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UNIV RS SITY LIBRARIE

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31293 00914 007

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the

dissertation entitled

Regulation of Growth in Deepwater Rice

presented by ‘

Susanne Hoffmann-Benning

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Generics

 

Dr. Hans Kende

 

Major professor

Date 3-19-1993

 

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REGULATION OF GROWTH IN DEEPWATER RICE
By

Susanne Hoffmann-Benning

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Genetics Interdepartmmtal Doctoral Program
MSU-DOE Plant Research Laboratory

1993

ABSTRACT
REGULATION OF GROWTH IN DEEPWATER RICE
By

Susanne Hoffmann-Benning

Submergence induces rapid elongation of rice coleoptiles (Otyza sativa L.) and
of deepwater rice internodes. The growth response of deepwater rice plants is
mediated by ethylene and gibberellin (GA). Submergence and treatment with ethylene
led to a 75 % reduction in the level of ABA in the intercalary meristem and the
growing zone of deepwater rice internodes, while the level of GAl increased fourfold.
An interaction between GA and ABA could also be shown by applieation of these
hormones. My results indieatc that the growth rate of deepwater rice intemodes is
determined by the ratio of an endogenous growth promoter (GA) and a growth
inhibitor (ABA).

Fluridone, an inhibitor of ABA biosynthesis, and submergence led to a
reduction of the level of ABA and a promotion of growth in rice ooleoptiles,
indieating an involvement of ABA in determining the growth rate of rice coleoptiles.

The epidermis limits intemodal growth in rapidly growing deepwater rice.
Osmiophilic particles with a diameter of 80 nm in rice, 200 nm in cucumber, and up
to 300 nm in corn appear between the plasma membrane and the outer epidermal
wall. They are associated with rapid growth and, in deepwater rice, with the zone of
cell elongation. Monensin inhibits the appearance of these particles, indicating that

they are derived from the Golgi apparatus. Results of enzyme-gold labeling and

digestion experiments using proteinase K indicate that the osmiophilic particles are, at
least in part, proteinaeeous. Antibodies against lipid transfer protein, an extensin—like
protein, an arabinogalactan protein, and "expansin" did not bind to the osmiophilic
particles. Staining for peroxidase showed that enzyme activity is at least in close
proximity to the osmiophilic particles.

Further experiments showed that in GA—treated, rapidly growing rice stem
sections the cuticle is a growth-limiting structure. GA treatment leads to an increase
in the incorporation of [“C]palmitic acid and [“C]oleic acid into the cuticle of
growing intemodes and to increased levels of at least one cuticular component, a
dihydroxyhexadecanoic acid. I conclude that the activities of enzymes that catalyze

hydroxylation of fatty acids may be enhanced.

To

Christoph and Urs

iv

ACKNOWLEDGMENTS

I thank Hans Kende for giving me this project to work with. He provided me with
excellent advice and taught me how to approach scientific questions. I am grateful for
the support and advice I received from the. members of my advisory committee:
Rebecea Grumet, Natasha Raikhel, Chris Somerville, and Jan Zeevaart. I thank Karen
Klomparens, who, even so she joined my committee only at the end of my work, was
always there to give advice and encouragement.

Iwould alsoliketothankallthemembers ofthelabandallmy friendsinthePRL,
the Center for Electron Optics, and the genetics program for their advice and their
helpful discussions. I thank Doug Gage, Bev Chamberlin, and Christiane Nawrath for
their help analyzing cutin samples by GC-MS, and Drs. Alice Bonnen, Ray
Hamerschmitt, P. Kolattukudy, John Ohlrogge, Chris Rock and Manuel Talon for
their advice and help during various steps of this thesis.

Ithankmyparents forallowingmetogo mywayandfortheirsupportevenagainst
the advice of friends who said: “She is only a girl and will be married and housewife
some day, so why doesn’t she get a real job for now?” And - most of all - I thank
my husband Christoph and my son Urs for their love, patience and support in trying

to prove these friends wrong.

TABLE OF CONTENTS

Page
LIST OF TABLES .......................................................................... ix
LIST OF FIGURES ........................................................................ xi
LIST OF ABBREVIATIONS ............................................................. xiv
CHAPTER 1: INTRODUCTION ........................................................ 1
DEEPWATER RICE AND ITS GROWTH RESPONSE ................... 1
THE ROLE OF THE PLANT GROWTH SUBSTANCES
ABSCISIC ACID (ABA) AND GA IN THE GROWTH OF
DEEPWATER RICE ...................................................... 5
ULTRASTRUCTURAL CHANGES IN DEEPWATER RICE IN
RESPONSE TO SUBMERGENCE .................................... 6
WHAT IS THE CUTICLE AND HOW DOES GROWTH AFFECT
ITS BIOSYN'THESIS AND COMPOSITION ? ..................... 8
REFERENCES ..................................................................... 12
CHAPTER 2: THE ROLE OF ABA IN THE GROWTH OF
RICE COLEOPTILES ............................................................. 18
ABSTRACT ........................................................................ 18
INTRODUCTION ................................................................. 19
MATERIALS AND METHODS ................................................ 22
RESULTS AND DISCUSSION ................................................. 23
CONCLUSIONS ................................................................... 36

CHAPTER 3: THE ROLE OF ABSCISIC ACID AND GIBBERELLIN IN
THE REGULATION OF GROWTH IN DEEPWATER RICE

INTERNODES ..................................................................... 42
ABSTRACT ........................................................................ 42
INTRODUCTION ................................................................. 43
MATERIALS AND METHODS ................................................ 44
RESULTS AND DISCUSSION ................................................. 47
CONCLUSIONS ................................................................... 52
REFERENCES ..................................................................... 59

CHAPTER 4: CHARACTERIZATION AND PARTIAL IDENTIFICATION
OF GROWTH-RELATED OSMIOPHILIC PARTICLES IN CORN AND

DEEPWATER RICE .............................................................. 61
ABSTRACT ........................................................................ 61
INTRODUCTION ................................................................. 62
MATERIALS AND METHODS ................................................ 63
RESULTS AND DISCUSSION ................................................. 68
CONCLUSIONS .................................................................. 99
REFERENCES ..................................................................... 103

CHAPTER 5: CUTICLE BIOSYNTHESIS AND COMPOSITION IN
GIBBERELLIC ACID-TREATED DEEPWATER RICE

INTERNODES .................................................................... .106
ABSTRACT ....................................................................... 106
INTRODUCTION ................................................................ .107
MATERIALS AND METHODS ............................................... 110
RESULTS AND DISCUSSION ................................................ 113
CONCLUSIONS ................................................................. .129
REFERENCES ................................................................... .130
CHAPTER 6: GENERAL CONCLUSIONS .......................................... 134
REFERENCES ................................................................... .136

LIST OF TABLES

Page
Table 2.1. Effect of IAA and GA, on growth rate of intact rice coleoptiles ..... 25
Table 2.2. Effect of GA, and of CCC and AMO 1618, inhibitors of GA
biosynthesis, on the growth of rice coleoptiles ............................... 25
Table 2.3. length of coleoptile, mesocotyl, and root and ABA level in rice
coleoptiles plus primary leaves and in coleoptiles alone .................... 33

Table 2.4. Ethylene and fluridone act independently on rice coleoptile growth . 35

Table 3.1. Amlysis of variance on the effect of submergence on ABA levels in
deepwater rice intemodes ........................................................ 51

Table 3.2. Analysis of variance on the effect of ethylene on ABA levels in
deepwater rice intemodes ........................................................ 54

Table 3.3. Levels of GAl and GA20 in the intercalary meristem and cell
elongation zone of intemodes of adult deepwater rice plants grown in

air or submerged for various times ............................................. 55
Table 4.1. Distribution of osmiophilic particles along the rice stem .............. 75
Table 4.2. Number of gold grains in osmiophilic particles and other cell

compartments after labeling with proteinase K gold ......................... 79
Table 4.3. Number of osmiophilic particles after digestion with proteinase K,

cutinase, glueanase, or treatment with buffer (control 1) ................... 84
Table 4.4. Effect of incubation in 20 RM monenesin for 12 h on the

appearance of osmiophilic particles in corn and rice ........................ 88
Table 5.1. Thickness of the epidermal cell walls and the cuticle .................. 114

Table 5.2. Effect of cutinase on the tissue tension in the lowest 3 cm of the
youngest internodc ............................................................... .117

Table 5.3. Effect of cutinase or buffer on the tissue tension of the lowest 3 cm
in the youngest internode ........................................................ 117

LIST OF FIGURES

Page

Figure 1.1. Chain of events linking submergence to accelerated growth of

deepwater rice as known in 1988 ............................................... 4
Figure 1.2. Proposed path of cutin biosynthesis (from Holloway, 1980) ......... 10
Figure 2.1. Growth of rice, wheat, cat, and barley coleoptiles in response to

treatment with 30 EM fluridone ................................................. 26
Figure 2.2. Response of rice seedlings to ABA and fluridone ...................... 28
Figure 2.3. ABA levels in rice seedlings at various times after imbibition ...... 29

Figure 2.4. Coleoptile lenth of rice seedlings grown as described in Figure 2.3 31
Figure 2.5. Rice seedlings grown on vermiculite ..................................... 32

Figure 3.1. Growth of deepwater rice stem sections submerged for three days
in water containing one of several concentrations of ABA ................. 48

Figure 3.2. Growth of deepwater rice stem sections submerged for three days
inwater, in3pMABA,in3uMABAplusoneofseveral

concentrations of GA,, or in 10 “M GA, alone .............................. 49
Figure 3.3. Effect of submergence on the level of ABA in deepwater rice ...... 50
Figure 3.4. Effect of ethylene on the level of ABA in deepwater rice ............ 53

Figure 3.5. Chain of events linking submergence to accelerated growth of
deepwater rice ...................................................................... 58

Figure 4.1. Transmission electron micrograph of a cross section through the
stem of a GA,-treated deepwater rice plant (high pressure frozen) ........ 70

Figure 4.2. Transmission electron micrograph of a cross section through a corn
coleoptile epidermis cell treated with 10 RM IAA for l h ................. 71

Figure 4.3. Transmission electron micrograph of a cross section through a
cucumber hypocotyl epidermis cell ............................................. 72

Figure 4.4. Time course of appearance of osmiophilic particles in the outer
epidermal wall of submerged deepwater rice stems ......................... 73

Figure 4.5. Scheme of enzyme-gold labeling as adapted from Bendayan et a1.
(1984) ................................................................................ 76

Figure 4.6. Transmission electron micrograph of cross sections of auxin-
treated corn coleoptile epidermal cells labeled with proteinase K gold . . 77

Figure 4.7. Transmission electron micrographs of corn coleoptile cross
sections labeled with controls for proteinase K gold specificity ........... 81

Figure 4.8. Transmission electron micrographs showing the effect of monensin
on the Golgi apparatus ............................................................ 86

Figure 4.9. Transmission electron micrographs visualizing peroxidase activity
in corn coleoptile epidermis cells ............................................... 89

Figure 4.10. Transmission electron micrographs of cross sections of corn
coleoptiles labeled with an antibody against THRGP (1:1000; A) ........ 92

Figure 4.11. Transmission electron micrographs of cross sections of corn
coleoptile epidermis cells labeled with an antibody against LTP
(1:50; A) ............................................................................ 94

Figure 4.12. Transmission electron micrographs of cross sections of corn
coleoptile epidermis cells labeled with an antibody against a barley
cell wall peroxidase (1:100; A) ................................................. 97

Figure 4.13. Transmission electron micrographs of cross sections of corn
coleoptile epidermis cells labeled with a monoclonal antibody against
AGPs ................................................................................. 98

Figure 4.14. Transmission electron micrographs of cross sections of corn
coleoptile epidermis cells labeled with an antibody against expansin S1
(C; 1: 25 dil.) and 82 (A; 1:400 dil.) ......................................... 100

Figure 5.1. Angle of curvature in tissue segments displaying tissue tension . . . . 111

Figure 5.2. Effect of cutinase on tissue tension in the lowest 3 cm portion of
the youngest intemode of deepwater rice ..................................... 116

Figure 5.3. Length of coleoptile segments incubated in water or 10 ptM IAA
with or without cutinase, .respectively, for various times .................. 118

Figure 5.4. Incorporation of [“C]pa1mitic acid into the lowest l-cm zone of the
youngest intemode of deepwater rice .......................................... 120

Figure 5.5. Incorporation of [“C]palmitic acid into the second lowest 1-cm zone of
the youngest intemode of deepwater rice ..................................... 121

Figure 5.6. Phosphor-imager printout indieating the radioactivity in cutin
monomers produced by reductive hydrolysis and separated by TLC . . . . 123

Figure 5.7. Incorporation of [“C]oleic acid into the lowest l-cm zone of the
youngest intemode of deepwater rice .......................................... 125

Figure 5.8. Phosphor-imager printout indieating the radioactivity in cutin
monomers produced by reductive hydrolysis and separated by TLC . . . . 127

ABA
ACC
AGP
AMO 1618

BSA
CCC
ELISA

fluridone

GA
GC
HPLC
IAA
LTP
PBS
PCIB
PEG
PMSF
TCH
'I'EM
THRGP
TLC

ABBREVIATIONS

abscisic acid
l-aminocyclopropane-l-carboxylate
arabinogalactan protein
4—hydroxy-5-isopropyl-2-methylphenyltri-
methylammonium chloride l-piperidine
bovine serum albumine
(2-chloroethyl)trimethy1ammonium chloride
enzyme-linked immunosorbent assay
l-methyl-3-phenyl-5-[3-(trifluoromethyl)-phenyl]—
4(4H)pyridonone

gibberellin

gas chromatography

high performance liquid chromatography
indole-3-acetic acid (auxin)

lipid transfer protein

phosphate buffered saline
p-chlorophenoxy-isobutyric acid
polyethylene glycol

phenylmethylsulfonyl fluoride
thioearbohydrazide

transmission electron microscope
threonine hydroxiproline—rich glycoprotein
thin layer chomatography

xiv

CHAPTER 1

INTRODUCTION
DEEPWATER RICE AND ITS GROWTH RESPONSE

Rice (Otyza sativa) is grown on 11% of the world’s arable land and almost
exclusively for human consumption. There are three types of rice: upland rice, which
is grown in high-lying areas and is rainfed; lowland rice, which is grown as paddy-
rice, and deepwater rice. Deepwater rice is grown predominantly in Southeast Asia in
areasthatarefloodedduring themonsoonseason. Ithasdeveloped several adaptive
mechanisms that allow it to survive flooding: first, it has an aeration system which
transportsozfromtheatmosphereviatheleaves andtheintemodalcavitiesallthe
waytotherootstomaintainaerobic metabolisminsubmerged organs (Raskinand
Kende, 1985). Second, submergence induces rapid internodal growth. Planted at least
onemonthbeforethemonsoonrains start, plantsbegintoelongateastheybecome
partly submergedintherising floodwaters. Growthratesofupto25cmperdayhave
beenobserved, andplantseanreachatotalhightofupto7m(Vergaraetal.,1976).
Flowering in deepwater rice is under photoperiodic control to ensure that panicle
emergenceoccursafterthewaterhasstoppedrising. Thisisimportantsinceplants
lose the ability to elongate once panicles have formed. Seeds are harvested from boats
orafterthewaterhasreceded. 'I’heabilityofdeepwaterriceplantstosurviveflooding

makesitasubsistencecropinareashkeBangladesh,whereseverefloodsoccm

frequently.

Deepwater rice is not the only rice variety that elongates when submerged. All
rice varieties show an increase in coleoptile elongation when their seedlings are
submerged, and an increase in stem growth once the intemodal elongation has started
prior to flowering. However, the eapacity for rapid elongation starts in non-deepwater
rice cultivars at approximately ten weeks of age while deepwater rice is responsive
when it is ca. four weeks old (Keith er al. , 1986). Some other semiaquatic plants also
show an induction of growth after submergence. One is Callitriche platycarpa in
which stem elongation is controlled by ethylene and gibberellin (GA, Musgrave at at. ,
1972). In Ranunculus sceleratus (Smulders and Horton, 1991) and some Runner
species, waterlogging and ethylene promote petiole growth (V oesenek and Blom,
1989; Voesenek et al. , 1990a; Voesenek er al. , 1990b).

The inducibility and rapid elongation of deepwater rice makes it a good system
to study intemodal growth. Elongation occurs predominantly in the youngest intemode
and ean also be seen in isolated deepwater rice stem sections (Raskin and Kende,
1984a). Submergence leads to a decrease in the endogenous oxygen tension (as low as
2% during the dark period) and an increase in endogenous CO, level (to ca. 6%; for
a review see Kende, 1987). The low 0, tension leads to an increase in l-amino-
cyclopropane-l-earboxylic acid (ACC) synthase activity (Cohen and Kende, 1987)
and, thus, to an increase in the ethylene biosynthesis (Raskin and Kende, 1984a).
Ethylene by itself promotes growth of non-submerged plants. (Métraux and Kende,
1983). Two enzymes of polyamine biosynthesis, S-adenosyl-L-methionine
deearboxylase and arginine deearboxylase, are also stimulated within 4 h of

3
submergence and appear to be related to accelerated cell division in the intercalary

meristem (Cohen and Kende, 1986).

The submergence response can also be induced by GA. Raskin and Kende
(1984c) have shown that ethylene increases the responsiveness of the intemodal tissue
to GA. This, in turn, leads to intemodal elongation and is associated with an
enhancement of amylolytic activity and increased translocation of photosynthates in
the growing zone (Raskin and Kende, 1984b; Smith et al., 1987). This rise in the
growth rate can be measured 180-220 min after submergence, 60 min after treatment
with a gas mixture containing 3% Oz, 6% C02, 91% N2, 1 all] ethylene, and 40—60
min after treatment with GA, (Rose-John and Kende, 1985). Growth is based on an
approximately three-fold increase in the production of new cells in the interealary
meristem and on a three- to fourfold increase in the final size of cells emerging from
the meristem into the cell elongation zone (Métraux and Kende, 1984; Bleecker et al. ,
1986). Recent findings of Sauter and Kende (1992a) indieate that GA first promotes
cell elongation in the interealary meristem and that cell growth triggers an increase in
the rate of cell divisions. In parallel, there is a decreased incorporation of Si into the
cell wall (Rose-John and Kende, 1984), a delay in lignification (Sauter and Kende,
1992b), and a delay in cellulose microfibril reorientation from transverse to oblique
(Sauter and Kende, 1993). In addition, Kutschera and Kende (1989) observed the
appearance of osmiophilic particles between the plasma membrane and the outer
epidermal wall of rapidly growing, submerged plants. The chain of events leading
from submergence to intemodal elongation, as it was known at the beginning of this

thesis, is summarized in Figure 1.1.

SUBMERGENCE

I

REDUCED OXYGEN TENSION IN SUBMERGED INTERNODES

I

INCREASED ETHYLENE BIOSYNTHESIS

I

INCREASED RESPONSIVENESS TO GIBBERELLIN

I

INTERNODAL GROWTH

figure 1.1. Chain of events linking submergence to accelerated growth of deepwater

rice as known in 1988.

