The effects of cycocel (CCC) on tomato under water stress

TYAITE

t-^+rySJFUlE

TIBRARY

THE EFFECTS OF CYCOCEL (CCC) ON TOl',tATO UNDER !'IATER STRESS b

Solomon Amoabin B.Sc. Agric' ^{Hons (Ghana)}

Department of ^{Plant PhYsiologY}

!,laite Agriculcural ^Research Institute

The UniversiÈY of ^Adelaide

South Australia

Thesis ^submitted for the ^{Degree of} Master of Agricultural ^Science

December, ^1983.

TABLE OF ^CONTENTS

STATBMBNT

ACKNOWLEDGEMENTS SUI',IMARY

ABBREVIATIONS AND ^SYI'{BOLS FIGURES

TABLBS

INTRODUCTION CCC AND PLANT

Page ^No_.

i ii iii

v

vii

x

CFIAPTER

I

r.1

r.2

CHAPTER

II II.1

TT.2

II.3 II.4 II.5 II

.6

r.1.1

T.L,2

r.1.3 r.1.4

GROWTH

The use of cycocel in ^general agriculture

Physical, chemical and physiological properties of ^cycocel

CCC (and other compounds) as plant ^growth retardants Effects of ^{CCC on} Plant ^growth

cif t.citv

Effect of ^CCC on vepetative ^and

reDroductive ^prowth

CCC and nutrient content ^{and uptake} Uptake and metabolism of ^CCC

Mode of Action

CCC-induced resistance to pests and diseases

PHYSIOLOGY OF ^PLANT I^/ATER STRESS ^RESISTANCE Development of ^{l^/aEer stress}

Indicators of ^{plant water status}

Mechanism of water stress ^resisEance

Drought avoidance

Stomatal resPonse to water stress Chemical induction of stomatal closure Drought tolerance

r.2.7.r

OsnoresuTation

r.2. 7.2

^{Ti eTasticitv}

I.3.1 ^{CCC (and} other retardants) ^and drought resistance

MATERIALS AND I.,ÍETHODS

PLANT MATERIAL

1 1 1 2 2 4 4

6

r.1 .4. ¹

r.r

.4.2

B

I

9 11

I.1.5

r.1 .6 T.T.7

r.1.8

T,2.L

r.2.2 I.2.3 r.2.4 r.2.5 r.2.6 r.2.7

11 11 13 T4 15 16 19 20 20 22 22

Ir.6.

¹

TT.6.2

rr .6.3

PLANT GROWTH ENVIRONI"IENT CUJ,TTVATION OF ^PLANT

PREPARAT]ON AND APPLICATION OF ^CCC

METHODS OF STRESS I},IPOSITION

I',IEASUREMENT OF ^PLANT WATER STATUS

Leaf water potential Leaf osmoLic potential Relative water ^content

25 25 25 25 26 26 27 27 28 28

II

.7 MEASUREMENT OF GROh]TH PARAMETERS

Plant Ileight Leaf ^Area

Fresh wej-ght

Dry weight

ÏI.

^{B LEAF DIFFUS]VE} RESTSTANCE. STOMATAL COUNTS

AND TRANSP]RATI^ON

II.8.1 Leaf diffusive ^resistance

II. ^{B. I} .l CaTibration of di usive resistance ^Poronteter

rI.

^{B. 1 .2} Measurenertt of Teaf diffusive ^resistance

II

.8. 2.L StonataT counts TI.

II.

II. II.

II.8.3 II

.9

rr.9. ¹ rr.9.2 7.r

7,2 7.3 7.4

Page No.

28 2B 29 29 29

29 29 29 30 33 33 34 34

34 35

36

36 36 36 36 37 39

42 42 43 47

4B 48 49 49

Transpiration

CHEMICAL PROCEDURES

Determination of _Proline

Extraction and assay of quaternary amlnonium compounds

using the ^{N .1"1.} R. technique

II.

