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This is to certify that the

dissertation entitled

STUDIES ON THE BIOSYNTHESIS
AND ACTION OF ETHYLENE

presented by
Anthony B. Bleecker
has been accepted towards fulfillment

of the requirements for

Ph.D. degreein Botany and Plant
Pathology

Lie?“ 4 ALL—L

Major professor

Date Eebruary 25, 1987

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

 

 

 

'MSU

 

 

RETURNING MATERIALS:
Place in book drop to

 

 

 

 

 

LJBRARJES remove this checkout from
.—;‘—-. your record. FINES will
be charged if book is
returned after the date
stamped below.
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7 9 522:

 

STUDIES ON THE BIOSYNTHESIS AND ACTION OF ETHYLENE

By

Anthony B. Bleecker

A DISSERTATION

Sumbitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1987

ABSTRACT
STUDIES ON THE BIOSYNTHESIS AND ACTION OF ETHYLENE

By
Anthony B. Bleecker

l-Aminocyclopropane-l-carboxylate (ACC) synthase (S-adenosyl-L-
methionine methylthiodenosine-lyase; EC 4.4.1.14), extracted from tomato
pericarp tissue, was purified 6500-fold by conventional and high-
performance liquid chromatography. Two-dimensional gel electrophoresis
of this preparation indicated that ACC synthase activity was associated
with a protein band at 50 kDa, a value consistent with size
determinations by gel filtration. Monoclonal antibodies (MAb) against
ACC synthase were obtained from murine hybridoma cell lines. These MAbs
recognized the native enzyme, as shown with an immunoprecipitation assay.
Immunoaffinity purification of ACC synthase with a Sepharose gel coupled
to either of two different MAbs yielded a 50 kDa polypeptide as shown by
sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An
enzyme-linked immunosorbent assay (ELISA) capable of detecting 1 ng of
antigen was developed. The ELISA system was used to demonstrate that two
of the MAbs recognize different epitopes on the ACC synthase protein.
Hound-induced increases in ACC synthase activity in tomato fruit tissue

were correlated with changes in ELISA-detectable protein. In-vivo

 

labeling of wounded tissue with [35$]methionine followed by extraction
and immunopurification in the presence of various protease inhibitors

yielded a single major radioactive band that migrated at 50 kDa with

SDS-PAGE. Pulse-labeling with [35$]methionine at various times after
wounding indicated that the wound-induced increase in ACC synthase
activity involved g3 Egyg synthesis of a rapidly turning over 50 kDa
polypeptide.

Partial submergence of deepwater rice (Oryza sativa L. cv. Habriganj

 

Aman II) elicited 3 responses: enhancement of internodal elongation,
inhibition of leaf growth and promotion of adventitious root formation.
All three responses could be induced in isolated stem sections by
treatment with ethylene. Dose-response curves indicated that the
responses were linearly related to the logarithm of the ethylene
concentration over 2 orders of magnitude. Application of 2,5-
norbornadiene (N80) to ethylene-treated sections resulted in a parallel
shift in dose-response curves to higher ethylene concentrations. When
NBD was applied to GA3-treated sections, increased ethylene production
resulted. The increase in ethylene production was reversed by applying
either ethylene or propylene, indicating a negative feedback control of
ethylene synthesis in rice stems.

A comparative anatomical study of internodes isolated from air-grown
and partially submerged deepwater rice plants was undertaken to localize
and characterize regions of growth and differentiation in the stem.
Microscopic analysis revealed three zones of internodal development: a
zone of cell division at the base of the internode, a zone of cell
elongation above the division zone, and a zone of cell differentiation
without cell elongation. The effects of submergence were a threefold
increase in the rate of cell division, an expansion of the cell

elongation zone and a suppression of tissue differentiation.

ACKNOWLEDGEMENTS

I would like to thank all the members of the PRL, including
Faculty, Staff and fellow Graduate Students for all their help and
support during the course of my work here. I would particularly like to
thank the members of my committee for their input into my research.
Special appreciation goes to: Gail Robinson, who was responsible for
much of the technical work on ACC synthase; Jane Schuette, who performed
the tissue sectioning and staining of deepwater rice; Shauna Somerville,
whose help with the MAb technology was invaluable; Dawn Kirby who helped
in the preparation of this manuscript. Finally, I would like to thank
my advisor, Hans Kende, whose insight into ethylene biology provided the

foundation for the work presented here.

ii

TABLE OF CONTENTS

LIST OF FIGURES ............................ v
LIST OF TABLES ............................ vii
LIST OF ABBREVIATIONS ........................ viii
GENERAL INTRODUCTION .......................... 1
CHAPTER I. USE OF MONOCLONAL ANTIBODIES IN THE PURIFICATION AND

CHARACTERIZATION OF l-AMINOCYCLOPROPANE-l-CARBOXYLATE SYNTHASE,

AN ENZYME IN ETHYLENE BIOSYNTHESIS ................... 4
I. Abstract .......................... 5
11. Introduction ........................ 6
III. Materials and Methods .................... 7
IV. Results ........................... 13
V. Discussion ......................... 23
VI. References ......................... 26

CHAPTER II.

THE ROLE OF PROTEIN SYNTHESIS IN THE REGULATION OF WOUND-

INDUCED I-AMINOCYCLOPROPANE-l-CARBOXYLATE SYNTHASE IN TOMATO FRUIT

TISSUE INVESTIGATED WITH MONOCLONAL ANTIBODIES ............. 28
I. Abstract .......................... 29
II. Introduction ........................ 30
II. Materials and Methods .................... 31
IV. Results ........................... 35
V. Discussion ......................... 56
VI. References ......................... 62

CHAPTER III. AN EVALUATION OF 2,5-NORBORNADIENE AS A REVERSIBLE

INHIBITOR OF ETHYLENE ACTION IN DEEPWATER RICE ............. 66
I. Abstract .......................... 67
II. Introduction ........................ 68
III. Materials and Methods .................... 69
IV. Results ........................... 71
V. Discussion ......................... 78
VI. References ......................... 81

CHAPTER IV.

ANATOMICAL ANALYSIS OF GROWTH AND DEVELOPMENT PATTERNS

IN THE INTERNODE OF DEEPWATER RICE ................... 84
I. Abstract .......................... 85
II. Introduction ........................ 86
III. Materials and Methods .................... 87
IV. Results. . .\ ........................ 89
V. Discussion ........................ 100
VI. References ........................ 105
CONCLUSIONS ............................. 109

iv

LIST OF FIGURES
Figure

1.1 Two-dimensional gel of a highly purified ACC-synthase
preparation ........................

1.2 Immunoprecipitation of ACC synthase using monoclonal
antibody purified from ascites fluid produced with
hybridoma line 5b .....................

1.3 SOS-PAGE of tomato-pericarp proteins stained with
Coomassie blue .......................

1.4 Gel filtration HPLC of an ACC synthase preparation in the
presence and absence of IgG from hybridoma line 5b .....

2.1 Elution profiles of four of the chromatographic procedures
used in the purification of ACC synthase ..........

2.2 Immunoprecipitation assays of supernatants from selected
culture wells containing original hybridoma cell lines. . .

2.3 A second immunoprecipitation assay using supernatants from
culture wells which tested positive in the first screen . .

2.4 SDS-PAGE of proteins stained with Coomassie blue .......

2.5 Time course of the conversion of SAM to ACC catalyzed
by free and MAb-bound ACC synthase .............

2.6 ACC-synthase activity as a function of substrate
concentration for the free and MAb-bound enzyme ......

2.7 Lineweaver-Burke plots for free and MAb-bound ACC synthase. .

2.8 The effects of MAb type (hybridoma line 2b or 5b) and
presence or absence of 1 unit of ACC synthase on the
signal generated in the ELISA system used to quantify
ACC synthase .......................

2.9 The relationship between ACC synthase concentration and
ELISA signal .......................

.10 Time course of wound-induced increases in both
ACC-synthase activity and immunodetectable protein
from ripening tomato pericarp tissue ...........

P‘.)

2.11 Fluorograph of [35$]methionine-labeled, immunopurified
protein extracted from wounded tomato pericarp tissue. . .

Page

15

22

24

36

39

41

43

46

47
48

SO

51

53

.12 Flugggraph of an SOS-PAGE fractionation of
[ S]methionine-labeled, immunopurified protein from
wounded tomato fruit tissue ................ 55

.13 Time cggrse of extractable enzyme activity, incorporation

of [ S]methionine into protein, and the percentage of
radioactivity in the immunopurified 50 kDa polypeptide
in wounded tomato pericarp tissue ............. 56

.1 Dose-response curves relating the logarithm of the C H4
concentration to internodal elongation, leaf growtfi,

and adventitious root initiation and the effect of

increasing levels of NBD on these relationships ...... 72

.2 The effect of GA dosage on internodal elongation and
endogenous CZHg levels in the presence and absence
0 .....................

‘0

of 2000 ul/L N 74
.1 The relationship between cell size and distance from the

base of the uppermost, elongating internode ........ 90
.2 Photograph of the basal region of the uppermost,

elongating internode of deepwater rice .......... 92
.3 Light micrograph of a longitudinal section through the

intercalary meristem of the uppermost internode of a

rice stem grown in air . . . . . ............. 94
.4 Light micrograph of a longitudinal section through the

intercalary meristem of the uppermost internode of a

submerged rice stem .................... 95
.5 Light micrograph of a cross section through the

intercalary meristem of the uppermost internode of

an air-grown rice stem .................. 96
.6 Scanning electron micrograph of a longitudinal section

through the region of the uppermost internode just above

the elongation zone of an air-grown plant ......... 97
.7 Light micrograph of a longitudinal section through a

region 5 cm up from the base of the uppermost internode

of a submerged plant ................... 98
.8 Light micrograph of a longitudinal section through the

second-highest node of an air-grown plant ......... 101

Light micrograph of a longitudinal section through the
second-highest node of a submerged plant ......... 102

vi

LIST OF TABLES

Purification of ACC synthase from the homogenate of
10 kg of tomato pericarp tissue .............

Level of ACC-synthase activity and of radioactivity in
the immunoaffinity-purified fraction from wounded
tomato pericarp disks ..................

Purification of ACC synthase from the homogenate of
3.5 kg of tomato pericarp tissue ............

Immunization schedule and serum titers for ACC-synthase
specific antibodies ...................

Recovery of ACC synthase activity after
immunoprecipitation ...................

The effect of exogenouse ethylene and N80 on internodal
growth of stem sections treated with 0.5 uM
gibberellin A3 .....................

Reversible inhibition of NBD-stimulated ethylene
production in stem sections by ethylene and propylene. .

vii

Page

15

22

37

38

42

75

77

ACC

DTT
ELISA

GA

IM

MAb

NBD

PLP

SAM
SOS-PAGE

LIST OF ABBREVIATIONS

I-aminocyclopropane-l-carboxylic acid
dithiothreitol

enzyme linked immunosorbent assay
gibberellin

intercalary meristem

monoclonal antibody

2,5-norbornadiene

pyridoxal phosphate
S-adenosyl-L-methionine

sodium dodecylsulfate - polyacrylamide gel electrophoresis

viii

GENERAL INTRODUCTION

 

While a large body of information exists today on a multitude of
biochemical and physiological effects of plant hormones on growth and
development in higher plants, we still know very little concerning the
mechanisms by which plant hormones exert these effects at the molecular
level. If we view the role of plant hormones as an intermediate one in
the transduction of developmental ,or' environmental signals along
(presumably) biochemical pathways, we must not only try to understand how
each hormone interacts with its target tissue but also how these
environmental and developmental signals produce alterations in hormone
concentrations. While it is assumed 3 951951 that plant hormones exert
their effects via proteinaceous receptors, no in 31539 demonstration of
such a receptor has been made for any of the 5 major classes of plant
hormone. We have faired little better in our understanding of the
regulation of hormone levels. We still cannot state with any certainty
the biochemical pathways for the synthesis of auxin or abscisic acid in
higher plants. It is onTy with the cytokinins, ethylene, and the
gibberellins that the biosynthetic pathways are known. Armed with this
knowledge, we are beginning to ask specific questions concerning the
regulation of the latter two classes of plant hormones in response to

developmental and environmental stimuli.

The focus of this dissertation will be on the mechanisms by which
the plant hormone ethylene exerts its influence on plant growth and
development. In chapters 1 and 2, I examine the biosynthesis of ethylene
in tomato fruit tissue. This work consists primarily of the purification
and characterization of the enzyme l-aminocyclopropane-l-carboxylate
(ACC) synthase. As outlined in the introductions to Chapters 1 and 2,
existing evidence indicates that this enzymatic step in the pathway of
ethylene biosynthesis represents the rate-limiting step and, thus, is a
key regulatory point in determining ethylene levels within a particular
tissue. All evidence points to g; 3919 protein synthesis of the enzyme
as the mechanism by which ACC synthase activity is induced. It was
considered that purification of the enzyme would allow raising of
antibodies specific to ACC synthase. These antibodies would be useful
both as specific probes for studying the properties of the enzyme and as
a useful tool with which to obtain nucleotide sequences specific for ACC
synthase. With these tools, the role of genetic regulation in the
control of ethylene biosynthesis could be elucidated.

In Chapter 3, I turn to the next step in the signal transduction
chain: the interaction of ethylene with ethylene responsive tissues. For
this work, the involvement of ethylene in the submergence response of a
deepwater cultivar of rice is further explored. This system is
particularly attractive because the stem of deep water rice contains 3
ethylene-responsive tissues on which the effects of ethylene are easily
quantifiable. Since the receptor(s) for ethylene have not yet been
biochemically defined, our approach to characterizing their interaction
with ethylene must be limited to studying the relationship between the

ethylene dose and the ultimate physiological responses. The data

obtained should at least be consistent with the kinds of dose-response
relationships observed in the well characterized hormonal systems of
animals if the latter systems are to provide useful paradigms for
understanding ethylene action. The study of animal hormone systems has
benefited from the availability of chemical agonists and antagonists. In
Chapter 3, I also examine the effects of the ethylene antagonist
2,5-norbornadiene on the ethylene responses in deepwater rice.

In the final chapter, I move further down the response pathway and
examine at the anatomical level the ultimate manifestations of the
submergence response 'hi deepwater rice. The submergence response
involves not only dramatic changes in internodal growth, but also major
changes in fundamental developmental processes within the stem. A better
understanding of the cellular basis for the submergence response should
provide important insights into the mechanisms by which the plant
hormones mediate the response. The signal transduction pathways for
plant hormones are undoubtedly complex. It is only by approaching this
question from both ends of the pathway that the yet uncharacterized

intermediate steps will ultimately be revealed.

CHAPTER I

USE OF MONOCLONAL ANTIBODIES IN THE PURIFICATION AND
CHARACTERIZATION OF l-AMINOCYCLOPROPANE-l-CARBOXYLATE
SYNTHASE, AN ENZYME IN ETHYLENE BIOSYNTHESIS

ABSTRACT

I-Aminocyclopropane-l-carboxylate (ACC) synthase, extracted from tomato
pericarp tissue, was purified 6500-fold using conventional and high-
performance liquid chromatographic methods. Two-dimensional gel
electrophoresis of this preparation indicated that ACC synthase activity
was associated with a protein band with a molecular weight of 50 kDa, a
value consistent with molecular-weight determinations by gel filtration.
Monoclonal antibodies against ACC synthase were obtained from murine
hybridoma lines. These antibodies recognized the native enzyme as shown
with an immunoprecipitation assay. A monoclonal IgG-immunoaffinity gel
was used to isolate, from a relatively crude enzyme preparation, a single
protein which migrated at 50 kDa on a SDS polyacrylamide gel. lflinXQ
labeling of wounded tomato pericarp tissue with [35S]methionine followed
by immunoaffinity purification of ACC synthase yielded a radioactive
protein with a molecular weight of 50 kDa. We conclude that the 50 kDa
protein represents ACC synthase in extracts of wounded tomato pericarp

tissue.