5

. The questions asked in this thesis were:
1. How does ethylene change the responsiveness of the growing tissue to GA?
2. What are the osmiophilic particles and how are they associated with rapid growth?
3. Do cuticle biosynthesis and composition change in rapidly growing deepwater rice

intemodes?

THE ROLE OF THE PLANT GROWTH SUBSTANCES ABSCISIC

ACID (ABA) AND GA IN GROWTH OF DEEPWATER RICE

What are plant hormones? ”A plant hormone is an organic compound
synthesized in one part of the plant and translocated to another part, where in very
low concentrations it causes a physiological response. " (From Salisbury and Ross,
1992). Plant hormones that promote growth are auxin and GA, those that inhibit
growth are ABA and ethylene. Usually, ethylene is considered an inhibitor of growth
in terrestrial plants. However, in some semiaquatic plants, it has been shown to
induce stem or petiole elongation. Applied GA also induces growth in deepwater rice
(Raskin and Kende, 1984c; Suge, 1985, Suge and Tiirkan, 1990). Yamaguchi (1973)
and Tiirkan and Sugc (1991) found, using a bioassay, that the level of biologieally
active GAs is higher in submerged rice than in non—submerged rice. When plants
were pretreated with inhibitors of GA biosynthesis, neither submergence not ethylene
induced intemodal growth (Raskin and Kende, 1984c; Suge, 1985). Raskin and Kende
(19840) showed that ethylene increases the responsiveness of the intemodal tissue to

GA.

6
How does ethylene change the responsiveness of the intemodal tissue to GA?

Zeevaart (1983) showed that applied ethylene increases ABA metabolism and,
thereby, eauses a decrease of biologically active ABA in leaves of Xanthiwn
smanan’um. Ethylene could have a similar effect in deepwater rice. It could eause a
decrease in the endogenous content of ABA, which is a potent inhibitor of GA action
in many systems. With reduced ABA levels, endogenous or applied GA would be
more effective in promoting growth. In other words, the rate of growth could be
determined by the ratio of a growth inhibitor (ABA) to a growth promoter (GA). A
similar antagonistic action of ABA and GA occurs e.g., in cereal seeds where GA
enhances amylolytic activity, while ABA inhibits it (see Jacobson and Chandler,
1988). The possibility of ABA regulating intemodal growth in deepwater rice is
addressed in chapters 2 and 3 and summarized in Hoffmann—Benning and Kende

(1992a).

ULTRASTRUCTURAL CHANGES IN DEEPWATER RICE IN
RESPONSE TO SUBMERGENCE

Submergence of deepwater rice leads to an increase in intemodal growth.
Along with this increased growth, several ultrastructural changes appear: cell division
and cell elongation increase (Sauter and Kende, 1992a), lignifieation is retarded
(Sauter and Kende, 1992b), the reorientation of cellulose microfibrils is delayed
(Sauter and Kende, 1993), and the intemodes exhibit tissue tension (Kutschera and

Kende, 1988). Tissue tension has been described by Sachs (1865) as follows: “the

7
thick outer epidermal wall maintains the thin inner walls in a state of compression and

is the expansion-limiting structure of the whole organ”. It is displayed as a rapid,
elastic outward bending upon transfer of the stem sections into water. Kutschera and
Kende (1988) suggested that an increase in the plastic extensibility of the cell wall
takes place at the growth-limiting outer epidermal wall and examined the
ultrastructure of the epidermis. They observed the appearance of osmiophilic particles
between the plasmamembrane and the outer epidermal wall of submerged deepwater
rice intemodes (Kutschera and Kende, 1989); this was subsequently also seen in
ethylene- and GA—treated intemodes (Hoffmann-Benning and Kende, 1992b). These
particles are 80-120 nm in diameter in rice and have been described previously in
rapidly growing corn coleoptiles (Kutschera er al. , 1987). Similar but smaller and less
distinct particles have been observed by Robards (1969) and Olesen (1980). In these
eases, they were thought to be linked to cell wall growth. However, Barckhausen and
Rosenstock (1973) saw similar particles in wounded potato tubers, where they
appeared to be associated with suberization. Since the osmiophilic particles appear
only on the outer epidermal wall, they could either be linked to cell wall or cuticle
biosynthesis.
Thus, the following questions were addressed in chapter 4 of this thesis:

- Where are the osmiophilic particles localized and how are they related to

submergence-induced growth?
- Are they transported through the secretory pathway?

- What is their composition (polysaccharides, fatty acids, proteins)?

8
WHAT IS THE CUTICLE AND HOW DOES GROWTH AFFECT

ITS BIOSYNTHESIS AND COMPOSITION ?

How does GA affect intemodal growth? Usually the epidermis is thought of as
the growth-limiting tissue with the outer epidermal wall playing a major role in
restraining growth (i.e. Kutschera and Kende, 1988). In promoting growth, GA
should, therefore, have an effect on the outer epidermis and, particularly, on the outer
epidermal wall. However, the cuticle on the outer epidermal wall could also
contribute to tissue tension and play a role in limiting growth, and GA could influence
cuticle biosynthesis and/or composition. Enhancement of cuticle biosynthesis at the
level of its monomers has already been observed in GA-treated peas by Bowen and
Walton (1988).

The plant cuticle is a lipid layer on the outside of the outer wall of epidermal
cells. Its structural component is cutin, a wax composed of a family of C“ and Cu
fatty acid monomers. These monomers often contain one or more hydroxy- or epoxy-
groups. The major C1, component of cutin is dihydroxypalmitic acid; the major C"
components are lS-hydroxyoctadeeanoic acid, 9,10-epoxy-hydroxyoctadeeanoic acid,
9,10,18-trihydroxy-octadecanoic acid, and their A12 unsaturated counterparts
(Kolattukudy, 1980). It appears that about half the monomers are cross-linked through
hydroxy groups (Deas and Holloway, 1977; Kolattukudy, 1977 ; Kolattukudy, 1984).
Small amounts of phenolic acids and possibly other types of ligands are covalently
attached to the hydroxy groups of the polymer (Riley and Kolattukudy, 1975).

The existence of a cuticle is not restricted to higher plants. Frey-Wyssling and

9
Miihlethaler (1965) proposed that the cuticle of terrestrial plants replaces the mucilage

layer of aquatic plants. It can be found in angiosperms, gymnosperms, psilophytes,
liverworths, bryophytes and some pteridophyta, with ferns and lycopods seeming to
have the chemically simplest cutin (for review see Holloway, 1980). Even though the
cuticle of higher plants has been presumed to be an evolutionary adaptation to a
nonaquatic environment, cutin-like surface compounds have been found in some
bacteria and fungi (Caldicott and Eglinton, 1973), and in some higher aquatic plants
such as Zoestra marina (Kolattukudy, 1978).

How is the cuticle synthesized? According to an early hypothesis, 'procutin" is
secreted through the outer wall of the differentiating epidermal cell as semisolid
material (i.e. unsaturated fatty acids) where it gradually polymerizes (Frey-Wyssling
and Miihlethaler, 1965). A more refined picture is shown in Figure 1.2 (from
Holloway, 1980): Synthesis of monomeric fatty acid derivatives takes place in the
cytoplasm. These are then transported through the cell wall to the outermost surface
of the cell where polymerization takes place. Enzymes for monomer synthesis
have been characterized by Kolattukudy and coworkers (C16: hydroxylase, Soliday
and Kolattukudy, 1977 , 1978; mixed-function oxidase, Walton and Kolattukudy,
1972; Soliday and Kolattukudy, 197 8; C18: mixed-function oxidase, Croteau and
Kolattukudy, 1975a; epoxide hydratase, Croteau and Kolattukudy, 1975b). In
addition, Croteau and Kolattukudy (1974) found that a 1000 g fraction from Vicia
faba leaves incorporated labeled hydroxy acids when ATP and CoA were present.
This hydroxyacyl transferase enzyme seems to be responsible for the polymerization

of cutin from monomers,which is thought to occur outside the epidermal cells. A role

10

 

 

C E alrocelluIOr
W Transport
phase
,( I
C 7 iii. Cytoplasmic
. p.103.

 

 

 

 

 

Epidermal cell

Figure 1.2. Proposed path of cutin biosynthesis (from Holloway, 1980). Monomer
synthesis occurs in the cytoplasm (Cy). These predominantly fatty acid derivatives are
mennansportedthmughthecenwaHMandpolymefizedeidrerdufingnansponor
at the cuticular matrix (C).

11
for peroxidases in the polymerization of phenolic compounds to cutin and suberin has

also been proposed (Ferrer et al. , 1991; Roberts and Kolattukudy, 1989).
Polymerization of cutin could take place on the inner face of the outer epidermal wall
(Roberts et al. , 1959) or is, more likely, associated with the cuticular matrix
(Kolattukudy and Espelie, 1985).

The form in which the cuticle precursors are transported through the epidermal
wall is unknown. Some investigators have seen osmiophilic droplets migrate through
the outer epidermal wall but provided no convincing evidence that these are composed
of cutin precursors (C1, and C1, fatty acids and their hydroxy- and epoxy derivatives)
rather than of cell wall components (Bollinger, 1959; Heide-Jorgensen, 1978, 1991).
Paul and McWorther (1990) observed osmiophilic particles associated with wax
filament formation in Sorghum halepense L. Thus, the osmiophilic particles in rice
may, indeed, be involved in cuticle biosynthesis, especially since formation of the
cuticle appears to be associated with active growth in other plants (Hull et al., 1978;
Martin and Juniper, 1970).

What is the role of the plant cuticle? It has been shown to constitute a major
diffusion barrier for water and gas exchange (SchOnherr, 1976) and is also thought to
play a role as barrier between plants and microbes (van den Ende and Linskens,
1974). Kolattukudy (1965 , 1970) found that cutin biosynthesis occurs most rapidly in
expanding Vicia faba and Brassica oleracea leaves. The relative composition of the'
cuticle appears to change under various environmental conditions and at different
physiological ages of the plant (Skoss, 1955; Kolattukudy et al. , 1974).

Chapter 5 is an account of investigations on cuticle biosynthesis in GA-treated,

l2

rapidly growing deepwater rice intemodes and the potential role of the cuticle as a

growth-limiting structure.

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13

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14

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15

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16

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17

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CHAPTER 2
THE ROLE OF ABA IN THE GROWTH OF RICE

COLEOPTILES

ABSTRACT

In contrast to other cereals, rice seedlings and adult plants of some rice
varieties ean survive flooding. In seedlings, submergence promotes coleoptile growth,
a response that ean also be induced by a gas mixture containing 3% 0,, 15% C0,,
and 1 all] ethylene in N,. I investigated whether abscisic acid (ABA) is involved in
regulating the growth of rice coleoptiles. Rice seedlings were grown on solutions
containing fluridone, an inhibitor of earotenoid and, indirectly, of ABA biosynthesis.
Treatment with fluridone reduced the level of ABA in coleoptiles and first leaves by
more than 75% and promoted coleoptile growth by more than 60%. Little or no
enhancement of coleoptile growth by fluridone was observed in barley, cat, or wheat.
Applied ABA counteracts fluridone—induced growth. Seedlings grown in the above gas
mixtureorinaircontaining3t05ul/lethyleneaswellas submergedorfluridone—
treated seedlings show not only increased coleoptile growth but also a reduction in the
level of endogenous ABA. This indieates that ABA may be involved in regulating rice
coleoptile growth. The involvement of ABA in determining the growth rate of rice

coleoptiles may be related to the semiaquatic growth habit of this plant.

18

19
INTRODUCTION

Rice (Oryza sativa, L.) has a number of physiological and metabolieal
adaptations that enhance its chances for survival under conditions of temporary
flooding. One of these is the capacity of plants to elongate rapidly when they become
submerged. This feature helps rice plants to emerge from the water and to avoid
drowning. In adult deepwater rice plants, submergence stimulates elongation of the
internode (Métreaux and Kende, 1983; Vergara et al. , 1976). In seedlings, it
promotes coleoptile growth and reduces root growth (Turner et al. , 1981; Wada,
1961; Yarnada, 1954). These morphological changes in response to submergence can
also be seen with seedlings grown under low partial pressures of O, (Kordan, 1977;
Ohwald, 1967; Ranson and Parija, 1955) or in air containing low concentrations of
ethylene (Ku et al., 1970; Miller and Miller, 1974; Suge, 1971). Atwell et al. (1982)
have suggested that accumulation of ethylene is responsible for the stimulation of rice
coleoptile elongation in stagnant water and that CO2, an antagonist of ethylene,
inhibits coleoptile growth. However, Ishizawa and Esashi (1984a) proposed a
stimulatory role of CO, and ethylene in rice coleoptile growth. Raskin and Kende
(1983) showed that coleoptile growth is promoted independently and to the same
extent by low levels of O, (3%), high levels of CO, (15%), and by l ul/l ethylene.
This regulatory mechanism allows rice to grow out of shallow water where O, is
limiting and where CO, and ethylene accumulate in the submerged organs of the
plant.

How do these conditions alter growth of rice seedlings? One possibility is that

20

submergence leads to an increase in the endogenous level of plant growth promoters.
Both auxin (indole-3-acetic acid, IAA) and gibberellin (GA) have been examined for
their role in inducing rice coleoptile growth. However, no clear picture concerning
the role of GA and IAA in submergence-induced growth has emerged so far. Imaseki
and Pjon (1970) found that ethylene enhances auxin-induced growth in rice coleoptile
segments. Similarly, Ishizawa and Esashi (1983, 1984) suggested that auxin is at least
necessary to maintain long-term growth of rice coleoptile segments and that ethylene
enhances the IAA-induced growth response. However, Katsura and Suge (1979)
concluded that ethylene may act independently from auxin.

Kefford (1962) found that p-chlorophenoxy-isobutyric acid (PCIB), a presumed
antiauxin, inhibited rice coleoptile growth in seedlings grown under various light
conditions, while IAA and GA promoted it. Inhibition by PCIB could be overcome by
IAA. In addition, PCIB reduced GA-induced growth. Kefford concluded that there is
an interaction between IAA and GA in the control of rice coleoptile growth and that
the depth of submergence might influence the endogenous auxin concentration. This
suggestion is consistent with a later observation of Mapelli (1986) who detected an
increase in auxin levels in rice seedlings under anaerobiosis. But Hoson et al. (1992)
detected lower IAA levels in submerged rice coleoptiles than in air—grown ones.
However, Gopalakrishnan and Sircar (1974) found no effect of GA on the growth of
rice shoots, and Suge (1974) observed only a very small effect of applied GA on
coleoptiles in dwarf rice but a strong growth promotion of first leaf sheaths.
Murakami (1962) also observed a small induction of coleoptile growth in rice

seedlings cultured on GA for six days. Horton (1991) reported that application of IAA

21
and especially of GA in air promoted coleoptile growth of rice seedlings which had

been irradiated with a short pulse of light. The results described above are
contradictory and do not establish the role of plant hormones in regulating elongation
of rice coleoptiles. Even so, it appears that applied IAA and GA may stimulate rice
coleoptile growth under certain conditions. However, their role with respect to
submergence-induced growth is not yet clear. Up to now, the possibility that
reduction in the level of a growth inhibitor during submergence could lead to
increased rice coleoptile growth has not been considered. In maize seedlings, the
abscisic acid (ABA) content is increased and shoot elongation is reduced at low water
potential (Saab et al. , 1990). Inhibition of ABA biosynthesis with fluridone relieved
the inhibitory effect of water stress on growth. Thus, increased growth of submerged
rice coleoptiles may result from a decrease in the level of ABA. Promotion of rice
coleoptile growth by ethylene may also be related, at least in part, to a reduced ABA
content as Zeevaart (1983) has found that treatment with ethylene reduces the level of
biologically active ABA in leaves of Xanthium stmmarium.

This chapter is a report of experiments on the role of ABA in the growth
response of rice coleoptiles. ABA levels were either increased by application of the
hormone or decreased by environmental factors (submergence; gas composition of the
ambient atmosphere) or by treatment with fluridone, an inhibitor of earotenoid
biosynthesis. The role of ABA in influencing the elongation of wheat, oat, and barley
coleoptiles has also been determined for comparative purposes. Results of this work
have been reported earlier (Hoffmann and Kende, 1990; Hoffmann-Benning and

Kende, 1991).

22
MATERIALS AND METHODS

Growth of plant material. Seeds of rice (Oryza sativa L., cv. M-9), wheat
(Triticum vulgare, cv. Ionia), oat (Avena sativa L. , cv. Korwood), and barley
(Hordeum vulgare L. , cv. Lakeland) were allowed to imbibe in darkness at 29° C in a
Petri dish containing 15 ml sterile distilled water. Germinated seeds were transferred
to sterile l-liter glass jars containing 150 ml of vermiculite wetted with 80 ml of
sterile distilled water, varying concentrations of ABA (Sigma, St. Louis, MO), IAA
(Sigma, St.Louis, MO), gibberellin A, (6A,, Calbiochem-Behrin , CA), 30 M
fluridone (1-methy1-3-phenyl-5-[3-(trifluoromethyl)-phenyl]4(4H)pyridonone, a gift of
Eli Lilly, Indianapolis, IN), 150 mg/l AMO 1618 (4-hydroxy-5-isopropyl-2-
methylphenyltri-methylammonium chloride l-piperidine earboxylate, from
Calbiochem-Behring, CA), or 250 mg/l CCC [(2-chloroethyl) trimethylammonium
chloride, from American Cyanamide, Princeton, NJ]. Fluridone was dissolved in
acetone, which, upon dilution, had a final concentration of 0.5 %; in these
experiments, the water used for control treatment and treatment with the other
chemicals also contained 0.5 % acetone. The jars, which had gas inlet and outlet
ports, were sealed, and the seedlings were grown in the dark at 29° C under a
continuous flow (60 ml/min) of humidified air with or without 3 to 5 all] ethylene or
in a gas mix containing 3.5% 0,, 19.3% C0,, and 5-6 ul/l ethylene in N2. To
prepare ethylene-free air, the air was passed through a 25-cm-long column ('7 cm
I.D.) packed with purafil (Purafil Inc., Atlanta, GA). Ethylene was determined by gas

chromatography (GC) of l-ml samples (Kende and Hanson, 1976). For submergence,

23
the seedlings were grown in the same jars containing 600 ml sterile distilled water.

Four days after start of imbibition, growth of the seedlings was measured, and the
coleoptiles or coleoptiles plus first leaves were harvested and frozen in liquid N, for

further analysis.

ABA extraction and determination. ABA from coleoptiles was extracted
according to Walker-Simmons (1987), and either phase partitioned with ethyl acetate
for ELISA (enzyme-linked immunosorbant assay) or purified by HPLC (high
performance liquid chromatography) and measured by GC (Zeevaart et al. , 1989).
The ELISA was performed as described by Walker-Simmons (1987) with the
monoclonal antibody of Mertens et al. (1983), which was purchased from Idetek (San
Bruno, CA). The ABA-4’-conjugate was prepared according to Weiler (1980).
Belefont and Fong ( 1989) observed that organic acids may interfere with the ELISA. I
verified that the ABA determinations made by ELISA were consistent with those
made by HPLC/GC (not shown). The amounts of ABA determined by either method

showed the same changes after the various treatments.

