was adjusted to 2 mg
protein/ml as measured by absorption at 280 nm (22). A heat treatment
at 500 had no influence on enzyme activity. For experiments on
changes in enzyme activity with plant age, assays for total activity of
ribulose-Pz Carboxylase/oxygenase were run on the leaf extracts prior
to passing the crude homogenate over a G-25 Sephadex column.

Before assay, the enzyme was activated by incubation for at least
30 min with 10 mM NaHC03 and 1 nM dithiothreitol in the dissolving
buffer. The ribulose-Pz Carboxylase assays were initiated by the
addition of 10 pl of 12.5 mM ribulose-Pz to 240 u] of assay mixture
containing 100 mM Bicine at pH 8.3, 20 mM MgClz. 0.25 mM EDTA, 10 mM
NaH14C03. 1 nM dithiothreitol and 20 ug of ribulose-Pz
carboxylase/oxygenase and were run at 300 for 0.5 or 1 min. For Km
determinations, the ribulose-Pz carboxylase assays were initiated by
the addition of 10 ul aliquots of activated enzyme to the same reaction
mixture, except that the concentrations of NaH14c03 and ribulose-
P2 were varied. Activity was determined as 14C acid-stable

product as measured by scintillation counting.

67

The ribulose-Pz oxygenase assay was based on ribulose-Pz depen-
dent oxygen uptake as measured in a Rank Brothers oxygen electrode.
Assays were initiated by the addition of 20 “1 of 12.5 mM ribulose-PZ
to a reaction mixture consisting of 480 pl of 100 11M Bicine at pH 8.3,
20 mM MgC12, 0.25 mM EDTA, 1 nM dithiothreitol, and 40 ug of
ribulose-PZ carboxylase/oxygenase which had been activated as
described above and placed in the reaction mixture for temperature
equilibration for approximately 1 min. The reactions were run at 30°
and monitored for 50-60 5 with a chart recorder; rates were taken only
from the initial linear portion of the curves. These rates were not
greatly lower than rates measured in reactions initiated by the addition
of C02 activated enzyme.

After the initial experiments were run, another protocol for the
oxygenase assay was developed (15), and was used when determining Km
(oxygen) values. Reactions were initiated by the addition of 20 ul of
activated enzyme to 480 pl of reaction mixture containing 100 MM Bicine
at pH 8.3, 20 mM MgC12, 0.25 mM EDTA, 1 mM dithiothreitol, 0.5 mM
ribulose-Pz and variable amounts of 02. The buffer was made from
boiled distilled deionized water and was further degassed under vacuum
and stored under nitrogen. Precise oxygen concentrations were obtained
by aerating the reaction solution in the oxygen electrode chamber with
oxygen and nitrogen gasses through a rubber serum stopper prior to addi-
tion of the enzyme. Calibrations were made using 1.12 11M as 100% oxygen
saturation and 0.235 mM as the concentration obtained from 21% 02
(24). There was a small amount of HC03' in this assay due to carry
over with the activated enzyme and also due to K2C03 in the KOH used

to adjust the pH of the reaction buffer, however, Lorimer et a1. (15)

68

have demonstrated that the amount carried over is not enough to signifi-
cantly compete with the oxygenase activity.

For comparisons of polyphenol oxidase activity from the heterozy-
gous mutant 33733 and wild-type 33733 plants, leaves were taken from
plants of the same age grown either in a growth chamber or greenhouse as
indicated. Leaves were ground with a mortar and pestle in a 2:1 ratio
of grinding buffer to plant fresh weight. The grinding buffer was 50 11M
Tris-HCl at pH 7.4, l M NaCl, 1 nM EDTA, 2 mM MgC12 and 2% PVPP. This
is the same buffer used to grind tobacco leaves for the crystallization
of ribulose-PZ Carboxylase/oxygenase . The crude extracts were either
centrifuged at 27,000 X g for 30 min and used immediately or aged for
approximately 40 h at 4° prior to centrifugation during which time the
amount of polyphenol oxidase activity increased (23).

Polyphenol oxidase was assayed spectrOphotometrically (23) and also
as L-dihydroxyphenylalanine (L-DOPA) dependent oxygen uptake in a Rank
Brothers oxygen electrode at 30°. Assays were initiated by the addi-
tion of 20 ul of extract to 980 pl of 50 nM phosphate and 50 11M citric
acid buffer at pH 7.0, and 6 "M L-DOPA. Reactions were monitored fbr 1
to 2 min.

Polyacrylamide gel electrophoresis (5) was run with 10 to 50 ug of
protein applied per gel tube (100 x 5 mm). Gels consisted of 5% acryla-
mide and were run at 3.5 mA/tube until the tracking dye (bromphenol
blue) reached the bottom of the tube. SOS-polyacrylamide gel electro-
phoresis (25) was run in tube gels (100 x 5 mm). Gels consisted of 10%
acrylamide, and 10 to 50 ug of protein were placed on each gel with an
applied current of 8 mA/tube until the tracking dye reached the bottom

of the tube. Nondenaturing isoelectric focusing was done in

69

polyacrylamide tube gels according to the procedure of O'Farrell (17),
except that urea was excluded from all buffers. Gels consisted of 7%
acrylamide and were run for a total of 600-700 volt-h/tube. All gels
were fixed and stained overnight in 0.1% Coomassie Blue R, 10% (w/v)
trichloroacetic acid, and 25% (v/v) isopropanol, and then destained in
7.5% (v/v) acetic acid and 20% (v/v) methyl alcohol containing Rexyn
1-300 mixed-bed ion exchange resin (Fisher Sci. Co.). Gels were scanned
at 600 nm with a Gilford gel scanner.

Protein concentrations in crude extracts were determined using a
modified Lowry procedure (1). Ribulose-Pz was prepared enzymatically
(9). Sephadex G-25 was from Pharmacia Fine Chemicals, NaH14c03
from Amersham, and acrylamide gel materials from BioRad. Other reagents

were from Sigma.

70

Results

Our initial objective was to examine the ratio of ribulose-Pz
carboxylase to oxygenase activity in leaves from the wild-type and
mutant plants. In preliminary experiments, widely varying specific
activities were obtained. Therefore the effect of plant age, the
effect of growing plants in the growth chamber versus the greenhouse,
and the effect of polyphenol oxidase content were examined. For the
ribulose-Pz carboxylase/oxygenase experiments, leaves were taken from
a group of plants of the same age which had been germinated and grown
under identical conditions (Figs. 1,2 and 4) Each weekly repetition of
this experiment with one generation of plants was done with the same
plants. A t0p leaf was removed at each cited age. This leaf was
approaching a stage of full expansion and was approximately the fourth
visible leaf from the apex. However, fer the two earliest ages (20 and
40 days from planting), it was necessary to harvest entire plants to
obtain enough material fbr crystallization of the enzyme. Thus, ages
cited are for the whole plant, but the leaf examined was near the top of
the plant and was of the same age, about 1 week old. Crystalline enzyme
was prepared at each plant age for comparison of its specific activity

from leaves of the mutant Su7su and the wild-type su7su plants.

Effect of Age and Growth Condition 0n Total and Specific Activity of
Ribulose-Pz Carboxylase/Oxygenase.