INTRODUCTION

Biosynthesis of ethylene, a regulator of plant growth and development, is
subject to developmental and environmental regulation (1-3). During the
early stages of tomato fruit development, for example, the immature green
fruit produces little ethylene. One of the earliest observable events in
tomato ripening is a sharp increase in the rate of ethylene formation
(4). A number of subsequent ripening-related processes are controlled by
ethylene (5). Application of an environmental stress to tomato fruit
tissue, e.g. wounding, causes an additional increase in ethylene

production (6,7).

In higher plants, ethylene is synthesized from 1-aminocyclopropane-1-
carboxylic acid (ACC) (8,9). A cell-free preparation of ACC synthase, the
enzyme catalyzing the conversion of S-adenosyl-L-methionine (AdoMet) to
ACC, was first obtained from ripening tomato fruit (10). In most plant
tissues, the rate of ethylene synthesis is limited by the activity of ACC
synthase. Hence, conditions and chemicals that induce ethylene formation,
e.g. stress or auxin at high concentrations, often enhance the activity
of ACC synthase (for a review see ref. 3). In tomato fruits,
developmental and wound-induced increases in ethylene prodUCtion are
reflected in the levels of extractable ACC-synthase activity (6,7).
Induction of ACC synthase in tomato fruit tissue appears to involve
93 £919 synthesis of the enzyme as indicated by the ability of

cycloheximide to inhibit wound-induced increases in the activity of ACC

synthase (6,7) and by an increase in the buoyant density of the enzyme in

wounded tomato-fruit tissue incubated on 2

H20 (11).

The activity of ACC synthase often determines the developmental fate of
plant tissues; hence, there is considerable interest in understanding how
this enzyme is regulated. To reach such an understanding, molecular
probes for ACC synthase and its antecedent mRNA and DNA sequences will be
required. We report here on the isolation of monoclonal antibodies
against ACC synthase and their use in the purification and

characterization of the enzyme from tomato fruit tissue.

MATERIALS AND METHODS

Plant Material. Fruits from glasshouse-grown tomato plants

 

(Lycopersicon esculentum Mill., cv. Duke) were harvested at the pink to

 

red stage. Enzyme induction and preparation of crude homogenates from the
pericarp were as described previously (12). Hollowed out tomato fruit
halves were filled with 50 mM LiCl and incubated at room temperature
overnight. Fruit tissue was then sliced into 1 cm pieces and homogenized

at low speed with a polytron.

ACC-Synthase Assay. Appropriate aliquots of enzyme were incubated at

 

30° C in 20 mM K-phosphate buffer (pH 8) containing 100 uM AdoMet and 5
uM pyridoxal phosphate (PLP). The amount of ACC formed was determined by

chemical conversion of ACC to ethylene followed by gas chromatographic

quantitation (13). One unit of enzyme is defined as the conversion of 1

nmol of AdoMet to ACC per hour at 30° C.

 

Enzyme Purification. A 40-95% (NH4)ZSO4 fraction of the homogenate was
obtained as described (11). The protein precipitate was either used
immediately or stored at -80° C. After resuspension of the precipitate in
dialysis buffer consisting of 10 mM K-phosphate buffer (pH 8), 0.1 mM
dithiothreitol (DTT) and 5 uM PLP, the enzyme was dialyzed overnight.

Purification by HPLC was performed using a SP 8700 HPLC system (Spectra
Physics, San Jose, CA). A protein sample of up to 500 mg was loaded onto
a preparative stainless steel HPLC column (1.5 x 25 cm) packed with
Synchroprep AX-300 support (SynChrom, Linden, IN) and equilibrated with
dialysis buffer. Protein was eluted from the column over 120 min with a

linear NaCl gradient (0 - 700 mM) at a flow rate of 3 ml min'l.

Fractions
of 9 ml were collected, and those showing highest specific enzyme
activity were combined. Enzyme purified by preparative HPLC anion
exchange chromatography was dialyzed against dialysis buffer and loaded
(up to 40 mg of protein) onto a 1 x 20-cm hydroxylapatite column at a

flow rate of 0.5 ml min-1.

The column was washed with dialysis buffer
until the eluate showed no absorbance at 280 nm. ACC synthase was eluted
in 100 ml of a K-phosphate gradient (pH 8, 10 - 300 mM) at the above flow
rate. Fractions of 4 ml were collected, and those showing highest
specific enzyme activity were combined. Enzyme from this purification
step was further purified on a 1 x 20-cm column packed with

phenyl-Sepharose as previously described (11). Additional purification

was achieved with a 1 ml Affi-Gel Blue (Bio-Rad, Richmond, CA) column

according to the procedure of Boller (personal communication). The column
and enzyme were equilibrated with 10 mM K-phosphate buffer (pH 6.8)
containing 5 uM PLP. The enzyme was loaded onto the column in dilute
solution (200 ug protein ml'l) at a flow rate of 0.5 ml min'l. The column
was washed with starting buffer until the eluate showed no absorbance at
280 nm. The enzyme was eluted with 50 mM K-phOSphate buffer (pH 8)
containing 5 uM PLP at the above flow rate. This enzyme preparation of
relatively high specific activity was further purified on an analytical
HPLC anion exchange column (0B4 x 250 cm, Nugel DE-300, Separation
Industries, Metuchen, NJ). Enzyme ( 2 mg protein) was injected onto this

1 and eluted over 40 min with a NaCl

column at a flow rate of 1 ml min'
gradient (0 - 700 mM). Fractions of.1 ml were collected, and those with
high specific enzyme activity were combined. A final purification step
was performed using an analytical HPLC hydroxylapatite column (Bio-Rad).
Enzyme was injected onto the column at a flow rate of 0.4 ml min"1 and
eluted over 40 min with a gradient of K-phosphate buffer (pH 8, 10 - 300

mM) at the above flow rate. Buffer compositions were as recommended by

the manufacturer.

Molecular-Weight Determination by Gel Filtration. A partially purified

 

preparation of ACC synthase (400 units per mg protein) or ACC synthase
bound to monoclonal antibody was fractionated on a TSK-250 (7.5 x 600 mm)

1

HPLC column (Bio-Rad) at a flow rate of 0.4 ml min' using 10 mM

K-phosphate buffer (pH 7) containing 0.15 mM NaCl as the mobile phase.

Gel Electrophoresis and Fluorography. Gradient (8-14%) PAGE 'hi the

 

presence of SDS was performed according to Laelmnli (14). Gels were

10

stained with Coomassie blue as described (14). Gels were prepared for
fluorography by presoaking in sodium salicylate (1 M). Dried gels were
placed onto Kodak SB-5 X-ray film and exposed at -80° C for 6-15 days.
Radioactivity was quantified by densitometric scanning using a Response
Spectrophotometer (Gilford). For two-dimensional PAGE, the first
dimension was run in non-denaturing, discontinuous, 8% polyacrylamide
tube gels using a GT 3 apparatus (Hoefer, San Francisco, CA) according to
manufacturer's recommendations. Enzyme activity was located in tube gels
by slicing the resolving gel into 0.5-cm segments and incubating the
slices in the ACC-synthase assay mixture for 1 hr. The second dimension

was run in 8-14% gradient polyacrylamide gels.

Preparation of Antigen and Immunization. ACC synthase was purified from

 

tomato pericarp by (NH4)2504 precipitation (40 - 95% fraction) followed
by preparative 'Hni exchange, hydroxylapatite, phenyl-Sepharose, and
analytical ion exchange chromatography. Enzyme with a specific activity
of 1-3x104 units per mg protein was routinely obtained. The purified
preparation was coupled to lipopolysaccharide (LPS) (15). CNBr-activated
LPS (80 ug) was incubated with 100 ug of protein in 20 mM K-phosphate
buffer (pH 8.5) at 4° C for 12 hr. The LPS-protein conjugate was
collected by centrifugation in a Microfuge (Fisher, Springfield, NJ) for
15 min, and the pellet was resuspended in 0.2 M lysine to block residual
cyanate esters. The suspension was centrifuged again, and the pellet
resuspended in the original protein solution. The pH of the solution was
adjusted to 7.2 with HCl, diluted tenfold with sterile phosphate-buffered
saline and sonicated briefly. A female 20-week-old BALB/c mouse was

injected i.p. with 20 ug of conjugated LPS and was boosted with

11

additional 20 ug injections of the conjugate 14 and 40 days later. Two
additional i.p. injections with 30 ug each of conjugated LPS each were

made three and two days before fusion of the spleen cells (day 70).

Cell Culture and Hybridoma Selection. Murine myeloma cells (108) from
line SP2/O-Agl4 (16) were fused with 2x108 spleen cells from the
inmunized mouse (17). Hybridomas secreting antibodies against ACC
synthase were detected using an immunoprecipitation assay. They were
cloned twice by limiting dilution. All cultures were grown in DMEM medium
(Gibco, Grand Island, NY) supplemented with fetal calf serum (Hazelton

Res. Lab., Denver, PA).

Monoclonal Antibody Production, Purification and Characterization.
Ten-week- old BALB/c mice were injected i.p. with 0.5 ml pristane and two

weeks later with 3x106

hybridoma cells. After an additional 5 days,
ascites fluid was removed and either used inmediately or frozen at
-80° C. Monoclonal antibodies were purified from ascites fluid either by
hydroxylapatite chromatography (18) or by protein A affinity
chromatography (MAPS II Kit, Bio-Rad). Antibody subtypes were determined

using Ouchterlony immunodiffusion plates (Serotec, Blackthorn, UK).

Immunoprecipitation Assay for ACC Synthase. Specified dilutions of
either control mouse serum, mouse antiserum, tissue culture supernatant
or IgG purified from mouse ascites fluid were tested for binding to ACC
synthase according to the following procedure: 50 ul of dilute
antibody-containing solution was added to 50 ul of a dilute, partially

purified ACC-synthase preparation (1-5 units/50 ul). All dilutions were

12

made in 50 mM K-phosphate buffer (pH 8) containing 0.3 M NaCl, 1 mg ml'1
bovine serum albumin and 5 uM PLP. The mixture was incubated at room
temperature for 2 hr. Sufficient rabbit-antimouse IgG was added in 25 ul
to bind 3.5 ug of mouse IgG, and the mixture was incubated for an
additional 1 hr. Finally, 25 ul of a 1% S. agrggs cell suspension was
added and incubated fOr 1 in" at room temperature. The mixture was
centrifuged in a Microfuge for 15 min, and the supernatant transferred to
a tube containing ACC-synthase assay buffer. ACC synthase was determined
as described above. For initial screening of culture supernatants, the
entire immunoprecipitation assay was performed in 96-well microtiter

plates.

In-Vivo Labeling of ACC Synthase. Wounded tomato disks were prepared
from pink tomatoes as described (11). To enhance uptake of label, disks
were placed under a stream of air until they had lost 10% of their
original weight. An aqueous solution (0.5 ml) of 0.1-0.2 mCi
[35$]methionine (1127 Ci/mmol) was applied to 15 disks (10 9). After
incubation for 1.5 hr, the disks were homogenized with a teflon
homogenizer in 10 ml of buffer (100 mM K-phosphate, pH 8, 5 mM EDTA, 5 uM
PLP) and centrifuged at 18,000 g for 15min. The supernatant was

centrifuged once more at 100,000 g for 30 min.

Immunoaffinity Purification of ACC Synthase. An immunoaffinity matrix

 

was prepared by incubating 5-6 mg of purified monoclonal antibody with 1
ml of swollen CNBr-activated Sepharose 4B in 50 mM Na-borate (pH 8.5) and
0.5 M NaCl for 3 hr at room temperature. Residual cyanate esters were

blocked with 1 mM ethanolamine (pH 8.5). Purification of ACC synthase was

13

achieved by adding 30-50 ul of swollen immunoaffinity gel to either 15 ml
of crude extract (for the in yjyg labeling experiments) or to 1.5 ml of
partially purified ACC synthase (300-400 units per mg protein). The
suspension was incubated at room temperature for 3 hr, and the gel-bound
enzyme was separated from the solution by low-speed centrifugation (100
g, 5 min). The gel was washed twice with 1.5 ml of 100 mM K-phosphate
buffer (pH 8) containing 0.3 M NaCl; once with the same buffer at pH 6;
and once again at pH 8. For SOS-PAGE, the ACC-synthase protein was
extracted twice from the immunoaffinity gel with 50 ul of electrophoresis
sample buffer (0.1 M Tris-HCl, pH 6.8, 3% SDS, 4% glycerol).
B-Mercaptoethanol or DTT were added to this extract to a final

concentration of 5-10% and 5%, respectively, prior to electrophoresis.

RESULTS

Purification of ACC Synthase. Two major problems had to be overcome in
the purification of ACC synthase. Even in the pericarp of ripening tomato
fruits, one of the richest known sources of ACC synthase, the level of
enzyme activity is very low (0.2 nmol hr'1 9"1 fresh weight, ref. 7). In
addition, ACC synthase proved to be very labile during purification (11).
The first problem was ameliorated by overnight treatment of pericarp
tissue with LiCl which enhanced enzyme activity more than 100-fold (12).
The problem of instability was solved, in part, by using HPLC for

purification of the enzyme. This allowed several purification steps to be

performed in a short period of time. Losses of activity were also reduced

14

by storing the enzyme at -80° C at any stage of purification, the

recovery being greater than 70% upon thawing.

ACC synthase was purified 6,500-fold with a 5% recovery from 10 kg of
tomato pericarp tissue using a combination of conventional and
high-performance liquid chromatographic nethods (Table 1). ll major
portion of the purified enzyme preparation was further fractionated by
two-dimensional PAGE. Non-denaturing conditions were used in the first
dimension, and ACC synthase was localized by determining the activity of
the enzyme in slices of a second gel run in parallel. SOS-PAGE was
used in the second dimension. One major Coomassie blue-stained protein
band in the second dimension with, a molecular weight of 50 kDa
corresponded to the location of ACC-synthase activity in the first
dimension (Fig. 1). A very faint protein band of ca. 95 kDa molecular

weight was also evident.

Monoclonal Antibodies against ACC Synthase. When the titer of

 

ACC-synthase-specific antibodies in the serum of the immunized mouse was
sufficiently high to immunoprecipitate 25 units of enzyme per ul of
serum, spleen cells were fused with myeloma cells, and the resultant
hybridomas were screened for secretion of ACC-synthase-specific
antibodies. From an initial 26 wells giving positive results, 5 stable
hybridoma cell lines were obtained which produced monoclonal antibodies
directed against ACC synthase. Ascites fluid derived from each of these
cell lines completely removed ACC-synthase activity from enzyme
preparations at various stages of purity. Control solutions containing

normal mouse serum, ascites fluid obtained from an SP2 myeloma cell line,

15

Table 1. Purification of ACC synthase from the homogenate of 10 kg of

tomato pericarp tissue

 

 

Total Protein Specific Overall Recovery
Fraction activity activity purification
units mg units/mg prot. -fold %
40-95%
(NH4)2S04
fraction 10,500 2,500 4.2 0 100
Preparative
ion exchange
HPLC 9,000 320 28 7 85
Hydroxylapatite
column 8,600 48 179 43 82
Phenyl Sepharose
column 4,800 5.1 941 224 46
Analytical ion
exchange HPLC 2,400 0.7 3,428 816 23
Affigel Blue
column 1,600 0.15 10,666 2,539 15
Analytical
hydroxylapatite

HPLC 550 0.02 27,500 6,547 5

 

16

ACC Synthauhmol h")
n as

~ - 66.0 kDa

M r. ., u-q' - 45.0 kDa
' :1;

‘ _ ' . . .. d f «36.0 kDa

I “ , . . —-29.0koa
(L-.--.----.,, ' -. 1-24.0KDI
E. -s>;,_. 4. ”VJ..— .W— 13"... ....- ._-- .4 . :11..:~::—«- *M-m

WK r: «8%,; ~ ' .