RESULTS AND DISCUSSION

Submerged rice seedlings develop a much longer coleoptile than do air-grown
rice seedlings (see Fig. 2.5). Since information on the role of IAA and GA on rice
coleoptile growth is contradictory, I reexamined this question by determining the

effects of applied IAA and GA, and of two inhibitors of GA biosynthesis, AMO 1618

24
and CCC, on the elongation of intact rice coleoptiles. Under my conditions, applied

IAA had no effect on coleoptile growth (Table 2.1). This indicates that IAA levels do
not limit growth of intact rice coleoptiles and that addition of IAA does not mimick
the response to submergence. Treatment with 100 M GA, lead to a small but
significant increase in coleoptile length (17%) which, by itself, cannot explain the
submergence-induced growth increase (72%, see, e.g., Table 2.3). Treatment with 10
M GA, and the GA-biosynthesis inhibitors CCC or AMO 1618 did not affect
coleoptile growth (Table 2.2). Therefore GA does not seem to have a regulatory
function in coleoptile growth.

Because two growth promoters, IAA and GA, did not promote rice coleoptile
growth, it may be the decrease in the amount of an endogenous growth inhibitor, e.g.
of ABA that is responsible for the enhanced elongation of submerged rice coleoptiles.

To investigate the possible role of ABA as an inhibitor of coleoptile growth, I
compared the effect of fluridone on rice, oat, barley, and wheat seedlings. There is
strong evidence that ABA is synthesized via an indirect pathway from carotenoids
(Gage ct al., 1989; Li and Walton, 1990; Rock and Zeevaart, 1991). Because
fluridone is an inhibitor of carotenoid biosynthesis (Bartels and Watson, 197 8;
Gamble and Mullet, 1986), its effect on growth in rice coleoptiles can be explained in
terms of reduced ABA formation from carotenoids. Treatment with fluridone for four
days promoted the growth of wheat, oat, and barley coleoptiles by 14 % , 22 % , and
14% above control, respectively (Fig. 2.1). In the same experiment, rice grown on
fluridone for four days showed a 67% increase in coleoptile length. When fluridone

was added during the last two days of incubation, it had no effect on the growth of

25
Table 2.1. Effect of IAA (100 pM) or GA, (100 am on growth of intact rice

coleoptiles. The values represent the mean of the lengths of 42 (IAA/Control) and 54

(GA,/Control) coleoptiles :1; SE. determined four days after start of imbibition.

 

 

 

Coleoptile length (mm)
Treatment Control IAA or GA, n p
IAA 17.4 :1; 0.7 16.9 i 0.8 42 n.s.
GA, 16.1 :I: 0.6 18.8 i 0.7 54 0.05

 

Table 2.2. Effect of GA, (10 uM) and of CCC (250 mg/l) and AMO 1618 (150
mg/l), inhibitors of GA biosynthesis, on the growth of rice coleoptiles. The values
represent the mean length of 21 coleoptiles :1: SE. determined four days after start of

imbibition.

 

Coleoptile length (mm)

 

Exp Control GA, CCC CCC + GA, AMO AMO + GA,

 

I 17.0 :1: 0.8 18.3 i 1.2 15.4 :I: 0.8 16.6 :I: 1.3 n.d. n.d.

II 17.9 :I; 0.8 18.1 :I: 1.2 n.d. n.d. 15.8 :I: 1.0 17.7 i 1.1

 

26

50

 

Coleoptile length (mm)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rlce Wheat Oat Barley

Figure 2.1: Growth of rice, wheat, cat, and barley coleoptiles in response to
treatment with 30 M fluridone. Water control (El); fluridone present during the last
two days of growth (I); fluridone present during four days of growth (I). The
length of coleoptiles was measured four days after the start of imbibition. The bars

represent the means :1; SE. of 20 (control and two days of treatment) and 15 (four

days of treatment) coleoptiles.

27
cat and barley coleoptiles, and the effect on wheat was the same as that observed after

four days. Over a two-day period, fluridone promoted rice coleoptile elongation by
37% . Fluridone inhibited mesocotyl as well as root growth in rice seedlings.

To investigate further the role of ABA in regulating the growth of rice
coleoptiles, seedlings were grown on 1 uM ABA, 30 RM fluridone, or fluridone plus
ABA (Fig. 2.2). ABA treatment reduced growth of the rice coleoptile by 40% when
compared with the control, whereas fluridone promoted coleoptile elongation as found
before. When ABA and fluridone were added simultaneously, fluridonc-induced
growth was eliminated. The response of the mesocotyl was the opposite of that of the
coleoptile. Its growth was promoted by ABA as reported earlier by Takahashi (1972,
1973) and was reduced by fluridone (Fig. 2.2). Rice mesocotyls are the only known
organs of non-stressed, wild type plants whose growth is stimulated by ABA.
However, ABA has been shown to have a growth-promoting effect on ABA deficient
mutants, e.g. of Arabidopsis (Koomneef et al. , 1982) and a growth-maintaining effect
on the roots of water-stressed maize (Saab et al., 1990).

It had been shown that the level of endogenous ABA decreases during seed
germination, e.g. in wheat caryopses, while the growth rate of the seedling increases
(Morris and Walker-Simmons, 1989). I determined the ABA content of dry rice seeds
and seedlings that were grown for up to four days on water, 30 M fluridone or that
were submerged on day two as described in Materials and Methods (Fig. 2.3). In
control plants grown in air, the level of ABA dropped during the first day by 45%

and continued to decline over the next three days to 20% of the original level in dry

28

4o

 

30-

 

20-

Length (mm)

10-

 

 

 

 

 

 

M

Coleoptile Mesocotyl

 

Figure 2.2. Response of rice seedlings to ABA and fluridone. Treatment was started
at imbibition, and measurements were taken after four days. Water control (Cl);
1 uM ABA (I); 30 M fluridone (I); 30 M fluridone and 1 uM ABA (I). The

bars represent the means of 15 seedlings :|: SE.

29

 

SO I 7 I 1

ABA (ng/g fr wt)

 

 

 

 

Davs after imbibition

Figure 2.3. ABA levels in rice seedlings at various times after imbibition. Seedlings
were grown on vermiculite moistened with water (air grown, O) or 30 RM fluridone
(v), or they were submerged two days after start of imbibition (O). The points
represent the means :1; SE. of three (fluridone, submergence) and five (air grown)
experiments, respectively, with approximately 40 seedlings per experiment.

30
seeds. The ABA content of fluridone-treated seedlings was slightly lower than that of

control plants on days one and two, and approximately 60 96 that of control plants on
days three and four. The concentration of ABA was not reduced to zero with
fluridone, probably because of carry-over of ABA from the dry seeds. The ABA level
in seedlings that were submerged two days after start of imbibition decreased within
one day to that of the fluridone—treated plants. The change in the ABA concentration
was reflected in increased coleoptile growth in both submerged and fluridone-treated
seedlings (Fig. 2.4).

Figure 2.5 shows rice seedlings that had been submerged, treated with
fluridone, or with ethylene. Both, Figure 2.5 and Table 2.3 demonstrate that
- treatment with fluridone leads to a growth response similar to that caused by
submergence, 3-5 ul/l ethylene, or a gas mixture containing 3.5% 0,, 19% C0,, 5-6
ul/l ethylene in N,. All four treatments increased elongation of coleoptiles and
reduced root growth, with submergence having the most pronounced effect.

Table 2.3 also shows that the levels of ABA were reduced in coleoptile plus
primary leaf and coleoptile alone as a result of the above treatments. In rice seedlings
grown in 30 MM fluridone, coleoptile growth was stimulated by 63 % , and the ABA
content was reduced in both the coleoptile and the coleoptile plus primary leaf by
66% and 77 96 , respectively. Even though fluridone did not elicit the largest growth
response in coleoptiles, it did cause the most prominent reduction of endogenous ABA
levels. A possible reason for this may be that, besides reducing ABA levels, fluridone
may also have a secondary inhibitory effect on coleoptile growth. Submergence and

treatment with ethylene or the a gas mixture consisting of 3.5% 0,, 19% C0,,and 5-6

31

 

SO I I I ]

Coleoptile length (mm)

 

 

 

 

Days after imbibition

Figure 2.4. Coleoptile length of rice seedlings grown as described in Figure 2.3. The

symbols correspond to the ones used in Figure 2.3.

32

CONTROL FLURIDONE SUBHERGED ETHYLENE

 

Figure 2.5. Rice seedlings grown on vermiculite moistened with water (air control),
with 30 M fluridone, submerged, or treated with 4 ”III ethylene. The picture was

taken four days after start of imbibition.

33

 

6.3 :63 cab v.5 min 963 15:8 mo &

 

 

to H 3 H 3 H 3 H 3 H he H . a; a ”he
3. 3 3 3. in 3. page?
5 $2
3% 3x: 3m 3m ”.8 983 388 8 e
3 H 3 H 8 3. H g H .3 H a; e «85
2 3m 3. .2 3 2 :2 .83 +
03.38 5 <3.
3 H we 3 H e an 3 H 3“ h c H S n _ H v.8 hm H 3“ 38m
3. H 3 as H m a 2 H E v c H S. m c H 3 to H «a 88862
3. H 3.8 3 H c 2 3. H wen u 3 H 3.... e c H SN 2. H 3: 05.328
955 593
a: 0 825m :8 m u
x8585

 

.8353. 08 8 38:8 e Ba 9588 87% a? 580 385%

885 he .md H :88 05 .59: m_o>o_ <m< 05 e8 .829, 05. 35.828 9. .8 .md H 588 05 288.52 59.3

Bu .829, BE. 3:25.898 8858 a 5 once 83 E»: 85 :e 3828 9.3 85 5 8358 new 28 35:8 we eaten—E8 25.
.EE N2 5 08:5» 5: 9m 2s sou as so gen 8:35.50 2355 a» a s a .2535» S: 3 «5:85 as a .59
.883 E .8925...» .5 ha E 2535: 21 an no ADV ha 5 .883 .8 :38» 8.83 35:83 A8988 8.68 83328 not
5 2a @825 33 SEE Ba 83.38 8e 5 33 <m< “.5 ace 88 e5 .3882. .580? 8 593 .3 gen...

34
“Ill ethylene in N2 led to a reduction in ABA content and to a promotion of coleoptile

growth.

Ethylene alone eaused only a 21% reduction of ABA content in coleoptiles
which was far less than the reduction with fluridone or submergence. This can be
explained by the fact that the full submergence response in rice coleoptiles requires an
atmosphere of low 0,, high CO, and ethylene, with low 0,, high CO2 and ethylene
each contributing one third of the response (Raskin and Kende, 1983).In an
atmosphere of 3.5% 02, 19% C02, and 5-6 ul/l ethylene, the ABA content of rice
coleoptiles was reduced by 37% compared to the control. This is comparable to the
38% reduction in submerged coleoptiles.

In rice seedlings, submergence promotes growth, in part, through the action of
ethylene. It is important to know whether fluridone induces growth of rice seedlings
by promoting ethylene biosynthesis. Fluridone leads to an approximately 2.5-fold
increase in ethylene biosynthesis, namely from 16.9 pmol ethylene (g PW)" h'1 in
contol plants to 42.5 pmol ethylene (g FW)" h‘1 in fluridone-treated plants. To test
whether fluridone acts via stimulating ethylene biosynthesis, I measured the length of
rice coleoptiles of seedlings grown for four days on water in air, on water in 10 ulll
ethylene, on 30 “M fluridone in air, and on fluridone in ethylene. Table 2.4 shows
the combined result of two independent experiments. Both, treatment with ethylene
and fluridone led to increased coleoptile growth (36% and 78% , respectively). The
increase in the presence of ethylene and fluridone was 116%. Therefore, the effects of
ethylene and fluridone on growth are additive, and fluridone does not act by

increasing ethylene synthesis. In addition, the effect of fluridone on

35

Table 2.4. Ethylene (3-5 p.1/1) and fluridone (30 uM) act independently on rice

coleoptile growth. The values represent the means of ten measurements ( 2 exp.) :1:

 

 

S.E.
Control Fluridone Ethylene Ethylene + Fluridone
Coleoptile
length (mm) 18.0 :1: 1.0 32.1 3|; 1.6 24.4 :t 1.2 38.8 :t 1.9
Growth above
control (mm) 14.1 6.4 20.8
Growth above
control (96) 78 36 116

 

36
growth was comparable in closed systems, where ethylene accumulated (as above),

and in open systems, where ethylene-free air was continuously circulated through the
incubation flask, thereby removing ethylene produced by the seedlings. It is possible
that ethylene acts on ABA metabolism, while fluridone acts on ABA biosynthesis.
Indeed, Zeevaart (1981) has shown that in Xanthium ethylene increases ABA
metabolism to phaseic acid and ABA-glucose ester, both of which are biologically

inactive.

CONCLUSIONS

Fluridone, an inhibitor of earotenoid biosynthesis, induces coleoptile growth in
rice as does submergence (Fig 2.1, Table 2.3). The effect of fluridone ean be
reversed by ABA (Fig. 2.2). This indieates that ABA may be involved in the
regulation of rice coleoptile growth and is additional evidence that ABA is synthesized
from earotenoids. Figure 2.3 shows that the level of endogenous ABA drops during
germination of rice. This coincides with an increase in coleoptile length (Fig. 2.4).
Submergence and fluridone hasten this decline in ABA content. Our data indicate that
submergence- and possibly ethylene-induced coleoptile growth is based on a reduction
in ABA levels, and that ABA plays a role in regulating the growth of rice coleoptiles.
Promotion of shoot growth by fluridone was also described by Saab et a1. (1990) for
water-stressed corn seedlings. In that case, fluridone appears to prevent accumulation
of ABA at low water potentials.

I could not show an involvement of GA in the regulation of rice coleoptile

37

growth either by applying GA, or by examining the effect of gibberellin biosynthesis
inhibitors (Table 2.1, 2.2). I also could not find an effect of IAA on the growth of
intact seedlings, which indieates that endogenous auxin levels do not limit growth of
intact coleoptiles.

Therefore, submergence may only in part stimulate growth by increasing the
level of a growth promoter, e.g. auxin, but mostly by reducing the level of a growth
inhibitor, e.g. ABA. The rate of growth in rice coleoptiles may be determined by the

ratio of the promoter (IAA) and the inhibitor, (ABA).

The submergence response of rice seedlings may thus be regulated in the following

way:

reduced 0, coleoptile
submergence --> increased (12H. --> reduced ABA —-> growth

increased C02

38
REFERENCES

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on elongation of coleoptiles of submergence-tolerant and -intolerant rice
cultivars. J. Exp. Bot. 33, 1030-1044.

Bartels, P.G. , Watson, C.W. (1978) Inhibition of carotenoid biosynthesis by fluridone
and norflurazone. Weed Sci. 2, 198-203.

Belefant, H., Fong, F. (1989) Abscisic acid ELISA: organic acid interference. Plant
Physiol. 91, 1467-1470.

Gage, D.A., Pong, F., Zeevaart, J.A.D. (1989) Abscisic acid biosynthesis in isolated
embryos of Zea mays L. Plant Physiol. 89, 1039-1041.

Gamble, P.E. , Mullet, LE. (1986) Inhibition of carotenoid accumulation and abscisic
acid biosynthesis in fluridone-treated, dark-grown barley. Eur.J.Biachem. 160,
117-121.

Gopalakrishnan, S., Sircar, S.M. (1974) A comparative study of the effects of some
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120.

Hoffmann, S., Kende, H. (1990) The role of ABA in the growth of rice coleoptiles.
(Abstract No. 409). Plant Physiol. 93, S-71.

Hoffmann-Benning, S., Kende, H. (1991) On the role of abscisic acid and gibberellin
in the regulation of growth in rice. Plant Physiol. 99, 1156—1161.

Horton, R.F. (1991) The effect of ethylene and other regulators on coleoptile growth
of rice under anoxia. Plant Sci. 79, 57-62.

Hoson, T., Masuda, Y., Pilet, P.E. (1992) Auxin content in air and water grown rice
coleoptiles. J. Plant Physiol. 139, 685-689.

Imaseki, H. , Pjon, C.-J. (1970) The effect of ethylene on auxin-induced growth of
excised rice coleoptile segments. Plant Cell Physiol. 11, 827-829.

Ishizawa, K., Esashi, Y. (1983) Cooperation of ethylene and auxin in the growth
regulation of rice coleoptile segments. J. Exp. Bot. 34, 74-82.

Ishizawa, K., Esashi, Y. (1984a) Gaseous factors involved in the enhanced elongation
of rice coleoptiles under water. Plant, Cell, Environment 7, 239-245.

39

Ishizawa, K. , Esashi, Y. (1984b) Osmoregulation in rice coleoptile elongation as
promoted by cooperation between IAA and ethylene. Plant Cell Physiol. 25,
495-504.

Katsura, N. , Suge, H. (1979) Does ethylene induce elongation of the rice coleoptile
through auxin? Plant Cell Physiol. 20, 1147-1150.

Kefford, N .P. (1962) Auxin-gibberellin interaction in rice coleoptile elongation. Plant
Physiol. 37, 380-386.

Kende, H. , Hanson, AD. (1976) Relationship between ethylene evolution and
senescence in morning-glory flower tissue. Plant. Physiol. 57, 523-527.

Kordan, H.A. (1977) Coleoptile emergence in rice seedlings in different oxygen
environments. Ann. Bot. 41, 1205-1209.

Koornneef, M., Jorna, M.L., Brinkhorst-van der Swan, D.L.C., Karssen, CM.
(1982) The isolation of abscisic acid (ABM-deficient mutants by selection of
induced revertants in non-germinating gibberellin-sensitive lines of Arabidopsis
thaliana (L.) Heyn. Ihear. Appl. Genet. 61, 385-393.

Ku, H.S., Suge, H., Rappaport, L., Pratt, H.K. (1970) Stimulation of rice coleoptile
growth by ethylene. Planta 90, 333-339.

Li, Y., Walton, D.C. (1990) Violaxanthin is an abscisic acid precursor in water-
stressed dark-grown bean leaves. Plant Physiol. 92, 551-559.

Mapelli, S., Rocchi, P., and Bertani, A. (1986) ABA and IAA in rice seedlings under
anaerobic conditions. Biol. Plant. 28, 57-61.

Mertens, R., Deus-Neumann, B., Weiler, E.W. (1983) Monoclonal antibodies for the
detection and quantifieation of the endogenous plant growth regulator, abscisic
acid. FEBS Letters 160, 269-272.

Miller, J.H., Miller, P.M. (1974) Ethylene and the responses to light of rice
seedlings. Physiol. Plant. 30, 206-211.

