ETHYLENE BIOSYNTHESIS Jean-Claude PECH. ETHYLENE ORIGINATING FROM LIPIDS? Until 1964, ethylene was though to derive from the enzymatic disruption of lipids.

ETHYLENE BIOSYNTHESIS

Jean-Claude PECH

ETHYLENE ORIGINATING FROM LIPIDS?

Until 1964, ethylene was though to derive fromthe enzymatic disruption of lipids involving free radicals formation

Arguments:-Oxygen is required for the biosynthesis ofethylene in plants;

-Free radical scavengers (antioxidants that prevent lipid oxidation, e.g. propyl gallate ...) inhibited ethylene production.

DISCOVERY OF METHIONINE AS A PRECURSOR OF ETHYLENE

Lieberman and Mapson, 1964* *Methionine

NH3+

CH3-S-CH2-CH2-CH-COO-

OTHER INHIBITORS OF ETHYLENE SYNTHESISLieberman and Owens, 1971

CH2-CH-CH2-O-C=C-CH-COOH

OH NH2 H NH2

H

Rhizobitoxine from Rhizobium japonicum

CH2-CH2-CH2-O-C=C-CH-COOH

NH2 H NH2

H

AVG= Amino ethoxy vinyl glycine from Streptomyces

Shang-Fa YANGDISCOVERY OF ACC AND METHIONINE CYCLE

« Knight of Roquetaillade », Ethylene Congress Agen, 1992

Radio-chromatograms of ethylene extracts of apple plugs

14C-Methionine

Plugs in air, 12h

Plugs in N2, 6h

Plugs in N2, 6hthen in air, 6h

* *Methionine

NH3+

CH3-S-CH2-CH2-CH-COO-

?

?CH2 CH2 * *

Ethylene

1-aminocyclopropane-1-carboxylic acid

CH2 CH2

CH

NH3+ COO-

* *S-Adenosyl Methionine

* *

NH3+

CH3-S-CH2-CH2-CH-COO-

Ade

+

L-methionine

S-adenosyl-methionine(AdoMet)

Yang CycleYang Cycle

HH22C NHC NH33

CCHH22C COOC COO--

ACCACC

EthyleneEthylene

5’-methylthioadenosine5’-methylthioadenosine(MTA)(MTA)

5’-methylthioribose (MTR)5’-methylthioribose (MTR)

5’-methylethioribose-1-phosphate 5’-methylethioribose-1-phosphate (MTR-1-P)(MTR-1-P)

2-keto-4-methylthiobutyric acid (KMBA)2-keto-4-methylthiobutyric acid (KMBA)

Ethylene biosynthesisEthylene biosynthesis

C=CC=CHHHH

HH HH

ATP+H2OATP+H2O

PPii+PP+PPii

11//22 O O22

HCN+HHCN+H22O+COO+CO22

HH22OOadenineadenine

ATPATPADPADP

OO22PPii+HOOCH+HOOCH transaminationtransamination

Abstract

« Knight of Roquetaillade », Ethylene Congress Agen, 1992

Don GRIERSON, DISCOVERY OF ACC OXIDASE

DISCOVERY OF A METHOD FOR MEASURING EFE IN A SOLUBLE FORM

Ververidis and John, 1991: Complete recovery in vitro ofethylene forming enzyme activity. Phytochemistry, 30:725-727

1: Highly impureextract

2: Highly pure extract

Silver staining Pre-immuneUnpurifiedserum

Purified serum

ACC SYNTHASE PURIFICATION FROM WOUNDED ZUCCHINI

L-methionine

S-adenosyl-methionine(AdoMet)

ACC synthaseACC synthase

Yang CycleYang Cycle

HH22C NHC NH33

CCHH22C COOC COO--

ACCACCACC oxidaseACC oxidase

EthyleneEthylene

5’-methylthioadenosine5’-methylthioadenosine(MTA)(MTA)

5’-methylthioribose (MTR)5’-methylthioribose (MTR)

5’-methylethioribose-1-phosphate 5’-methylethioribose-1-phosphate (MTR-1-P)(MTR-1-P)

2-keto-4-methylthiobutyric acid (KMBA)2-keto-4-methylthiobutyric acid (KMBA)

Ethylene biosynthesisEthylene biosynthesis

C=CC=CHHHH

HH HH

ATP+H2OATP+H2O

PPii+PP+PPii

11//22 O O22

HCN+HHCN+H22O+COO+CO22

HH22OOadenineadenine

ATPATPADPADP

OO22PPii+HOOCH+HOOCH transaminationtransamination

GACCGACCMACCMACC

Inhibitors of ACC synthase: Rhizobitoxine, AVG

Factors stimulating ACS gene expression:Auxins, chilling, wounding, drought, pathogens,ethylene

