Regulation of ethylene biosynthesis in higher plants by carbon dioxide

Postharvest Biologyand Technology

Postharvest Biology and Technology 7 (1996) 1-26

Review

Regulation

of ethylene biosynthesis by carbon dioxide

in higher plants

Francis M. Mathooko *I* Laboratory

of Postharvest Horticulture, Faculty of Agriculture, Okayama University, Okayama 700, Japan Accepted 7 April 1995

Abstract Elevated CO2 levels used with or without reduced O2 levels regulate many biochemical and physiological processes in higher plants, among them ethylene biosynthesis. The mode of action of elevated CO2 in the regulation of ethylene biosynthesis is still a subject of much debate. Various hypotheses have been put forward to explain its mode(s) of action and most of them have pointed out that CO2 regulates ethylene biosynthesis, at least in part, by counteracting ethylene action. This is thought to be mainly through the regulation of l-aminocyclopropane-1-carboxylate (ACC) synthase, presumably the rate-limiting enzyme in the ethylene biosynthetic pathway, and in some instances ACC oxidase. The present review brings together recent developments on the biochemical, physiological and molecular bases for the regulation by CO:! of ethylene biosynthesis in higher plants. The mode of activation of ACC oxidase by CO2 is also discussed. Keywords:

ACC oxidase; ACC synthase;

Carbon

dioxide;

Ethylene

production;

MACC

1. Introduction

Since the classical work of Kidd and West (1927) on the beneficial effects of low 02 and/or high CO2 levels on the storage of fruits and vegetables, considerable research has been conducted on controlled atmosphere storage of these commodities. However, much of the research had previously been directed towards determination of the optimum conditions of storage for the various fruits and vegetables with only a limited effort being directed towards understanding the basic biochemical and physiological effects (Kader, 1986). The modification of ethylene biosynthesis and/or action and manipulation of the level of ethylene in plants by either stim*Fax: +254 151 21764. l Present address: Department Jomo Kenyatta

University

of Food Science and Postharvest Technology, Faculty of Agriculture,

of Agriculture

09255214/96/$15.00 0 1996 Elsevier SSDI 092S-5214(95)00026-7

and Rchnology,

Science

P.0. Box 62000, Nairobi,

B.V. All rights reserved

Kenya.

2

EM. Mathooko / Postharvest Biology and Technology 7 (1996) l-26

ulation or inhibition of its production is indeed of great potential to agriculture. In recent years, considerable research has been conducted on understanding the biochemical and physiological bases for the effects of controlled atmospheres on ethylene biosynthesis and/or action among other metabolic processes. Given that ethylene biosynthesis is induced by an array of factors both internal and external, it is plausible that CO2 may have differential effects on ethylene biosynthesis induced by these stimuli. Both beneficial and detrimental effects of CO;! have been demonstrated, but the mode(s) of CO2 action on plant tissues is still a mystery. CO:! may act both as an inducer and as a suppressor of ethylene production. The differential effects of CO2 on ethylene production and/or action in particular depend on the commodity, variety, physiological age, initial quality, in situ CO2 concentration, temperature and duration of exposure to such conditions (Kader, 1986; Kubo et al., 1990; ChavezFranc0 and Kader, 1993; Pesis et al., 1994) and probably the mode of induction of ethylene production such as wounding, stress, auxin, ripening or autocatalytic (Mathooko, 1995). The regulation of ethylene biosynthesis has been reviewed previously (Yang and Hoffman, 1984; Mattoo and White, 1991) and proceedings of symposia devoted to ethylene biosynthesis have been published (Fuchs and Chalutz, 1984; Clijsters et al., 1989; Pech et al., 1993). Recently, Kende (1993) published a review on the regulation of ethylene biosynthesis at the molecular level. The present review brings into perspective the recent developments on the biochemical, physiological and molecular bases for the effects of elevated CO2 levels on ethylene biosynthesis as well as its possible modes of activation of l-aminocyclopropane-lcarboxylate (ACC) oxidase, the enzyme that catalyzes the last step in the ethylene biosynthetic pathway. 2. The ethylene biosynthetic pathway Ethylene, one of the simplest organic molecules with biological activity, is a plant hormone that regulates many aspects of plant growth, development and senescence (Yang and Hoffman, 1984; Mattoo and White, 1991; Abeles et al., 1992). It plays an important regulatory role in the physiology of plants and, in particular, the senescence and postharvest physiology of fruits, flowers and vegetables. Therefore, depending on where and when it occurs, it may be beneficial or harmful to harvested horticultural crops (Yang, 1985, 1987). Because of its effects on plant senescence, large losses of fruits and vegetables are incurred in both developed and developing countries, with the losses being much higher in developing countries because of the lack of sufficient postharvest handling systems (Theologis et al., 1992). The efficiency of horticultural systems, therefore, can be improved by the ability to regulate ethylene synthesis and/or responses to suit specific practical needs (Yang and Hoffman, 1984; Yang, 1985; Mattoo and White, 1991). Thus, an understanding of ethylene biosynthesis and action is of fundamental as well as applied significance towards its regulation. Ethylene in higher plants is synthesized via the following pathway: L-methionine -+ S-adenosyl-L-methionine (AdoMet) + l-aminocyclopropane-1-carboxylic acid