Morris, C.F. , Walker-Simmons, M. (1989) ABA-inducible cDNAs in embryos of
dormant wheat grain. (Poster No. 1020) Plant Physiol. 89, p.171.

Murakami, Y. (1962) The promoting effect of gibberellin on the elongation of rice
radicle. 11w Bot. Mag. 75, 102-106.

Ohwaki, Y. (1967) Growth of rice coleoptiles in relation to oxygen concentrations.
Sci. Rep. Tah. Univ. 33, 1-5.

4O

Ranson, S.L., Parija, B. (1955) Experiments on growth in length of plant organs. 11.
Some effects of depressed oxygen concentrations. J. Exp. Bot. 6, 80-93.

Raskin, I. and Kende, H. (1983) Regulation of growth in rice seedlings. J. Plant
Growth Regal. 2, 193-203.

Rock, C.D., Zeevaart, J.A.D. (1991) The aba mutant of Arabidopsis is impaired in
epoxy-earotenoid biosynthesis. Proc. Natl. Acad. Sci. USA 88, 7496-7499.

Saab, I.N., Sharp, R.E., Pritchard, J., and Voetberg, 6.8. (1990) Increased
endogenous abscisic acid maintains primary root growth and inhibits shoot
growth of maize seedlings at low water potentials. Plant Physiol. 93, 1329-
1336.

Suge, H. (1971) Stimulation of oat and rice mesocotyl growth by ethylene. Plant Cell
Physiol. 12, 831-837.

Suge, H. (1974) Synergistic action of ethylene with gibberellins in the growth of rice
seedlings. Crop. Sci. Soc. Japan. Proc. 43, 83-87.

Takahashi, K. (1972) Abscisic acid as a stimulator of rice mesocotyl growth. Nature
New Biol. 238, 92-93.

Takahashi, K. (1973) Interaction between ethylene, abscisic acid and gibberellic acid
in elongation of rice mesocotyl. Planta 109, 363-364.

Turner, F.T., Chen, C.-C., McCauley, G.N. (1981) Morphologieal development of
rice seedlings in water of controlled oxygen levels. Am. J. Bot. 29, 721-738.

Vergara, B.S., Jackson, 3., DeDatta, S.K. (1976) Deep-water rice and its response to
deep-water stress. In: Climate and Rice. International Rice Research Institute,
Los Bafios, Philippines, pp 301-319.

Wada, S. (1961) Growth patterns of rice coleoptiles grown on water and under water.
Sci. Rep. Tahoku Univ. 27, 199-207.

Walker-Simmons, M. ( 1987) ABA levels and sensitivity in developing wheat embryos
of sprouting resistant and susceptible cultivars Plant Physiol. 84: 61-66.

Weiler, E.W. (1980) Radioimmunoassay for the differential and direct analysis of free
and conjugated abscisic acid in plants. Planta 148, 262-272.

Yamada, N. (1954) Auxin relationships of the rice coleoptile. Plant Physiol. 29, 92-
96.

41

Zeevaart, J.A.D. (1983) Metabolism of abscisic acid and its regulation in Xanthium
leaves during and after water stress. Plant Physiol. 71, 477-481.

Zeevaart, J.A.D., Heath, T.G., Gage, D.A. (1989) Evidence for a universal pathway
of abscisic acid biosynthesis in higher plants from "O incorporation patterns.
Plant Physiol. 91, 1594-1601.

CHAPTER 3
THE ROLE OF ABSCISIC ACID AND GIBBERELLIN IN THE

REGULATION OF GROWTH IN DEEPWATER RICE INTERNODES

ABSTRACT

Submergence induces rapid elongation in rice coleoptiles (Otyza sativa L.) and
of deepwater rice intemodes. This adaptive feature helps rice to grow out of the water
and to survive flooding. Earlier, we found that the growth response of submerged
deepwater rice plants is mediated by ethylene and gibberellin (GA). Ethylene
promotes growth, at least in part, by increasing the responsiveness of the intemodal
tissue to GA. In the present work, I examined the possibility that increased
responsiveness to GA was based on a reduction in endogenous abscisic acid (ABA)
levels. Submergence and treatment with ethylene led, within three hours, to a 75 96
reduction in the level of ABA in the intercalary meristem and the growing zone of
deepwater rice intemodes. The level of GAl increased fourfold during the same time
period. An interaction between GA and ABA could also be shown by application of
these hormones. ABA inhibited growth of submerged intemodes, and GA
counteracted this inhibition. Our results indieate that the growth rate of deepwater rice
intemodes is determined by the ratio of an endogenous growth promoter (GA) and a

growth inhibitor (ABA).

42

43
INTRODUCTION

Rice (Oryza sativa L.) has a number of physiological and metabolic
adaptations that enhance its chances for survival under conditions of temporary
flooding. One of these is the capacity of plants to elongate rapidly when they become
submerged. This feature helps rice to emerge from the water and to avoid drowning.
In seedlings, submergence promotes coleoptile growth (Kordan et al. , 1977 ; Turner et
al. , 1981; Yamada, 1959) and in adult deepwater rice plants, elongation of the
internode (Métraux and Kende, 1983; Vergara et al., 1976). In both instances, the
plants respond to the altered gas composition of their submerged organs, namely to
reduced partial pressure of 02, to increased partial pressure of C02, and to the
accumulation of ethylene (Ku et al., 1970; Ohwaki, 1967; Ranson and Parija, 1955;
Raskin and Kende, 1984 a,b; for a review, see Jackson and Pearce, 1991). In
deepwater rice, low partial pressures of 0, promote ethylene biosynthesis (Raskin and
Kende, 1984a) by enhancing the activity of the ethylene-biosynthetic enzyme 1-
aminocyclopropane-l-carboxylate (ACC) synthase (Cohen and Kende, 1987). Ethylene
promotes growth, at least in part, by increasing the responsiveness of the intemodal
tissue to GA (Raskin and Kende, 1984b). This is very different from what has been
found earlier in rice seedlings (see Chapter 2; Hoffrnann and Kende, 1990), where
ethylene also promotes coleoptile growth. GA does not seem to be involved in
regulating rice coleoptile growth, while rice intemodal growth is induced by GA and
inhibited by GA-biosynthesis inhibitors (Raskin and Kende, 1984b).

In the studies described below, I investigated one possible mechanism by

44
which ethylene may modulate the responsiveness of deepwater rice to GA. Zeevaart

(1983) found that applied ethylene reduced the level of ABA in leaves of Xanthium
smonariwn. Thus, increased responsiveness to GA in deepwater rice may be based on
an ethylene-mediated reduction in the level of endogenous ABA, a potent inhibitor of
growth in rice. To examine this possibility, I measured the level of ABA in the
interealary meristem and the growing zone of deepwater rice plants that had been
induced to grow rapidly by submergence or treatment with ethylene. I also determined
the level of GAl and GA” in the same tissue. These experiments were designed to
test my working hypothesis that the rate of intemodal growth in deepwater rice is
determined by the balance of GA, a growth promoter, and of ABA, a growth
inhibitor. Earlier, I had shown that coleoptile growth in rice seedlings is inhibited by
ABA and stimulated by fluridone, an inhibitor of ABA biosynthesis. In addition, both,
ethylene treatment and submergence led to a reduction of the endogenous ABA

content in rice coleoptiles (Hoffmann and Kende, 1990; chapter 2).

MATERIALS AND METHODS

Growth of plants. Rice plants (Otyza sativa L., cv. Habiganj Aman II) were
grown as described by Stiinzi and Kende (1989), except that the day temperature was

27° C for 11 h centered within a 13-h photoperiod.

45
Treatment of whole plants. Adult plants were submerged as described by

Metraux and Kende (1983). For ethylene treatment, they were placed in plastic
cylinders through which air containing 3 to 5 ul/ml ethylene was passed at a rate of
400 ml/ min. These treatments were performed in the same growth chamber in which
the plants had been grown. At various times, the basal l-cm portion of the youngest
intemode containing the interealary meristem and part of the elongation zone above it
was harvested and frozen in liquid N2. Submergence and treatment with ethylene were

performed with 8- to l2-week-old plants.

Isolation and treatment of stem sections. Stem sections containing the
youngest intemode were excised from 9- to ll-week-old plants as described by Raskin
and Kende (1984a). They were either submerged in glass cylinders containing 4 1 of
water with or without GA, and/or ABA or kept in plastic cylinders through which
humidified air was passed at a rate of 80 mllmin. The increase in intemodal length

was measured after three days of growth under continuous light.

ABA extraction and determination. ABA from the basal l-cm portion of
intemodes was extrated and an ELISA performed as described by Walker-Simmons
(1987) using the monoclonal antibody of Mertens et al. (1983) which was purchased
from Idetek (San Bruno, CA). The ABA-4’-conjugate was prepared according to

Weiler (1980).

46
GA extraction and determination. The basal l-cm portions of intemodes

from submerged and air-grown plants were collected over a period of several weeks
and frozen. Tissue from 40 to 100 plants was lyophilized yielding 1 to 3 g (dry wt) of
material. GAs were extracted, purified, and seperated according to Talon and
Zeevaart (1990). In brief, GAs from rice tissue were extracted overnight, first in
100% and then in 80% (v/v) methanol. They were purified by phase partitioning with
hexanes and ethyl ether, by absorption chromatography with Charcoal:Celite (1:2),
another phase partitioning with ethyl acetate, and chromatography on a QAE-
Sephadex A-25 column. They were separated by reverse-phase HPLC. Fractions
corresponding to GA, and GA” were analyzed by GC-selected ion monitoring as
described by Talon and Zeevaart (1990). The ions monitored were for GA,/[’Hz]GA-
methyl ester trimethylsilyl ether (m/z 508, 506, 450, 448) and for GAz/PHJGA-
methyl ester trimethylsilyl ether (m/z 420, 418, 377, 375). GA concentrations were

quantified as in Talon and Zeevaart (1990).

Statistical analysis. The data shown in Figures 3.3 and 3.4 were subjected to
analysis of variance for a complete randomized block design with three (submergence)
or four (treatment with ethylene) replicates (Steele and Torrie, 1960). One block
consisted of one series of ABA determinations from plants that were air grown,

submerged, or treated with ethylene for various time periods.

47
RESULTS AND DISCUSSION

Stem sections isolated from adult rice plants were either kept in air or
submerged in cylinders containing water and one of several concentrations of ABA
(Fig. 3.1). At a concentration of 1 pM, ABA reduced submergence-induced growth
by 30%; at 100 “M, it eliminated it completely. This inhibition was reversed by the
addition of GA, to sections submerged in 3 uM ABA (Fig. 3.2).

Two sets of experiments were performed to assess the effect of submergence and
ethylene treatment on the ABA content of the interealary meristem and the elongation
zone of deepwater rice intemodes. In one set, plants were immersed in 300-1 plastic
tanks filled with water or were kept in air in the same growth chamber. In the second
set, deepwater rice plants were placed in plastic cylinders through which ethylene-free
air or air containing 3 to 5 pl/l ethylene was passed. Within 1 h of submergence, the
ABA level in the interealary meristem and the cell elongation zone above it decreased
by more than 50% (Fig. 3.3). After 3 h, it was reduced by 75% and it decreased
further during the next 21 h. In air-grown control plants, the ABA content remained
at its original level. These data were subjected to analysis of variance for a
randomized complete block design with three replieates (Table 3.1). The differences
in ABA levels between different treatments were highly significant (F test;

P < 0.005) with a highly significant effect of submergence (F test; P < 0.005).

48

 

 

 

 

 

 

 

 

 

 

 

 

 

ppppppp

 

00000000

rW////////////////////////////
I%/////////////////////////////w

A
0

Figure 3.1. Growth of deepwater rice stern sections submerged for three days in
water containing one of several concentrations of ABA. The air-grown sections were

keptinaglasscylinderwithanairflowof80ml/min.Thevaluesrepresentthe

means :1: SE of 22 to 30 sections.

49

 

 

 

 

 

 

 

Figure 3.2. Growth of deepwater rice stem sections submerged for three days in

water,in3uMABA,in3uMABAplusoneofseveralconcentrationsofGA,,orin

10 M GA, alone. The values represent the means :1; S.E. of 22 to 30 sections.

50

 

 

 

 

25
Air-grown
20 -
:3
E 15 -
q—
9
O)
E.
< 10 r
m
<
Submerged
5 _
o J l L l l l l J l

 

O 3 6 9 12 15 18 21 24

Duration of Treatment (h)

Figure 3.3. Effect of submergence on the level of ABA in deepwater rice. Plants
were submerged in 300-1 tanks filled with water (V) or kept in air as control (B).
One-centimeter portions containing the interealary meristem and part of the cell
elongation zone above it were excised for extraction from the intemodes at the times

indicated. Fach point represents the mean :I: S.E. of three independent experiments.

51

Table 3.1. Analysis of Variance on the effect of submergence on ABA levels in

deepwater rice intemodes (Data from Fig.3.3). df Degrees of freedom;

** Signifieant at the 0.01 level; *** Significant at the 0.005 level.

 

 

Source df Mean squares
Replieates 2
Treatments 7 197.0 (“‘9

a (submergence) 1 1303.9 P“)

b (time) 3 15.0 C")

a x b 3 15.4 (")
Error 13 2.7

 

52
In deepwater rice plants treated for 3 h with ethylene, the ABA level in the

interealary meristem and the cell elongation zone was 75 % lower than that of the
corresponding control plants at the same time (Fig. 3.4). This difference in the ABA
concentration was maintained over the next 21 h. Again, the data were evaluated by
analysis of variance for a randomized complete block design with four replicates
(Table 3.2). There were significant differences in ABA content between different
treatments (F test; P = 0.025) with a highly signifieant effect of ethylene (F test;

P < 0.005).

I determined the amount of the endogenous GA, and GA” in the interealary
meristem and intemodal elongation zone of deepwater rice plants that had been
submerged for 0, 1, 3, and 24 h (Table 3.3). The level of GA, increased fourfold
within 3 h of submergence. The level of GA”, which is the immediate precursor of
GA,, also increased but more slowly than that of GA,. After 24 h of submergence, it
was 3 times higher than the original value. The GA extractions and quantifieations

were repeated with a second batch of plants and obtained similar results.

CONCLUSIONS

The results described above support the notion that the growth rate of
deepwater rice intemodes is determined not only by a growth-promoting plant
hormone, GA, but also by a growth inhibitor, ABA. Elongation of deepwater rice
intemodes, like that of rice coleoptiles, is stimulated by submergence (Métraux and

Kende, 1983; Raskin and Kende, 1984a; Vergara et al., 1976). There are, however,

53

 

N
01
I

N
O
I

Air-grown

 

ABA (ng/g fr wt)
5."
r
i—-—-
1

d
O
I

Ethylene-treated

5+- -—§— %

0' I l 1 l I J l l
0 3 6 91215182124

Duration of Treatment (h)

 

 

 

 

Figure 3.4. Effect of ethylene on the level of ABA in deepwater rice. Plants were
grown in plastic cylinders through which ethylene-free air (V) or air containing 3 to
5 ulll ethylene (D) was circulated at a flow rate of 400 mllmin. One-centimeter
portions containing the interealary meristem and part of the cell elongation zone above
it were excised for extraction from the intemodes at the times indieated. Each point

represents the mean 1: S.E. of four independent experiments.

54

Table 3.2. Analysis of Variance on the effect of ethylene on ABA levels in deepwater
rice intemodes (Data from Fig. 3.4.). df Degrees of freedom;

"' Signifieant at the 0.025 level; “* Significant at the 0.005 level.

 

 

Source df Mean squares
Replicates 3
Treatments 5 132.5 (*)
a (submergence) 1 586.1 0"")
b (time) 2 14.1
a x b 2 24.2

Error 15 29.8

 

55

Table 3.3. Levels of GA, and GA” in the interealary meristem and cell elongation
zone of intemodes of adult deepwater rice plants grown in air or submerged for
various times. Tissue for this experiment was harvested over a period of several
weeks (ca. 60 plants per time point). A repetition of the GA, determinations with a
second batch of tissue lead to similar increases with similar amounts of GA,.

n.d. not determined.

 

Duration of GA, Relative GA” Relative

treatment (h) (ng/ g dry wt) level (ng/ g dry wt) level

 

0 15.2 1.0 12.5 1.0
1 18.9 1.2 n.d. n.d.
3 62.3 4.1 19.4 1.6

24 63.0 4.1 40.9 3.3

 

56
distinct differences in the growth response of seedlings and adult plants. Both are

stimulated to grow at low partial pressures of 0,, high partial pressures of C0,, and
in the presence of ethylene (Raskin and Kende, 1983; Raskin and Kende, 1984a). In
coleopiles, these gases act independently of each other, each eliciting about one-third
of the overall response (Raskin and Kende, 1983). In adult plants, on the other hand,
there is an interaction between 0,, C0,, and ethylene (Raskin and Kende, 1984a).
The hormonal basis for coleoptile and intemodal growth appears to be different as
well. Elongation of rice coleoptiles is not reduced by inhibitors of GA biosynthesis
(Hoffmann and Kende, 1990; Hoffmann-Benning and Kende, 1992) and is thought to
require auxin (Jackson and Pearce, 1991). In contrast, intemodal growth is completely
dependent on the presence of GA (Raskin and Kende, 1984b). I hypothesized that
ethylene may cause a reduction in endogenous ABA levels, as it does in Xanthium
leaves (Zeevaart, 1983) and that responsiveness to GA may be a function of ABA
content. My data support this view. The level of ABA decreased in the the interealary
meristem and the cell elongation zone of submerged and ethylene-treated intemodes
by 75% (Figs. 3.3 and 3.4), and the inhibition of intemodal growth by ABA was
fully reversed by GA (Fig. 3.2). The lag phase for the induction of growth by
submergence is between 3 and 4 h (Stfinzi and Kende, 1989); that for the reduction in
ABA content is less than 3 h (Figs. 3.3 and 3.4). Therefore, the change in ABA
levels precedes the growth response.

The level of GA, in the interealary meristem and cell elongation zone increases
fourfold within 3 h of submergence, and that of GA” threefold within 24 h (Table

3.3). GA, is the active GA in rice, and GA” is its immediate precursor (Kobayashi et

57
al., 1989). My results are in agreement with those of Suge (1985) and Tfirkan and

Suge (1990) who, using a bioassay, found an increase in GA levels upon submergence
of deepwater rice plants. It is not known whether submergence or ABA affect GA
content directly or whether the level of GA increases as a function of enhanced
growth, e. g. because of the formation of new cells in the interealary meristem.

On the basis of the results reported above, I can postulate two new links in the
chain of events that leads from submergence to accelerated growth in deepwater rice
(Fig. 3.5). The reduced 0, tension in submerged intemodes promotes ethylene
synthesis (Raskin and Kende, 1984a) by enhancing the activity of ACC synthase
(Cohen and Kende, 1987). In addition, ethylene is physieally trapped in submerged
intemodes beeause of its low diffusion rate in water. Ethylene inhances the
responsiveness of deepwater rice intemodes to GA (Raskin and Kende, 1984b), at
least in part beeause of a reduction in ABA content. While the level of ABA
decreases in submerged intemodes, that of GA increases. Thus, rapid intemodal
growth of deepwater rice may result from an altered balance between a growth-

promoting (GA) and a growth-inhibiting (ABA) hormone.