¹⁰ EXPERIMENTAL DESIGN ^AND STATISTICAL ^ANALYSIS RESULTS AND DISCUSSION

CHAPTER

III

III. l

^CCC AND WATER ^STRESS EFFBCTS ON WATER STATUS AND

OF TOMATO

1.1 Introduction

I.2 Effects of differenL concentrations of ^{CCC and} water sLress on l{ater potential ^{and growth}

III.1 .2.I Introduction

III.

¹ .2.2 ^ResuTts

Irr.

^{1 .2. 3 Discussion}

III.1.3 ^The effects of ^{1000 ppm CCC on} leaf, ^{stem and} root ^growth

III.l .3.I Introduction III.1 .3,2 ^ResuTts

rIr.

¹ .3. 3 ^Discussion

III.1.4 The effecrs of ^{1000 ppm} ccc applíed at different

stages of ^{growth and water stress on rrrater potential}

and growth

III.1 .4.I Introduction

III.1.4.2

^ResuTts

III.1

.l+,3 Discussion

TIT.2 A COI',IPAR]SON OF PEG_INDUCBD \^IATER STRESS AND SOIL ^{I^/ATBR}

DEPLETION BY WITH-HOLDING

GRO\^ITH

III III

( TREATI'4ENT

IT,T,2.1 Introduction

III.2.2

^Results

ITT.2.3 Discussion

)¿

52 53 56

\^/ATER) COMBINED \^/ITI{ CCC

III.3

^CCC AND hIATER RELATIONS OF TOMATO

III.3.1 Introduction

III.3.2 ^{Relative water content} III .3 .2.I Method

III.3 ,2,2 nesults

III.3.3 ^Osnrotic potential and turgor ^potential III.3 .3.L Method

III.3.3.2 ^ResuTts III.3.3.3 ^Discussion

III.4

^{CCC AND} ACCUI'4ULATION OF PROLINB AND OTHER QUATERNARY AI'IMONIUI.{ ^COMPOUNDS

I]I

IIT

III III.5

III II]

III III.6

DIFFUSIVE RESISTANCE

III.6.1

Introduction

III.6.2

^Results

III.6.3

^Discussion

II].7

EFFECTS OF CCC AND hIATER STRESS ON TOTAL WATER LOSS

.4.1 InLroduction .4.2 ^Results

l4i3

^Discussion

lege ^l{o.

5B 5B s9 s9 s9 63 63 63 67

70 70 7T 79

BO BO

81 87

B8 88 8B

9I

96 96 96 99

to2

106

119 EFFBCT OF CCC ON LEAF DIFFUSIVE RES]STANCE

.5.1 IntroducLion .5.2 ^Results .5.3 Discussion

EFFECTS OF CCC AND WATER STRESS

III.7 III.7

TIT.7 .1) .3

Introduction Results Discussion

CHAPTER

IV

GENERAL DISCUSSION BIBLIOGRAPHY

APPENDICBS

I

STATEMENT

This thesis ^has not been previously subrnitted

for a degree at this or any other University,

and is the original work of the writer except where due reference is ^made in ^t.he text.

IN

l_ l-

ACKNO\{LEDGEMENTS

I wish to ^{express my} sincere ^thanks to Professor ^L.G.

Paleg for his ^guidance ' constructive criticisms ^and

encouragenent in the course of the programne. I ^am also grateful to Dr. D. Aspinall for acLing as ^a second supervisor.

I am also indebted to Dr. ^{C.F. Jenner} for his ^guidance during the absence of ^my suPervisors, ^and also to Dr. P.E. Kriedemann for his invaluable advice. I ^am also grateful to Dr.T.J. ^Douglas for his help and

advice during ^my early ^days in Australia. lly ^sincere

thanks also ^go to Ehe technical staff ^and all ^the other ^members of uhe Department of Plant Physiology

for their cooperation and help in diverse ^v/ays.

I arn grateful Lo Carol ^{Arnoabin who helped} in typing and showed a lot of ^{patience and} understanding ^duri-ng the preparation of this ^thesis.