When analyzed on a weekly basis, the specific activity of isolated
crystalline ribulose-PZ carboxylase (Fig. 1A) and ribulose-PZ

oxygenase (Fig. 2A) had highly significant differences (P of 0.01 for

71

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ACrystolline enzyme

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8. Leaf extract

su /su

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40.60.80.10011201
Days from Planting

73

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Days from Planting

75

the carboxylase and 0.05 for the oxygenase). Specific carboxylase
activity of the enzyme from these top leaves was greatest in young
plants, 44 days old, which were grown in the growth chamber, and then
declined until values 1/3 to 1/2 the peak specific activity were
measured wnth enzyme crystallized from plants 66 days old. At this
time, the plants were transferred to the greenhouse; subsequently the
specific activity of the crystalline enzyme from the top leaves
increased again, reaching maximum values at 80 to 85 days of plant age.
After this peak, there was a slow decrease in specific activity of the
crystalline enzyme from plants of increasing age. These changes in
specific activity of the crystalline enzyme were essentially the same in
33733 and 33733 plants. Similar changes with age and growth conditions
were also observed in the activity of the enzyme in the crude leaf
extracts, expressed as activity per mg of total soluble protein, for
both the carboxylase (Fig. 18) and the oxygenase (Fig. 28). These
changes in activity indicate that a comparison between 33733 and 33733
plants must be done with leaves from plants of the same age. Likewise,
any changes in the ratio of carboxylase to oxygenase activity must be
evaluated in leaves developed under exactly the same growth conditions.

In spite of a significant change in specific activity on a weekly
basis, there was no significant difference in the specific activity of
ribulose-P2 carboxylase when comparing all the data from the hetero-
zygous mutant 33733 with the wild-type 33733 by an f-test. The mean
carboxylase specific activity was 371 1_121 and 330 :_101 nmol 002 x
min'1 x mg protein'1 for 33733 and 33733 respectively. The

mean oxygenase activity from the two plant types averaged 53 :_12 nmol

76

1 x mg protein"1 for Su/su and 45 :_13 for su/su,

02 x min'
under the assay conditions (air) employed. While the differences for
the oxygenase could be judged significant by an f-test, there was wide
variablity as shown by the overlap in standard deviations of the means.
The oxygenase assay was subject to more variability than the carboxylase
assay, especially since the enzyme was rapidly losing activity in the
COZ-free nedium necessary for the oxygenase assays.

Carboxylase activity in crude homogenates of these tobacco leaves
averaged 189 :_90 nmol 602 x min"1 x mg protein'1 from 33733
plants and 276 :_156 for 33733. Total oxygenase activity in the crude
extracts was 13.2 :_8.5 nmol 02 x min"1 x mg protein‘1 for the
33733 mutant and 15.2 1_12.8 for the 33733 wild-type plants. Although
these values varied widely, there was no significant difference in total
activity on a protein basis between 33733 and 33733 plants when the data
were analyzed with an f-test.

Two anomalies were observed. At 80 to 90 days, total activity in
the crude extract from the wild-type 33733 plants was as high on a
protein basis as was the specific activity far the crystalline enzyme
from the same extract (Figs. 1A and 18). This might be explained by
postulating activators in the crude extract or partial inactivation of
the enzyme during crystallization, but the reason for low specific
activity in the crystalline enzyme remains unknown. Another difficulty
encountered was the endogenous rate of oxygen uptake in the oxygenase
assay. Ribulose—Pz Oxygenase activity in the crude extract from
plants older than 107 days is not given because the endogenous rate of

02 Uptake became so high that accurate measurements were impossible.

77

In fact, when ribulose-Pz was added to leaf extracts from these older
plants, although the leaves were newly developed, the rate of 02

uptake was often depressed over the high endogenous rate.

Effect of Age and Growth Conditions on Polyphenol Oxidase.

Measurable polyphenol oxidase activity in the leaf extracts appears
to be affected by the developmental stage of the plant. In the wild-
type 33733 plant grown in the growth chamber, soluble polyphenol oxidase
activity first appeared in freshly prepared homogenates from top leaves
when the plants were about 66 days old (Fig. 3). The appearance of
activity in 33733 lagged behind the first appearance in 33733 plants by
approximately one week. However, when based on the morphological and
develOpmental state of the plants rather than their chronological age,
the time of appearance of soluble polyphenol oxidase is about the same
in both plants.

Soluble polyphenol oxidase activity appeared at an earlier age in
plants grown in the growth chamber than in those grown in the green-
house. Activity was first present in aged extracts from 33733 plants
raised in the growth chamber for 40 days; by 47 days the activity ( 60
nmol 02 uptake x min '1x ml '1extract) was present in top
leaves from both 33733 and 33733 plants. .33733 and 33733 plants of
identical age, but grown in the greenhouse, had no soluble polyphenol
oxidase activity at these ages.

It has been shown that polyphenol oxidase activity in leaf extracts
is responsible for some enzyme inactivation (14), and that it modifies
the polypeptide pattern of the large subunit of ribulose-Pz

carboxylase/oxygenase (7). In this study, correlation of changes in the

78

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79

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DAYS FROM PLANTING

su/su

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80

activity of ribulose-Pz carboxylase/oxygenase with induction and

amount of soluble polyphenol oxidase activity in the crude extract was
noted. The large decline in specific activity of the crystalline
ribulose-P2 carboxylase/oxygenase from plants groWn in the growth
chamber from 44 to 66 days occured at the same time as the rise in
soluble polyphenol oxidase activity (Figs. 1 and 3). When these plants
were then transferred to the greenhouse, there was a concomitant drop in
soluble polyphenol oxidase activity (data not shown) and a rise in the
total and Specific activity of the ribulose-Pz Carboxylase/oxygenase
(Figs. 1 and 2). Thereafter, the gradual decline in specific activity
of the ribulose- P2 Carboxylase/oxygenase from the top leaves of older
plants correlated with the gradual increase in activity of soluble
polyphenol oxidase. These fluctuations in the ribulosePz
carboxylase/oxygenase activity occurred in spite of the fact that the
homogenizing medium contained 2% PVPP to remove the naturally occurring
phenolic substrates of polyphenol oxidase, whose quinone oxidation
products in part account far enzyme inactivation. Such extracts did not
turn dark brown, but PVPP treatment did not remove the polyphenol

oxidase which has been shown to modify other proteins(14).

Effects of Age and Growth Conditions on the Ratio of Ribulose-Pz
Carboxylase to Ribulose-Pz Oxygenase from 33733 and 33733 Plants.

The data in Figure 4 that were used in calculating these ratios
were taken from Figures 1 and 2. The assays had been run in identical
buffers, at the same pH and temperature, under air and as near in time
as was possible (within 4 h of each other). The mean carboxylase/

oxygenase ratio for the crystalline enzyme from the mutant Su7su was 7.1

81

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A. Crystalline enzyme

su/su
, Su/s

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Carboxylase/Oxygenase Ratio
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Days from Planting

83

:_2.0 and 7.5 :_2.0 for the wild-type 33733. This difference was judged
not significant based on an f-test. The variations in the ratio from
week to week were highly significant (P=.01) ranging from 4.4 to 12.2
(Fig. 4A).