  
 
 

near
qj - 20.1 kDa

‘ " 14.2 kDa

‘ it '1 .
'. p:

1 Al,

. .‘ 1',

. _.

Eig__1. Two-dimensional gel of a highly purified ACC-synthase
preparation. Enzyme activity was localized in the first dimension by
incubating slices of a parallel-run gel in ACC synthase assay buffer.
The arrowheads indicate the locations of protein bands in the second
dimension (NaDodSO4-PAGE run with 5% B-mercaptoethanol) which correspond
to the enzyme activity in the first dimension (non-denaturing

discontinuous PAGE).

17

and ascites containing monoclonal antibodies to other plant proteins did
not immunoprecipitate ACC synthase. An example of a titration curve
using monoclonal antibody from line 5b is given in Figure 2. The
monoclonal antibodies from all cell lines were of the IgG1 subclass as
determined by the Ouchterlony immunodiffusion assay (data not shown). ACC
synthase bound to nitrocellulose could not be detected with any of the

five monoclonal antibodies.

Immunoaffinity Purification of ACC Synthase. An immunoaffinity gel was
prepared by covalently coupling purified IgG from hybridoma line 5b to
Sepharose 48. Based on the disappearance of enzyme activity from an
ACC-synthase preparation, the immunoaffinity gel had a binding capacity
of 2.5x104 units of ACC-synthase activity per ml of swollen gel. When the
bound enzyme was resuspended in ACC-synthase assay buffer, it exhibited
30-40% of the activity removed from the original enzyme solution (data
not shown). The immunoaffinity gel (50 ul) was used to isolate ACC
synthase from a partially purified enzyme preparation (1,200 units, 300
units per mg protein). The gel with bound enzyme was washed in a series
of buffers to remove unspecifically absorbed protein. When specifically
bound protein was extracted from the gel with buffer containing 505
and subjected to SOS-PAGE in the presence of 5% DTT, a major
polypeptide of molecular weight 50 kDa, in addition to the two IgG
subunits, was detected (Fig. 3, lane B). In the absence of DTT, the
protein migrated as a diffuse band with an apparent molecular weight of
80-100 kDa (data not shown). When a second aliquot of affinity gel was

added to the protein solution from which all enzyme activity had been

removed, no additional protein became specifically bound to the gel (Fig.

18

 

 

 

 

 

 

1CKDP
é
a 80-
E
‘6
<1 6K)-
0
an .
G
£5 4!)—
§. .
(D
Q 20-
(J
< l"
0..
l l 1 I 1 l
I) 1CK) 20K) 30K)

fig;_2. Immunoprecipitation of ACC synthase using monoclonal antibody
purified from ascites fluid produced with hybridoma line 5b. Two units
of enzyme (40 units per mg protein) were preincubated in 100 ul with the
indicated amount of 1961 from line 5b, (0); or of mouse 1961 in dilutions
of control mouse serum, (0). ACC-synthase activity remaining in the
supernatant after immunoprecipitation is expressed as % of control (no

added mouse IgG).

l9

Elg;_3. Lanes A-C: NaDodSO4-PAGE of tomato-pericarp proteins stained with
Coomassie blue. A, ACC-synthase-enriched protein fraction (300 units per
mg protein) after purification by preparative anion exchange HPLC; 8,
proteins eluted from the immunoaffinity gel (line 5b 196) after
incubation of the gel with 2.5 mg of the protein preparation used in lane
A; C, proteins eluted from a second aliquot of immunoaffinity gel added
to the protein preparation shown in lane A after all enzyme activity had
been removed by the first aliquot of the immunoaffinity gel; 2, purified
1961 from hybridoma line 5b. Lanes E-G: Fluorograph of [35$]protein
extracted from wounded tomato pericarp tissue and fractionated by

NaDodSO -PAGE. The tissue was labeled with 200 uCi of [35$]methionine

4
during the second 1.5 hr after wounding. E, (NH4)2504-precipitated

protein from a crude extract of tomato tissue; 5, radioactive protein
purified by immuno- affinity gel and run on NaDodSO4 polyacrylamide gel
in the presence of 5% DTT; G, as in lane B but run on NaDodSO4
polyacrylamide gel in the absence of DTT. Lane E represents the protein

from 0.25 9, lanes F and G from 10 g of tomato tissue.

20

 

i kDa

 

 

21

3, lane C). Based on the amount of enzyme activity present in the
original solution and the amount of protein in the 50 kDa molecular

5

weight band, a specific activity of 4x10 units per mg protein was

calculated for ACC synthase.

In-Vivo Labeling of ACC Synthase. When wounded tomato pericarp disks

 

(10 g) were fed 200 uCi of [35S]methionine between 1.5 and 3 hr after
wounding, ca. 3x105 cpm of radioactivity were incorporated into
(NH4)ZSO4-precipitable material. ACC-synthase activity was purified
directly from crude homogenates using immunoaffinity gel with 196 from
either hybridoma line 2b or 5b. 505 eluates of the gel, containing
200-300 cpm of radioactivity, were fractionated by SDS-PAGE. One
major radioactive band, migrating at 50 kDa in the presence of 5% DTT
(Fig. 3, lane E) and at 80-95 kDa in the absence of DTT (Fig. 3, lane F),
was detected by fluorography. In some preparations labeled during the
second 1.5 hr after wounding, a trace of a radioactive band was found at
56 kDa (results not shown). Densitometric scans of the X-ray films
indicated that this band contained less than 1% of the radioactivity

present in the major 50-kDa band.

The relationship between the induction of ACC-synthase activity by
wounding and the appearance of the 50-kDa radioactive protein band is
shown in Table 2. During the first 1.5 hr after wounding, pericarp tissue
developed less ACC-synthase activity and incorporated less radioactivity

into the 50-kDa protein band than during the second 1.5 hr.

22

Table 2. Level of ACC-synthase activity and of radioactivity in the
immunoaffinity-purified fraction from wounded tomato pericarp

disks.

 

Labeling time and hybridoma line

 

o-1.5 hr 1.5-3 hr 1.5-3 hr 1.5-3 hr

* 'k + *
line 5b line 5b line 5b line 2b

 

Enzyme activity

(units per 10 g tissue) 16.6 91.2 7.4 86.5
Radioactivity
(densitometer integral) 0.21 3.66 0.16 3.36

 

Tomato pericarp disks were fed [35$]methionine for the first or second
1.5 hr following wounding.
*Enzyme activity was determined in a dialyzed crude extract prior to the
addition of immunoaffinity gel. Imunoaffinity-purified protein was
fractionated by SOS-PAGE. Radioactivity was localized by
fluorography and quantitated by densitometry. +Residual enzyme
activity and radioactivity associated with the 50-kDa protein band were
determined once more in the extract from column 2 following the initial

removal of ACC synthase with the immunoaffinity gel.

23

Molecular-Weight Determinations by Gel Filtration. Previous results
based on gel filtration using Sephadex G-100 indicated a molecular weight
of 57 kDa for ACC synthase (11). Gel filtration of partially purified ACC
synthase using HPLC both in the presence and absence of DTT showed a
single peak of activity corresponding to a molecular weight of 55 kDa
(Fig. 4). When ACC synthase was preincubated with a molar excess of
monoclonal IgG from line 5b prior to gel filtration, the activity eluted
as one peak corresponding to a molecular weight of 170 kDa. Since the
free monoclonal antibody eluted at a molecular weight of 123 kDa, the
shift in the molecular weight of the enzyme activity indicates a

stoichiometry of 1:1 for the monoclonal antibodyzACC-synthase complex.

DISCUSSION

Our results represent strong evidence that ACC synthase in extracts of
tomato pericarp tissue consists of a 50-kDa polypeptide. Two independent
means of purification based on chromatographic and immunological methods
lead to the isolation of a single major protein band of this molecular
weight. Additionally, a 50-kDa protein isolated with an immunoaffinity
gel became labeled when ACC-synthase activity was induced by wounding of
tomato tissue. The estimated specific activity of the immunopurified
enzyme (2-4x105 units per mg protein) and the turnover number (k3)
derived therefrom (3-6 sec-1) fall within the range observed for a number
of enzymes (19) and are consistent with the values reported previously

for ACC synthase (20). From these values, we calculated that ACC synthase

24

 

 

 

g .5 a a
no a: a:
£3 .“3 3 E:
.0! t 4 4
"' A
h
F. 30—
'8
E
55
g 20- #
fl
.2
fl
2
5'
o 10
o
<
o

 

 

 

 

Fraction Number

fig;_4, Gel filtration HPLC of an ACC synthase preparation (300 units per
mg protein) without DTT in the elution buffer in the presence (0) and
absence (0) of IgG from hybridoma line 5b. The elution profiles of ACC
synthase with DTT in the elution buffer or with control mouse serum added‘
to the enzyme preparation were very similar to the one shown here (a).
The center of the ACC-synthase peaks were determined by plotting the data

points on Gaussian graph paper.

25

represents less than 0.0001% of the total protein in the pericarp of

ripening tomato fruits.

The behavior of the immunopurified protein on SOS-PAGE was
drastically altered when DTT was omitted from the sample buffer. Both
Coomassie-blue-stained and in yiyg radiolabeled protein migrated as a
diffuse band with an apparent molecular weight of 80-100 kDa, indicating
the presence of at least one disulfide bond in the molecule. When
SOS-PAGE was performed in the presence of B-mercaptoethanol instead
of DTT, both the high- and lowemolecular-weight protein bands were
detected, indicating incomplete reduction of the protein. These disulfide
bonds may be intramolecular, preventing complete denaturation of the
enzyme and causing anomalous behavior during SOS-PAGE, or
intermolecular, in which case the slower migrating band may represent a
dimer of two 50-kDa polypeptides. The latter possibility appears unlikely
because the molecular-weight determinations by. gel filtration in the
absence of reducing agent indicate that the native enzyme in tomato
extracts exists as a single protein of approximately 50 kDa molecular
weight. The shift in molecular weight of ACC synthase bound to monoclonal
antibody is additional evidence that the active enzyme exists as a

monomer possessing only one epitope for the antibody.

Our work affirms the power of monoclonal antibody technology when
applied to the isolation and characterization of low-abundance proteins.
Using partially purified enzyme, the immunoaffinity gel allowed an over
2000-fold purification of ACC synthase in a single step. Use of

monoclonal antibodies will also permit investigations into the role of

protein synthesis and turnover in the regulation of ACC synthase activity

I

vivo. Ultimately, we hope that these studies will lead to an

understanding of the mechanisms by which ethylene-mediated processes are

regulated during plant development and stress.

10.
11.
12.

13.

REFERENCES

. Abeles, F. B. (1973) Ethylene in Plant Biology (Academic Press, New

York), pp. 30-102.

. Lieberman, M. (1979) Ann. Rev. Plant Physiol. 30, 533-591.

 

. Yang, S. F. 8 Hoffman, N. E. (1984) Ann. Rev. Plant Physiol. 35,

 

 

155-189.

. Workman, M. & Pratt, H. K. (1957) Plant Physiol. 32, 330-334.

 

. Jeffery, D., Smith, C., Goodenough, P., Prosser, I. 8 Grierson, D.

(1984) Plant Physiol. 74, 32-38.

 

. Boller, T. & Kende. H. (1980) Nature 286, 259-260.
. Kende, H. & Boller, T. (1981) Planta 151, 476-481.
. Adams, 0. O. & Yang, S. F. (1979) Proc. Natl. Acad. Sci. USA 76,

 

170-174.

. Lurssen, K., Naumann, K. & Schroder R. (1979) 2. Pflanzenphysiol. 92,

 

285-294.
Boller, T., Herner, R.C. & Kende, H. (1979) Planta 145, 293-303.
Acaster, M. A. & Kende, H. (1983) Plant Physiol. 72, 139-145.

 

Ramalingam, K. Lee, K.-M., Woodard, R. W., Bleecker, A. B. & Kende,
H. (1985) Proc. Natl. Acad. Sci. USA 82, 7820-7824.

 

Lizada, M. C. C. & Yang, S. F. (1979) Anal. Biochem. 100, 140-145.

 

14.
15.

16.
17.
18.

19.
20.

27

Laemmli, U. K. (1970). Nature 227, 680-685.

Lange, M., Guern, C. L. & Casenave, P.-A. (1983) g. Immunol. Methods

 

63, 123-131.
Shulman, M., Wilde, C. D. & Kohler, G. (1978) Nature 276, 269-270.
Galfe, G. & Milstein, C. (1981) Methods Enzymol. 73, 3-46.

 

Stanker, L. H., Vanderlaan, M. & Juarez-Salinas, H. (1985) g.
Immunol. Methods 76, 157-169.

 

Stryer, L. (1981) Biochemistry (Freeman, San Francisco), p. 114.

 

Kende, H., Bleecker, A. B., Kenyon, W. H., Mayne, R. G. (1986) in
Plant Growth Substances 1985, ed. M. Bopp (Springer, Berlin), pp.
120-128.

CHAPTER II

THE ROLE OF PROTEIN SYNTHESIS IN THE REGULATION OF
WOUND-INDUCED l-AMINOCYCLOPROPANE-l-CARBOXYLATE
SYNTHASE IN TOMATO FRUIT TISSUE INVESTIGATED
WITH MONOCLONAL ANTIBODIES

28

29

ABSTRACT

A partially purified preparation of I-aminocyclopropane-l-carboxylate
(ACC) synthase (EC 4.4.1.14) from tomato fruit tissue was used to
generate monoclonal antibodies (MAb) specific for the enzyme.
Immunoaffinity purification with either of two different MAbs yielded a
50 kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel
electrophoresis. An enzyme linked immunosorbent assay (ELISA) capable of-
detecting <1 ng of antigen was developed. The ELISA system was used to
demonstrate that two of the MAbs recognize different epitopes on the ACC
synthase protein. Wound-induced increases in ACC-synthase activity in
tomato fruit tissue were correlated with changes in ELISA detectable
protein. 1_ vi_vo labeling of wounded tissue with [35$]-methionine
followed by extraction and immunopurification in the presence of various
protease inhibitors yielded a single radioactive band that migrated at 50
kDa molecular mass. Pulse labeling with [35$]methionine at various times
after wounding indicated that the wound-induced increase in ACC-synthase
activity involved gs £219 synthesis of a rapidly turning over 50 kDa

polypeptide.

30

INTRODUCTION

An increase in the rate of ethylene biosynthesis in higher plants is
characteristic of a number of developmental processes and responses to
environmental stresses. From seed germination (Abeles 1986) to such
terminal events as abscission (Sexton et al. 1985) and fruit ripening
(McGlasson et al. 1978), shifts in tissue development are often
accompanied by elevated ethylene levels. Physical stresses such as
wounding (Boller and Kende 1980), drought (Apelbaum and Yang 1981) and
flooding (Jackson 1985) are known to stimulate ethylene biosynthesis.
Biotic stresses, including invasion by pathogens (Montalbini and Elstener
1977) and treatment with elicitors (Chappell et al. 1985) also cause
increases in ethylene production.

In most of the cases cited above, the increases in ethylene
biosynthesis can be attributed to increased activity of the enzyme
I-aminocyclopropane-l-carboxylate (ACC) synthase (S-adenosyl-L-methionine
methylthioadenosine-lyase, EC 4.4.1.14.) (Yang and Hoffman 1984). This
enzyme catalyzes the conversion of S-adenosylmethionine (SAM) to ACC in
higher plants (Boller et al. 1979; Yu et al. 1979). The subsequent
oxidation of ACC to ethylene by the ethylene-forming enzyme is considered
to be constitutive in most plant tissues (Yang and Hoffman 1984). Thus,
ACC synthase is the rate-limiting enzyme and, therefore, represents the

regulatory point in ethylene biosynthesis.