Finally, the financial support ^provided by the

Commonwealth Scholarship and Fellowship Plan ^{and the} extra support from The University of Adelaide are

gratefully acknowledged.

IAI

SUMMAT{Y

CCC (2-chloroethyltrirnethyl ^ammonium chloride) a synthetic plant ^grorvth retardant, has been pleviously ^founcl to increase the resistance of various plants to clrought. ^{l{o::k clone on} this subject mostly attributes the CCC effect to ^reduced leaf area and i:rcreased root/shoot ratios ^due to the retardation of shoot grorvth though there are a few cases where ^CCC did not retard shoot groivth but stil1 induced drought resistance. A few workers have mentioned the involvement of CCC-induced stonatal closure in the drought resistance of

CCC-t,reated plants. ^Sone workers ^have associated the CCC-induced drougl'rt

resistance with netabolic changes of ^CCC per se, ^and CCC-mediated netabolic

changes in ^{plants under} vlateï stress, without melrtioning how these changes are

related to the CCC-induced d:rought resistance.

This study was conducted to cletermine whether CCC affected the drought resistance of tomato, which is relatively sensitive to water stress, and, if so, to explore some of the possible nechanisms underlying the effect. Attention

was directed towards the effects of ^CCC on the tonato plants under stress independent of its retardation effect on growth.

At a concentration of ^{100@prn, CCC retarded the growth} of the tomato

plants 6 days after its application as soil drench. ^{CCC reduced} height, leaf area, fresh and dry weights of leaves and stem, without ^any retarclation effect

on root growth, resulting in an increased root/shoot ratio. Whether ^CCC retarded the ^growth of ^the plant or not before inducing water stress, ^the

CCC-treated plants maintained higher water potential especially within ^the first ^{few days} after with-holding water, ^such that they did not wilt as quickly as the non-CCC-treated plants. Despite this ^prolonged survival under water

stress due to ^CCC treatment, ^{growth was} not sustained. ^{When PEG was used to}

induce stress, CCC did not have any effect ^on water potential ^and it could ^not reduce the toxic effect of ^PEG, in the forrn of leaflet margin chlorosis.

Under soil water depletion induced by with-holding water from the plants,

RWC was higher in the CCC-treated plants but osmotic potential did ^not

decrease as rnuch in the CCC-treated plants as the non-CCC-treated plants.

The relationships between water potential and RWC, osmotic potential and turgor potential were not altered by ^CCC which indicated that ^CCC did not enhance the

treated plantst ability to adjust osrnotically. This was supporte<l by the apparent lack of effect of ^{CCC tTeatment} in pronoting solute accumulation.

In normal well-watered plants, ^CCC caused a rapid differential ^increase in adaxial leaf diffusive resistance but not in abaxial resistance indicating

a CCC-induced closure of the adaxial ^stomata, independent of its effects ^{on growth.}

AV

This was consistent rvith a marked decrease in transpirational water loss from the adaxial leaf surface of ^the CCC-treated plants. ^Water stress per se ^(induced by wi-th-holding water fron the plants) also caused stomatal closure but this was quicker in the non-CCC-treated plants than in the CCC-treated plants. The same hrater potential threshold was found for stonatal closure and effective control of further water loss under stTess, and this was unaffected by CCC. This indicated that, because of the

initial CCC-induced adaxial stomatal closure and the concommitànt reduced

transpiration, the water potential of the CCC-treated plants declined less

rapidly as the stress progressed, and that the time required to reach the water potential threshold for stomatal closure and effective control of

I^Jater loss was prolonged by the CCC treatment.

It was concluded that, independent of its ^growth retardation effect,

CCC enabled plants to delay the onset of severe internal water deficit and,

therefore, prolonged survival through CCC-induced adaxial stonatal closure and the attendant decreased transpirational water loss. This did not ^seem to involve any CCC-induced osrnotic adjustnent. ^{When CCC retarded growth,}

however, the increased root/shoot ratio and the reduced leaf area could be

additional factors contributing to the CCC-induced resistance to ^water stress.