The reliablility of the ratio determination using activity in crude
extracts was not great due to high endogenous rates of oxygen uptake in
extracts from increasingly older plants. The difference between the
average ratios of 23.1 i 9.3 for 33733 and 17.0 i 6.7 for 33733 (Fig.
4B) is significant according to an f-test; however this may simply be a
reflection of the earlier induction of polyphenol oxidase activity in
.33733 than in 33733 which would produce higher endogenous rates of
oxygen uptake at an earlier age (previous section). Differences in the
ratio of activity in crude extract from week to week are highly
significant (Fig. 4B).

A much higher ratio of ribulose-Pz carboxylase to ribulose-Pz
oxygenase activity was measured in crude extracts compared with crystal-
line enzyme preparations as we previously reported (Ref. 8, Fig. 4).
The reason for this change is not known. However, our data indicate
that it occurs because ribulose-Pz Carboxylase in crude extracts is
50-85% as active as the crystalline enzyme, while ribulose-PZ Oxygen-
ase is only 25-35% as active in crude extracts as is the crystalline

enzyme, when each is measured on a per mg protein basis.

Kinetic Parameters of Ribulose-PZ Carboxylase/Oxygenase from Su/su,
su7su and Three Revertant Plants Recovered from Haploid 33.
Vmax and Km values far the crystalline enzyme from leaves of the

mutant Su/su, its wild-type sibling, su7su, and three revertant plants

84

isolated from haploid 33 (10) are presented in Table I. There were no
differences found in any of the kinetic parameters when comparing the
carboxylase/oxygenase from 33733 plants with the mutant 33733 or two of
the revertants. The only exception was with the revertant 33. This
plant has a chromosome number of 87.0 :_1l.l, is homozygous for 33, and
phenotypically variant, although it is still green (10). The enzyme
from the revertant 33 had the same apparent Km(COZ) and Km(ribulose-

P2) as did the wild-type 33733, but its Vmax for C02 was 50% lower.

This lower Vmax was observed in numerous preparations of the carboxylase
from leaves of_33, but because of enormous changes which we have seen in
the specific activity of the carboxylase/oxygenase from leaves of
different developmental stages, and under different growth conditions,
the reason for the apparently variant Vmax is uncertain. At present,
the increased ploidy level of_33, which is a nejor difference between
this plant and 33733, may be postulated to be related to the altered

Vmax of the carboxylase.

Physical Parameters of Ribulose-PZ Carboxylase/Oxygenase from 33733,
33733, and Three Revertants of Haploid 33.

Since our 33_tobacco plants had a high ploidy level and variant
ribulose-PZ Carboxylase activity, and since Garrett (6) had demon-
strated altered kinetic and physical parameters for the enzyme in rye-
grasses differing in ploidy level, several physical properties of
ribulose-P2 carboxylase/oxygenase from 33733 and 33733 were compared

with those of enzyme from the revertant 33.

.meewueeeemce mexnem

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85

 

 

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86

One hypothesis to explain Garrett's results is that the ratio of
large to small subunits may be different in the carboxylase/oxygenase
from the tetraploid plants as compared to the enzyme from diploid
plants. To examine this, the molecular weights of the holoenzymes from
several plants were compared on native gels(5% polyacrylamide). Enzyme
from all the tobacco plants migrated identically, indicating that the
molecular weights of the holoenzymes were the same (data not shown).
Carboxylase/oxygenase from tobacco leaves was dissociated with SDS and
the large and small subunits were separated by SOS-polyacrylamide gel
electrophoresis. The ratio of amount of large subunit to small subunit
was estimated based on the area under the respective protein peaks
obtained by scanning the gels at 600 nm after staining with Coomassie
blue. There were no differences in this
ratio for any of the plants (Table II). Quantifying the amounts of pro-
tein with this dye is not totally reliable, but our results are similar
to those reported by Kung et a1. (11) using three different methods.
Isoelectric focusing of the holoenzyme was also done under nondenaturing
conditions. Isoelectric points were identical for enzyme from all the
plants (data not shown).

The large subunit of the crystalline enzyme from tobacco leaves
dissociated into 2 to 3 bands when run on SOS-polyacrylamide gels with
or wfithout carboxymethylation. This occurred regardless of the method
used to purify the enzyme (crystallization (3,21) or the chromatographic
purification used by Ryan and Talbert (20)). Older preparations of the
enzyme from tobacco had a greater number of these anomalous bands.
Ribulose-Pz Carboxylase/oxygenase from spinach leaves exhibited only

one large subunit band.

87

Table 2

Protein Ratio of Large to Small Subunits of
Ribulose-PZ Carboxylase/Oxygenase

 

 

Plant Experiment Ratio£_
Spinach 3 3.01 i 0.25
su/su 1 3.13 i 0.24
3 2.97 1 0.16
R2 1 2.43 1 0.36
2 2.90 1 0.25
3 3.17 :_1.17
R3 2 2.81 i 0.15

 

1 Large and small subunits were dissociated with SOS-polyacrylamide
gel electrOphoresis on 10% gels. Gel scans were made at 600 nm and
ratios determined from relative areas under the peaks obtained.

Ratios are means t 5.0. of at least 2 detenninations.

88

Discussion

Crystalline ribulose-PZ carboxylase/oxygenase from the heterozy-
gous tobacco mutant 33733 and its wild-type sibling 33733 had identical
kinetic and physical parameters. Specific activities of enzyme prepara-
tions were examined weekly from a population of plants, over a genera-
tion, and found to be identical for both plants. Kung and Marsha (12)
have shown that there are no differences in the isoelectric points of
polypeptides of denatured enzyme from the two plants. Therefore, a
reevaluation of previous literature about the yellow 33733 tobacco
mutant is in order. Zelitch and Day (26) reported that heterozygous
mutant 33733 leaves had a higher rate of photorespiration than wild-type
.33733. However, Chollet (4) has questioned the validity of the light/
dark 14c02 assay of photorespiration which they used. Kung and
Marsha (12) reported differences in specific activity for both ribulose-
P2 Carboxylase and oxygenase, but they did not detail growth condi-
tions and plant age. As reported here, large differences in specific
activity can be attributed to differences in plant age, growth condi-
tions, and levels of soluble polyphenol oxidase activity in tobacco
leaves. Thus a comparison of ribulose-Pz Carboxylase/ oxygenase
preparations from 33733_with 33733 plants at different developmental
stages could produce widely different activities, whereas no change
actually would exist if the plants were grown under identical conditions
and assayed at developmentally equivalent ages. Reasons far changes in
total and specific activity of the enzyme during development are unknown
but can probably be accounted far by artifacts. Our data indicated

that a decrease in activity of the isolated enzyme correlated with an

89

increase in soluble polyphenol oxidase in the leaf extract. Addition of
PVPP to remove phenolic substrates does not remove the polyphenol
oxidase which may partially alter or inactivate the
carboxylase/oxygenase protein. However, no physical or kinetic change
was apparent in the isolated enzyme. Thus, it seems unlikely that a
change in ribulose-PZ Carboxylase/oxygenase is responsible for the 33
phenotype as Okabe has Speculated (18,19).