31

All available evidence indicates that increases in ACC-synthase
activity involve gg £919 protein synthesis. Cycloheximide prevented the
induction of ACC-synthase activity by wounding (Kende and Boller 1981)
and by auxin (Yoshii and Imaseki 1982). In addition, wounded tomato
tissue (Acaster and Kende 1983) and elicitor-treated parsley cells

(Chappel et al. 1985), which had been incubated on 2H 0, yielded ACC

2
synthase of increased buoyant density. This indicated that the enzyme

formed during the induction incorporated 2

H-labeled amino acids and was,
at least in part, d_e ngvg synthesized. More recently, monoclonal
antibodies (MAb) for ACC synthase have been used to show that wounded
tomato fruit tissue incorporated [35$]methionine into a 50 kDa
polypeptide which copurified with ACC-synthase activity (Bleecker et al.
1986).

A better understanding of how various developmental and '
environmental signals can cause an increase in ACC-synthase activity
could provide valuable information about the molecular processes involved
in the transduction of these signals in higher plants. With this goal in
mind, we report herein on the properties of MAbs for ACC synthase and

their use in investigations of the role of protein synthesis in the

induction of ACC synthase in wounded tomato fruit tissue.

MATERIALS AND METHODS

Plant material. Fruits from greenhouse-grown tomato plants (Lycopersicon

 

 

esculentum Mill., cv. Duke) were harvested at the pink to red stage.

 

Crude extracts were prepared as previously described (Bleecker et al.

32

1986). Two kg of LiCl-treated tomato pericarp tissue was chapped into
I-cm cubes and homogenized with 2 l of extraction buffer (100 mM potasium
phosphate buffer, pH 8.5, 5% (w/v) polyvinylpyrrolidone, 1 mM DTT, 5 uM

pyridoxal phosphate) using a polytron (Brinkmann Instr., Westburg, N.Y.).

Chemicals. All reagents were purchased from Sigma Chemical Co., (St.

Louis, Mo.) unless otherwise specified.

ACC-synthase assay. Enzyme solutions were incubated at 30°C in 20 mM

 

potassium phosphate buffer (pH 8) containing 50 uM SAM and 5 uM pyridoxal
phosphate. The quantity of ACC formed was determined by chemical
conversion of ACC to ethylene followed by gas chromatographic
quantification (Lizada and Yang 1979). One unit of enzyme converts 1

nmol of SAM to ACC per h at 30°C.

 

Enzyme Purification. A 40-95% (NH4)2SO4 fraction of the crude homogenate
was obtained as previously described (Acaster and Kende 1983). The
protein precipitate was either used immediately or stored under liquid
nitrogen. After resuspension of the precipitate in dialysis buffer (10
mM potassium phosphate, pH 8, 5 uM pyridoxal phosphate), the enzyme was
dialyzed overnight. The dialyzed enzyme was initially purified on a
preparative anion exchange HPLC column (Bleecker: et al. 1986).
Subsequent column purification steps were performed as described
(Bleecker et al. 1986). Immunoaffinity purifications using MAb from
either hybridoma line 2b or line 5b antibody were carried out as

described (Bleecker et al. 1986).

33

Preparation of antibody reagents. Murine hybridoma cell lines secreting

 

MAb specific to ACC synthase were generated from spleen cells of a mouse
that had been immunized with an ACC synthase-enriched protein preparation
covalently coupled to lipopolysaccharide (Bleecker et al. 1986).
Monoclonal antibodies to ACC synthase were obtained from ascites fluid
(Bleecker et al. 1986) or from culture supernatants. Hybridoma lines
were initially cultured in medium containing 10% serum (Bleecker et al.
1986). These lines were adapted to HL-I serum-free medium (Ventrex,
Portland, Me.) by slowly weening the cultures from the serum-based

6

medium. Cells were cultered in 50-ml flasks to a density of 2x10 cells

'1 at which time (5-10 days after subculture) the cells were removed by

ml
centrifugation (8,000 RPM) in a clinical centrifuge. Culture supernatant
was either frozen and stored at -20°C or used imediately. The
supernatant (1 l) was concentrated to a small volume (10 ml) with an
Amicon pressure cell (Danvers, Mass.). Monoclonal antibody was purified
from the concentrated supernatant using Protein A affinity chromatography
(MAPSII, BioRad, Richmond, Ca.). Purified monoclonal antibody was
coupled to Sepharose 48 as previously described (Bleecker et al. 1986).
Biotinylated antibody was prepared by reacting 3 mg of purified antibody
with a 30-fold molar excess of NHC-LC-Biotin (Pierce, Rockford, Ill.) in
1.5 ml buffer (20 mM sodium borate, pH 8.5) for 2 h at room temperature.
The product was dialyzed overnight against 10 mM sodium phosphate, pH

7.3, 150 mM NaCl (PBS) containing 0.05% NaN The biotinylated antibody

3.
was stored at 4°C with 10 mg ml'1 bovine serum albumin (BSA) as

stabilizer.

34

In-vivo labelinggof ACC synthase. Wounded tomato disks were prepared
from pink tomatoes and fed [35$]methionine (30.5 GBq mmole'l, New England
Nuclear, Boston, Mass.) as previously described. After incubation for
the indicated lengths of time, the tissue was extracted with 100 mM
potassium phosphate pH 8, 5 mM EDTA, 5 uM pyridoxal phosphate and then
centrifuged first at 30,000 g and then at 135,000 g (Bleecker et al.
1986). In the case of the experiments with protease inhibitors, the
inhibitors were present in the extraction buffer at the indicated

concentrations.

Enzyme-linked immunosorbent assay ELISA). Assays were performed in

 

96-well PVC culture plates (Dynatech, Alexandria, Va.). Two ug of
capture antibody in 10 mM sodium carbonate buffer (pH 9.2) was added to
each well, and plates were incubated overnight at 4°C. Wells were washed
three times with PBS containing 0.05% BSA. Wells were then blocked with
200 ul 3% BSA in PBS for 2 h at 37°C. After four additional washes, 50
ul of ACC synthase solution diluted in 1% BSA/PBS were added to each
well, and plates were incubated on shaker at room temperature for 1 h.
Plates were then washed four times in 0.05% BSA in PBS. One ug (50 ul)
of biotinylated second antibody in 1% BSA in PBS was then added to each
well. Plates were washed four times in wash buffer containing 0.2% Tween
20. Fifty ul of a IOOO-fold dilution of streptavidin-peroxidase reagent
(BRL, Bethesda, Md.) in PBS + 1% BSA were added to each well, and plates
were incubated for 1 h. Plates were washed six times with wash buffer
plus 0.2% Tween 20. Fifty ul of substrate solution (3.2 mg 2,2-azinodi-
[3-ethybenzthiazoline sulfonic acid], six ul 30% peroxide in 6 ml of

sodium citrate, pH 4.5) were added, and plates were covered in foil and

35

incubated on a shaker for 30 to 60 min. Reactions were stopped by the
addition of 50 ul 5% $05 in H20. After shaking for 5 min, A405 was

measured in an ELISA plate reader (Biotek).

RESULTS

Enzyme purification. A number of chromatographic procedures were
developed for the purification of ACC synthase from tomato fruit tissue.
Figure 1 depicts the elution profiles for four of these procedures.
Various combinations of these purification steps yielded ACC synthase
with a specific activity of up to 30,000 units per mg protein (Bleecker
et al. 1986). Table 1 shows the results of the purification procedure
used to obtain the ACC synthase preparations that were subsequently used
to induce antibody production in Balb/c mice.

The immunization schedule and serum titers are provided in Table 2.
The spleen was taken from the mouse 70 days after the first immunization.
At this time, 10 ul of a 300-fold dilution of serum immunoprecipitated 1
unit of ACC-synthase activity. Based on our estimates that the minimum
specific activity for the ACC synthase protein is 400,000 units per mg
protein (Bleecker et al. 1986), it was calculated that there was
sufficient antibody present in 1 ml of serum to precipitate about 100 ug
of ACC synthase protein.

8 cells with less than

The spleen of the immunized mouse yielded 2x10
10% cell death. Fused cells were plated out in seven 96-well culture
plates and, after two weeks, each well was screened for production of

antibodies to ACC synthase using an immunoprecipitation assay (Bleecker

36

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1
0.8 to
2 . 0 . Hydronylepatite
Preparative Anion ‘. cmmmwamy
Exchange HPLC ,’
,I ~o.e
/
r-d Z
c ,’ m o
a 1 0 0' '04 Q 8 0 5
< 1' 2 <
0" V
I “-0.2
a
l
I:
I
I
0 i i i i i ‘fi i o e ; 1 : f 4 . - I - - , - - , -
0 20 40 60 80 100120140 0 so 120 180 240 300
Time (min) Time (min)
0.2 0.50
”WINE!“ N150" Mil-Gel Blue Cinematography
Exchange HPLC
"-0.7
>05
..05 E :3. 0.264
' Q <
~0.4 g
10.3
>0.2
~o.1 00 s {a 1'5 270
Fraction Number

1M NaCl

Figure 1. Elution profiles of four of the chromatographic procedures
used in the purification of ACC synthase. Shaded portions of the graphs
indicate fractions containing enzyme activity. Details of these

purification steps are provided in Bleecker et al. (1986).

(w) eieudsoud- w

37

 

 

h fimm ooo.~m Nmo.o ooo.~ mmcegoxmncov pmu_uzpe:<
NH Na” oom.m Hm.o coo.m emeeeeaam_»eaea
em OH coo mm coo.m~ ee=_oe eu_aeaa_axeee»=
no u omm Km ooo.o~ one: mmcmguancow m>_umeeamea
so“ H am wmm oofi.om eommfiezzv Nma-oa
a ape»- :_muoeq ms\mpwcz as maps:
copumupe_gza
acm>ouma p—cgm>o xuw>wuum u_mvumam cameos; xuv>Puu< _muo» =o_uumem

 

.msmmmu acouwemq cameo» do ox m.m do

mumcmmoEo; may seem mmmgp=5m uu< eo :o_umuweweaa .H mppmh

38

 

 

 

n.d.

Table 2. Immunization schedule and serum titers for ACC-synthase-
specific antibodies. Titers are expressed as the dilution
of serum required to immunoprecipitate 1 unit of ACC synthase
using 10 ul of diluted serum and 10 ul of a 30 units mg'1
protein enzyme solution.

Days after Amount of Titer

first injection antigen injected

(ug LPS)

0 20 n.d.1
I4 20 n.d.
34 -- 0.1
38 20 n d
60 -- 0.005
67 30 n d
68 30 n d
70 (fusion date) -- 0.003
1

not determined

39

 

100-

50-

ACC Synthase Activity (% Control)

 

 

 

 

 

 

 

 

 

 

 

 

 

0
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12
Well Number

 

Figure 2. Immunoprecipitation assays of supernatants from selected
culture wells containing original hybridoma cell lines. One unit of ACC
synthase was incubated with 50 ul of supernatant. Immunoprecipitation
was achieved by the addition of rabbit anti-mouse immunoglobin and
Staphylococcus aureus cell suspension (Bleecker et al. 1986). Hybridoma

line 5b was cloned from cells in well A5.

40

et al. 1986). Figure 2 shows an example of an immunoprecipitation screen
of initial hybridoma cultures. Wells containing supernatant capable of
precipitating more than 60% of the ACC synthase activity (e.g. well No.
A5) were screened again. Of the 28 wells which gave positive results, 26
wells tested positively in a second screening (Fig. 3). Only 14 of these
26 cell lines continued to test positively after 3 weeks of subculturing.
These lines were cloned out twice by limiting dilution. For five of the
cell lines, designated 1b though 5b, all single colonies wells tested
positively after the second cloning. This indicated that the hybridoma
cells present were monoclonal in origin.

Several methods for the production and purification of monoclonal
antibodies from the five hybridomonas cell lines were investigated.
Ascites fluid, obtained from mice injected with hybridoma cells,
contained up to 5 mg of IgG per ml (Bleecker et al. 1986). All five cell
lines were also adapted to serum-free medium from which secreted antibody
could be obtained directly. Culture supernatant from 2-week-old cultures
generally contained about 5 to 10 mg of 196 per liter and represented up
to 25% of the total protein present in the supernatant. Purification of
antibody by Protein A affinity chromatography using the BioRad MAPS II
system resulted in highly purified antibody from both ascites fluid and
culture supernatant. A 3-ml column of affinity gel could bind up to 10
mg of IgG1 antibody. Figure 4 shows an SOS-PAGE fractionation of ascites
fluid and purified MAb from hybridoma line 5b, and culture supernatant

and purified MAb from hybridoma line 2b.

ACC synthase/antibody interactions. The interaction of ACC synthase with

 

Mab from two of the five hybridoma cell lines (2b and 5b) was examined.

41

 

saesé

50~

ACC Synthaee
Activity (96 Control)

 

 

 

 

 

 

 

 

 

 

 

 

 

30"

20"

10

O 'l- n l lull: nlll- h
N

QWNPNMQIDGO FQOOVPNUVD
'- HPPPPPMPPPPNMNNNNN

ChflbuuaVVeHa

2-
i
5-
3%
7-
35

Figure 3. .A second immunoprecipitation assay using supernatants from
culture wells which tested positive in the first screen.
Immunoprecipitation was carried out as in Fig. 1. Bars 30-36 represent

originally negative wells chosen at random as controls.

42

Table 3. Measurement of ACC synthase activity bound to immunoaffinity
gel. Bound activity is the amount of enzyme activity observed
when washed MAb-Sepharose gel was resuspended in enzyme assay
buffer. Residual activity is the amount of enzyme activity

remaining in original solution after immunoaffinity

 

 

adsorption.
Hybridoma line Initial activity Bound activity Residual
activity
units units units
2 b 1600 580 39

5 b 1600 410 20

 

43

Figure 4. SOS-PAGE of proteins stained with Coomassie blue. Lanes: 1,
ascites fluid containing MAb 5b; 2, affinity purified MAb 5b; 3,
immunoaffinity purified ACC synthase eluted from MAb 5b Sepharose gel; 4,
immunoaffinity purified ACC synthase eluted from MAb 2b Sepharose gel; 5,
affinity purified MASb 2b; 6, supernatant from hybridoma line 2b
cultures; molecular weight standards (phosphorylase B, 92.5 kDa; BSA,
66.2 kDa; ovalabumin, 45 kDa; carbonic anhydrase, 31 kDa; soybean trypsin
inhibitor, 21.5 kDa; lysozyme, 14.4 kDa; 2 ug per proteins band per
lane.) For immunopurification of ACC synthase, 1600 units of enzyme (80

1 protein), was incubated with 30 ul of immunoaffinity Sepharose

units mg'
gel. After elution for the Sepharose gel with SDS buffer, 80% of the

sample was subjected to SOS-PAGE.

 

44

“mgvv If"

a .
. ; ', ‘
a‘ivxtgfifigfi’i “ T
' : 4.? '°‘
. a' g

Q
5.
.

t " ‘
,‘ 4:44

-. to -‘-.-’;~ - .

 

45

Immunoprecipitation experiments indicated that 90% of the ACC-synthase
activity present in crude extracts could be removed from solution by
either 2b or 5b antibody (Bleecker et al. 1986). When the
immunoprecipitate was resuspended and assayed for ACC-synthase activity,
25-30% of the original enzyme activity was recovered (data not shown),
indicating that the enzyme retains activity while bound to the antibody.

The activity of bound enzyme was clearly demonstrated with the
immunoaffinity-Sepharose gel. ACC synthase bound to the resuspended gel
showed enzyme activity even after four washes with salt buffer (Table 3).
Again, about 25-35% of the original activity was present on the gel.