58

SUBMERGENCE

i

REDUCED OXYGEN TENSION

i

INCREASED ETHYLENE BIOSYNTHESIS

/\

REDUCED ABA LEVELS INCREASED GA LEVELS

i

INCREASED RESPONSIVENESS TO GA

\

INTERNODAL GROWTH

Figure 3.5. Chain of events linking submergence to accelerated growth of

deepwater rice.

59
REFERENCES

Cohen, E. , Kende, H. (1987) In vivo l-aminocyclopropane—l-earboxylase synthase
activity in intemodes of deepwater rice. Enhancement by submergence and low
oxygen levels. Plant Physiol. 84, 282-286.

Hoffmann, S., Kende, H. (1990) The role of ABA in the growth of rice coleoptiles
(Abstract No. 409). Plant Physiol. 93, S-7l.

Hoffmann-Benning, S. , Kende, H. (1992) On the role of abscisic acid and gibberellin
in the regulation of growth in deepwater rice. Plant Physiol. 99, 1156-1161.

Jackson, M.B., Pearce, D.M.E. (1991) Hormones and morphologieal adaptation to
aeration stress in rice. In: Plant Live under Oxygen Deprivation. M.B.
Jackson, D.D. Davies, H. Lambers, eds, SPB Academic Publishing, The
Hague, Netherlands, pp. 47-67.

Kordan, H.A. (1977) Coleoptile emergence in rice seedlings in different oxygen
environments. Ann. Bot. 41, 1205-1209.

Kobayashi, M., Sakurai, A., Saka, H., Takahashi, N. (1989) Quantitative analysis of
endogenous gibberellins in normal and dwarf cultivars of rice. Plant Cell
Physiol. 30, 963-969.

Ku, H.S., Suge, H., Rappaport, L., Pratt, H.K. (1970) Stimulation of rice coleoptile
growth by ethylene. Planta 90, 333-339.

Mertens, R., Deus-Neumann, B., Weiler, E.W. (1983) Monoclonal antibodies for
the detection and quantification of the endogenous plant growth regulator,
abscisic acid. FEBS Lett. 160, 269-272.

Métraux, J.-P., Kende, H. (1983) The role of ethylene in the growth response of
submerged deepwater rice. Plant Physiol. 72, 441-446.

Ohwaki, Y. (1967) Growth of rice coleoptiles in relation to oxygen concentrations.
Sci. Rep. 701:. Univ. 33, 1-5.

Ranson, S.L., Parija, B. (1955) Experiments on growth in length of plant organs.
11. Some effects of depressed oxygen concentrations. J. Exp. Bot. 6, 80-93.

Raskin, I. and Kende, H. (1983) Regulation of growth in rice seedlings. J. Plant
Growth Regal. 2, 193-203.

60

Raskin, I. , Kende, H. (1984a) Regulation of growth in stem sections of deepwater
rice. Planta 160, 66-72.

Raskin, 1., Kende, H. (1984b) Role of gibberellin in the growth response of
submerged deep water rice. Plant Physiol. 76, 947-950.

Steele, R.G.D., Torrie, J .H. (1960) Principles and procedures of statistics. McGraw-
Hill, New York.

Stiinzi, J .T., Kende, H. (1989) Gas composition in the internal air spaces of
deepwater rice in relation to growth induced by submergence. Plant Cell
Physiol. 30, 49-56.

Suge, H. (1985) Ethylene and gibberellin: regulation of intemodal elongation and
nodal root development in floating rice. Plant Cell Physiol. 26, 607-614.

Talon, M., Zeevaart, J.A.D. (1990) Gibberellins and stem growth as related to
photoperiod in Silene armert'a L. Plant Physiol. 92, 1094-1100.

Tiirkan, 1., Suge, H. (1991) Localization of high concentration of gibberellins in
elongating intemodes of floating rice. Japan. J. Breed. 41, 553-559.

Turner, F.T., Chen, C.-C., McCauley, G.N. (1981) Morphologieal development of
rice seedlings in water of controlled oxygen levels. Am. J. Bot. 29, 721-738.

Vergara, B.S., Jackson, B., DeDatta, SK. (1976) Deep-water rice and its response
to deep-water stress. In: Climate and Rice. International Rice Research
Institute, Los Bafios, Philippines, pp. 310-319.

Walker-Simmons, M. (1987) ABA levels and sensitivity in developing wheat
embryos of sprouting resitant and susceptible cultivars. Plant Physiol. 84, 61-
64.

Weiler, E. (1980) Radioimmunoassay for the differential and direct analysis of free
and conjugated abscisic acid in plants. Planta 148, 262-272.

Yamada, N. (1954) Auxin relationships of the rice coleoptile. Plant Physiol. 29, 92-
96.

Zeevaart, J.A.D. (1983) Metabolism of abscisic acid and its regulation in Xanthium
leaves during and after water stress. Plant Physiol. 71, 477-481.

CHAPTER 4
CHARACTERIZATION AND PARTIAL IDENTIFICATION OF
GROWTH-RELATED OSMIOPI-IILIC PARTICLES IN CORN AND

DEEPWATER RICE

ABSTRACT

The epidermal cell layer appears to limit intemodal growth in deepwater rice
as it does in other rapidly growing plant organs. Ultrastructural examination of the
growing zone of deepwater rice intemodes showed the appearance of osmiophilic
particles between the plasma membrane and the outer epidermal wall of plants
induced to grow rapidly by submergence or by treatment with ethylene or gibberellin.
The diameter of the osmiophilic particles in deepwater rice is about 80 nm, in
cucumber200nm,andinauxin-treatedcomcoleoptilesupto300nm. Inalleases,
they are associated with rapid growth and, in deepwater rice, with the zone of cell
elongation. Monensin inhibits the appearance of these particles, indicating that they
are derived from the Golgi apparatus. In excised tissue and ultrathin sections treated
with proteinase K, the number of osmiophilic particles was reduced, while glueanase
and cutinase treatment were without effect. Cutinase-gold accumulated mostly in the
layer on the inside of the cuticle. The osmiophilic particles beeame densely labeled
after incubation with proteinase K-gold, indieating that they are, at least in part,
proteinaceous. Antibodies against lipid transfer protein (LTP), an extensin-like

61

62
protein, an arabinogalactan protein, and 'expansin' bound to the cell wall or plasma

membrane but not to the osmiophilic particles.

INTRODUCTION

Deepwater rice is grown in Southeast Asia in areas that are flooded during the
monsoon season. Its most distinct feature is its ability to grow very rapidly in
response to submergence. This enables the plant to keep part of its foliage above
water and to survive flooding (V ergara et al. , 1976). Submergence induces intemodal
growth by increasing ethylene biosynthesis which, in turn, eauses a decrease in
endogenous abscisic acid (ABA) level and an increase in sensitivity to gibberellins
(GA; see Kende, 1987 ; Hoffmann-Benning and Kende, 1992a). Kutschera and Kende
(1988) showed that, in rapidly growing intemodes, the epidermis is under tension and
suggested that the outer epidermal wall is a growth-limiting structure. They further
showed by electronmicroscopy the appearance of osmiophilic particles between the
plasma membrane and the outer epidermal wall of submerged intemodes (Kutschera
and Kende, 1989). Similar particles had been described by Kutschera et al. (1987) in
rapidly growing corn coleoptiles. In both eases, the particles were thought to be
associated with cell wall biosynthesis. Robards (1969, in Salix and Fagus) and Olesen
(1980, in Euphorbiaceae, Pedicularis, Phyllodoce, Batrachium, Samulus, Gentiana,
Epilobium, and Salsola) observed osmiophilic particles and reported that they are
involved in cell wall biosynthesis. However, Barckhausen and Rosenstock (1973)

considered similar osmiophilic particles in wounded potato tubers to be related to

63
suberization. Osmiophilic particles were thought to be associated with wax filament

formation in Sorghum (Paul and McWorther, 1989).

Since the osmiophilic particles seem to be associated with rapid growth in
deepwater rice intemodes and appear only between the plasma membrane and outer
epidermal wall of the outer epidermis, they could contain:

(1) cell wall components

(2) . enzymes mediating growth of the cell wall
(3) cuticle components

(4) enzymes of cutin biosynthesis

I determined the time course of appearance and the loealization of the
osmiophilic particles along the rice intemode and analyzed their composition by

enzyme-gold labeling, selective digestion, staining, and immunocytochemistry.

MATERIALS AND METHODS

Growth of plants. Rice plants (Oryza sativa L., cv. Habiganj Aman II) were
grown as described by Stiinzi and Kende (1989), except that the day temperature was
27° C for 11 h centered within a 13-h photoperiod.

Corn (Zea mays L.) kernels were soaked in aerated water overnight and
planted in wet vermiculite. They were grown for four days in total darkness at 20° C.
On the last two days, a red-light pulse was given for 10 min to reduce mesocotyl

growth.

64
Treatment of whole plants. Nine- to 12-week-old adult deepwater rice plants

were submerged as described by Métraux and Kende (1983). For ethylene treatment,
they were placed in plastic cylinders through which air or air containing 3 to 5 all ml
ethylene was passed at a rate of 400 ml] min. These treatments were performed in the

same growth chamber where the plants had been grown.

Isolation and treatment of stem sections. Stem sections containing the
youngest intemode were excised from 9- to ll-week-old plants as described by Raskin
and Kende (1984a). They were placed in water (control) or in 10 M GA, solution
and incubated in plastic cylinders through which humidified air was passed at a rate of
80 ml/ min or were completely submerged (Raskin and Kende, 1984a). For most

experiments, plants were treated for three days.

Treatment of coleoptiles. After four days of growth, the coleoptiles were
separated from the first leaves, and l-cm sections were isolated 2 to 3 mm below the
tip. These sections were first incubated in water for 1 h and then in either water

(control) or 10 uM IAA for an additional hour (Kutschera et al., 1987).

Monensin treatment. Deepwater rice stem sections were placed into a beaker
containing water or 10 M GA, with or without 20 M monensin for the indicated
times prior to fixation. Corn coleoptile sections were incubated in water or 10 M

IAA with or without monensin.

65
Preparation of tissue for electron microscopy. Rice stem segments of 2 mm

length were excised, unless mentioned otherwise, 5 mm above the nodal septum of
the youngest, growing intemode and fixed in 4 % glutaraldehyde in 0.1 M sodium
phosphate buffer, pH 7.2, for 1.5 h on ice. The tissue was subsequently washed three
times in the same buffer on ice. Secondary fixation followed in 1% 030, in the above
buffer for 1.5 h at room temperature. The tissue was then washed twice in phosphate
buffer, twice in water, dehydrated in a graduated series of acetone and embedded in
Spurr’s epoxy resin. Ultrathin sections ( ~ 80 nm) were cut, stained with uranyl
acetate and lead citrate (Reynolds, 1963), and viewed on a Philips 201 transmission
electron microscope (TEM).

One-mm corn coleoptile segments were excised from the center of the

coleoptile sections and treated as described for rice stem sections.

High-pressure freezing and freeze substitution. Rice stem segments of less
than 2 mm length were excised 5 mm above the nodal septum of the youngest
intemode, vacuum infiltrated in hexadecene, and frozen in a Balzers High Pressure
Freezer at Miami University, Oxford, Ohio. The frozen tissue pieces were freeze
substituted in 1% OsO4 in acetone at -80° C for 3 days, at -20° C for 24 h, washed
four times with acetone for 15 min each, and embedded in Spurr’s epoxy resin.
Ultrathin sections were cut and stained with uranyl acetate and lead citrate and viewed
on a Philips 201 transmission electron microscope.

Tissue pieces of less than 1 mm length were excised from the center of corn

coleoptile sections and prepared as described for rice stem sections.

66
Digestion experiments. Tissue samples fixed chemically as described above

were incubated in cutinase or proteinase solution (0.1 mg/ml) or 0.01 M Na,HPO,
(pH 9) for 1 h either between primary and secondary fixation or after embedding,
sectioning, and etching with 0.5 % sodium metaperiodate. Ultrathin sections were

stained as described above. The experiments were done at least twice.

Enzyme-gold labeling. The method used was adapted from Bendayan et al.
(1984). For the preparation of the enzyme gold complex, all steps were performed on
ice. The pH of a 5-ml colloidal gold suspension (Ted Pella, Redding, CA) containing
5 to 10 drops of phosphate buffered saline (PBS) was adjusted to a value higher than
the pl of the respective enzyme (for proteinase K, pH 10.5; for cutinase, pH 11).
This suspension was added to 0.2 mg enzyme dissolved in 200 pl distilled water and
mixed. Two drops of 1 % polyethylene glycol (PEG, MW 20000) were added,
mixed, and the preparation centrifuged at 28000 rpm for 35 min at 4° C (Beckrnann
ultracentrifuge, Ti 50 rotor). The supernatant was disearded, and the pellet
resuspended in 1.5 ml 0.01 M phosphate buffer (pH 9), containing 0.02% PEG and
stored in a refrigerator.

For the labeling procedure, fixed tissue was sectioned and collected to nickel
grids (silver to light gold sections), etched with sodium metaperiodate for 45 min, and
washed thoroughly with water. The grids were then incubated for 20 min in PBS, for
1 h in enzyme-gold solution, washed with PBS and distilled water, stained for 1 h
with uranyl acetate and viewed in the TEM. All labeling steps were performed at

room temperature. The labeling with proteinase K gold plus controls was performed

67
five times with two independent sets of blocks. Control labeling was done at least

three times.

Antibody-gold labeling. Ultrathin sections of conventionally fixed corn tissue
mounted on nickel grids were etched for 45 min with 0.5 % sodium metaperiodate
and washed thoroughly with water. For the detection of threonine hydroxyproline-rich
glycoprotein (THRGP) they were blocked in 0.01 M PBS containing 2 % bovine
serum albumine (BSA) for 15 min. For the detection of lipid transfer protein (LTP),
arabinogalactan protein (AGP) and peroxidase sections were blocked in 0.01 M PBS
containing 2% BSA, 0.2% Triton X 100, and 0.2% New 20 for 15 min.
Subsequently, grids were incubated in the appropriate antibody solution in the above
buffers for 1 h, followed by a wash with buffer, and an incubation in protein A gold
(1:50; EY Laboratories, San Mateo, CA) in PBS containing 1% BSA for 30 min. The
sections were again washed in buffer and water, stained with uranyl acetate for l h,
and viewed in the TEM. All labeling steps were performed at room temperature. The

experiments were repeated three times.

Histocbernical staining. Staining for peroxidase activity was performed
according to Sexton and Hall (1991). Staining for glycol groups was adapted from
Thiery (1967) and Roland and Vian (1991). Ultrathin sections were collected on
nickel or titanium grids, treated with 1% periodic acid for 25 min, washed, and then
incubated in 0.2% thiocarbohydrazide (TCH) in 20% acetic acid for 24 h. They were

washed in water with decreasing concentrations of acetic acid followed by a wash in

68
water. The grids were transferred to 1% silver proteinate, incubated in the dark for

30 min and, after several washes in water, viewed in the TEM.

RESULTS AND DISCUSSION

CHARACTERIZATION OF ULTRASTRUCTURAL CHANGES ASSOCIATED

WITH RAPID GROWTH

Rapidly growing rice intemodes display a phenomenon called "tissue tension"
(Kutschera and Kende 1988). When they are cut longitudinally after one day of
treatment with GA, and then floated on water for 10 min, they curve outwards with
the epidermis on the concave side of the section (see Chapter 5). This curvature,
which has also been seen in other rapidly growing tissues (Kutschera et al. , 1987), is
not seen in slowly growing rice. Tissue tension has been interpreted as the
manifestation of two conflicting forces in a rapidly growing stem: the epidermis is
under tension because it is growth limiting while the inner tissue does not limit
growth and is under compression (Kutschera, 1989). To study the role of the
epidermis in regulating rapid growth, its ultrastructure was examined.

Kutschera and Kende (1988) had observed the appearance of osmiophilic
particles between the plasma membrane and the outer epidermal wall of submerged
rice plants. These particles were also seen in GA,-treated rice plants (Fig. 4.1) which
also grew rapidly. They were 80 to 100 nm in size and appeared only in the outer
epidermis at the interface betwee the plasma membrane and the outer epidermal wall.

69

They were not seen in other cells of the rice intemodes.

Such osmiophilic particles had been described previously by Barckhausen and
Rosenstock (1973) in suberizing potato tubers and by Kutschera et a1. (1987) in
rapidly growing corn coleoptiles. In corn coleoptiles that were induced to grow by
treatment with auxin, the size of the particles could reach up to 300 nm (Fig. 4.2).
They were visible between the plasma membrane and the outer epidermal wall and
predominantly, but not exclusively, on the outside of the cell.

The conclusion that the osmiophilic particles are associated with growth is
based on the following three observations:

(1) Corn coleoptiles induced to grow rapidly had 10-14 osmiophilic particles (Fig.
4.2, Table 4.3 on p. 84) per cell in cross sections while their number in slowly
growing corn coleoptiles was 3 per cell in cross sections.

(2) Osmiophilic particles were also observed in intemodes of deepwater rice (Fig.
4.1) which were induced to grow rapidly by submergence, treatment with GA, or
ethylene, but they were not evident in slowly growing plants (Table 4.1, p. 75).

(3) Particles were also visible in the rapidly growing region of cucumber hypocotyls
(0.5 cm below the hook; Fig. 4.3) but not in the slowly or non-growing regions of
the hypocotyl (0.5 cm above the base).

To correlate further the presence of osmiophilic particles with the submergence-

induced growth response of deepwater rice, their distribution and time course of

appearance was analyzed. The number of osmiophilic particles per cell and section
increased with time after submergence from 0.5 to 3.2 on day 3 (Fig. 4.4). This

paralleled the growth of rice plants which began ca. 4 h afier submergence.

 

70

 

 

Figure 4.1. Transmission electron micrograph of a cross section through the stem of
a GA,-treated deepwater rice plant (high pressure frozen and freeze substituted). In
this and all further electron micrographs the osmiophilic particles (OP) are indieated
by arrowheads and following abbreviations are used: C = cuticle, CW = cell wall,

PM = plasma membrane, Cyt = cytoplasm, V = vacuole. Bar = 200 nm.

71

 

Figure 4.2. Transmission electron micrograph of a cross section through a com
coleoptile epidemris cell treated with 10 uM IAA for 1 h. Tissue used for this and all

further electron micrographs and experiments has been chemically fixed. Bar = 200 nm.

 

 

Figure 4.3. Transmission electron micrograph of a cross section through a cucumber
hypocotyl epidermis cell. The tissue was taken from 5 mm below the hook. Bar =
200 nm.