Properties of ribulose-PZ carboxylase/oxygenase were identical in
crystalline preparations from 2 phenotypic revertants isolated from
haploid 33 plants, which were homozygous far the mutant 33 gene (10).
For the enzyme from another revertant, 33, that had a high chromosome
number of 87.0 :_11.1, the Vmax for the carboxylase activity was about
half that for the enzyme from 33733. The other physical and kinetic
properties of the enzyme from this-33 revertant were similar to the
enzyme from 33733. In view of wide variations in activity of the enzyme
when isolated, the reasons for, and significance of, the lower Vmax in
this revertant will require further examination. Genetically it is
difficult to explain a kinetic change in an enzyme from a change in
ploidy level, but since the carboxylase is encoded in both the nucleus
and chloroplast, it could conceivably be modified by the nuclear ploidy
level. On the other hand, as demonstrated in this paper, enormous
variations in specific activity of the isolated enzyme nay be artifacts
of isolation. Gray et a1. (7) have shown that changed isoelectric
focusing patterns may be artifactual as well. To demonstrate an altered
ribulose-PZ Carboxylase/oxygenase, the change in kinetic pr0perties

must be correlated with changes in associated physiological processes,

9O

namely rates of photosynthesis, photorespiration and plant growth, as

attempted by Garrett (6) with ryegrasses of different ploidy level.

91

Literature Cited

Bensadoun A, and D Weinstein 1976 Assay of proteins in the presence

of interfering materials. Anal Biochem 70:241-250

Burk LG, and HA Menser 1964 A dominant aurea mutation in tobacco.

Tobacco Sci 8:101-104

Chan PH, K Sakano, S Singh, SG Wildman 1972 Crystalline fraction 1

protein: Preparations in a large yield. Science 176:1145-1146

Chollet R 1978 Evaluation of the light/dark 14c assay of photo-
respiration. Plant Physiol 61:929-932

Davis 80 1964 Disc electrophoresis-II method and application to

human serun proteins. Ann NY Acad Sci 121:404-427

Garrett MK 1978 Control of photorespiration at RuBP carboxylase/

oxygenase level in ryegrass cultivars. Nature 274:913-915

Gray JC, SD Kung, SG Wildman 1978 Polypeptide chains of the large
and small subunits of fraction 1 protein from tobacco. Arch

Biochem Biophys 185:272-281

Hall NP, PJ Koivuniemi, NE Talbert 1978 The ratio of RuBP
carboxylase to RuBP oxygenase in crude and purified preparations

from tobacco. Plant Physiol 615:99

10.

11.

12.

13.

14.

15.

92

Horecker BL, J Hurwitz, A Weissbach 1958 Ribulose diphosphate.
Biochem Prep 6:83-90

Koivuniemi, PJ 1980 Isolation and characterization of green
revertant plants from the dominant tobacco aurea mutant 33;
Genetic, physiologic and enzymologic studies. Ph.D. thesis.

Michigan State University, E. Lansing, MI 48824

Kung SD, K Sakano, SG Wildman 1974 Multiple peptide composition of

the large and small subunits of Nicotiana tabacum fraction 1

 

protein ascertained by fingerprinting and electrofocusing. Biochim

BiOphys Acta 365:138-147

Kung SD, TV Marsha 1976 Regulation of RuBP carboxylase/oxygenase
activity and its relationship to plant photorespiration. Nature

259: 325-326

Linsmaier DH, F Skoog 1965 Organic growth factor requirements of

tobacco tissue cultures. Physiol Plant 18: 100-127

Loomis WD 1974 Overcoming problems of phenolics and quinones in
the isolation of plant enzymes and organelles. Methods Enzymol

31A:528-544

Lorimer GH, MR Badger, TJ Andrews 1977 D-ribulase-1,5-bisphospate
carboxylase-oxygenase: Improved methods for the activation and

assay of catalytic activities. Anal Biochem 78: 66-75

16.

17.

18.

19.

20.

21.

22.

92- 5

Mayor AM, E Harel 1979 Polyphenol oxidases in plants.
Phytochemistry (Oxf) 18:193-215

O'Farrell, PH 1975 High resolution two-dimensional electrOphoresis

of proteins. J Biol Chem 250:4007-4021

Okabe K 1977 Pr0perties of ribulose diphOSphate carboxylase/

oxygenase in the tobacco aurea mutant Su/su Var. Aurea. Z

 

Naturforsch 32c:781-785

Okabe K, GH Schmid 1978 Pr0perties of the tobacco aurea mutant
Su/su var. Aurea. on photorespiration and on the structure and
function relationship in chloroplasts. In G Akoyunoglou, JH
Argyroudi-Akoyunoglou, eds, Chloroplast Development, Elsevier/

North-Holland Biomedical Press, Amsterdam, pp 501-506

Ryan FJ, NE Talbert 1975 Ribulose diphosphate carboxylase/
oxygenase III. Isolation and properties. J Biol Chem

250:4229-4233

Sakano K, JE Partridge, LM Shannon 1973 Absence of carbohydrates
in crystalline fraction 1 protein isolated from tobacco leaves.

BiOChim BiOphys Acta 329:339-341

Singh S, SG Wildman 1973 Chloroplast DNA codes for the ribulose

diphosphate carboxylase catalytic site on Fraction 1 proteins of

Nicotiana species. Mol Gen Genet 124:187-196

23.

24.

25.

26.

93

Tolbert NE l973 Activation of polyphenol oxidase of chloroplasts.
Plant Physiol 51: 234-244

Umbreit ww l972 Constant volume manometry-the "Warburg." In uw

Umbreit, RH Burris, JF Staffer, eds, Manometric and Biochemical

 

Techniques, Ed 5 Chap 1. Burgess, Minneapolis, pp l-19

 

Weber K, JR Pringle, M Osborn 1972 Measurement of molecular
weights by electrophoresis on SOS-acrylamide gel. Methods Enzymol
26:3-27

Zelitch 1, PR Day l968 Variation in photorespiration. The effect
of genetic differences in photorespiration on net photosynthesis in

tobacco. Plant Physiol 43: 1838-1844

CHAPTER III

Characterization of the Thylakoid Membranes of the Tobacco Aurea

Mutant Sufisu and ofThree Green Revertant Plants

Paul J. Koivuniemi , N.E. Tolbert and Peter S. Carlson

Program in Genetics, Department of Biochemistry and Department
of Crop and Soil Sciences, Michigan State University,

East Lansing, Michigan 48824

Manuscript-received:

94

1.

95

This research was supported by National Science Foundation Grants
PCM 78-l5891 to N.E.T. and AER 75-20882 to P.S.C. and is published
as Journal Article No. of the Michigan Agricultural Experiment

Station.

This paper is part of a Ph.D. thesis by P.J.K. in the Program in

Genetics.