The kinetic properties of ACC synthase bound to 5b MAb were compared
to the properties of the free enzyme. The rate of product formation at a
saturating SAM concentration (200 uM) was linear for at least 1.5 h for
both the MAb-bound (hybridoma line 5b) and free enzyme (Fig. 5). The
reaction was saturated at about 20 uM SAM for the free enzyme, while the
MAb-bound enzyme was not saturated until 100 uM SAM was used (Fig. 6).
From a Lineweaver-Burke plot, the Km values were calculated to be 9 and
25 uM for the free and MAb-bound enzyme, respectively (Fig. 7).

Efforts to elute active ACC synthase protein from the immunoaffinity
gel have not been successful. Use of low-pH buffers, urea, and MgCl2 all
resulted in loss of activity and reduced yield of protein compared to
elution with 2% SDS. Elution of the protein with SDS resulted in the
recovery of a single major polypeptide of 50 kDa (Bleecker et al. 1986).
A SOS-PAGE fractionation of eluted protein (Fig. 4) shows that both line
2b and 5b immunoaffinity gels yielded similar quantities of the 50 kDa
protein per unit bound activity. Based on the recovered protein and the

initial activity of the enzyme solution, up to 6,000-fold purification of

46

 

.A .4
O M

ACC(nmol)
v t 2 °°
+SAM

 

 

 

15 3o 45 so 75 so 105
Time(min)

Figure 5. Time course of the ACC-synthase-catalyzed conversion of SAM to
ACC for the free (0) and MAb-bound (e) enzyme. The reactions were run at
a substrate concentration of 200 uM. The arrow indicates the time at
which SAM was added to a reaction mixture after preincubation of the

enzyme for 60 minutes.

47

 

 

8- Free
7 )-
4
6’ Bound

UT

03

 

ACC Synthase Activity (units)
to «5

out

 

 

 

O

 

0 1'0 zo 3'0 4b so 66 7o 8‘0 9‘0 160
[SAMHMMI

Eigg:g_§. ACC-synthase activity as a function of substrate concentration

for the free (0) and MAb bound (0) enzyme.

48

 

 

.5
0|
.5
UI

 

     

   

 

 

 

 

   

 

 

.. Free Bound
T ;r
m r-
ztpt lain
C .2
3 c
7 05 34x5
> I
>
01 A a O . I I A g
-o.5 o 0.5 1.0 1.5 -o.05 o 0.05 0.1 0.15 0.2
[SAM1“ mM“) [SAMI‘l min“)

Figure 7. Lineweaver-Burke plots for free and MAb bound ACC synthase.
The calculated Km was 9 and 25,uM fer the free and MAb bound enzyme,

respectively.

49

the enzyme was achieved. A final specific activity of the enzyme was

calculated to be >500,000 units per mg protein.

Enzyme-linked immunosorbent assay (ELISA). To further characterize the
MAbs and to develop a sensitive technique for the quantification of
ACC-synthase protein, an immunoassay (ELISA) was developed. The ELISA
required the use of 2 MAbs which recognized non-overlapping epitopes on
the ACC-synthase protein. The first MAb was fixed to the plastic wells
of 96-well plates and was used to "capture" ACC synthase from solutions
applied to the wells. The second Mab, to which biotin had been
covalently coupled, was added to the wells, and the amount of bound
biotinylated MAb was quantified with a strepavidin-peroxidase conjugate.
Figure 8 shows the results of ELISAs performed using various combinations
of 2b and 5b antibodies and 1 unit of ACC synthase. A strong ELISA
signal was obtained only when ACC synthase was present and only when MAb
2b and 5b were used alternately for capture and detection of ACC
synthase. These results demonstrate that the two antibodies recognized
non-overlapping epitopes on the enzyme and that the active enzyme
possesses only one epitope for each antibody. The sensitivity of the
ELISA is demonstrated in Figure 9. Using a serial dilution of ACC
synthase, it was found that as little as 0.05 units of enzyme produce a
signal above background and that the response is linear up to 2 units of
enzyme per well. The ELISA response was similar when the serial dilution
of ACC synthase was made in buffer with 1% BSA or with crude tomato
extract as diluent (Fig. 9). Thus, except for some background signal,
there did not appear to be any interference by the crude extract in the

detection of ACC synthase. Figure 9 shows that the crude extract also

50

 

A

0.5 ’- g

0.4 -

A405

0.2 -

0.1r

 

 

 

 

 

 

 

, a m In 11
ACC Synthase +- — .4...

Biotinylated MAb 2b 5b 2b 5b
CaptureMAb 5b 5b 2b 2b

Figure 8. The effects of MAb type (hybridoma line 2b or 5b) and presence

or absence of 1 unit of ACC synthase on the signal generated in the ELISA

system used to quantify ACC synthase.

51

 

1.2“

1.0

0L8

A405

0L6

CL4»

     

Oh2-

O

 

 

 

0 0:5 1 .o 115 2:0
ACC Synthase ( units/well)

Figure 9. The relationship between ACC synthase concentration and ELISA
signal when a partially purified enzyme preparation (350 units/mg
protein) was diluted to the indicated concentrations using either crude

tomato extract (0) or 0.1% BSA (0) as diluent.

52

yielded the expected amount of enzyme activity when increasing amounts of
ACC synthase were added.

The ELISA system was used to examine the change over time in
immunodetectable protein in extracts of wounded tomato pericarp disks
(Fig. 10). After the first hour, both ACC synthase activity and
immunodetectable protein increased several fold, reaching a plateau or
even slightly decreasing between 3 and 5 h after wounding; following
this, a second increase in both enzyme activity and immunoreactive
protein was observed. The amount of ACC synthase present at any time as
calculated from the ELISA standard curve was up to 50% greater than the

amount of enzyme activity measured in the extracts.

In-vivo labeling. Labeling of wounded pericarp tissue with
[35$]methionine followed by immunoaffinity purification resulted in the
isolation of a single major labeled polypeptide which migrated at 50 kDa
on SOS-PAGE (Fig. 11). Addition of various protease inhibitors
(phenylmethylsuflonyl fluroride, leupeptin, pepstatin) during extraction
and purification of the enzyme did not alter the molecular mass of the
immunopurified polypeptide (Fig. 11). In many experiments, a small
amount of radioactivity was also observed at around 90 kDa on SOS-PAGE
(Fig. 11). In some gels, a very faint radioactive band was also present
at 56 kDa (Bleecker et al. 1986). The 90 kDa protein may be the result
of incomplete disulfide reduction of the 50 kDa polypeptide (Bleecker et
al. 1986).

When wounded pericarp tissue was labeled with [35]S-methionine for 1

h at various times after wounding, the total amount of radioactivity

present in the immunopurified 50 kDa protein increased with time after

53

 

T
E 15L ELISA
g 0
c:
3 e
E
.2 10' °
3 - Enzyme
° Assay
To
«1
in
'5
I: '5”
>
m 0
(J a
(J
‘I

 

 

 

o i e :3. 4 5 o i s
Time after Wounding (h)

Figure 10. Time course of wound-induced increases in both ACC-synthase
activity and immunodetectable protein from ripening tomato pericarp
tissue. ACC-synthase activity was calculated from the ELISA signals
using a standard curve generated from a serial dilution of partially

purified enzyme (Fig. 9).

54

Figure 11. Fluorograph of [35$]methionine-labeled, immunopurified
protein extracted from wounded tomato pericarp tissue. Each lane
represents protein from 10 g of tissue labeled with 150 uCi of

[35$]methionine during the 5th and 6th h after wounding. Extraction and
immunopurification were performed without protease inhibitor (lane 1) or

with 0.1 mg ml'1 phenylmethylsulfonyl fluoride (lane 2), 50 ug ml'1
leupeptin (lane 3), or 10 ug ml'1 pepstatin (lane 4).

55

<93

<66

 

‘31

<21
‘14

Figure 12. Fluorograph of an SOS-PAGE fractionation of [35$]methionine-
labeled, immunopurified protein from wounded tomato fruit tissue. Ten

[35$]methionine

grams of tissue were labeled for one hour with 200 uCi of
before extraction. The number above each lane represents the hour at

which tissue was extracted.

56

 

 

I 3°"
25' a _
g 5‘ 45‘; 3
3 20" 5 a a
v 0.05 0 . o
g 2 - :3.
z 15- 0.04 3 410 °. '6'
2 a 0"
n ° .-
0 (h03." :, a
g 10- g 1.3
*5 0.02 5’ «5 "' 3’
>4 3’ I 51
0’ 5P 001 A 9-
C) ' 32 a
o v
< O l l t J l I 1‘0 I :0

 

 

 

 

O 1 2 :3 4 5 £5 7
Time after Wounding (h)

Figure 13. Time course for extractable enzyme activity (0),
incorporation of [35$]methionine into protein (A), and the percentage of
radioactivity in the immunopurified 50 kDa polypeptide (o) in wounded
tomato pericarp tissue. The pericparp tissue was fed 200 001
[355]methionine for 1 h before extraction at the times indicated. These

results have been obtained with the immunopurified protein shown in

Figure 12.

57

wounding (Fig. 12). However, incorporation of radioactivity into total
protein also showed substantial increases with time after wounding (Fig.
13). The percentage of radioactivity incorporated into the 50 kDa
polypeptide reached a maximum (0.04%) during the third hour after
wounding. Thus, expressed as a percentage of total incorporation, the
incorporation of label into the 50 kDa polypeptide correlated with the
increase in extractable enzyme activity as a function of time. The
transient plateau in the accumulation of enzyme activity was accompanied
by a transient decrease in the relative incorporation of radioactivity

into the 50 kDa protein.

DISCUSSION

The purification of ACC synthase from tomato tissue was problematic for
two reasons: the amount of ACC synthase protein in crude extracts was
very small (Kende et al. 1986), and the enzyme was unstable during
purification (Acaster and Kende 1983). Initially, a number of substrate
analogs and inhibitors were used to prepare potential affinity columns in
the hope that a a high degree of purification could be accomplished in a
single step (Acaster and Kende 1983). However, none of these columns
proved to be effective in the purification of the enzyme.

Using various traditional and high-performance liquid chromatography
procedures, a substantial purification of the ACC synthase was achieved
(Bleecker et al. 1986). However, the yield was too low and the purity
insufficient to provide the material needed to generate polyclonal

antibodies and/or a partial sequence of the protein. Monoclonal

58

antibodies specific to ACC synthase have proved to be very effective
reagents for the isolation of ACC synthase protein. The immunoaffinity
gel provides, in a single step, up to 20,000-fold purification of ACC
synthase from an (NH4) SO4 fraction of a crude extract.

The success in obtaining monoclonal antibodies to such a
low-abundance protein may be attributed to two important factors. The
level of extractable enzyme was increased up to 100-fold by wounding
and/or treatment of the tissue with LiCl. In addition, the protein
preparation used to generate antibodies was enriched in ACC synthase
several hundred fold over the crude homogenate such that about 10% of the
purified protein was represented by ACC synthase protein. The use of a
lipopolysaccharide (LPS) conjugate of ACC synthase to stimulate antibody
production may also have contributed to the successful outcome. It is
known that LPS possesses a number of immunological activities (Bradley
1979). Recent reports indicate that conjugation to LPS increased the
antigenic responses to various proteins and haptens (Lange et al. 1983).
Lipopolysaccharide is known to activate B lymphocytes in the absence of
T-cells (Bradley 1979), and it has been reported that the proportion of
activated or "blast“ B-lymphocytes present in a spleen correlates with
the yield of antibody producing hybridomas following cell fusion (Stahli
et al. 1980). In this regard, we observed a high proportion (10%) of
enlarged blast cells from the spleen of the ACC syntase-LPS immunized
mouse.

Since pure ACC-synthase protein was not available for selection of
ACC-synthase-specific MAbs, we chose an immunoprecipitation assay to
select our hybridoma lines. This was possible because the assay for ACC

synthase is very sensitive permitting us to use one unit (ca. 2 ng) of

59

ACC synthase in a relatively crude protein mixture. This screen provided
us with monoclonal antibodies with high affinity and specificity for the
the native enzyme. However, none of the five monoclonal antibodies
recognize ACC synthase protein bound to nitrocellulose (Bleecker et al.
1986). The immun0precipitation assay used would tend to select for
assembled topographic (i.e. tertiary) sites over segmental (i.e.
secondary) sites on the antigen since native conformation of the enzyme
was a prerequisite for detecting antibody binding (Berzofsky 1986). The
physical nature of ACC synthase may also be a contributing factor. The
interaction of ACC synthase protein with nitrocellulose may, e.g., result
in a protein structure with little or no native conformation. In support
of this idea, it has been observed that spotting of native ACC synthase
protein on moistened nitrocellulose completely eliminated enzyme activity
(data not shown).

It is clear from the kinetic analysis of immunoaffinity-gel-bound
enzyme activity that the Km and Vmax are altered relative to the free
enzyme. The recovery of only 25-30% of the enzyme activity could be
attributed either to EN) actual loss of active sites resulting from
antibody binding or to an alteration in the catalytic constant or
turnover number (k3) for the enzyme. The data presented cannot
discriminate between these possibilities.

The stability of both free and MAb-bound enzyme in the presence of
200 uM SAM is in contradiction to recent published reports that ACC
synthase from mung bean (Satoh and Esashi 1986) and winter squash
(Nakajima and Imaseki 1986) showed time-dependent substrate inactivation.
A similar phenomenon was also reported in crude extracts of tomato

(Boller et al. 1979). On the other hand, a second report indicated no

6O

substrate inhibition in tomato extracts (Yu et al. 1979). It has been
suggested that substrate inactivation may be the cause of the rapid
turnover of ACC synthase activity 111 M (Satoh and Esashi 1986).
However, the lack of substrate inactivation observed in the present
experiments suggests that the effect may not be a general one but rather

a consequence of the specific in-vitro assay conditions (e.g.

 

temperature, pH, purity of substrate).

During the course of the work presented here, Nakajima and Imaseki
(1986) reported on the purification of ACC synthase from winter squash.
The protein from winter squash has very different physical and enzymatic
properties from that of tomato fruit. We have examined two local
varieties of winter squash and have found that the ACC synthase activity
from squash could not be immunoprecipitated by MAb 2b and 5b, nor by
polyclonal antiserum from the immunized mouse (data not shown).

The ELISA system developed for ACC synthase provides a useful
technique for quantifying as little as 50 pgrams of ACC synthase protein
in crude extracts of tomato fruit tissue. Since the ELISA signal depends
on the binding of two different MAb proteins to separate epitopes on the
enzyme, it should be considered an extremely specific as well as
sensitive assay for the protein. Because of the extremely low levels of
the protein in non-wounded tomato tissue, the maximum amount of ACC
synthase present in ripening tomato is just at the limit of sensitivity
for the assay. Thus, this technique cannot as yet be used to monitor
developmental changes in enzyme levels during ripening.

The ELISA of crude extracts performed at various times after
wounding indicates that the amount of antigenic protein is consistently

higher than measured by the enzyme assay using the same extracts. This

61

result indicates that immunologically reactive protein is present which
is enzymatically inactive. The enzymatically inactive protein may
represent an inactive form of ACC synthase in the tomato tissue, e.g. an
inactive precursor or inactivated product of the enzyme. Alternatively,
inactivation could have occurred during extraction and/or overnight
dialysis of the crude extracts. It should be remembered that binding of
both line 2b and line 5b MAbs is apparently very dependent on native
conformation of the enzyme since nitrocellulose-bound ACC synthase was
not recognized by either MAb. Thus, the inactive form of antigenically
recognized protein in the tomato extracts must exist in a conformation
that is very similar to that of the active enzyme. In addition, it seems
likely that the inactive form of the enzyme is present as the 50 kDa
polypeptide since the 50 kDa protein represented 90% of the radiolabeled
protein immunopurified from crude extracts using either line 2b or line
5b MAb.