2

ABA ATP 14c Ca

OC

CCC cm cm2

c0^z D^0Z:

dn?

F.C.

Fig.

g or-gm

I

H,

HC1

H^0¿

hr(s) i. e.

ins.

K

J-

K.

KCl kg

I

Me0H Mg M.!'1.

I'fl,JC

_, _-1 pE.m

"s

^-

ug

¡r1 mg

ml

mm

v

ABBREVIATI ONS AND ^SYIVIBOLS

abscicic ^acid

adenosine triPhosPhate

radioactive ^carbon calciun

degrees centiglîade cycocel

centimetre ^{( s} )

square cent-imetre(s) carbon dioxide

deuterium oxide square decimetre(s)

field ^caPacitY figure

gram( s )

hydrogen ion hydrochloric ^acid

water hours

that is

inches potassium potassiurn ion potassium chloride kilogram(s)

litre ^(s ) methanol magnesium

molecular ^weight

r:nethanol : chloroform : ^water

microeinstein per square meLre per second microgram(s )

microlitre ^{( s} )

milligram(s) rnillilitre ^{( s} )

millimetre (s)

vl_

NZ ^niLrogen

N ^normal

NMR nuclear magneLic resonance

No. ^number

P ^phosphorus

32P radioactive ^phosphorus PEG polyethylene glycol ppm parts _Per mÍ-llion

86nu radioactíve ^rubidium rpm revolutions Per minute

R\,\lC relative !ùater contenL

Sec. second(s)

Si silicone

t ^time

t-buOH (tert-Bu0H) tertiary butanol

^t ^change in time

7" ^percent

tl h¡ater potential

Us ^osmotic (solute) _Potential üp turgor ^potential

{rm matric potential

Fig. I. ¹

Fig. I.2 Fig. II.1

Fie. III.1

Fig. IIl.2

Fig. III.1.3 Fig. III.1.4

Fig. III.1.5

Fig. III.

^{2. 1}

Fig. III.3.1 Fig. III.3.2 Fig. III.3.3 Fie. III.3.4

vl_r

FIGURES

Structural representation of ^some plant grorvth

retardants ^(Cathey, 1964).

Metabolic scheme for the ^mode of action of ^CCC by Stoddart ^(1964).

Relationship between the time lapse (Ai) and

diffusive resistance in the calibration of ^the

porometer at 20"C and 24"C.

Effects of different levels of ^CCC preEreatment on waLer potential during 4 days of with-

holding water.

Photographs showing CCC-treated and control plants under well-watered condition and under stress induced by with-holding 'iúater. ^The CCC-treated

plants were supplied with 1000 ppm CCC.

Retardation effects of 1000 ^{ppm CCC} and water stress.

Effect of 1000 ppm CCC on (a) leaf area and (b)

leaf length, with respect to leaf ^position, 5 days and 16 days after ^{t.he CCC} treatment.

Effects of ^{1000 ppm CCC supplied} at different stages of growth and water stress on water

potential.

Effect of ^{CCC on water} potential under PEG-induced

stress and soil ^moisLure deplet.ion induced by

with-holding water.

Effect of ^CCC and water stress on water potential.

Effects of ^CCC and water stress on ^RtrtlC.

Relationship between Rl'/C and waLer potent.ial (moisture release curve).

Effect of ^CCC on water potential under sËress and recovery.

Effect of ^{CCC on} osmotic poEenLial under stress and recovery.

PaRe No.

I2

32

55 60 61 5

3B

40

44

46

50

62

64

Fig. III.3.5

65

vlal-

Relationship ^between water potential ^and

osmotic potential.

Effect of ^{CCC on turgor} potential ^{under stress}

and recovery.

Relationship ^between water potential and turgor potential.

Effect of ^CCC and water stress on water poLential.

Effects of ^CCC and water stress on proline

accumulation.

Effects of ^CCC and water stress on h¡ater Potential.

stress on proline Effects of ^CCC and water

accumulation.