96

ABSTRACT

Phenotypic characterizations of the semidominant aurea tobacco

(Nicotiana tabacum L.) mutation Su/gu, the homozygous mutant §u[§u and 3

 

green revertants (_1,.3g, and 3;) are presented. The leaf color pheno-
type of Sufisu varies from yellow to light-green when grown under high
and low light intensity conditions respectively. The change in visual
phenotype under high light intensity conditions is correlated with
decreased content of chlorophyll per leaf area, agranal chloroplast
ultrastructure, changes in the number of chlorOphyll-protein complexes,
and absence of one or more of the light harvesting chlorophyll-
polypeptides of 25,000-29,000 Daltons. The homozygous mutant grown
under low light intensity was shown to be completely lacking in grana
stacks and to be deficient in chlorOphyll-protein complexes. Revertant
Rl_was found to be identical to wild-type plants in all parameters
examined (visual phenotype, chloroplast ultrastructure, chlorophyll-
protein complexes, chlorophyll-protein complex polypeptides) except in
chlorophyll content. Rl_did not show an increased chlorophyll and
carotenoid content as did the wild-type plants when exposed to high
light. The other 2 revertants, Rg_and 3;, were similar to the heterozy-
gous mutant §g[§u in most of the parameters examined. They yellowed due
to a loss of chlorophyll and an increase in the amount of carotenoids,
had agranal chlorOplasts, and had variant chlorophyll-protein complexes
when grown under high light intensities. However, each appeared to con-
tain some of the light harvesting pigment-protein complex polypeptide(s)
found to be absent in §u[§u when grown under high light intensity

conditions.

97

INTRODUCTION

 

The sulfur mutation IE9) is a semidominant tobacco aurea mutation
first described by Burk and Menser (1964). Schmid and coworkers (see
Schmid, l97l) have done extensive ultrastructural and physiological
analyses of both the heterozygous mutant Sngu and its wild-type sibling
‘§_[§u. They have shown that the heterozygous mutant plants lack the
grana lamellae characteristic of nonnal green plants (Schmid, et al.,
1966; Schmid and Gaffson, l964; Homann and Schmid, l967) and that they
also bleach under high light, and green under low light intensity
conditions (Schmid, l967).

Recently, Remy and coworkers (l974,l976) have reported that the
heterozygous mutant SuAsu contains only small amounts of the light
harvesting pigment-protein complex, which is thought to be associated
with photosystem II (Arntzen, 1978).

We have isolated several revertants of the Su mutation (Koivuniemi,
et al., 1980b), and have done extensive characterization of the primary
C02 fixing enzyme, ribulose-l, S-biSphosphate carboxylase/oxygenase,
from these plants since the enzyme had been reported to be variant in
the heterozygous mutant, §u[§u, (for a review, see Koivuniemi, et al.,
1980a). As a continuation of the characterization of 3 of the revertant
plants, we now report the analysis of the phenotypes of the revertants,
the heterozygous mutant SuLsg, the wild-type.§u[§g, and also a partial
characterization of the homozygous mutant Su/Su, The analysis is
described in a reductionistic manner beginning with a characterization
of the plants' visual phenotypes and continuing through (a) measurement

of pigment complement, (b) examination of chloroplast ultrastructure,

98

(c) analysis of chlorophyll-protein complexes, and (d) analysis of
chlorophyll-protein complex polypeptides. Particular emphasis is placed

on the effects of high and low light intensities on these parameters in

the various genotypes.

99

MATERIALS AND METHODS

 

Plant Material

 

Plants used included the semidominant aurea mutant of Nicotiana

 

tabacum L., §_[§u, first described by Burk and Menser (l964), the 2
homozygous siblings, Su/Su (yellow lethal mutant) and §u[§u (green
wild-type), and 3 revertant clones of plants derived from haploid mutant
§u_plants which are designated as 31) Rg_and 33, For a description of
the origin of the revertant plants and growth conditions used for all

plants, see Koivuniemi, et al. (l980a, l980b).

Light Conditions

 

Plants used for these experiments were grown under 3 different
light conditins. High light intensity plants were grown in the growth
chamber at 22°C on light regimes of l6 h light and 8 h dark at a light
intensity of 330 uE m'2 sec'l. Most plants referred to as low
light intensity plants were grown in the greenhouse in Michigan during
the whnter under the prevailing natural light conditions supplemented
with artificial light to achieve a 16 h light 8 h dark photoperiod.
Some control plants were grown in the growth chamber under conditions
identical to the high light conditions except that the light intensity
was adjusted to 32 uE m'2 sec'l.

Pigment Measurement
Pigments were extracted from leaf tissue in 80% acetone.

Chlorophyll content was calculated according to the equations of Arnon

lOO

(l949) and carotenoid content was calculated according to the method of

Lianen-Jensen and Jensen (l97l).

Electron Microscopy

 

Leaves to be examined by electron microscopy were washed with water
and fixed with gluteraldehyde in phosphate buffer (pH 7.2) for 2.5h at
4°C and then postfixed in 2% 0504 for 16h. The tissue was dehydrated
in a graded ethanol series and embedded in Spurr's (l969) resin. Silver
sections were cut on a Porter-Blum ultramicrotome and stained with 2%
uranyl acetate followed by 0.2% lead citrate. Photomicrographs were

made on a Phillips 84 200 transnission electron microscope.

Chlorophyll-protein Complexes

 

Chlorophyll-protein complexes were prepared and run on SDS-
polyacrylamide gels according to the method of Markwell et al. (l978).
Five grams of washed-deveined leaf tissue was homogenized unth a mortar,
pestle, and sand in a medium containing 400 mM mannitol, 20 mM Tricine
(pH 7.6) and 10 mM NaCl and then filtered through a single layer of
Miracloth. The homogenate was centrifuged briefly at 270 x g to remove
whole cell debris, and the supernatant was centrifuged at ll,000 x g fbr
8 min. The chloroplast pellet was resuspended in buffer containing 50
"M Tris (pH 8.0) and l ”M EDTA and centrifuged at 19,000 x g for l0 min.
This step was repeated once with the pellet, and an aliquot was set
aside for chlorophyll detennination. The pellet was resuspended in
buffer containing 6.2 nM Tris (pH 8.3), 48 mM glycine and l% (w/w) SDS
at a ratio of $05 to Chl (w/w) of lO/l. The manbrane pellet was

homogenized and then centrifuged at 25,000 x g for l5 min.

lOl

Insoluble material was discarded and the SDS-extracted supernatant
material was made l0% v/v with glycerol. Aliquots containing 10-40 ug
of chlorophyll were loaded on gels consisting of 5% polyacrylamide.
Gels were run at room temperature for 60 to 80 min at l00 volts. The
gel reservoir buffer contained 6.2 mM Tris (pH 8.3), 48 mM glycine, and
0.l% SDS.

Chlorophyll-Protein Complex Polypeptides.

The SDS-extracted sample from above was either boiled for 2 to 3
min and loaded on gels or extracted with 5 volumes of acetone, and then
centrifuged at 5,000 x g for 3 min to produce a protein pellet. The
pellets were suspended in a sample buffer containing 0.025 M Tris-HCl
(pH 7.0), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.00l% bromo-
phenol blue, at a 10 to 1 ratio (w/w) $05 to chlorophyll. The equiva-
lent of 5 to 30 ug of chlorOphyll were loaded on discontinuous SDS-
polyacrylamide slab gels (5 mm thick) prepared according to the proce-
dure of Laemmli (l6). Gels were run for l.5h at 80 volts and then at
l80 to 200 volts until the tracking dye reached the bottom of the gels.
Gels were fixed and stained overnight in 0.l% Coomassie Blue R, 5% (v/v)
acetic acid and 20% (v/v) methyl alcohol, and then destained in a

solution containing 5% (v/v) acetic acid and 20% (v/v) methyl alcohol.