The results of the L v_iv_o protein labeling experiments are
consistent with the previous evidence that: wound induction of
ACC-synthase activity involves gg ggyg protein synthesis (Acaster and
Kende 1983; Bleecker et al. 1986; Kende and Boller 1981). These results
also support the notion that ACC-synthase protein is subject to rapid
turnover 1g yiyg. Substantial incorporation of radioactivity into the 50
kDa polypeptide was observed even during the 5th hour after wounding, a
time when no net accumulation of enzyme activity or of ELISA-detectable
protein was occurring.

The question whether induction of ACC synthase involves
transcriptional activation of the gene was not addressed in the

experiments reported here. Acaster and Kende (1983) found that 100 UN

62

cordycepin, an inhibitor of RNA synthesis, reduced wound-induced ACC
synthase accumulation by 50%. Chappell et al. (1985) reported that
neither cordycepin nor actinomycin D inhibited elicitor-induced
ACC-synthase accumulation in parsley cell cultures, although both
inhibitors effectively prevented phenylalanine ammonia lyase and
chitinase accumulation. Similarly, Wang and Adams (1982) found that
actinomycin D was ineffective in preventing ACC-synthase accumulation in
pre-chilled cucumber plugs. Thus, the role of gene transcription in ACC-
synthase accumulation remains an open question. Future work in our

laboratory will approach this question using the techniques of 1g vitro

 

translation/immunoprecipitation and direct measurements of ACC-synthase-

specific mRNA once nucleic acid probes for the gene become available.

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63

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64

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ghaseoli (Pers) wint. Planta 12g, 301-306

Nakajima, N., Imaseki, H. (1986) Purification and properties of

1-aminocyclopropane-I-carboxylate synthase from mesocarp of

 

Cucurbita maxima Duch. fruits. Plant Cell Physiol. 22, 969-980

Satoh, 5., Esashi, Y. (1986) Inactivation of I-aminocyclopropane-I-
carboxylic acid synthase of etiolated mung bean hypocotyl segments
by its substrate, S-adenosyl-L-methionine. Plant Cell Physiol. 22,
285-291

Sexton, R., Lewis, L.N., Trewavas, A.J., Kelly, P. (1985) Ethylene and
abscission. In: Ethylene and plant development, pp. 173-196,
Roberts, J.A., Tucker, G.A., eds. Butterswoths, London

Stahli, C., Staehelin, T., Miggiano, v., Schmidt, 0., Haring, P. (1980)
High frequency of antigen specific hybridomas. Dependence on
immunization parameters and prediction by spleen cell analysis.
J. Immunol. Meth. 22, 297-304

Yang, S.F., Hoffman, N.T. (1984) Ethylene biosynthesis and its regulation
in higher plants. Ann. Rev. Plant Physiol. §§, 155-190

65

Yoshii, H., Imaseki, H. (1982) Regulation of auxin induced ethylene
biosynthesis. Repression of inductive formation of
l-aminocyclopropane-l-carboxylate synthase by ethylene. Plant
Cell Physiol. 22, 639-649

Yu, Y.B., Adams, 0.0., Yang, S.F. (1979) 1-aminocyclopropanecarboxylate
synthase, a key enzyme in ethylene biosynthesis. Arch. Biochem.
Biophys. 192, 280-286

Wang, C.Y., Adams, 0.0. (1982) Chilling-induced ethylene production in
cucumbers (Cucumis gggiyg L.). Plant Physiol. g2, 424-427

CHAPTER III

AN EVALUATION OF 2,5 NORBORNADIENE AS A

REVERSIBLE INHIBITOR OF ETHYLENE
ACTION IN DEEPWATER RICE

66

67

ABSTRACT

Partial submergence 0f deepwater rice (Oryza sativa L. cv. Habiganj

 

Aman II) elicits three responses: enhancement of internodal elongation,
inhibition of leaf growth and promotion of adventitious root formation.
All three responses can be induced in isolated stem sections by treatment
with ethylene. Dose-response curves indicate that the responses are
linearly related to the logarithm of the ethylene concentration over 2
orders of magnitude. Application of the cyclic olefin 2,5-norbornadiene
(N80) to ethylene-treated sections results in a parallel shift in
dose-response curves to higher ethylene concentrations, indicating that
NBD behaves as a competitive inhibitor of ethylene action. Internodal
elongation of stem sections is promoted by gibberellic acid (GAB) in the
absence of exogenous ethylene. Endogenous ethylene levels do not
increase in GA3-treated sections, and application of NBD does not prevent
GA3-promoted elongation. To the contrary, NBD treatment results in
increased growth at intermediate GA3 concentrations. These results
support the idea that ethylene acts through endogenous GA in promoting
growth in deepwater rice. NBD applied to GA3-treated stem sections
results in increased ethylene production. This enhancement of ethylene
formation is reversed by application of either ethylene or propylene,
indicating that ethylene biosynthesis in rice internodes is under

negative feedback control.

68

INTRODUCTION

Deepwater rice, like a number of other semiaquatic plants (7),
responds to partial submergence with a great increase in internodal
elongation (23). This rapid growth response, which is based on an
increased rate of cell division and elongation in the intercalary
meristem 0f the internode (13), is mediated by an interaction of the
plant hormones ethylene (C2H4) and gibberellin (GA) (12,15). Regulation
by altered hormone levels and changes in responsiveness to hormones are
involved in this response. Submergence promotes ethylene biosynthesis in
the internode (12). Ethylene alone stimulates internodal elongation in
non-submerged plants (12) and stem sections (16), and rapid elongation is
reversibly inhibited by inhibitors of 02H4 biosynthesis (12,16). Thus,
growth in partially submerged deepwater rice clearly is a response to
environmental stress and is mediated by an increase in the level of a
plant hormone.

Ethylene does not appear to promote growth directly but through the
action of GA. Raskin and Kende (15) showed that C2H4 failed to promote
rapid elongation in rice stem sections pretreated with tetcyclacis, an
inhibitor of GA biosynthesis. 0n the other hand, application of 10 uM
GA3 elicited the full growth response in the absence of added C2H4.

Since 1 ul/L CZH4 enhanced the effectiveness of low 0A3 concentrations in

promoting the growth of tetcyclacis-treated sections, it appears that

69

02H4 acts, at least in part, by increasing the sensitivity of the
internodal tissue to endogenous GA.

To extend the work described above and to define more clearly the
qualitative and quantitative role of C2H4 in the submergence response of
deepwater rice, a series of experiments were performed using the
reversible inhibitor of C2H4 action, 2,5-norbornadiene (NBD). The
inhibitory effects of NBD on C2H4 action in plants was first described by
Sisler and Pian (20) and more recently characterized by Sisler and Yang
(21). A number of physiological studies with NBD confirm the ability of
this cyclic olefin to block C2H4 action in a number of plants (19,20,21).
It is generally accepted that NBD competes with ethylene for binding at
the ethylene receptor site. Thus, high concentrations of CZH4 can
reverse the inhibitory effect of NBD (19,21).

The work reported here examines the effectiveness of NBD as a
reversible inhibitor of C2H4 action in three C2H4-responsive tissues of
rice stems. In addition to the promotion of internodal elongation, 02H4
inhibits leaf growth (16) and promotes the development of adventitious
roots at the nodes (2,22). NBD was also used to evaluate the role of

endogenous C2H4 in GA3-promoted internodal elongation of stem sections.

MATERIALS AND METHODS

Chemicals. GA3 was a gift from Merck and Co., Inc. (Rathway, NJ).
2,5-Norbornadiene (bicyclo[2.2.1]hepta-2,5-diene) was purchased from

Aldrich Chemical Co. (Milwaukee, WI) and L-[3,4-14C]methionine (45

7O

mCi/mmol) was purchased from Research Products International Corp. (Mount
Prospect, IL).

Plant Material. Seeds of deepwater rice (Oryza sativa L., cv

 

“Habiganj Aman II") were obtained from the Bangladesh Rice Research
Institute (Dacca).

Growth Conditions. Rice was germinated and grown as described by
Metraux and Kende (12). Stem sections containing the youngest elongating
internode were prepared from 10- to 12-week-old plants according to
Raskin and Kende (16). Sections (15 per treatment) were incubated in
sealed plexiglas cylinders (2.5 l) under continuous light with the basal
2 cm of the sections standing in 30 ml of either distilled water or a
GA3 solution. The appr0priate volumes of C2H4, propylene (C3H6) and/0r
NBD were injected through serum caps into each cylinder to yield the
required gaseous concentration of each compound. The cylinders were
purged with air for 5 min every 12 h, resealed, and C2H4 and/or NBD was
injected again.

Analysis of Growth. Growth of internodes and leaves was determined
after 3 days as previously described (16). The number of adventitious
roots at the lower node was determined by counting all roots 22 mm in
length.

Determination of Ethylene Concentration. At the end of the
incubation period, gas samples were collected from the internodal lacunae
as described (18), and the ethylene concentration was determined by gas
chromatography (10). In the radioisotope labeling experiment, sections
treated with 5 uM GA3 were incubated in cylinders as above for 48 h at
which time 5 uCi of L-[3,414C]- methionine was added to the 0A3 solution.

Sections were returned to cylinders and incubated for an additional 24 h.

71

Gas from the internodal lacunae was collected and transferred to a 40-ml
syringe. Unlabeled CZH4 was added to give a final concentration of 100
ul/L. The C2H4 was trapped by addition of HgClO4 to the syringe (1).
Syringes were shaken overnight at 4°C. The HgClO4 solutions were then
transferred to 18 ml of Safety Solve scintillation fluid (Research
Products International, Mount Prospect, IL). The radioactivity was
determined by scintillation counting and expressed as cpm above

background.
RESULTS

Effects ofcaHg‘and NBD Dosage on Rice Stem Sections. Internodal
elongation of stem sections increased with increasing levels of applied
C2H4 (Fig. 1A). The extent of growth was linearly related to the
logarithm of the C2H4 concentration over a range of two orders of
magnitude with maximum growth occurring between 5 and 10 ul/L C2H4. In
the presence of increasing concentrations of NBD, both the
threshold level of CZH4 needed for the growth response and the level of
C2H4 required to saturate it were shifted to higher C2H4 concentrations,
while the maximum growth attained and the slopes of the dose-response
curves were essentially unaffected.

Ethylene inhibited leaf growth in isolated stem sections. Figure 18
shows the dose-response curves obtained in the presence of increasing
concentrations of N80. The curve generated in the absence of NBD
indicates that the C2H4 response is saturated between 0.5 and 1 ul/L

CZH4, a value substantially lower than that obtained for internodal

72

 

100

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A INTERNODE
A
E
E
V
:5
3
o
h
(D
G 0 "01 1:0 1° 10°
02H4 (pill)
100 B LEAF
A
E
E
v
:5
3
o
\-
(D
O‘LF/IL' ' ‘ , 1V 0
0 on L0 10 °
02H4 (pl/l)
g 20 C ROOT
o
o
a:
m 15-
D
.9.
‘2 10
o
>
2
._ 5’}
°. o
o 9
2 °‘ 0 V3, 1.0 10 ‘00
02H4 (pl/I)

Fig. 1. Dose-response curves relating the C2H4 concentration (on a

logarithmic scale) to internodal elongation (A), leaf growth (8) and

adventitious root initiation (C) and the effect of increasing levels of

NBD on these relationships. NBD concentrations were 0, 500, 1000, 2000

ul/L. Each point is the mean of 15 measurements t standard error.

73

elongation. Increasing concentrations of NBD again shifted the
dose-response curve to higher C2H4 values.

In the course of these experiments, it was observed that CZH4 also
increased the number of adventitious roots developing at the lower node
of the stem section. Figure 1C shows that root development is saturated
at 0.2 ul/L C2H4 and that inhibition of this process by NBD was
reversed with increasing C2H4 concentrations.

Effect of NBD on GA3-Pr0moted Internodal Elongation. Since GA3
elicits the full growth response in stem sections that had not been
treated with 02H4, we used N80 to examine the possible involvement of
endogenous C2H4 in GA3-promoted growth. The dose-response curves for
GA3-promoted growth in the presence or absence of 2,000 ul/L NBD are
shown in Figure 2. NBD did not inhibit the promotion of growth by GAB“
To the contrary, NBD-treated sections showed a small but consistent
enhancement of growth over air controls at intermediate 0A3
concentrations.

Two possible explanations were considered for the NBD-stimulated
growth of GAB-treated sections: the enhanced growth was either due to
some intrinsic growth-promoting activity of N80, or resulted from the
NED-stimulated increase in endogenous 02H4 levels (see Fig. 2). To
distinguish between these alternatives, sections were incubated in 0.5 uM
GA

2000 ul/L NBD; and O, 10 or 100 ul/L CZH The effects of these

3’ 4'
treatments on internodal growth are presented in Table 1. Treatment with
NBD enhanced growth by 26% over controls (Table I) and increased C2H4
concentration in the lacunar space to 0.18 ul/L (Fig. 2). Addition of 10
ul/L C2H4 to this system did not result in additional enhancement of

growth, indicating that the endogenous CZH4 concentration was not

74

 

 

 

 

 

100-
I 80-
?? ‘*14155
E it
v 60'“ 03 A
{5 ‘t
3 S
g 40‘ 4412 7'
CD .

2°” ~04

0 0
Fig. 2. The effect of GA3 dosage on internodal elongation and

endogenous 02H4 levels in the presence and absence of 2000 ul/L NBD.
Growth values represent the mean of 10 determinations t standard error.
02H4 concentration was determined from the pooled interlacunar gas of 10
samples.

75

Table 1. The effect of exogenous 02H4 and N80 on internodal growth of
stem sections treated with 0.5 uM 6A3

 

 

 

Treatment Internodal
02H4 NBD Growth
(Ul/L) (mm 1 SE)
0 0 54 i 6
O 2000 68 i 7
10 2000 69 i 8

100 2000 85

H-
CD

 

76

sufficient to account for the N80- promoted enhancement of growth.
Addition of 100 ul/L CZH4 did enhance growth showing that the sections
were capable of responding.

The Effect of NBD on Endogenous Ethylene Levels. The concentration
of ethylene in the lacunar space was measured in stem sections treated
with GA3 (Fig. 2). In the absence of NBD, CzH4 levels remained very low
(0.05 ul/L) even at high (5 uM) concentrations of GA3. However, in the
presence of 2000 ul/L NBD, increasing amounts of C2H4 were correlated
with increasing 0A3 concentrations, such that at 5 uM GA3 02H4 levels
were tenfold higher in NBD-treated sections than in air-grown controls.
If one interprets these results in terms of the inhibitory properties of
NBD on CZH4 action, the data indicate a negative feedback effect of 02H4
on C2H4 biosynthesis. To investigate this possibility, two kinds of
experiments were performed. Sections were treated with 5 uM GA3, 2000
ul/L N80, and either no or 100 ul/L C2H4. To determine endogenous CZH4
synthesis, [3,4-14C]methionine was fed to the plants during the third day
of growth. Table II shows the effect of N80 and N80 plus C2H4 on CZH4
synthesis. HBO-treated plants incorporated three times more
radioactivity into CZH4 than did air-grown controls. Addition of 100
ul/L CZH4 to NBD-treated plants substantially reduced the amount of
radioactivity in C2H4. To allow direct measurement of endogenous
ethylene levels, 03H6 was used to reverse the effects of NBD on C2H4
synthesis. Propylene, a biologically active analog of 02H4 (3), is
capable of stimulating the growth response in deepwater rice and of
reversing the physiological effects of NBD (data not shown). The results
in Table 11 show that C3H6 reversed the HBO-stimulated synthesis of C2H4

in GA3 treated sections. These results are consistent with a negative

77

Table 2. Reversible inhibition of HBO-stimulated C2H4 production in
stem sections by CZH4 and 03H6

 

 

Treatmenta Radioactivity Total
in C2H4 C2H4
b
(cpm) (ul/L)
Air 8.3 -
NBD 28.6 -
NBD + 02H4 14.8 -
Air - 95
N80 - 470
N80 + C3H6 - 165

 

aAll stem sections were treated with 5 uM GA3, and some with 2000 ul/L

NBD, 100 ul/L CZH4

and 2000 ul/L C3H6 as indicated.

bStem sections were fed L-[3,414C]methionine.