73

3.5

 

2.5 —
2.0 r
1.5 -

§/i

0.5 -

Number of Particles

 

 

0.0 l l I l

 

Days of Submergence

l“figure 4.4. Time course of appearance of osmiophilic particles in the outer epidermal
Wall of submerged deepwater rice stems. The number of osmiophilic particles was
determined 5 mm above the nodal septum. The values represent the means :I; S.E. of

osmiophilic particles in 30 cells.

74

The distribution of osmiophilic particles along rice intemodal sections grown
in water (control), 10 11M GA, or submerged for three days is given in Table 4.1. In
control plants, the number of osmiophilic particles was less than one per cell and
section. However, in both GA,-treated and submerged plants, their number started to
increase 4 mm above the nodal septum and reached more than three particles per cell
and section between 5 and 15 mm above the nodal septum. At 30 mm above the nodal
septum, their number was again reduced to the control level. The region between 4
and 15 mm above the nodal septum corresponds to the zone of increased cell
elongation (Bleecker et al. , 1986). This indicates that the osmiophilic particles may
play a role in cell elongation.

The number of osmiophilic particles in rice was approximately three per cell
and cross section and 14 per cell and longitudinal section after three days of
submergence. Treatment with ethylene (not shown) and GA, (Table 4.4), which also
promote growth of deepwater rice plants, caused a similar increase in the number of
osmiophilic particles as did submergence. In corn coleoptiles, where growth was
induced by 10 uM auxin, the number of osmiophilic particles increased to about 12
per cell and cross section and to 30 per cell and longitudinal section. They were up to
300 nm in diameter, which made them easier to observe and analyze than the particles

in rice.

 

In

I!-

75

Table 4.1. Distribution of osmiophilic particles along the rice stem. The tissue was
taken three days after start of the indicated treatments. The values represent the means

1: S.E. of 60 cells.

 

 

 

Distance from Number per cell and section
nodal septum
(mm) Control Submerged + GA

2.5 0.4 :I: 0.1 0.9 :1: 0.1
3 0.8 i 0.1 1.0 :1: 0.1 0.5 :I: 0.1
4 0.8 :I: 0.1 2.0 i 0.2 2.3 :1: 0.2
5 0.6 :I: 0.1 3.2 :I: 0.2 2.7 :1; 0.2
6 0.9 :l: 0.1 3.1 :1: 0.1 3.11: 0.2
7 0.6 :1: 0.1 2.1 :l: 0.2 2.2 :1: 0.2
15 0.9 :I: 0.1 3.5 d: 0.2
30 0.5 :l: 0.1 0.6 :I: 0.1

76
ENZYME-GOLD LABELING

The method of enzyme-gold labeling was developed by Bendayan et al. (1984).
It is based on the assumption that an enzyme may still recognize its substrate even in
a fixed section. If the enzyme is bound to colloidal gold, one can localize the
substrate by observing the distribution of the gold grains which are associated with the
enzyme. Figure 4.5 shows a scheme of this binding as modified from Bendayan et al.
(1984). To determine the general composition of the osmiophilic particles, I tested the
binding of cutinase gold and of proteinase K gold to ultrathin sections of auxin-treated
corn coleoptiles. Corn was used since the osmiophilic particles in corn are much
larger and more abundant than in rice. Corn sections labeled with proteinase K gold
showed dense labeling of the particles at all stages of secretion (Fig. 4.6). Particles
appeared in vesicles within the cytoplasm (Fig. 4.6.A), between the plasma-membrane

and the cell wall (Fig. 4.6.B-D), and within the cell wall (Fig. 4.6.E).

V A q/ gold particle
<1

D4“ enzyme
5

d
i ‘1

WW “—subslrare

   

    
    
 

   
  

  
    
 

   

  

“53.03 —-p
section

ENZYME-GOLD
TECHNIQUE

l“cure 4.5. Scheme of enzyme-gold labeling as adapted from Bendayan et al. (1984).

77

Figure 4.6. Transmission electron micrograph of cross sections of auxin-treated corn
coleoptile epidermal cells labeled with proteinase K gold. The particles occurred
within the cytoplasm (A), at the plasmamembrane-cell wall interface (B-D), and

withinthecellwall(E).Bar = 200nm.

78

 

79

Table 4.2. Number of gold grains in osmiophilic particles and other cell
compartments after labeling with proteinase K gold. The gold grains were counted in
13 areas of equal size for all cell components. Values followed by different letters are

significantly different from each other (LSD 0.01)-

 

 

Gold grains
Location per 10‘ um2
Osmiophilic particles 20.4 1: 2.0 a
Cell Wall 5.5 :l: 0.4 b
Vacuole 1.4 :l; 0.2 c
Cytoplasm/Organelles 3.6 :I: 0.4 bc

Resin 1.2 :I: 0.2 c

80

To confirm that the distribution of gold grains was more dense in the
osmiophilic particles, I counted the number of gold grains within the particles and in
areas of the same size in the cytoplasm, vacuole, cell wall, and resin. The number of
gold grains in the osmiophilic particles was four times higher than in the cell wall, six
times higher than in the cytoplasm, and approximately 20 times higher than in the
resin (Tab. 4.2). In the vacuole, the number of gold grains was at background level,
either because of loss of the vacuolar contents during fixation or beeause of the low
level of protein in the vacuoles. I conclude that the gold grains accumulate in the
osmiophilic particles, indicating that they are proteinaceous. To test whether the
binding of proteinase K gold was specific, various controls were performed (Figure
4.7):

(1) Incubation with proteinase K gold plus BSA eliminated the labeling almost
completely (Fig. 4.7 C). This was expected since BSA competes with the
substrate in the cell.

(2) Incubation with proteinase K gold plus unlabeled proteinase K also eliminated
gold labeling because free proteinase K competed with the gold-labeled
enzyme (Fig. 4.7 E).

(3) When sections were incubated in proteinase K gold at pH 3, there was, as
expected, no gold labeling because the enzyme is not active at this pH (Fig.
4.7 F).

(4) PMSF, an inhibitor of proteinase K that binds covalently to the active site of
the enzyme, prevented binding of proteinase K to the osmiophilic particles
(Fig. 4.7 G). Instead, there seemed to be an aggregation of the enzyme.

81

Figure 4.7. Transmission electron micrograph of corn coleoptile cross sections
labeled with proteinase K gold (A), cutinase gold (B), proteinase K gold plus BSA
(C). not labeled (D), proteinase K gold plus unlabeled proteinase K (E), proteinase K
801d at pH 3 (F), proteinase K gold plus PMSF (G). The osmiophilic particles are

indieated with arrowheads. Bar = 200 nm.

‘I fill is!!! I

.Ii .I I- llell

82

 

83
(5) An unlabeled, unstained section showed again two osmiophilic particles (Fig.

4.7 D). One appeared to be within a vesicle in the cytoplasm. The other

appeared to be in a vesicle fusing with the plasma membrane.

These controls, when compared to the proteinase K gold labeled section (Fig. 4.7 A),
showed that the binding of proteinase K gold was indeed specific.

Labeling of sections with cutinase gold (Fig. 4.7 B) showed a distribution of
gold grains different from that found with proteinase K gold (Fig. 4.7 A). Cutinase is
an enzyme that degrades polymerized cutin. With cutinase gold, the label was most
dense in the layer inside of the cuticle, with decreasing amounts of gold towards the
inner part of the cell wall; no labeling of the osmiophilic particles was found with
cutinase gold. This supports the hypothesis of Kolattukudy that the cuticle monomers
become increasingly polymerized as they are transported across the cell wall. It also

shows that the osmiophilic particles do not contain polymerized cutin.

DIGESTION EXPERIMENTS

To confirm the above result, digestion of the osmiophilic particles was
attempted using proteinase K, cutinase, a combination of both, or glueanase. These
digestions were done either betrveen primary and secondary fixation or after
embedding. In both, rice and com, the number of osmiophilic particles was reduced
to about 60% of the control when the sections were incubated in proteinase K after
embedding (Table 4.3 A). Incubation with cutinase, buffer, or glueanase resulted in

no change in the number of particles. A combination of proteinase K and cutinase

84
Table 4.3. Number of osmiophilic particles after digestion with proteinase K,

cutinase, glueanase, or treatment with buffer (Control 1). Incubation occured for 1 h
after embedding (A) or between primary and secondary fixation (B). Control 2

represents an untreated section. The values represent the means :l: S.E. of 30 cells.

 

A Number of osmiophilic particles per cell and section

 

Proteinase K

Controll Proteinase K Cutinase plus cutinase Glueanase

 

Corn 9.5 :I: 0.4 5.8 :1: 1.4 8.6 i 5.3 5.3 :1: 0.3 11.4 :I: 0.8
Rice 4.3 :I: 0.2 2.6 :l: 0.2 4.2 i 0.2 2.1 d: 0.2

 

 

B Number of osmiophilic particles per cell and section

 

Control 1 Proteinase K Cutinase Control 2 Glueanase

 

Corn 14:1:1 31:0.3 16:1:1 1411 141:1

 

85
reduced the number of osmiophilic particles to the same extent as did proteinase K

alone. The decrease in the number of osmiophilic particles was probably not larger
beeause the enzyme can digest only those particles loealized on the surface of the
resin section. Particles that are deeper in the resin are not exposed to the enzyme and
remain undigested.

In corn coleoptiles incubated with proteinase K after primary fixation, the
number of osmiophilic particles was reduced from 14 to 3 per cell and cross section,
while incubation with buffer (control 1) or cutinase had no effect on their number
(Table 4.3 B). A mixture of proteinase K plus cutinase showed the same reduction in
the number of osmiophilic particles as did proteinase K alone. The fact that the
osmiophilic particles were digested with proteinase K confirms that they are, at least

in part, proteinaceous.

MONEN SIN EXPERIMENTS

Monensin is an ionophore that inhibits Golgi transport. Beeause it appeared
that the osnriophilic particles were transported in vesicles to the cell wall, I tested the
effect of monensin on their appearance. After 12 h of monensin treatment, some
vesicles of the Golgi apparatus in rice were larger in size than at 0 h, while others
had retained their original size and shape (Fig. 4.8). It appeared that monensin was
starting to affect Golgi transport. This was confirmed by the reduction of osmiophilic
particles from 3 per cell and cross section to 0.5 per cell and cross section. After 17

h of monensin treatment, the Golgi apparatus appeared to deteriorate, its vesicles

86

Monensin Number of

treatment _ ‘ OPs

 

0 h 3.0 :l: 0.4
12 h 0.5 i 0.2
17 h 0.7 :I: 0.2

 

Figure 4.8. Transmission electron micrographs showing the effect of monensin on the
Golgi apparatus. Deepwater rice stems were grown in 10 M GA, for three days :1:
20 p.M monensin for the indieated times. Bar = 200 nm. Numbers accompanying the
electronmicrographs indieate the number of osmiophilic particles counted in cross

sections of intemodes i monensin (mean of 20 cells :1; SE).

87

were very large, and some cells showed a reduction in membrane structures. The
number of osmiophilic particles was reduced to 0.7 per cell and section.

In rapidly growing rice intemodes and corn coleoptiles, treatment with
monensin for 12 h reduced the number of osmiophilic particles from 2.6 and 8.0 to
0.5 and 3.8, respectively. This resembles closely the number of particles in the slowly
growing control plants (0.6 in rice; 2.1 in corn; Table 4.4). The results of the
monensin experiments indicate that the osmiophilic particles are transported through

the Golgi secretory pathway and that their contents may be glycosylated.

ENZYIWE ASSAYS AND HISTOCHEMICAL STAINS

I used a technique described by Thiéry (1967) to stain glycol groups, e.g. 1,2-
glycol groups, that occur in the cell wall. I could not detect any positive staining in
the osmiophilic particles. However, even the staining of the cell wall was very weak.
Thus, I eannot yet determine whether the osmiophilic particles contain glycol groups.

Next, I tried to stain for peroxidase since this enzyme appears to be involved
in cell wall and cutin biosynthesis (Kerby and Somerville, 1992; Ferrer et al., 1991).
Staining for peroxidase (Sexton and Hall, 1991) yielded positive staining between the
plasma membrane and outer epidermal wall (Fig. 4.9). The stain appeared to be
darker in areas where osmiophilic particles usually occur. However, even though it
seems possible that the osmiophilic particles contain peroxidase activity, I was unable

to pinpoint the stain to the osmiophilic particles.

88

Table 4.4. Effect of incubation in 20 uM monensin for 12 h on the appearance of
osmiophilic particles in corn and rice. Particles were counted 5 mm above the nodal
septum of rice intemodes grown in water (control) or 10 uM GA, and in the middle
of l-cm segments of corn coleoptiles incubated in water (control) or 10 M IAA. The

values represent the means :I; S.E. of 20 cells.

 

Number of osmiophilic particles per cell

 

 

- Monensin + Monensin

Corn Control 2.1 d: 0.4 2.3 :I: 0.6
IAA 8.0 :l: 0.5 3.8 :l: 0.6

Rice Control 0.6 :l: 0.2 0.6 :l: 0.2

GA 2.6 :l: 0.4 0.5 :I: 0.2

 

89

Figure 4.9. Transmission electron micrographs visualizing peroxidase activity in corn
coleoptile epidermis cells. Corn coleoptiles were incubated in 10 “M IAA, chemically
fixed, and stained according to Sexton and Hall (1991) for 15 min (A, B) or 1 h (C,
D). A and C correspond to tissue pieces incubated in diaminobenzidine and H20,
(peroxidase stain), B and D correspond to tissue pieces incubated in KCN (negative
control). Bar = 500 nm.

90

 

9 1
ANTIBODY LABELING

To further analyze the nature of the osmiophilic particles, I tested antibodies
against several proteins which may be secreted into the cell wall.
(1) Threonine-hydroyproline-rich glchprotein from corn (THRGP)
(2) Lipid transfer protein (LTP)
(3) Arabinogalactan protein (AGP)
(4) Peroxidase
(5) EXPansin

Threonine—hydroxyproline-rich glycoprotein from corn is an extensin-like
protein that has been purified by Kieliszewski et al. (1991). It is localized in the cell
wall and comprises less than 1% of the total cell wall. The antibody was provided by
Dr. Marcia Kieliszewski (Michigan State University, E. Lansing). It bound to the
outer wall of epidermal cells of corn coleoptiles but not to the osmiophilic particles,
the cytoplasm or vacuole (Fig. 4.10 A). Preimmune serum (Fig. 4.12 B) or protein A
gold alone (Fig. 4.10 C) showed only very few gold grains in the cell wall. Thus, the
osmiophilic particles do not appear to contain THRGP.

Lipid transfer proteins transfer lipids between membranes in in vitro assays.
They are divided into specific and non-specific LTPs. Plant LTPs are non-specific
LTPs. They are soluble basic proteins with a molecular mass of about 9 kDa. They
are able to transfer phospholipids and chloroplast glycolipids but not neutral lipids
such as triacyl-, diacyl- or monoacylglycerols, sterols or acylsterols (for a review see

Yamada, 1992). Madrid and von Wettstein (1991) suggested that LTPs

92

 

Figure 4.10. Transmission electron micrograph of cross sections of corn coleoptiles

labeled with an antibody against THRGP (1:1000; A). B: preimmune; C: protein A

gold alone. Bar = 200 nm

93
occur in two classes, one (ca. 10 kDa size) that is synthesized and translocated

through the secretory pathway via the endoplasmatic reticulum and Golgi complex
(e.g. in corn). The other (ca. 30 kDa size) is synthesized on free ribosomes and
modified in glyoxisomes (e.g. in castor bean). A monospecific antibody against the
corn LTP has been isolated by and obtained from Dr. J .-C. Kader (Université Pierre
et Marie Curie, Paris, France; Sossountzov et al. , 1991). These authors found LTP to
be localized in the epidermis of corn coleoptiles. Similarly, Thoma et al. (1992) also
loealized LTP in the epidermal cells of Arabidopsis. This confirmed earlier results of
Sterk et al. (1991) who found expression of LTP mRNA in epidermal cells of carrot
by in situ hybridization. Sterk et al. (1991) and Thoma et al. (1992) discussed the
possible role of LTPs in either cutin biosynthesis or response to pathogen infection
since its localization was mostly in the outer epidermal wall and mostly in tissues
where the cuticle is synthesized. While the sections were not labeled with either
control serum (Fig. 4.11 B, E) or protein A gold (Fig. 4.11 C), labeling of the cell
walls was seen with anti-LTP antibodies as described by Thoma et al. (1992) for
Arabidopsis. The osmiophilic particles showed only occasional gold labeling, but
dense labeling was seen in the outer corners of some epidermal cells in an area of
amorphous, electron-dense material. An anti-barley-LTP antibody of von Wettstein et
al. (1989; obtained from Dr. P. von Wettstein-Knowles, Carlsberg Laboratory,
Copenhagen, Denmark) showed the same distribution as did the antibody against corn
LTP (not shown). It appears that the osmiophilic particles do not contain LTP, but

that LTP may be localized in an amorphous area of the wall (Fig. 4.11E).

94

Figure 4.11. Transmission electron micrograph of corn coleoptile epidermal cells
labeled with an antibody against LTP (1: 50; A, D). B, E: control serum; C: protein

A gold alone. Bar = 200 nm

95

 

96
As could be seen from the cytochenrical stain, some peroxidase activity is

localized between the plasma membrane and the outer epidermal wall. An anti-
peroxidase antibody from Kirby and Somerville (1992; Michigan State University, E.
Lansing) raised against a barley cell wall peroxidase was used to further study the
localization of this enzyme. This antibody cross-reacts with a doublet band from rice
on a Western blot (not shown). It bound specifically to corn coleoptile epidermis cells
(Fig. 4.12). The antibody bound to the cell wall but not to the osmiopilic particles
(Fig. 4.12 A). Preimmune serum showed no labeling (Fig. 4.12 B). It appears that
the osmiophilic particles do not contain the peroxidase which is recognized by this
antibody. However, since there are several peroxidase isozymes in plants, I eannot
exclude the possibility that the osmiophilic particles contain a peroxidase.

Based solely on light microscopy staining, Schopfer (1990) proposed that the
osmiophilic particles contain arabinogalactan proteins (AGPs). AGPs are cell wall
components that are thought to anchor the cell wall to the plasma membrane. 1 used a
rat monoclonal antibody (MAC 207) against an epitope containing arabinose and
glucuronic acid to test whether the osmiophilic particles contain AGPs. The antibody
was obtained from Dr. Keith Roberts (John Innes Institute, Norwich, UK; Pennel et
al., 1989). MAC 207 bound as described by Pennell et al. (1987). The gold grains
were loealized along the plasma membrane but not in the osmiophilic particles,
indicating that they are not AGPs containing these specific carbohydrate residues (Fig.
4.13 A). A negative control, JIM4, an antibody recognizing AGPs specific of certain
cells of Daucus carota (Knox et al. , 1989), showed no specific binding to the sections

(Fig. 4.13 a).