102

RESULTS

Table 1 contains a summary of the parameters investigated and of
the results. As described in the materials and methods section, plants
were grown in the greenhouse at a relativley low light intensity during
the winter and also in a growth chamber at a high constant light

intensity of 330 uE m"2 sec'1 over a l6 h day.

Pigment Complement

 

Under winter greenhouse conditions of relatively low light inten-
sity, the heterozygous mutant Sufisu had approximately half the chloro-
phyll and carotenoid of its wild-type sibling §u[§u (Table 2). The
relative proportions of chlorophyll a to chlorophyll b and the ratio of
total chlorophyll to total carotenoids were the same. This was consis-
tent with the light green color of the mutant under those growth condi-
tions. However, under the relativley high light intensity conditions in
the growth chamber, the mutant plant bleached considerably to its
characteristic yellow-green color. The change in visual phenotype
resulted from a 3-fold increase in the amount of carotenoid on a leaf
area basis and from a decrease in the amount of total chlorophyll,
primarily a 50% reduction in the amount of chlorophyll b. In contrast,
both chlorophyll g_and b and total carotenoids increased simultaneously
in wild-type plants and their visual phenotype remained unchanged (Table
2).

The 3 revertants responded to the change in light intensity to
varying degrees but similarly to the mutant in each case. 3;, which has

been shown to be a closely linked, second site revertant (Koivuniemi, et

103

 

 

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106

al., l980b), was virtually indistinguishable from wild-type when grown
in the greenhouse in winter (Table 1). However, under high light inten-
sity conditions, it did not increase its pigment complement on a leaf
area basis as did the wild-type plants. There was only a small decrease
in the amount of chlorOphyll b in RI, but other than that, it remained
stable in both its visual and quantitative pigment phenotype (Table 2).

32, which is most likely a polyploid maintaining the SE gene
(Koivuniemi, et al., 1980b), was green when grown under low light inten-
sity conditions, although not as green as wild-type plants. It bleached
under high light conditions, but not as severely as did §u[§u. Unlike
.§u[§u,.32 showed a decrease in the amount of chlorophyll a rather than
chlorOphyll b, coupled with a small increase in the amount of total
carotenoid, which accounts for its relatively less severely bleached
phenotype (Table 2).

33, which is most likely monosomic for chromosome S and carrying
the S3 gene on that chromosome (Koivuniemi, et al., l980b), was most
similar to the Sufigu plants. Under greenhouse conditions, §u[§u and 33
were virtually indistinguishable in terms of color (although as would be
expected with a monosomic, there were morphological differences). High
light intensity conditions induced pronounced bleaching in R§_plants
which was caused by a reduction in the amounts of both chlorophyll g_and
.2 and a slight increase in the amount of total carotenoids (Table 2).
Like 32, R§_had a lower chlorOphyll.a[b ratio when grown in the growth
chamber than did §u[§u, thus a relatively greater loss of chlorOphyll a

was found in both revertants.

107

Chloroplast ultrastructure

 

All plants used for this section were grown under greenhouse condi-
tions. The only exception was the homozygous mutant §u[§u and Sufsu and
._g[§_ control plants which were grown heterOphically under aseptic
conditions for some experiments.

Observations made on wild-type and the heterozygous mutant showed
ultrastructural characteristics identical to those seen by Schmid, et
al. (l966). Grana stacks in wild-type plants were typical of those
found in higher green plants, while SgLsu had incomplete stacking and
only occasional evidence of a few (2-8) appressed membrane configura-
tions. The amount of grana stacking in chloroplasts of Sufsu plants
varied somewhat depending on light intensity; however, under no
conditions were chloroplasts seen with a completely normal
ultrastructure.

In the revertants, chloroplast ultrastructure correlated well with
the visual phenotype of the revertant plants and quantitative determina-
tions of their pigment complement on a leaf area basis as presented in
Table 2. As would be predicted from those results, 3; had a normal
chloroplast morphology (Fig. 1). Its grana lamellae were often seen in
stacks consisting of l5 to 20 appressed membranes which appeared to be
identical to wnld-type grana (Schmid, et al. l966).

ChlorOplasts from 32 plants showed variation in ultrastructure. As
was indicated by their chlorophyll content, they did have some stacking
of the lamellae into grana, sometimes consisting of as many as l0
membranes (Fig. 2). However, the grana were most often not nearly so
well defined and consisted of dispersed single thylakoids of 2 to 3

membranes appressed over most of the length of the chloroplast (Fig. 2).

108

Aoco.m¢ xv .5: omm AFmsam
emu .anums ummcmsmpqqsm mmoeusm m :o AFPuowzoeuoemum;

czoem =m\mw.ucmpzs ezwpzm Anoma~osoc me» see; umwpaoeopsu

 

mooo.mm xv .5: cm, mpmacmlemm .mcowuwvcoo mmzozcmmea
emuc: x_Pmow;Qoemou:m :Zoem mm ucmuem>me Eoee umopaoeoFsu

Aooo.m¢ xv .5: omm m_m:cm emm .mcowpwucoo mmaoccmmem
emmca x__mowsaoeuou:m czoem Mm.mcmuem>me seem Ame—aoeopsu

Aooo.mm xv .E: om_ mpmavmlemm .mcowuwucou mmaogcmmem
emucs a—_mow;aoepo=um czoem Hm pcmuem>me seem ummpaoeopzu

.e meamwu

.m meamwm

.N mezmwm

._ mezmwm

109

 

110

Additionally,_32 chloroplasts contained a large number of apparently
membrane bounded vesicles of varying shapes and sizes (Fig. 2) and
unknown origin. There was some indication that they arose from the
inner portion of the chloroplast envelope (Fig. 2 arrow).

Chloroplasts from revertant R§_were essentially identical in their
appearance to §u7§u chlorOplasts (Fig. 3 and Ref. 7), as would be
predicted from pigment analysis (Table 1). The lamellae were fbund
mostly as single structures, with occasional appression of 2 to 5
membranes into what could be termed rudimentary grana. Unlike R2 no
inclusion vesicles were found and with the exception of the lack of
grana, the chloroplasts had a normal appearance.

Figure 4 shows a chlorOplast from a homozygous mutant plant (§_/§u)
grown heterotrophically. Its leaves were pale yellow in appearance
whereas sufisu controls, grown under identical conditions had normal
green leaves and chloroplasts with typical grana stacks (data not
shown). The homozygous mutant was seen to be completely lacking in
grana stacks with only occasional overlapping of 2 lamellae (Fig. 4)
which is similar to heterozygous plants grown under high light
intensities. Large numbers of mitochondria were in close proximity to
the SufiSu chloroplasts (Fig. 4). This confirms an observation by
Schmid and Gaffron (l967) on the heterozygous Su/su genotype. The Su/Su
plants bleached to a completely albino condition and died when grown
heterotrOphically under growth chamber conditions (i.e. high light

intensity).