Inhibitors of ACC oxidase: Anoxia, uncouplers, free radical scavengers, Co++, Ni++, T°>35°C,

Factors stimulating ACO gene expression:Pathogens, ethylene, wounding

EFFECTORS OF ACC SYNTHASE AND ACC OXIDASE

At-ACS1 (induced by cycloheximide)

At-ACS5 [induced by Li, cycloheximide(CH) and low concentration of cytokinin(CK)] At-ACS4 (induced by CH, IAA and wounding)

At-ACS2 (induced by Li, CH, wounding)

At-ACS4 induced by CH, IAA, wounding)

At-ACS4 (induced by CH, IAA, anaerobisis and wounding)

Adult plant Etiolated seedling

At-ACS5 (basal level stimulated by Li and CK

At-ACS8 not yet characterized

At-ACS10 in response to light in plants overexpressing CONSTANS

At-ACS2 in young leaves and flowers

At-ACS7 (induced by CH)

At-ACS9 = eto1

At-ACS6 (induced by O3, Cu2+, IAA, CH, CN-, anaerobiosis, wounding, touching and ethylene)

At-ACS1

At-ACS2

Le-ACS2

Le-ACS4

At-ACS6

Le-ACS6

Le-ACS1A

Le-ACS1B

At-ACS7

At-ACS11

Le-ACS5

At-ACS4

At-ACS8

At-ACS5

At-ACS9

Le-ACS7

Le-ACS3

Le-ACS8

At-ACS10

At-ACS12

Branch I

Branch II

Branch III

*

****

ACC SYNTHASE GENE FAMILY IN ARABIDOPSIS

Le-ACO1, 3

Le-ACO1 in senescentor wounded leaves

Le-ACO2

Le-ACO1,2,3 and 4induced by O3

Le-ACO3 (breaker stage)

Le-ACO4

Le-ACO4

Le-ACS7 induced early by wounding

Le-ACS6 (in MG fruit) negativ. regul. by ethylene

Le-ACS4 induced by wounding

Le-ACS1A (breaker stage) negativ. regul. by ethylene

FLOWER

Le-ACS7 induced early by flooding

Le-ACS3 induced by flooding

Le-ACS2 and 4 positiv. regul. by ethylene

Le-ACS3 (not expressed in fruit, present in tomato cell suspension)

Le-ACS2 (stamens, mature and senescent anthers)

Le-ACS2 (senescent petals)

ACO AND ACS GENE EXPRESSION IN TOMATO

Regulation of ethylene biosynthetic genesRegulation of ethylene biosynthetic genes

ACC synthase Arabidopsis Rapidly induced by auxin (ACS4).

Induced by low levels of cytokinin (ACS5).Rumex Flood resistant species downregulates ACS,

flood sensitive does not. Rice Enzyme activity induced by hypoxia/flooding.Mung bean Induced by mechanical stress (AIM1).Tomato Induced by fruit ripening.

Induced by flooding (ACS3).Induced by ozone (ACS2).

Phaseolus Induced by auxin (ACS1).Synergistic induction with auxin (ACS2).

Winter squash Induced by auxin (accA).Induced by wounding (accW).

Phalaenopsis Induced by ethylene (ACS1)Induced in stigma after pollination or auxin treatment(ACS1, ACS2).Induced in ovary after pollination or auxin treatment(ACS3).

Carnation Late induction by ethylene, pollination induces expression after 12-24 hours in petals (CARACC3).

Stellaria Regulated by photoperiod.Potato Rapidly induced by ozone, infection and Cu2+ (ACS5).

Slowly induced by ozone, infection and Cu2+ (ACS4).Zucchini Induced by wounding and auxin only in fruit (ACC1A).

ACC oxidase

Arabidopsis Induced by ethylene (ACO2/E1305).Tomato ACO1 antisense transgenic plants have reduced

ethylene production and epinasty after flooding.Induced during flower development (ACO1-3).Induced by wounding (ACO1).Induced during senescence (ACO, ACO3).

Phalaenopsis Induced by ethylene.Unpollinated flowers cannot convert ACC toethylene.

Cantaloupe Induced by ethylene, wounding, NaCl, drought andripening (ACO1).Induced by NaCl and drought (ACO3).

Petunia Induced by ethylene (ACO3).Induced in senescing floral organs (ACO1, ACO3).Induced in developing pistils (ACO3, ACO4).

Winter SquashInduced by wounding, induction blocked by ethyleneinhibitor, endogenous ethylene accelerates induction.

Carnation Induced by pollination in styles and petals (SR120).Mung bean Induced by ethylene(ACO1, ACO2).Peach Induced by wounding and fruit ripening.Apple Induced during fruit ripening.Broccoli Induced after harvest (ACC Ox1).

Induced in reproductive organs postharvest (ACC Ox2).