97

 

figure 4.12. Transmission electron micrograph of corn coleoptile epidermal cells

labeled with an antibody against a barley cell wall peroxidase (1:100; A). B:

preimmune serum. Bar = 200 nm

98

 

 

Figure 4.13. Transmission electron micrographs of corn coleoptile epidermal cells
labeled with monoclonal antibodies against AGPs. The antibodies used were MAC
207 (A) and JIM4 (B). Bar = 200 nm

99
Expansins are two proteins that mediate cell wall extension without detectable

hydrolytic breakdown of the wall. They were isolated from the walls of the growing
region of cucumber hypocotyls and mediate wall extension in other dicots as well and,
to a lesser extent, in monocots (McQueen-Mason et al. , 1992). The two proteins, 81
and $2 (29 and 30 kD, respectively), do not have endoglyeanase activity. Both, total
serum and monospecific antibodies against them, were obtained from Dr. Daniel
Cosgrove (Pennsylvania State University, USA; Zhen-Chang Li et al. , 1993). The
monospecific antibodies against both proteins bound to the cell wall, predominantly of
the epidermal cells, but not to the osmiophilic particles in either corn (Fig. 4.14) or
cucumber (not shown). The level of labeling was higher in rapidly growing corn
coleoptiles than in slowly growing ones and could sometimes also be seen over Golgi

vesicles (not shown).

CONCLUSIONS

The osmiophilic particles in rice and corn were predominantly localized
between the plasma membrane and the outer epidermal wall in rapidly growing
intemodes and coleoptiles, respectively (Fig. 4.2, 4.3). In rice, they appeared during
rapid intemodal elongation (Fig. 4.4) and were localized in regions of increased cell

elongation (Table 4.2).

100

Figure 4.14. Transmission electron micrographs of corn coleoptile epidermis cells
labeled with antibodies against expansin 82 (A: 1:400 dilution) and 81 (C: 1:25
dilution). Binding of the corresponding control sera is given in B (for 82) and D (for
81). Bar = 200 nm

101

-, ~ vmu‘m,, v.
._,r~. , "
,.

 

102
Enzyme-gold labeling showed that the osnriophilic particles are, at least in

part, proteinaceous (Figs. 4.6, 4.7, Table 4.3). This result was confirmed by
digestion experiments performed either between primary and secondary fixation
(Table 4.4 B) or after embedding (Fig. 4.4 A).

Treatment of rice or corn plants with monenSin, an inhibitor of Golgi
transport, led to an almost complete disappearance of the osmiophilic particles. This
indieates that they were translocated through the secretory pathway of the Golgi
complex (Fig. 4.9, Table 4.4).

Immunogold labeling showed that the osmiophilic particles did not contain
extensin-like THPRG (Fig. 4.10), LTP (Fig. 4.11), arabinogalactan proteins
recognized by JIM4 and MAC 207 (Fig. 4.13), a peroxidase recognized by an
antibody obtained from Kirby and Somerville (1992; Fig. 4.12), or expansins (Fig.
4.14). However, a peroxidase stain (Fig. 4.9) was loealized in.» the area where
osmiophilic particles appeared, indicating that they may contain peroxidase, but an
isozyme that is different from the one of Kirby and Somerville (1992). The
osmiophilic particles could contain other cell wall components, e. g. , phenolics, acetyl
CoA, or acetyl CoA-cutin transacylase. The latter two may be involved in cutin
biosynthesis, but no antibodies exist against them. The osmiophilic particles could

also consist of several components similar to multi-enzyme complexes.

103
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traumatogenen Subcrinisierung beim Knollenparenchym von Solanum
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Bendayan, M. (1984a) Protein A gold electron microscopic immunocytochemistry:
methods, applications, and limitations. J. Electron Microsc. Tech. 1, 243-270.

Bendayan, M. (1984b) Enzyme-gold electron microscopic cytochemistry: a new
affinity approach for the ultrastructural localization of macromolecules. J.
Electron. Microsc. Tech. 1, 349-372.

Ferrer, M.A., Munoz, R., Ros Barcelo, A. (1991) A biochemical and cytochemieal
study of the cuticle-associated peroxidases in Lupinus. Ann. Bot. 67, 561-568.

Hoffmann-Benning, S. , Kende, H. (1992a) On the role abscisic acid and gibberellin
in the regulation of growth in rice. Plant Physiol. 99, 1156-1161.

Hoffmann-Benning, S. , Kende, H. (1992b) Characterization of osmiophilic particles
associated with rapid growth in corn and rice. Plant Physiol. 99, Abstract #
177 .

Kende, H. (1987) Studies on intemodal growth in deepwater rice. In: DJ. Cosgrove,
D.P. Knievel eds. , Physiology of Cell Expansion during Plant Growth.
Ameriean Society of Plant Physiologists, Rockville, MD, pp. 227-238.

Kieliszewski, M., Lamport, D.T.A. (1987) Purification and partial characterization of
a hydroxyproline-rich glycoprotein in a graminaceous monocot, Zea mays.
Plant Physiol. 85, 823-827.

Kirby, K., Somerville SC. (1992) Purification of an infection-related, extracellular
peroxidase from barley. Plant Physiol. 100, 397-402.

Knox, J.P., Day, S., Roberts, K. (1989) A set of cell surface glycoproteins forms an
early marker of cell position, but not cell type, in the root apical meristem of
Daucus carota L. Development 106, 47-56.

Kutschera, U., Bergfeld, R., Schopfer, P. (1987) Cooperation of epidermis and inner
tissues in auxin-mediated growth of rice coleoptiles. Planta 170, 168-180.

Kutschera, U. , Kende, H. (1988) The biophysical basis of elongation growth in
intemodes of deepwater rice. Plant Physiol. 88, 361-366.

104

Kutschera, U. , Kende, H. (1989) Particles associated with the outer epidermal wall in
intemodes of deepwater rice. Ann. Bot. 63, 385-388.

Kutschera, U. (1989) Tissue stresses in growing plants. Physiol Plant. 77, 157-163.

McQueen-Mason, S., Durachko, D.M., Cosgrove, DJ. (1992) Two endogenous
proteins that induce cell wall extension in plants. Plant Cell 4, 1425-2433.

Metreaux, J.-P., Kende, H. (1983) The role of ethylene in the growth response of
submerged deepwater rice. Plant Physiol. 72, 441-446.

Madrid, S. , von Wettstein, D. (1991) Reconciling contradictory notions on lipid
transfer proteins in higher plants. Plant Physiol. Biochem. 29, 705-711.

Olesen, P. (1980) The visualization of wall-associated granules in thin sections of
higher plant cells: occurrence, distribution, morphology and possible role in
cell wall biogenesis. Z. filanzenphysiol. 96, 35-48.

Pennell, R.I., Knox, J .P., Scofield, G.N., Selvendran, R.R., Roberts, K. (1989) A
family of abundant plasma membrane-associated glycoproteins related to the
arabinogalactan proteins is unique to flowering plants. J. Cell Biol. 108, 1967-
1977.

Raskin, I, Kende, H. (1984) Regulation of growth in stem sections of deepwater rice.
Planta 160, 66-72.

Robards, A.W. (1969) Particles associated with developing plant cell walls. Planta
88, 376-379.

Roland, J.C., Vian, B. (1991) General preparation and staining of thin sections. In:
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Press Limited, 1991.

Sexton, R., Hall, J .L. (1991) Enzyme cytochemistry. In: Electron Microscopy of
Plant Cells, J.L. Hall and C.Hawes eds. Academic Press Limited, 1991.

Sossountzov, L., Ruiz-Avila, L., Vignols, F., Joillot, A., Arondel, V., Tschang, F.,
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Sterk, P., Booij, H., Schellekens, G.A., Van Kammen, A., De Vries, S.C. (1991)
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105

Thoma, S., Yasuko, K., Somerville, C. (1992) The non-specific lipid transfer protein
from Arabidopsis is a cell wall protein.

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Physiol. 33, 1-6.

CHAPTER 5
CUTICLE BIOSYNTHESIS AND COMPOSITION IN GIBBERELLIC

ACID-TREATED DEEPWATER RICE INTERN ODES

ABSTRACT

Submergence induces rapid elongation of rice coleoptiles (Otyza sativa L.) and
of deepwater rice intemodes. This adaptive feature helps rice to grow out of the water
and to survive flooding. Earlier, I found that three plant hormones, ethylene,
gibberellin (GA), and abscisic acid (ABA) participate in regulating the growth
response of submerged deepwater rice plants. Here I show that in GA-treated, rapidly
growing rice stem sections the cuticle is a growth-limiting structure. In addition, I
show that GA treatment leads to an increase in the incorporation of
[“C]palmitic acid and [“C]oleic acid into the cuticle of growing intemodes and to
increased levels of at least one cuticular component, a dihydroxyhexadecanoic acid.
From the changes in the chromatographic patterns of cutin monomers I conclude that

the activities of enzymes that lead to hydroxylation of fatty acids may be enhanced.

106

107
INTRODUCTION

Deepwater rice (Oryza sativa L.) is a rice type that elongates rapidly when the
plants become submerged. This feature helps the plants to emerge from the water and
to avoid drowning. In seedlings, submergence promotes coleoptile growth (Kordan et
al., 1977; Tamer et al., 1981; Yamada, 1959) and in adult deepwater rice plants,
elongation of the intemode (Métreaux and Kende, 1983; Vergara et al. , 1976). In
both instances, the plants respond to the altered gas composition of their submerged
organs, namely to reduced partial pressure of 0,, to increased partial pressure of
C0,, and to the accumulation of ethylene (Ku et al., 1970; Ohwaki, 1967; Ranson
and Parija, 1955; Raskin and Kende, 1984 a,b; for a review, see Jackson and Pearce,
1991). In adult deepwater rice plants, the reduced 0, concentration leads to an
increase in ethylene biosynthesis and, subsequently, to an increase in responsiveness
of the intemodal tissue to GA. Growth of deepwater rice can also be induced by
application of GA (Raskin and Kende, 1984 a,b).

How does GA promote intemodal growth? Hofmeister (1859) found that
separated outer and inner tissues of plants changed their dimensions upon separation:
the isolated epidermal layer shrinks while the inner tissue expands. He concluded that,
in intact shoots, the epidermis is under tension and the inner tissues under
compression. This can be shown by splitting isolated stem segments longitudinally.
They will bend outwards showing that the epidermis contracts and the inner tissue
expands. Sachs ( 1865) called this phenomenon tissue tension. It appears that the

plastic extensibility of the inner tissues is much larger than that of the outer epidermal

108
wall (Kutschera and Briggs, 1987; Kutschera et al. , 1987; Cosgrove, 1989) which

makes the outer epidermal wall the more rigid structure of the growing organ.
Kutschera (1989) proposed that ”the relatively inextensible, thick outer epidermal wall
maintains the cells with extensible, thin walls in a stage of compression. "
Consequently, the longitudinal tensile stress of inner walls is low and that of the outer
epidermal wall is high, creating tissue tension. Since the epidermis is the cell layer
responding to growth-inducing hormones (e.g. auxin, Went and Thimann, 1937), the
appearance of tissue tension was seen as an indication that the epidermis is the
growth-limiting structure. In rice intemodes, where GA promotes growth, the
epidermis and, in particular, the outer epidermal wall also limits growth (e. g.
Kutschera and Kende, 1988). Therefore, it has to be assumed that GA acts, at least in
part, on the outer epidermis to enhance growth. While most research focuses on the
effect of plant hormones on the cell wall, little attention has been given to the role of
the cuticle in restricting growth and to the effect of plant hormones on the
biosynthesis of the cuticle. An exception to this is the work of Bowen and Walton
(1988) who found an up to 2.5-fold increase of [“C]palmitic acid incorporation into
cutin monomers of GA-treated pea intemodes.

The plant cuticle is a lipid layer on the outside of the outer wall of epidermis
cells. Its structural component is cutin, a biopolyester, which is composed of a
family of C,, and C,, monomers (Kolattukudy, 1980a). These monomers often contain
one or more hydroxy or epoxy groups. The major C,. component of cutin is
dihydroxypalmitic acid; the major C , , components are l8-hydroxyoctadecanoic acid,

9,10—epoxy-hydroxyoctadecanoic acid, 9,10,18-trihydroxy-octadecanoic acid, and their

109
A‘2 unsaturated counterparts (Kolattukudy, 1980b). It appears that half the monomers

are cross-linked through hydroxy groups (Deas and Holloway, 1977 ; Kolattukudy,
1977 ; Kolattukudy, 1984). Small amounts of phenolic acids are covalently linked to
the polymer (Riley and Kolattukudy, 1975). Croteaux and Kolattukudy (1974) found
that a particulate fraction sedimenting at 1000 g from Vicia faba leaves incorporated
labeled hydroxy acids into cutin when ATP and coenzyme A were present. This
hydroxyacyl transferase activity seems to be responsible for the polymerization of
cutin from monomers and is thought to occur outside the epidermal cells.

The cuticle is a major diffusion barrier in water and gas exchange (Schonherr,
1976) and also impedes the entry of pathogenic microorganisms into the plant (van
den Ende and Linskens, 1974). Kolattukudy (1965 , 1970) found that cutin
biosynthesis occurs more rapidly in expanding Vicia faba and Brassica oleracea
leaves than in slowly expanding tissues. The C,, family of monomers predominates in
rapidly expanding plant organs whereas a mixture of the C,, and C,, families of
monomers is found in slower growing organs with thicker cuticle. In addition, the
relative composition of the cuticle appears to change under various environmental
conditions and at different ages of the plant (Kolattukudy et al. , 1974).

In this chapter, I report on cuticle biosynthesis in rapidly growing, GA-treated
deepwater rice intemodes and on the potential role of the cuticle as a growth-limiting

structure.

l 10
MATERIALS AND METHODS

Growth of plants. Rice plants (Oryza sativa L., cv. Habiganj Aman II) were
grown as described by Stiinzi and Kende (1989), except that the day temperature was

27° C for 11 h centered within a 13-h photoperiod.

Treatment of whole plants. Eight- to 12-week-old adult plants were
submerged as described by Métraux and Kende (1983). These treatments were
performed in the same growth chamber in which the plants had been grown. After
two days, the basal 1-cm portion of the youngest intemode containing the intercalary
meristem and part of the elongation zone above it was excised and frozen in liquid

N,.

Isolation and treatment of stem sections. Stem sections containing the
youngest intemode were excised from 9- to ll-week-old plants as described by Raskin
and Kende (1984a). They were placed in water (control) or in 10 M GA, solution
and incubated in plastic cylinders through which humidified air was passed at a rate of

80 mllmin.

Treatment with cutinase. Cutinase, which had been purified as described in
Sebastian and Kolattukudy (1988), was obtained from Dr. P.E. Kolattukudy (Ohio
State University). Stem sections were grown in either water or GA, for a total of 24 h

as described above. After 20 or 22 h, they were injected between the leaf and the

1 l 1
youngest intemode with 50 ul of either buffer (0.05 M NaHCO/NaCO, pH 9.0,

0.05% Tween 20) or 40 ug/ml cutinase in the above buffer and incubated for the last
2 or 4 h in water or GA,. One set of water- and GA-treated stem sections were not
injected as further controls. Twentyfour h after start of the GA treatment, the leaf
sheaths were peeled off, the lowest 3-cm zone of the intemode was excised and cut
longitudinally into quarters. These segments were incubated in distilled water for ca.
15 nrin, photocopied, and the angle of curvature was determined as shown below in

Figure 5.1.

 

outer epidermis

 

 

 

Figure 5.1. Angle of curvature in tissue segments displaying tissue tension.

112
Labeling of the rice cuticle. For labeling of the cuticle, 0.5 pCi of [1-

l‘C]palmitic acid (57.1 mCi/mmol, ICN, Irvine, CA) or [l-“Cloleic acid (50
mCi/mmol, ICN, Irvine, CA) in 25 pl aquaeous solution were injected between the
leaf and the youngest intemode at various times. Two h later, the leaf sheath was
removed, the lowest two centimeters of the intemode were excised, rinsed with water,
and frozen in liquid N,. For large-scale cuticle preparations to be used for thin layer

chromatography, the cuticle was labeled for 20 h prior to excision.

Preparation of rice cuticle. Cuticle extraction was modified from Kolattukudy
(1970). Intemodal tissue was ground in liquid N,. Ten to 20 ml of distilled water
were added to the ground tissue, mixed, and subsequently centrifuged at 27000 g for
10 min at 4° C. The pellet was extracted several times in chloroform/methanol (2:1,
v/v) until the tissue was totally white and all membrane lipids extracted. The
remaining tissue contained only cell wall polysaccharides and cuticle. It was dried,
and the radioactivity determined by liquid scintillation counting. For cutin analysis,
the dried sample was refluxed under N, for 24 h in distilled tetrahydrofuran
containing LiAlH. The amount of LiAlH was three times the sample dry weight.
After 24 h, some drops of water were carefully added to react with the remaining
LiAlH, and the pH of the solution was lowered to ~ 3. At this point, the cuticle was
depolimerized and its fatty acid components reduced to fatty alcohols. These products
were in solution while the cell wall formed a precipitate. The solution was filtered
through Whatrnan No. 4 filter paper and extracted three times with ethyl acetate. The

ethyl acetate phase was collected, evaporated, and resuspended in a small volume of

1 13
chloroform/ methanol (2:1).

Thin layer chromatography (TLC) was performed according to Kolattukudy
(1970). For TLC separation of fatty alcohol derivatives of the cuticle, a silica TCL
plate was activated at 110° C for ca. 30 min. The chromatographic solvent was ethyl
ether - hexane - methanol - acetic acid (80 : 20 : 10 : 1.5, v/v). The TLC plates were
stained with iodine or sulfuric acid, and radioactivity in the bands determined using a

phosphor imager (Molecular Dynamics).

FJectron microscopy. For measurements of cell wall- and cuticle thickness
plants were treated and fixed as described in chapter 4. Ultrathin sections were
viewed in a Phillips 201 transmission electron microscope, and measurements were

taken from the center of the cells on photographic prints.

RESULTS AND DISCUSSION

Since the outer epidermis of deepwater rice intemodes is considered a growth-
limiting structure, I examined the ultrastructure of epidermis cells of rice plants that
had been submerged or treated with GA,. Kutschera and Kende (1988) have shown
that submergence leads to the appearance of osmiophilic particles between the plasma
membrane and the outer epidermal wall. These particles are associated with rapidly
growing tissue and are most likely involved in cell wall or cuticle biosynthesis. I

measured the dimensions of the epidermal walls and the cuticle of rice plants that

114

Table 5.1. Thickness of the epidermal cell walls and the cuticle. Tissue was taken 5
mm above the second highest node of deepwater rice plants that had been air-grown,

submerged or treated with 10 uM GA,. The values represent the means of 13

measurements :1: S.E.