111

Chlorophyll:protein complexes

 

An initial effort was made to determine if there were any differ-
ences in the chlorophyll-protein complexes of the sibling genotypes
§_[§u (wild-type), Su/su (heterozygous mutant), and SulSu (homozygous
mutant). Since the homozygous mutant is lethal under autotrophic growth
conditions, all 3 genotypes were grown under identical heterotrophic
conditions (Koivuniemi, et al., 1980b). Chlorophyll-protein complexes
(detected as green bands) were separated on SDS-polyacrylamide gels from
equal fresh weights of each of the 3 plant types. As shown in Figure 5,
the wild-type and heterozygous mutant were almost identical in the
content and characteristics of their respective chlorophyll- protein
complexes. With the homozygous mutant on the other hand, only an
amorphous smear of chlorOphyll was seen when the complexes were
solubilized at a ratio of $05 to chlorophyll of l0/1 (w/w).

ChlorOphyll-protein complexes were also isolated from the 3 rever-
tants Bl, Bg_and.33. However, since these plants grow autotrophically,
it was possible to grow them under both high and low light intensity
conditions (i.e. growth chamber and greenhouse respectively). The
greenhouse grown plants had nearly normal chlorophyll-protein compelxes
(Fig. 6). However, when placed under growth chamber conditions of
higher light intensity, 2 of the revertants, 3g and 33, and the
heterozygous mutant Sufisu, all had patterns on SDS-polyacrylamide gels
identical to the homozygous mutant §u7§u (Fig. 5). That is, there were
no clearly defined chlorophyll-protein complexes and most of the
chlorophyll seemed to be bound as a high molecular weight aggregate
which barely ran into the gel. Revertant Rl_did not show this behavior,

and had a complement of chlorOphyll-protein complexes identical to that

112

 

 

 

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can _\op mo __»;goeo~;m\mom eo oepme m cw um~___n:_om memz
mmxmpgaoollnwm:ozcmmem mgp cw czoem mucmpa Ammyme maxp-cpwz
Auv use :m\=m accuse maomeoemum; on .mm ucmuem>me Any .mm
ucmpem>me Amv seem umum_omw mmxmpaeoo :wmuoea-Fpacaoeopgu

.mapm mwmmmEoou ;u_z cwmuoeq

eoe cmcwmum :mmn m>ms mmxm_QEou ._\o_ mo __»;noeo_;u\mom mo
owume m cw um~___n:—om memz mmxm_qeou .E:_ume umucmem_aa:m
mmoeosm a co a_Fwo_;aoeuoemumg czoem mucmpn Amw\mwv pecans
maomANoEo; on ucm .Amm<mwQ accuse mzomx~oempmc Anv .Ammwmmv
qup-u_wz Amv Eoee vmum_0m_ mmxm_quo :_muoea-F~»caoeo~;u

.o meamwe

.m meamwm

113

 

114

of wild-type. The result outlined in this section held whether the gels
were stained for proteins with Coomassie blue or if the complexes were
identified as chlorophyll bands. Distinct proteins bands were visible
in all cases where distinct chloropyll bands were discernible. However,
in cases where chlorophyll had only smeared into the gels, staining fOr
proteins showed a pattern similar to that seen for rocket
immunoelectrophoresis, and identical to the pattern seen for
chlorophyll.

Three hypotheses were considered to explain these observations:
(a) The homozygous mutant plants (SuASu), and heterozygous mutant S2753
and 2 revertants 32, and R§_(when grown under high light intensity
conditions), might not have contained well defined chlorophyll-protein
complexes; (b) The ratio of chlorophyll/protein could have been
abnormal, with much more protein present relative to the amount of
chlorOphyll resulting in incomplete solubilization of the complexes
from the membrane milieu; or, (c) The combination of (a) and (b) could
also be responsible.

To examine this, §u7§u plants were grown in the growth chamber;
half were grown at 330 uE m"2 sec'1 and the other half at 32 uE
m'2 sec'1 (high and low light intensity respectively). The
chlorophyll-protein complexes isolated from high light intensity grown
.EEKEE plants were solubilized with an $03 to chlor0phyll ratio of 30/l
(w/w) rather than the usual 10/l ratio. The higher SDS/chlorophyll
ratio partially solubilized the chlorophyll, thus indicating that the
ratio of protein to chlorophyll was higher in these cases and that the

chlorophyll-protein association was abnormal.

115

However, even this treatment with higher 505 concentrations did not
lead to resolution of chlor0phyll bands on gels of dissociated thyla-
koids from Sufsu plants grown under high light intensity conditions.
Staining these same gels with Coomassie Blue indicated the presence of
an aberrant protein- complex pattern which was not visible using

chlor0phyll as a marker (Fig. 7).

Chlorophyllsprotein complex polypeptides

 

Since the homozygous mutant Sufigu (as well as the heterozygous
mutant Suf§u_and the 2 revertants 32_and R§_when grown at high light
intensity) showed aberrant chlorophyll-protein complexes, an attempt was
made to determine whether or not the individual polypeptides comprising
the holo-complexes were present in these plants. Initially,
chlorOphyll-protein complex samples, prepared as above, were boiled for
2 to 3 minutes prior to running them on discontinuous SDS polyacrylamide
gels. Nith boiling, it was possible to demonstrate that several of the
polypeptides present in wild-type plants (sufisu) were also present in
Sngu and revertant plants grown at high light intensities. However,
there was still a large amount of apparently nonspecific binding of
Coomassie blue to the tracks containing extracts from each of these
abberrant plants. Therefore, chlorophyll was extracted from each of the
samples with approximately 5 volumes of acetone. The acetone precipi-
tated polypeptides were then solubilized in sample buffer and boiled for
3 to 4 minutes. The acetone extraction procedure removed some of the
background binding of stain, though not all. Enough was removed to
demonstrate that most of the polypeptides present in wild-type plants

were also in Sgfisu and the Rg_and R; revertants. There were major

116

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mecca AAxceoeoAco oc mmeeome meAm mAmmeeoou IAAz cAoneq

eOA echeAm cmme m>eI mmxmAeEou .oAAee A\oA Anew: mcu can»

emcuee A\om Ao AAzccoeoc0\mom Ac oAAee e chm: emNAAAeerm

memz mmAAAmcmAcA AcmAA cmAI emece zoem mAceAe :J\:m

EoeA mmxmAanu .zAm>AAomamme e ece my mcoAAAecoo msem mcp

emece czoem muceAq uceAee meemz~oemumc ece - xAm>AAomcmme
-omm -5 wnmm ece -omm N-E m: omm

mcoAAAecoo qumcmAcA AcmAA Amv 30A ece An“ cmAc emece czoem

mAceAe maxA-eAAz eeeA emAermA mmxmAeEou cAonec-AAxccoeoAIu

.xmAchu cAonec-AcmEmAa chumm>eeI -AcmAA

UII .A-omm N- E mcmm Am czoem mem: I =J\:J ece I

:J\:m ece -omm E mnomm ue czoem memz I eJ\=J ece

I :J\:m .meocpmz ece mAeAemAez cA emeAeomme we mmxmAQEou
cAonec- AAAIcoeoAcu mIA soeA em~AAAeerm mmeAAemcaAoc

.A meemAm

.w meemAe

117

l I I

 

 

118

differences in the polypeptides of the heterozygous mutant §u7§u
however; at least 2 polypeptides of approximately 25,000-29,000 MW were
completely absent (Fig. 8). These polypeptides have been identified as
light harvesting chlorOphyll-proteins (Boardman, et al., 1978). The
polypeptides were present in varying amounts in all 3 revertants when
they were grown under the same high light intensity conditions. When
polypeptides from Su7§u plants grown under low light intensity condi-
tions were prepared in the same manner, the missing polypeptides were
found to be present. Preliminary results indicate that these

polypeptides may be absent in the homozygous mutant Su7§u as well.