78

feedback model of the regulation of CZH4 biosynthesis in internodes of

deepwater rice.

DISCUSSION

The three C2H4-dependent responses examined in this study occurred
over CZH4 concentration ranges of about 2 orders of magnitude. This is a
relatively narrow range for plant hormone interactions (9) but is typical
for such animal hormones as estrogen (8). Interestingly, the three
responses differed widely in their sensitivities to applied C2H4.
Initiation of adventitious roots was 80% saturated at 0.1 ul/L C2H4’
which was near the threshold concentration for the internodal elongation
response. We use the term sensitivity in the broadest sense (5) since
the three responses may involve different biochemical mechanisms. At
this point, we cannot say where in the signal transduction chain the
limiting factors for each response may occur.

The inhibition of all three CZH4 responses by NBD could be overcome
by increasing CZH4 concentrations, indicating the competitive nature of
the interaction of N80 with C2H4 at the receptor site. A competitive
interaction of N80 and 02H4 has been demonstrated in other plant systems.
Using Linweaver-Burke plots, Sisler and Yang (21) and Sisler et al. (19)
found an NED-dependent shift in the apparent Km 0f C2H4 responses in pea
seedlings (growth) and citrus leaves (abscission). These results were
interpreted as indicating a decrease in the affinity of the receptor for
02H4 with increasing NBD concentrations, i.e., a competitive mode of

inhibition.

79

The role of endogenous C2H4 in GA3-promoted growth is shown in Fig.
2. Even growth-saturating concentrations of GA3 did not alter the low
basal level of C2H4 present in the lacunae of air-grown sections,
demonstrating that GA action does not involve alterations in C2H4
biosynthesis. When we attempted to show that the basal level of 02H4
present in GA3-treated sections was not required for growth by applying
N80 to such sections, we actually observed an increase in growth. Since
this growth promoting effect of NBD could be attributed to a weak
C2H4-like activity of NBD, we cannot positively conclude that some low
level of C2H4 activity is not required for the growth response in
deepwater rice.

The idea that NBD may have both anti-ethylene and ethylene-like
activity is not a contradictory one if one considers two probable
components of 02H4 action: Binding of C2H4 (or an analog) to the receptor
and a biochemical (e.g. conformational) change of the receptor which
initiates the chain of events leading to the physiological responses.
Borrowing terminology from the field of pharmacology, the above
components of hormone action would be called affinity and intrinsic
activity, respectively (6). A compound which shows both affinity and
intrinsic activity is termed an agonist, while a compound which shows
affinity but no intrinsic activity is termed an antagonist (6). Partial
agonists are drugs which show some intrinsic activity but block the
action of other compounds with higher intrinsic activity. A good example
of a partial agonist in 02H4 biology is C3H6, which competitively
inhibites C2H4 action at lower concentration (4) but also shows intrinsic
CzH4-like activity at higher doses (3). Whether NBD behaves as a pure

antagonist or a very weak partial agonist may not be relevant to many

8O

physiological systems where, in practice, it acts as a very effective
inhibitor of CZH4 action. However, in studies which call for the
complete elimination of C2H4-like activity, the question whether N80 is
an antagonist or partial agonist becomes important. In their study on
the effects of NBD on pea seedlings, where CZH4 inhibits epicotyl
elongation, Sisler and Yang (21) found that supraoptimal NBD
concentrations led to a reduction in growth. They attributed this result
to non-specific, toxic effects of NBD since similar concentrations of
cyclohexane also showed inhibitory effects. However, the effect of
cyclohexane was much less pronounced than that of NBD. Therefore, the
possibility that NBD exerted some CZH4-like activity cannot be ruled out
in this system.

An alternative explanation for the growth-promoting activity of NBD
observed in these experiments would be that NBD enhances growth
independent of 02H4 action. NBD caused a rather large increase in pea
epicotyl elongation in seedlings grown in air (21). This effect was
attributed to an inhibition of endogenous CZH4 activity in the seedlings;
however no evaluation was made whether endogenous levels of CZH4 were
sufficient to create the observed difference in growth. In our own
experiments with leaf growth (Fig. 18), we found that NBD promoted leaf
growth over air growth controls. Again, we cannot say how much of the
growth promotion by NBD can be attributed to an anti-02H4 effect alone.

The NBD-promoted increase in CZH4 production in GA3 treated stem
sections can be interpreted in terms of feed-back inhibition of 02H4
synthesis. Such a regulatory system has been described in a number of
plant systems (see ref. 24). Feedback or autoinhibition has been noted

in fruit and vegetative tissues and has been ascribed to inhibition of

81

I-aminocyclopropane-l-carboxylic acid (ACC) synthase (17,25) and/or
increased conjugation of ACC (11,14). Ethylene biosynthesis increases
substantially in the internodes of deepwater rice when plants are
submerged (12). Evidence indicates that low 02 in the internode acts as
a signal for this increase (16). By whatever mechanism low 02 stimulates
ethylene biosynthesis, it must somehow circumvent the feedback regulatory

system observed here.

REFERENCES

1. Abeles F8 1973 Ethylene in plant biology. Academic Press, New York

2. Bleecker AB, 5 Rose-John, H Kende 1985 Ethylene action in rice:
Reversible inhibition by 2,5-norbornadiene. Plant Physiol 77:5-157

3. Burg SP, EA Burg 1967 Molecular requirements for the biological
activity of ethylene. Plant Physiol 42:144-152

4. Dollwet HA, RE Seeman 1975 Propylene - a competitor of ethylene
action. Plant Physiol 56:552-554

5. Firn RD 1986 Growth substance sensitivity: The need for clearer
ideas, precise terms and purposeful experiments. Physiol Plant
67:267-272

6. Goth A 1981 Medical Pharmacology. C.V. Mosby, pp 7-14

7. Jackson MB 1985 Ethylene and responses of plants to soil
waterlogging and submergence. Ann Rev Plant Physiol 36:145-174

10.

11.

12.

13.

14.

15.

16.

17.

82

Katzenellenbogen BS, J Gorski 1972 Estrogen action in vitro:
Induction of the synthesis of a specific uterine protein. J Biol
Chem 247:1299-1305

Kende H, G Gardner 1976 Hormone binding in plants. Ann Rev Plant
Physiol 27:267-290

Kende H, AD Hanson 1976 Relationship between ethylene evolution and
senescence in morning-glory flower tissue. Plant Physiol 57:523-527
Liu Y, NE Hoffman and SF Yang 1985 Ethylene-promoted malonylation of
1-aminocyclopropane-I-carboxylic acid participates in autoinhibition
of ethylene synthesis in grapefruit flavedo disks. Planta
164:565-568

Metraux J-P, H Kende 1983 The role of ethylene in the growth
response of submerged deep water rice. Plant Physiol 72:441-446
Metraux J-P, H. Kende 1984 The cellular basis of the elongation
response in submerged deep-water rice. Planta 160:73-77
Philosoph-Hadas S, S. Meir, N Aharoni 1985 Autoinhibition of
ethylene production in tobacco leaf discs: Enhancement of
I-aminocycl0propane-1-carboxylic acid conjugation. Physiol Plant
63:431-437

Raskin I, H Kende 1984 Role of gibberellins in the growth response
of deep water rice. Plant Physiol 76:947-950

Raskin I, H Kende 1984 Regulation of growth in stem sections of
deep-water rice. Planta 160:66-72

Riov J, S.F. Yang 1982 Autoinhibition of ethylene production in
citrus peel discs. Suppression of 1-aminocyclopropane-I-carboxylic

acid synthesis. Plant Physiol 69:687-690

18.

19.

20.

21.

22.

23.

24.

25.

83

Rose-John S, H Kende 1985 Short-term growth response of deep-water
rice to submergence and ethylene. Plant Sci 38:129-134

Sisler EC, R Goren, M Huberman 1985 Effect of 2,5-norbornadiene on
abscission and ethylene production in citrus leaf explants. Physiol
Plant 63:114-120

Sisler EC, A Pian 1973 Effect of ethylene and cyclic olefins on
tobacco leaves. Tobacco Sci 17:68-72

Sisler EC and SF Yang 1984 Anti-ethylene effects of cis-2-butene and
cyclic olefins. Phytochem 23:2765-2768

Suge H 1985 Ethylene and gibberellin: Regulation of internodal
elongation and nodal root development in floating rice. Plant Cell
Physiol 26:607-614

Vergara BS, 8 Jackson, SK DeDatta 1976 Deep-water rice and its
response to deep-water stress. 1g Climate and Rice, International
Rice Research Institute, Los Banos, Philippines, pp 301-319

Yang SF and NE Hoffman 1984 Ethylene biosynthesis and its regulation
in higher plants. Ann Rev Plant Physiol 35:155-189

Yoshii H, H Imaseki 1982 Regulation of auxin-induced ethylene
biosynthesis. Repression of inductive formation of
I-aminocyclopropane-l-carboxylate synthase by ethylene. Plant Cell

Physiol 23:639-649

CHAPTER IV

ANATOMICAL ANALYSIS OF GROWTH AND DEVELOPMENT
PATTERNS IN THE INTERNODE OF DEEPWATER RICE

84

85

ABSTRACT

Submergence of the stem induces rapid internodal elongation in deepwater

rice (Oryza sativa L. cv. “Habiganj Aman II"). A comparative anatomical

 

study of internodes isolated from air-grown and partially submerged rice
plants was undertaken to localize and characterize regions of growth and
differentiation in rice stems. Longitudinal sections were examined by
light and scanning electron microscopy. Based on cell-size analysis,
three zones of internodal development were recognized: a zone of cell
division and elongation at the base of the internode designated the
intercalary meristem (IM); a zone of cell elongation without concomitant
cell division; and a zone of cell differentiation where neither cell
division nor elongation occur. The primary effects of submergence on
internodal development were a threefold increase in the number of cells
per cell file resulting from a decrease in the cell-cycle time from 24 to
7 h within the IM; an expansion of the cell-elongation zone from 5 to

15 mm leading to a threefold greater final cell length; and a suppression
of tissue differentiation as indicated by reduced chlorophyll content and
a lack of secondary wall formation in xylem and cortical sclerenchyma.
These data indicate that growth of deepwater-rice internodes involves a
balance between elongation and differentiation of the stem. Submergence

shifts this balance in favor of growth.

86

INTRODUCTION

Deepwater or floating varieties of rice respond to partial submergence
with a great increase in the rate and total extent of internodal growth.
Growth rates of up to 25 cm per day have been reported in the field
(Vergara et al. 1976). A number of recent experiments indicate that
enhanced internodal elongation results from the action of the plant
hormones ethylene and gibberellin (GA) (Metraux and Kende 1983; Raskin
and Kende 1984a,b). The following sequence of events is initiated by
submergence of the plant: the 02 concentration within the internode is
lowered (Raskin and Kende 1984a); low 02 levels stimulate ethylene
synthesis and cause the ethylene concentration within the internode to
rise (Raskin and Kende 1984a); ethylene apparently increases the activity
of endogenous GA which, in turn, promotes both cell division and cell
elongation (Raskin and Kende 1984b).

The cellular basis of the submergence response was examined by Metraux
and Kende (1984). Cell division and subsequent cell elongation occur at
the base of the uppermost elongating internode. This region of the
internode is defined as the intercalary meristem (IM) and is bounded both
above and below by non-meristematic, more differentiated tissues (Esau
1965, Chpts. 5 and 11). Intercalary growth is characteristic for
monocotyledonous plants and has been the subject of a number of
physiological and anatomical studies (Buchholz 1921; Fisher I970a, 1970b;
Kaufman et al. 1965; Sharman 1942). Studies with applied plant growth

87

regulators indicate that GAs are the class of hormones primarily involved
in the control of intercalary growth in monocotyledonous plants (Fisher
1970b, Kaufman 1965).

The magnitude and localized nature of the cell division and cell
elongation response make submerged deepwater rice an attractive system to
investigate the biochemical and molecular bases of hormone action in
plant growth and development. Such investigations require a detailed
understanding of the location and anatomical nature of intercalary growth
and development. In particular, it is necessary to define the specific
regions of the internode which are involved in cell division, cell
expansion and tissue differentiation. In this report, we examine the
anatomical characteristics of intercalary growth and development and the

effect of partial submergence on these processes.

MATERIALS AND METHODS

Plant material and conditions of incubation. Seeds of deepwater rice

 

(Oryza sativa L. cv. "Habiganj Aman II") were obtained from the

 

Bangladesh Rice Research Institute, Dacca. Conditions of germination,
growth and submersion of whole plants were as described by Metraux and
Kende (1983). Plants used in these experiments ranged in age from 12 to

14 weeks.

Preparation of tissue and microscopy. Prior to submergence, the length

 

of the uppermost growing internode was marked on selected tillers.

Experiments were performed on tissue taken from internodes that were

88

5.5-6.5 cm in length at the beginning of the experiment. After 48 h of
additional growth in air or under conditions of partial sumbergence, the
total length of the uppermost internode was measured. Beginning with a
basal cut just below the second highest node of the stem, 1.5-cm segments
were excised along the entire uppermost internode. For light microscopy,
internode sections were fixed in Nawashin III (0.3% chromic acid, 2%
acetic acid, 10% formalin, all by vol.) for 40 h at 4°C and dehydrated in
a series of increasing ethanol concentrations. After a transition
through xylene, the sections were embedded in paraffin. They were
sectioned with a rotary microtome and triple stained with alcoholic iron
hematoxylin, safranin and fast green (Johansen 1940, Chpts. 7 and 12).
For scanning electron microscopy, 1.5-cm stem segments from the basal end
of the growing internode were sliced in half longitudinally with a
single-edged razor blade, fixed in 1% (v/v) glutaraldehyde for 2 h,
dehydrated in an ethanol series, critical-point dried, coated with gold,
and photographed with a JEOL (Tokyo) model JSM 35C scanning electron
microscope. Cell sizes were measured by counting the number of
parenchyma cells in successive IOO-um regions of longitudinal cell files.
Average cell length was calculated from data obtained from three cell
files, one in the central region of the stem, and one each from the
fourth file counted from the inner and outer periphery of the stem
tissue, respectively. Chlorophyll content was determined according to

Arnon (1949).

89

RESULTS

Localization of cell division and cell elongation in rice internodes.
One can clearly distinguish the zones of cell division and cell
elongation in the rice internode when one compares the cell sizes from
the basal towards the apical end of the growing internode (Fig. 1). The
region at the base of the internode showing a constant minimum cell size
corresponds to the zone of cell division (Fisher 1970). The location of
this zone is consistent with the region of [3H]thymidine incorporation
previously described by Metraux and Kende (1984), although the size of
this zone (air-grown: approx. 2 mm; submerged: approx. 3 mm) is about
twice the size estimated for air-grown and submerged stem sections by
Metraux and Kende (1984). The cell-division zone is located immediately
above the white tissue of the node (at the 10-mm mark in Fig. 2).
Parenchyma cells within this zone average 17 um in length in air-grown
plants and 28 um in submerged plants (Fig. 1), indicating that cell
division in submerged plants does not keep pace with the increased rate
of cell elongation.