NMR spectrum of an extract of ^a CCC-treated

plantl s leaf, ^{showitrg choline, CCC and proline}

peaks.

NMR spectrum of an extract of ^a non-CCC-treated plantl s leaf, showing choline and proline peaks '

Diurnal pattern of (a) ^{adaxial and} (b) abaxial

diffusive resistances in CCC-treated and control plants, in the glasshouse.

Diurnal pat-tern of (a) ^{adaxial and} (b) abaxial

diffusive resistances in CCC-treated and control plants, in the growth room.

Effect of ^{CCC on adaxial} diffusive ^resistance within a period of ^{7 daYs.}

Effect of ^{CCC on adaxial} diffusive ^resistance

under stress and recoverY.

Effect of ^{CCC on abaxial} diffusive ^resistance

under stress and recoverY.

Relationship between water potential and adaxíal

diffusive resistance.

Relationship ^{between v/ater} potential and abaxial

diffusive resistance.

Relationship between vraLer poEential and adaxial conductance.

lqge ^No.

,'I Èl

!r:

Fig. III.3.6

Fig

. III.3.7 Fie. III.3.B

66

6B

69 12

73 74

75

76

77

82

B3

B4

89

90

92

93

94

Fig.

Fig.

III.4.

¹

TTT.4.2

Fig. III.4.3 Fie. III.4.4 Fie. III.4.5

Fig. III.4.6 Fig.III.5.1

Fig. III.5.2

Fig. III.5.3 Fig. III.6.1 Fig. II'I.6.2 Fig. III.6.3 Fig. III.6.4

r

Fig.lII.6.5

Lx

I ,l

'iI I

'l.l

I l

l I I

I

Fig. III.6.6 Fig. III.7.1 Fig. IrI.7

^.2

Fig. III.7.3 Fig.

^TII.7.4

Fig.

l

Fig"2

Fig.3

Relationship ^beLween water potential ^{and abaxial}

conductance.

Effects of-CCC and water stress on !'Iater loss/day' Effects of ^CCC and water stress ^{on cumulative} water ^1oss.

Relationship ^{between hraÈer} potenLial and water loss/day.

Relationship between water potenti-al ^and cumulative water 1oss.

APPENDIX ^FIGURES

Photograph showíng the method of determination

of tránspiration rate in excised leaves ^as described in Section I1.8.3

Photographs showing sLomatal distribution ^on the adaxial surface.

Photographs showing stomatal distribution ^on the abaxial ^surface.

Page No.

95 97

100

101

119

722

L23

9B

fË

":r

i i í

I ( î'

T'

I

l

I

x

TABLES

Physical and Chemical Properties of ^CCC.

Calibration ^values of ^che diffusive ^resistance

porometer at 20"C and 24oC.

Effects of different levels of ^{CCC and rvater} stress on growth characteristics after ^{4 days} of with-holding ^water.

The effects of ^{1000 ppm CCC on various grok¡th} characteristics of well-watered ^{tomato plants} _' Effects of ^{1000 ppm CCC applied} at different ^times

(Early and LaLe) on growth characteristics ^before

and after stress.

EffecLs of ^CCC on various gror'rth characteristics before the imposition of stress.

Effects of ^CCC and PEG-induced stress ^{orl various} growth characteristics.

Proline, ^{choline and CCC content} of leaf

esLlmated by the ^NMR technique.

Relationship ^between plant si-ze, diffusive reistance and transPiration.

Diffusive resistance of intact ^{plant and trans-} piration rate of the Adaxial ^and Abaxial ^surfaces óf excised leaves. Measurements were taken

5 days after ^{CCC treatment.}

APPENDIX TABLB

Number of stomaLa/rnm²

Table I.1

Table II.1

Table III. l

Table I]J.2

Table

III.3

Table

III.2.1

Table

III.2.2

Table

III.4.1

Table III.5.1

Table

III.5.2

Table ¹

31

4L

45

51

57

78

86

r2r

5l+

85

I

r