119

DISCUSSION

 

Schmid (1967,1971) has demonstrated that the heterozygous tobacco
aurea mutant_§u[§u varies in phenotype depending on the light intensity
at which the plants are grown. Our data confirm these reports and also
demonstrate that the 3 green revertants which we have recovered from
haploid §u_somatic tissue (Koivuniemi, et al., l980b) respond similarly
to their environment, but to varying degrees. The second site revertant
31 responded only slightly and was only abnonnal in that it did not
increase its pigment complement as did the wild-type suAgg. .Rg_and R;
behaved more like the heterozygous mutant §u[§u, losing some of their
chlorOphyll and increasing their carotenoid content under high light
intensity.

The chloroplast ultrastructure as viewed by electron microscopy
followed the same pattern. Rg_and R§_had defective grana stacking, even
when grown at low light intensities, while 3; had grana lamellae
identical to those found in wild-type plants. We have also shown that
homozygous mutant Su/Su plants had chloroplasts completely lacking in
grana stacks as would be predicted from their pale yellow color.

Chlorophyll-protein complexes were affected in a parallel manner in
all the non-wild-type plants examined. The homozygous mutant Su7§u had
no distinct chlorOphyll-protein complexes under SDS-solubilization
conditions where both wild-type, suAgu, and heterozygous mutant §u7§u
were seen to have essentially normal patterns. The revertants, 31, 32_
and 33, also had essentially normal complements of chlorophyll-protein
complexes under low light intensity growth conditions. However, under

high light intensity conditions, 3g and 53, along with the heterozygous

120

mutant_§u[§u, did not show discrete complexes under the lO/l (w/w) $05
to chlorophyll ratio used for solubilization. It was found, however,
that an increased SDS/chlorophyll ratio released the complexes and that
most of the polypeptides present in wild-type chlorophyll-protein
complexes were also present in the plants which were subject to high
light intensity stress. However, at least two major polypeptides of ca.
25,000-29,000 MW were completely absent from the heterozygous mutant
under high light conditions but present under low light conditions.

The evidence presented here and in our previous paper on the
ribulose-P2 Carboxylase/oxygenase enzyme (Koivuniemi, et al., 1980a)
is contrary to the hypothesis of Kung and Marsho (1976) and Okabe and
Schmid (l977, 1978) that the SE mutation primarily affects the
ribulose-l,S-bisphosphate carbosylase/ oxygenase. Our evidence points
to the existence of a strong affect of the SU mutation on the light
harvesting chlor0phyll-protein. We have been able to trace this pheno-
type through 5 levels of increasingly more detailed analysis. These
are: (a) the yellow-green visual appearance of the mutant, (b) the
quantitative determination of differences in pigment complement of the
mutant plants, (c) the defective ultrastructural appearance of the
mutant chloroplasts (agrana), (d) the apparent abberant chlorophyll-
protein complexes and (e) the absence of at least one major polypeptide
associated wnth the light harvesting pigment- protein complex when
heterozygous SuLsu plants are grown under high light intensity.
However, there is no certain one to one link between the §E gene and
this polypeptide. We have demonstrated that there is a large environ-

mental impact on the production of the phenotype at all levels of

121

observation, and that the polypeptide differences may only reflect a
pleiotropic affect of Sp. One has, nevertheless, a much more discrete

and quantifiable phenotype than plant color.

122
REFERENCES

 

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Arntzen, C.J.: Dynamic structural features of chloroplast
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Boardman, N.K., Anderson, J.M., Goodchild, D.J.: Chlorophyll-
protein complexes and structure of mature and developing
chloroplasts. Curr. Top. Bioenerg. 8, 35-109 (1978)

Burk, L.G., Menser, H.A.: A dominant aurea mutation in tobacco.
Tobacco Sci. 8, 101-104 (1964)

Homann, P.H., Schmid, G.H.: Photosynthetic reactions of
chloroplasts with unusual structures. Plant Physiol. 42, 1619-1632
(1967)

Koivuniemi, P.J., Tolbert, N.E., Carlson, P.S.: Ribulose-l,
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Koivuniemi, P.J., Carlson, P.S., Tolbert, N.E.: Isolation and
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Kung, S.D., Marsho, T.V.: Regulation of RuBP carboxylase/oxygenase
activity and its relationship to plant photorespiration. Nature
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Laemmli, V.: Cleavage of structural proteins during the assembly

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Lianen-Jensen, S., Jensen, A.: Quantitative determination of
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Markwell, J.P., Reinman, S., Thornber, J.P.: Chlorophyll-protein
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Suf§u_Var. Aurea. on photorespiration and on the structure and

 

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Schmid, G.H.: Origin and prOperties of mutant plants: yellow
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SUMMARY

It is possible to recover true genetic revertants from vegetative
(leaf) tissues of plants, although many of the apparent revertant spots
are caused by changes in chromosome number rather than by an event in a
particular DNA gene sequence.

The enzymes, ribulose-1,5-bisphoshate carboxylase/oxygenase and
polyphenol oxidase are identical in the heterozygous mutant Su7§u, 3
green revertants (_1, 5g, and 33), and wild-type §u7§u with the excep-
tion of 3g which had a Vmax for the carboxylase activity approximately
half that of the other enzymes. Other variations in ribulose-P2
carboxylase/oxygenase activity correlated inversely with increases in
the activity of polyphenol oxidase which was higher in older plants and
in plants grown under high light intensity conditions.

The change in the §Ef§2 visual phenotype from light-green to yellow,
associated with growth at low and high light intensity conditions
respectively, can be followed through an increasingly more detailed
analysis. The yellow color correlates with a decreased chlorophyll
content, agranal chloroplast, aberrant chlorophyll-protein complexes and
finally with at least two missing polypeptides associated with the light
harvesting chlorOphyll-protein complex. The 3 revertants mimic this
phenotype to the extent predicted by changes in visual phenotype induced
by similar environmental conditions. The homozygous mutant §u7§u_shows
the same phenotype as Su/su grown under high light even when grown under

low light intensity conditions.

125

126

Therefore, the §! mutation does not primarily affect the enzyme
ribulose-1,S-bisphosphate carboxylase/oxygenase, but it has a strong

affect on the light harvesting chlorophyll-protein complex.

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