Cell length increases linearly with increasing distance above the
cell-division zone to a maximum of 40 um in air-grown and 150 um in.
submerged plants (Fig. 1). Thus, the main difference between the
patterns of normal and submergence-induced growth is an expansion of the
cell-elongation zone from less than 5 mm in air-grown plants to about
15 mm in plants submerged for 2 d. Above the zone of cell expansion,
mean parenchyma cell size remains constant over the remainder of the
internode in air-grown plants. In submerged plants, the cell size

plateaus at 150 um over the remainder of the internodal tissue formed

9O

 

 

 

 

'lGClt °0o :°j/%% o
140 I o :0 0000 o 0 000°
g 120 » .3 . .
3‘ 100) .5” 0,0,0
3 .
a 80 * o 000:0
3 60 » . ° . 0%
o °°° °°°° e. 0‘"... e .2. 0.. .°. . ° °°°°°°o
40 t 000 00° ...' “.‘o... o '. O."- 0 0 o. .
e o M0100.
20 ’ 5.".
o . . , , . ”LL . 4
0 1o 20 30 50 60 70

Distance from Node (mm)

Fig. 1. The relationship between cell size and distance from the base of
the uppermost, elongating internode. Points represent the average of
measurements from three separate longitudinal sections through the same

internode. Air-grown, (a); submerged, (o).

91

during submergence. In a transition zone spanning about 5 mm, which
separates growth prior to and during submergence, mean cell size
decreases from 140 to 40 um (Fig. 1). This confirms previous results
which indicate that cells formed prior to submergence do not undergo
additional elongation after submergence (Metraux and Kende 1984). The
average cell sizes observed for submerged plants in this study are
consistent with those reported for submerged ethylene- and GA-treated
stem sections (Metraux and Kende 1984; Raskin and Kende 1984b). However,
they are larger than the values reported by Metraux and Kende (1984) for
submerged whole plants. This discrepancy is very likely based on
differences in experimental procedure. In order to localize regions of
growth, Metraux and Kende (1984) removed a longitudinal segment of the
leaf sheath at the base of the internode and marked the underlying
internode with ink. These same internodes were used for cell-size
determinations. Subsequently, Raskin and Kende (1984b) showed that
removal of the leaf sheath inhibited stem elongation. In this study,
where the leaf sheath was left undisturbed, it appears that elongation of
internodes in submerged whole plants is both quantitatively and
qualitatively similar to hormone-induced elongation in stem sections.

The physical location of the zones of cell division and cell
elongation may be seen in Fig. 2. The node below the youngest growing
internode is between millimeters 6 and 9 (see scale, Fig. 2), with the
nodal septum and the origin of the leaf sheath at millimeter 7. The IM
is the zone just above the white tissue; in air-grown plants between
millimeters 10 and 12, in submerged plants between millimeters 10 and 13.

The cell-elongation zone in air-grown plants is between millimeters 12

92

'5’ 3
tWF’W

25

o
E!

-.
T"

’2:

f:
_

 

 

 

Fig. 2. Photograph of the basal region of the uppermost, elongating
internode showing (from left to right) the internode with surrounding
leaf sheath, the internode with leaf sheath removed, and the internode
without leaf sheath and cut open longitudinally. The intercalary

meristem is located between the 10- and 13-mm marks.

93

and approx. 17, and in submerged plants between millimeters 13 and

approx. 25.

The effect of submergence on cell division in the intercalary meristem.
The intercalary meristem of rice internodes is a rib meristem with cell
divisions occurring only in the transverse plane. Thus, cells are
organized into discrete cell files (Figs. 3 and 4) which provide a record
of the history of cell divisions within the meristem. The number of cell
divisions per cell file over the 2-d experimental period averaged 233 for
air-grown and 761 for submerged plants. Based on the respective
estimated sizes of the cell-division zones, the individual cell lengths
within this zone, and the total number of cells formed in air-grown and
submerged plants over the 2-d period, we have calculated that the average
duration of the cell cycle of cells within the IM was 24 h in air-grown
and 7 h in submerged plants. The rate of cell division in the IM of
submerged plants, while quite rapid for eukaryotic cells, is within the
range reported for meristematic tissues of other higher plants (Green

1976).

Effects of submergence on internodal development. The anatomical nature
of intercalary growth was examined in a series of light and
scanning-electron micrographs (Figs. 3-7). Longitudinal sections through
the IM of air-grown (Fig. 3) and submerged (Fig. 4) plants show a similar
organization of the ground parenchyma into discrete cell files. Vascular
bundles, which are arranged in two distinct bands within the internode
(Juliano 1937), are continuous through the intercalary meristem

(Figs. 3-5). Xylem continuity is maintained within the constantly

    

Set...“ 4.1.55.

axe-gag

 

  

94

Light micrograph (x 40) of a longitudinal section through the

Fig. 3.

intercalary meristem of the uppermost internode of a rice stem grown in

The region shown corresponds to the tissue at the 12-mm mark in

air.

100 um.

g, Metaxylem; p, protoxylem. Horizontal bar

Fig. 2.

95

A3,.

§ ‘ .
“a ‘9 .1 ,2 RI X"
I I

\3.
1

o“

. .f‘
. i3! -

, I
M _

i:
"w.

.desfiavsnk

 

Fig. 4. Light micrograph (x 40) of a longitudinal section through the
intercalary meristem of the uppermost internode of a submerged rice stem.
The region shown corresponds to the tissue at the 12-mm mark in Fig. 2.

m, Metaxylem; p, protoxylem. Horizontal bar = 100 um.

96

        
 

 
 
  
  
 
     
 

  

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k ,.,

f-vi’” -°:-£‘ 7”" ‘9 "'-'.'?°‘_-.“‘{{-.

w 61: .0..=“‘.=}}‘2§’:‘f7%ac 30"- 7‘4 3
egg/”5:. van-cl) $5.33“ infiggaz,

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‘ * U {a » :20 .o"f-'°gaie-
'fifiwmeg’géégwéziiém
”afiga

     

Q
~

Fig. 5. Light micrograph (x 40) of a cross section through the
intercalary meristem of the uppermost internode of an air-grown rice

stem. m, Metaxylem; p_, protoxylem. Horizontal bar = 100 um.

97

 

Fig. 6. Scanning electron micrograph (x 900) of a longitudinal section

through the region of the uppermost internode just above the elongation
zone of an air-grown plant. The region shown corresponds to the tissue
at 15-mm mark in Fig. 2. m, Metaxylem; s, starch grains. Horizontal bar

= 10 um.

98

  
 

H

.. -,__,___. «

 

a-F
vstsacanrqnqqui-ungunwuaq-IIIDIIIII

 

Fig. 7. Light micrograph (x 20) of a longitudinal section through a
region 5 cm up from the base of the uppermost internode of a submerged

plant. m, Metaxylem initial. Horizontal bar = 100 um.

99

growing IM through protoxylem elements which contain lignified annular
rings, allowing elongation of surrounding tissue while providing
structural strength for water conduction (Esau 1965, Chpt. 11). Also
present are metaxylem initials which remain undifferentiated within the
IM and adjacent zone of cell expansion but which, in air-grown tissue,
rapidly differentiate just above the expansion zone as evidenced by
presence of lignified, reticulate secondary wall thickenings (Fig. 6).
In internodes of submerged plants, metaxylem initials continue to
elongate up to a length of 750 um but fail to differentiate. Along the
entire length of the newly formed tissue, metaxylem elements show no
signs of secondary wall thickenings or loss of end walls (Fig. 7).
Lignification of cortical fibers, which is evident just above the
elongation zone in air-grown plants (Juliano 1937), is also absent from
the tissue formed after submergence of the stem (data not shown).

The developmental gradient inherent in intercalary growth in
monocotyledonous plants has been used in the study of chloroplast
biogenesis both at the ultrastructural (Wellburn 1984) and biochemical
levels (Mayfield and Taylor 1984). Chloroplast development in the IM of
air grown plants is evidenced by the presence of chlorophyll (133 ug (g
FW)'1) and the presence of compound starch grains in the cells of the IM
(Figs. 3, 6). During submergence-induced growth, chloroplast biogenesis
is suppressed or at least does not keep pace with the increased stem
elongation, as indicated by an eightfold lower level of chlorophyll in
the newly formed tissue and by the absence of compound starch grains in

or above the IM (Figs. 4, 7).

100

Effect of submergence on adventitious root development. Submergence of

 

deepwater rice results in the development of adventitious roots at the
nodes of the stem (Vergara et al. 1976). Nodal root development appears
to be under the control of ethylene (Bleecker et al. 1985; Suge 1985) and
possibly GA (Suge 1985). The anatomical evidence indicates that root
initials are present in the nodal tissues of air-grown plants (Fig. 8).
Ethylene apparently operates via an activation of these otherwise
quiescent root initials. Submergence-induced development includes the
emergence of the roots from the node and the development of additional

vasculature within the nodal plexus (Fig. 9).

DISCUSSION

Evidence from our laboratory has shown that the submergence response in
deepwater rice is hormonally regulated. The central role of GA in the
response is indicated by the fact that rice stems treated with inhibitors
of GA biosynthesis no longer respond to submergence or treatment with
ethylene (Raskin and Kende 1984b), and by the observation that the full
growth response is elicited in stem sections by the application of 6A3
alone (Raskin and Kende 1984b). The response to GA is morphologically
and anatomically indistinguishable from the response to submergence. In
the following, we shall discuss the role of GA in mediating the changes

in growth and development of rice internodes caused by submergence.

The hormonal control of cell division. Gibberellins enhance cell

 

division in subapical meristems of a number of plants (Loy 1977).

101

‘‘‘‘‘‘

 

         

       

“.1“. aw c. Anuflmué

    
      

  

        

so , .33.. , .
...........«....u...z ..

" ...
. .‘ui.
.iaigu . a, ._

... . . inns-away. If}-.. .

    

. o I
. ,
~ .

 

Ln.

1 \l ..

  

Light micrograph (x 20) of a longitudinal section through the

Fig. 8.

Horizontal

i, Root initial.

second-highest node of an air-grown plant.

bar = 100 um.

103

Results with excised tissue, on the other hand, indicate that GA promotes
only cell elongation in lettuce hypocotyl segments (Stuart et al. 1977)
and, at saturating concentration, inhibits cell division in the IM of
53323 stem segments (Kaufman 1965). In deepwater rice, submerged whole
plants and stem sections treated with GA or ethylene show similar, large
increases in cell division and elongation (Metraux and Kende 1984; Raskin
and Kende 1984b). This enhanced cell-division activity is reflected in
increased levels of polyamines and their biosynthetic enzymes
specifically in the IM of submerged, GA-treated and ethylene-treated rice
stems (Cohen and Kende 1986). Since the number of dividing cells within
the IM is the same in air-grown and submerged plants, increased cell
numbers in the internode resulting from submergence must be attributed
primarily to an acceleration of the cell cycle. Gibberellin-induced
decreases in cell-cycle time have been documented in seedlings of dwarf
watermelons where applied 6A3 causes a shortening of the G1 and 5 phases

of the cycle (Liu and Loy 1976).

Hormonal control of cell elongation. The role of GA in stimulating

 

plant-cell elongation is well documented (for a recent review, see Jones
1983). However, the molecular basis of this response is not well
understood. In lettuce hypocotyls (Stuart and Jones 1977) and in 51292
stem segments (Adams et al. 1975), GA-induced increases in cell-wall
extensibility were reported. Because submerged deep-water rice plants
attain such high growth rates, they are well suited for investigations
into the biochemical basis of GA-induced growth. Within the IM of the
rice internode, where GA stimulates both cell division and elongation,

two types of cell-wall biosynthesis should be considered: de-novo

104

synthesis of non-expanding transverse walls formed during cell division
and continuous addition of new wall material to expanding longitudinal
cell walls. As cells are forced up through the IN by the formation and
elongation of new cells below them, they enter a phase of cell elongation
without accompanying cell division. Within this elongation zone,
biosynthesis of the extensible longitudinal cell walls predominates.
Finally, at the upper end of the elongation zone, cells stop elongating
rather abruptly, becoming irreversibly unresponsive to GA in terms of
cell elongation.

The changes in responsiveness to GA as the internode cells mature
through the developmental sequence described above could be due to
changes in the levels of active GA within these cells or to changes in
hormone sensitivity. The former possibility seems unlikely because stem
sections given a continuous supply of GA3 show final cell lengths similar
to those of submerged plants (Raskin and Kende 1984b). Alternatively,
cessation of GA-induced elongation could be due to the gain of some
function which prevents elongation. For example, in air-grown tissue,
lignification of developing metaxylem and cortical sclerenenchyma is in
the region just above the elongation zone (Fig. 6). It is conceivable
that maturation of these specialized cells prevents further elongation of
these and surrounding tissues. Progressive silicification of the cell
walls in the internodes of air-grown plants could also be a contributing
factor (Rose-John and Kende 1984). In submerged plants, on the other
hand, secondary wall synthesis and silicification are not evident at the
end of the elongation zone. Less obvious changes in cell-wall
biochemistry may be responsible for cessation of cell elongation in the

tissues of submerged internodes.

105

CONCLUSIONS

Elongation of deepwater rice internodes involves a balance of growth and
differentiation of the stem. Submergence, through the action of ethylene
and GA, shifts this balance in favor of growth. At the same time, an
acceleration of the cell cycle within the IM is induced. The mechanisms
by which hormones elicit these responses in deepwater rice are at present
unknown. However, it is likely that differential changes in gene
expression form the basis of these hormonal responses. It is hoped that
studies of the biochemical and molecular processes related to growth and
differentiation of rice internodes will help us to understand the mode of

action of plant hormones.

REFERENCES

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F.B. (1975) Effect of gibberellic acid on the plasticity and
elasticity of Avgga stem segments. Plant Physiol. 56, 757-760

Arnon, 0.1. (1949) Copper enzymes in isolated chloroplasts. Polyphenol

oxidase in Beta vulgaris. Plant Physiol. 24, 1-15

 

Bleecker, A.B., Rose-John, S., Kende, H. (1985) Ethylene action in rice:
reversible inhibition by 2,5-norbornadiene. (Abstr.) Plant Physiol.
77,157

Buchholz, M. (1921) fiber die Nasserleitungsbahnen in den interkalaren

Wachstumszonen monokotyler Sprosse. Flora 114, 119-186

106

Cohen, E., Kende, H. (1986) The effects of submergence, ethylene and
gibberellin on polyamines and their biosynthetic enzymes in the
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Esau, K. (1965) Plant anatomy. Wiley, New York

Fisher, J.B. (1970a) Development of the intercalary meristem of Cyperus
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Fisher, J.B., French, J.C. (1976) The occurrence of intercalary and un-
interrupted meristems in the internodes of tropical monocotyledons.
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Green, P.B. (1976) Growth and cell pattern formation on an axis: critique
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Johansen, D.A. (1940) Plant microtechnique. McGraw-Hill, New York

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Juliano, J.B., Aldama, M.J. (1937) Morphology of 0ryza sativa Linnaeus.

 

Philipp. Agric. 26, 1-136
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Kaufman, P.B., Cassell, S.J., Adams, P.A. (1965) On the nature of inter-
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107

Kaufman, P.B., Dayanandan, P. (1983) Gibberellin-induced growth in Avena
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Liu, P.B., Loy, J.B. (1976) Action of gibberellic acid on cell
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Loy, J.B. (1977) Hormonal regulation of cell division in the primary
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Metraux, J.-P., Kende, H. (1983) The role of ethylene in the growth
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Metraux, J.-P., Kende, H. (1984) The cellular basis of the elongation
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Raskin, I., Kende, H. (1984b) Role of gibberellin in the growth response
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Sharman, B.C. (1942) Developmental anatomy of the shoot of

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108

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109

CONCLUSIONS

ACC synthase from wounded tomato fruit tissue consists of a 50 kDa
polypeptide which represents less than 0.0001% of the total protein

in the pericarp.

Wound-induced increases in ethylene synthesis in tomato fruit

result from gg-novo synthesis of the 50 kDa polypeptide.

Dose-response curves for ethylene induced growth in deepwater
rice is consistent with a simple ligand-receptor model of hormone

action.

2,5-Norbornadiene acts as a competitive antagonist of ethylene

action in deepwater rice.

The submergence response in deepwater rice reflects an ethylene-
mediated acceleration of cell division and elongation in the
intercalary meristem of the internode accompanied by a suppression

of tissue development.