 

 

 

 

Thickness (nm)
Control Submerged GA,

Cell Wall

Outer Epidermal 112.5 :I: 7.6 122.8 1; 4.2 116.6 :1: 8.5
Wall

Lateral Wall 20.4 :I: 4.8 24.3 :I: 2.7 20.0 :I: 2.7
Inner Epidermal 51.3 :1; 4.2 55.2 :1: 3.0 65.6 :I: 2.8
Wall

Cuticle 37.7 i- 4.1 42.5 :I: 3.9 52.2 i 4.9

 

l 15
had either been air-grown, submerged or treated GA, (Table 5.1). There was no

change in the thickness of the outer epidermal wall. The cuticle and inner wall of GA-
treated rice stems appeared thicker than that of air-grown stems. The value for the
submerged plants was intermediate. Since neither the cell wall nor the cuticle became
thinner, their biosynthesis had to be maintained during rapid growth. The observation
that GA, leads to an increase in cuticle thiclaress prompts two questions:

1) Is the cuticle not only a physical barrier but also a growth-limiting structure?

2) Does cuticle biosynthesis change during GA-induced growth in deepwater rice?

To examine the first question, I treated air-grown or GA,-treated rice
intemodes for various times with cutinase or buffer, sliced them longitudinally and
determined their curvature after incubation in water (Figure 5.2, Tables 5.2, 5.3).
The left half of Figure 5.2 shows differences in tissue tension between slowly versus
rapidly growing sections. Slowly growing tissue did not show any outward bending,
while segments of rapidly growing tissue displayed a rapid outward bending upon
transfer of the stern segments into water. This is interpreted as showing that the inner
tissue can expand freely while expansion of the epidermis or, more specifically the
outer epidermal wall is limited. Measurements of the internal angle of curvature
(Tables 5.2, 5.3; visualized in Figure 5.2) showed that application of cutinase had no
effect on the bending of intemodal segments from air-grown plants. However short-
term application of cutinase to rapidly growing intemodes led to a significant decrease
in tissue tension as indicated by a reduction in outward bending of the internode
segments. Treatment with buffer had no effect on tissue tension. I conclude that

enzymatic digestion of the cuticle leads to a reduction in tissue tension and that the

116

 

Cutinase treatment (h)

Figure 5.2. Effect of cutinase on tissue tension in the lowest 3 cm portion of the
youngest intemode of deepwater rice. Stem sections had been grown in water (C) or
in 10 FM GA, for a total of 24 h. Treatment with cutinase took place during the last

two or 21 h. The outer epidermis is on the concave side of each section.

117

Table 5.2. Effect of cutinase on the tissue tension in the lowest 3 cm of the youngest
intemode of deepwater rice. Stem sections had been grown for 24 h in water (control)
or in 10 M GA,. Treatment with cutinase took place during the last 2 h of this time
period. The values represent the means 3|: S.E. of the internal angle determined from

44 stem segments.

 

Duration of cutinase treatment (h)

 

 

Treatment 0 2
Water 166 j: 2 169 :1: 1
GA, 98 :l: 4 149 :l: 3

 

Table 5.3. Effect of cutinase or buffer on the tissue tension of the lowest 3 cm in the
youngest intemode of rice. Stem sections were grown in 10 M GA, for 24 h. The

values represent the mean i S.E. internal angle of 32 stem segments (16 at 4 h).

 

Duration of treatment (h)

 

Injected with o 2 4

 

Buffer 97:1:5 83:1:4 893:4

Cutinase 97 :l: 5 123 j: 4 131 j: 5

 

118

 

 

 

 

14 I I I I I I
E
E
~— II
g "/v
0 V/
g 12 — -
3 II
(0 v
.2
'5
O.
O
.9
8 .
u- 10 —
O
.C
the
D
C
0
_l
8 1 l I 1 L 1
O 5 10 15 20 25 30 35

Duration of treatment (h)

Figure 5.3. Length of coleoptile segments incubated in water or 10 “M IAA with or
without cutinase, respectively, for various times (+ IAA, + cutinase: v; + IAA, -
cutinase: El; - IAA, + cutinase: v; - IAA, - cutinase: O. The values correspond to

the means :1; S.E. three independent experiments with 4 coleoptile segments each.

119
cuticle may - at least in part - play a role in limiting growth. A further indication that
the cuticle may play a role in limiting growth is my finding that application of
cutinase leads to an increase of corn coleoptile growth (Fig. 5.3).

Since the cuticle does not become thinner during increased growth and may
contribute to the limitation of growth by the epidermis, there must be increased
cuticle synthesis. GA, could, thus, affect cuticle biosynthesis either indirectly by
inducing growth or, less likely, by acting on enzymes of cuticle biosynthesis directly.
I compared the incorporation of [“C]palmitic acid into the cuticle of control and GA,-
treated stem sections. Figures 5.4 and 5.5 show the labeling of the rice cuticle in the
lowest and second lowest centimeter of the youngest intemode. In both zones,
treatment with GA, led to an increase in the incorporation of [“C]palmitate when
compared to the control plants. This was already visible after the first 2 h of
treatment with GA, and had increased to a level 20- to 30- fold above that of control
plants after 48 h. It was slightly reduced after 72 h, presumably because the intemode
stopped growing. This result shows that cuticle biosynthesis increases in intemodes
induced to grow rapidly with GA,.

Cuticle from control intemodes and intemodes treated with GA, for 48 h was
labeled with palmitate and depolymerized using reductive hydrolysis with LiAlH
(Kolattukudy, 1970). Its monomeric compounds were separated by TLC, and the
presence and amount of radioactivity associated with individual bands was determined
using a phosphor imager (Figure 5.6). Ten radioactive bands were evident in the cutin
hydrolysate of intemodes treated with GA,. The most prominent band at the running
front corresponds to palmitic acid. The amount of radioactivity incorporated into this

120

 

8000

V

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\

5000

4000

cpm/mg dry wt

2000

 

 

 

 

2 6 12 24 48 72

Time after start of treatment (11)

Figure 5.4. Incorporation of [“C]palmitic acid into the lowest l-cm zone of the
youngest intemode of deepwater rice. Plants were grown in water (closed bars) or in
10 M GA, (hatched bars) for the indicated times. Labeling was performed during the

last 2 h of each period. Each value represents the means of four experiments.

121

 

7000
6000 29’
5000
4000

3000

1000 r
M /
0

2 6 12 24 48 72

cDm/mg dry wt

2000

 

 

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\

       

I \\\\\\\\\\\\\\\\\\\\\\\\\\\‘

Time after start of treatment (b)

Figure 5.5. Incorporation of [“C]palmitic acid into the second lowest 1-cm zone of
the youngest intemode of deepwater rice. Plants were grown in water (closed bars) or
in 10 uM GA, (hatched bars) for the indicated times. Labeling was performed during
the final 2 h of each time period. Each value represents the means of four

experiments.

122

band was calculated from a phosphor imager printout measuring the radioactivity in
every band and was found to be 59 % of the control level in GA,-treated plants. The
second most prominent band (R, = 0.63) appeared in both, control and GA,-treated
plants. However, it contained over three times more radioactivity in the cuticle
hydrolysate of plants treated with GA, than in that of control plants. In addition,
incorporation of radioactivity into six more bands (R, = 0.85, 0.81, 0.71, 0.55, 0.30,
0.10) was increased upon GA-treatment. These changes were not evident in plants
that had been treated with GA, for only 4 h and labeled for the entire time period as
compared to plants treated for 48 h (not shown). From the TLC data of Walton and
Kolattukudy (1972), it appears that the top three bands correspond to unmodified fatty
acids that were incorporated into the cuticle, while the bands below them correspond
to mono-, di-, or tri-hydroxy fatty acids (or their epoxy derivatives). This indicates
that the activity of enzyme(s) responsible for hydroxylation (Walton and Kolattukudy,
1972) is enhanced in GA,-treated plants.

The level of [“C]oleic acid incorporation into the rice cuticle was much lower
than that of palmitic acid but it also showed an increase in sections heated with GA,
(Figure 5.7). In this case, GA, lead to a two-fold increase in incorporation within 6 h.
At 72 h, there was a five-fold increase above the control. The control plants showed
no change in the level of oleic acid incorporation. The results of Figure 5 .4, 5 .5, and
5 .7 show that cuticle biosynthesis increased in GA-treated, rapidly growing deepwater
rice intemodes. However, far more palmitic (C,, fatty acid) than oleic acid (C,, fatty
acid) was incorporated. This confirms the findings of Kolattukudy et al. (1974) that

the C,, family of monomers predominates in rapidly growing plants.

123

Figure 5.6. Phosphor-imager printout indicating the radioactivity in cutin monomers
produced by reductive hydrolysis and separated by TLC. The cutin was obtained from
stem sections that were grown in water (Cl and C2) or 10 M GA, (GA) for 48 h
and that had been labeled with [“C]palmitic acid for the last 20 h of that time period.
Lane C1 contains the same amount of radioactivity as lane GA, lane C2 contains the
same amount of extract as lane GA. Percent of control stands for the amount of
radioactivity in lane GA as compared to the C1. The values were taken from a
phosphor-imager printout scan of each band. The arrowheads at the bottom and top

indicate the origin and the solvent front, respectively.

 

125

2000

 

1500

1000

cpm/mg dry wt

500

 

 

 

o . . //,

Time after start of treatment (h)

Figure 5.7. Incorporation of [“C]oleic acid into the lowest l-cm zone of the youngest
intemode of deepwater rice. Plants were grown in water (closed bars) or in 10 M
GA, (hatched bars) for the indicated times. Labeling was performed during the last 2

h of each time period. The values represent the means of three experiments.

126

To compare the incorporation pattern of oleic and palmitic acid and to identify
at least some of the cuticular components on the TLC plate, I separated the products
of reductive hydrolysis of the cuticle from stem sections that had been treated with
GA, or were incubated in water and that had been labeled with [“C]palmitic acid or
with [“C]oleic acid. The radioactivity of individual bands was determined using a
phosphor imager (Figure 5 . 8). There were no differences in the pattern of oleic acid
incorporation between cuticles of contol and GA,-treated stem sections (Fig. 5. 8 B).
However, the differences in the incorporation pattern of [“C]palmitic acid into of
stem sections treated with GA, or water were confirmed (Figure 5.8 A). The bars
between the two sets of radioactive cutin monomers represent the positions of the
monomers of apple cutin which had been obtained from Dr. Kolattukudy (Walton and
Kolattukudy, 1972) and were run as standards. From a comparison of the R, values
from the second-most prominent band visible in the cuticle of GA,-treated, palmitic
acid-labeled stem sections (R, ~ 0.63) and the standards I conclude that this band
corresponds to a hexadecatriol (C). Reductive hydrolysis converts fatty acid
components of the cuticle to their corresponding alcohols. Thus, the band from rice
cutin with an R, ~ 0.63 was derived from a dihydroxy-hexadecanoic acid in the
cuticle. Since it ran parallel with the band in the apple standard that corresponds to
10,16-dihydroxyhexadecanoic acid, I assume that it is the same compound. The above
results indicate that treatment with GA, leads to a change in the composition of only
the 0,, family of fatty acids while the relative amounts of the fatty acids from the C,,
family to each other remained the same. In addition, the increase in [“ijalmitic acid

incorporation occurred only into cutin components below R, 0.85.

127

Figure 5.8. Phosphor-imager printout indicating the radioactivity in cutin monomers
prepared by reductive hydrolysis and separated by TLC. The cutin was obtained from
stem sections grown in water (C) or 10 pM GA, (GA) for 48 h and had been labeled
with [“C]palmitic acid (A) or [“C]oleic acid (B) for the last 20 h of that time period.
Both lanes of one set contain the same amount of radioactivity. The arrowheads at the
bottom and top indicate the origin and the solvent front, respectively. The bars

indicate the positions of standards (A: monool, B: diols, C: triols, D: tetraols).

128

 

 

 

GA

 

129
According to the literature (Walton and Kolattukudy, 1972) these lower bands contain

derivatives of hydroxylated fatty acids. This indicates that the enzyme activity for
fatty acid hydroxylation (Walton and Kolattukudy, 1972) is enhanced in GA,-treated
plants. Because total cutin biosynthesis was increased as well, activity of the enzyme
responsible for polymerization (cutin transacylase, Croteau and Kolattukudy, 1974)
may be enhanced as well. However, I could not find a significant increase in in vitro
cutin-transacylase activity in GA,-treated plants as compared to air-grown ones (not

shown).

CONCLUSIONS

Gibberellin induces rapid growth in deepwater rice intemodes. This growth
and the accompanying tissue tension show that the epidermis is the growth-limiting
tissue. In the case of rice stem sections there is no thinning out of either the cuticle or
the cell wall. In order to maintain the thiclaress of the cuticle, there has to be an
increase in cutin biosynthesis. This indicates that GA may, at least indirectly,
influence cuticle biosynthesis and that the cuticle may play a role in influencing
growth. Treatment with cutinase shows that this is indeed the case (Figures 5.2, 5.3;
Tables 5.1, 5.2). Application of cutinase leads to a reduction of the tissue tension in
rice stems and leads to an increase in the growth of corn coleoptiles. I conclude that
the cuticle may be a growth-limiting factor.

I also found that GA,-induced rapid growth leads to an increase in cuticle

biosynthesis (Figures 5.4, 5.5, 5.7) in deepwater rice stems, with at least one

130

quantitative change in cuticle composition (Figures 5.6, 5 . 8). The cutin monomer
whose level increases is probably a dihydroxyhexadecanoic acid. In addition, all other
bands with increased incorporation of [“C]palmitic acid appear more hydrophilic on
the TLC plate than the free fatty acid derivative and may thus be hydroxylated or
otherwise modified fatty acids. Bowen and Walton (1988) had investigated the
possibility that GA-induced growth influences cuticle biosynthesis and composition by
comparing the incorporation of [“C]palmitic acid into three selected cutin monomers
of control and GA-treated stems of Pisum sativum. However, in contrast to my
results, they found a similar increase of palmitate incorporation into all monomers and
concluded that the enhancement of cutin production occurred at the level of monomer
biosynthesis. Our experiments indicate that, in GA-treated deepwater rice intemodes

overall cutin synthesis as well as hydroxylation of cutin monomers are promoted.

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Biochem. Biophys. Res. Commun. 46, 16-21.

Went, F.W., Thimann, K.V. (1937) In: Phytohormones, Macmillan Co., New York,
NY, pp. 54-55.

Yamada, N. (1954) Auxin relationships of the rice coleoptile. Plant Physiol. 29, 92-
96.

CHAPTER 6

GENERAL CONCLUSIONS

Deepwater rice (Oryza sativa L.) has a number of physiological and metabolic
adaptations that enhance its chances for survival under conditions of temporary
flooding. One of these is the capacity of plants to elongate rapidly when they become
submerged. This feature helps rice to emerge from the water and to avoid drowning.
In seedlings, submergence promotes coleoptile growth (Kordan et al. , 1977 ; Turner et
al., 1981; Yamada, 1959) and in adult deepwater rice plants, elongation of the
intemode (Métraux and Kende, 1983; Vergara et al., 1976). In both instances, the
plants respond to the altered gas composition of their submerged organs. In adult rice
plants, the reduced 0, tension promotes ethylene biosynthesis which, in turn,
increases the responsiveness of the intemodal tissue to GA, which is the ultimate
growth-promoting hormone. It causes an increase in cell division and elongation and
several ultrastructural changes (for a review see Kende, 1987).

The first question in this thesis was how ethylene causes the responsiveness of
the intemodal tissue to GA. Through application of inhibitors and measurements of
endogenous hormones concentrations, we found that ethylene may change the
responsiveness to GA by altering the ratio of a growth inhibitor (ABA) to a growth
promoter (GA). Upon ethylene-treatment and upon submergence, the level of
endogenous ABA is reduced and the level of GA is increased prior to the onset of
growth (Hoffmann-Banning and Kende, 1992a; Chapter 3). Similarly, a reduction of
endogenous ABA content by fluridone, an inhibitor of carotenoid biosynthesis, is also

134

135
correlated with increased growth of rice coleoptiles (Chapter 2). However, in

coleoptiles the growth-promoting hormone is not GA but probably IAA.

In response to submergence, osmiophilic particles appear between plasma
membrane and the outer epidermal wall of deepwater rice intemodes. These particles
were found in the rapidly but not in slowly growing corn coleoptiles (Kutschera et
al. , 1987), deepwater rice intemodes (Kutschera and Kende, 1989; Hoffmann-Benning
and Kende, 1992b), and in cucumber hypocotyls (Chapter 4). Experiments using
monensin indicate that the osmiophilic particles are secreted via the Golgi pathway.
Binding of proteinase K gold to the osmiophilic particles is evidence that they are, at
least in part, proteinaceous. They do not appear to be lipid transfer proteins,
threonine hydroxyproline-rich glycoproteins, arabinogalactan proteins, or expansin;
however, they may contain some peroxidase activity (Chapter 4). My hypothesis is
that they are involved in either cell wall or cutin biosynthesis.

To keep up with rapid intemodal elongation, there would have to be changes
in the rate of cutin biosynthesis or in cutin composition. Both appear to be the case.
In deepwater rice that was induced to grow rapidly with 6A,, the rate of
incorporation of [“CJpalmitic acid into the cuticle was more than 20-fold higher than
in control plants. In addition, some cutin monomers were incorporated preferentially
in GA,-treated plants, one being a dihydroxypalmitic acid. This indicates that
hydroxylation of cutin monomers and, possibly, their incorporation into cutin is

increased in GA,-treated deepwater rice plants (Chapter 5).

136
REFERENCES

Hoffmann-Benning, S., Kende, H. (1992a) On the role of abscisic acid and
gibberellin in the regulation of growth in rice. Plant Physiol. 99, 1156-1161.

Hoffmann-Benning, S. , Kende, H. (1992b) Characterization of osmiophilic particles
associated with rapid growth in corn and rice. Plant Physiol. 99, Abstract #
177.

Kende, H. (1987) Studies on intemodal growth using deepwater rice. In: Cosgrove,
D.J., Knievel, D.P., eds. Physiology of Cell Expansion during Plant Growth.
The American Society of Plant Physiologists. pp 227-238.

Kordan, H.A. (1977) Coleoptile emergence in rice mlings in different oxygen
environments. Ann. Bot. 41, 1205-1209.

Kutschera, U., Bergfeld, R., Schopfer, P. (1987) Cooperation of epidermis and inner
tissues in auxin-mediated growth of rice coleoptiles. Planta 170, 168-180.

Kutschera, U. , Kende, H. (1989) Particles associated with the outer epidermal wall in
intemodes of deepwater rice. Ann. Bot. 63, 385-388.

Métreaux, J.-P., Kende, H. (1983) The role of ethylene in the grth response of
submerged deepwater rice. Plant Physiol. 72, 441-446.

Tumer, F.T., Chen, C.-C., McCauley, G.N. (1981) Morphological development of
rice seedlings in water of controlled oxygen levels. Am. J. Bot. 29, 721-738.

Vergara, B.S., Jackson, B., DeDatta, S.K. (1976) Deep-water rice and its response
to deep-water stress. In: Climate and Rice. International Rice Research
Institute, Los Bafios, Philippines, pp. 310-319.

Yamada, N. (1954) Auxin relationships of the rice coleoptile. Plant Physiol. 29, 92-
96.

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