RM. Mathookol Postharvest Biology and Technology 7 (1996) 1-26

3

(ACC) -+ ethylene (Adams and Yang, 1979). Two enzymes that are unique to this pathway are ACC synthase (S-adenosyl-L-methionine methylthioadenosinelyase, EC 4.4.1.14) and ACC oxidase [formerly known as the ethylene-forming enzyme (EFE)] (Kende, 1989, 1993) which catalyze the conversion of AdoMet to ACC and ACC to ethylene, respectively (Yang and Hoffman, 1984; Imaseki, 1991; Kende, 1993). ACC synthase produces besides ACC, S-methylthioadenosine (Yu et al., 1979) which is utilized for the synthesis of new methionine via a modified methionine or Yang cycle (Miyazaki and Yang, 1987; Abeles et al., 1992; Theologis, 1992; Fig. l), making it possible for high rates of ethylene biosynthesis to be maintained even when the free methionine pool is small (Kende, 1993). ACC synthase and ACC oxidase are developmentally regulated and are expressed in response to diverse inducers such as ethylene, auxin, wounding, temperature and metal ions such as Cd2+ and Li+ (Yang and Hoffman, 1984; Abeles et al., 1992; Theologis, 1992). ACC synthase is thought as the rate-limiting enzyme in the ethylene biosynthetic pathway (Yang and Hoffman, 1984) since it is induced by many stimuli and, therefore, plays a key role in regulating ethylene production (Yu et al., 1979; Yang and Hoffman, 1984; Mattoo and White, 1991; Kende, 1993). However, a large body of evidence is available to indicate that ACC oxidase activity also increases in some plant tissues in response to internal or external factors that induce ethylene production (Ye and Dilley, 1992; Kende, 1993) and, thus, its activity may be limiting. In addition to serving as an ethylene precursor, ACC can be metabolized to a presumably biologically inactive end-product, l-(malonylamino)cyclopropane1-carboxylic acid (MACC) (Hoffman et al., 1982; Yang and Hoffman, 1984; Imaseki, 1991) a reaction catalyzed by ACC malonyltransferase (Liu et al., 1984). The rate of formation of MACC in plant tissues affects the endogenous levels of ACC in tissues which have low ACC synthase activity (Imaseki, 1991). Malonylation of ACC can, therefore, decrease the free ACC level inside plant tissues and thus may play an important role in the regulation of ethylene production (Liu et al., 1984, 1985; Abeles et al., 1992). Since ethylene biosynthesis is regulated at a point after the formation of AdoMet (Yang and Hoffman, 1984), CO2 can regulate ethylene biosynthesis at least at three points: (1) the conversion of AdoMet to ACC catalyzed by ACC synthase; (2) the conversion of ACC to ethylene catalyzed by ACC oxidase; and (3) the conjugation of ACC into MACC catalyzed by ACC malonyltransferase. Besides, AdoMet can be decarboxylated and serve as a precursor in polyamine biosynthesis. However, it is not known to what extent this decarboxylation of AdoMet may regulate ethylene biosynthesis. 3. Regulation of wound-induced

ethylene biosynthesis

Total ethylene and CO2

Fruits and vegetables are subject to mechanical wounding and stress during harvesting, sorting, packaging and transportation and these hasten the ripening and senescence processes through increased ethylene production (Yu and Yang, 1980; Yang and Hoffman, 1984; Hyodo, 1991). This enhancement in ethylene production

EM. Mathooko / Posthantest Biology and Technology 7 (1996) l-26 S-CH,-CH,-CH /

,1,

Arginine

y

-COO-

CH’

permiI#IcPut

1 S

r escineJthine

SperLe

’ Spermidine Fruit ripening Indole-3-a&c acid Calcium-cytokinin Physical wounding Chilling injury Drought stress Anaerobiosis Ethylene Flooding

Hz C,

/NH

R

~

;z’coA

J

I-

t

v&NH3

+

- C - CH,- COO-

Malonyltransferase

HZC‘\,,-

coo-

HZC (AC0

(MAW

f Anaerobiosis

Uncouplers Cobalt / Salicylic acid Temp. > 35’C

Free radical scavengers

I

‘

COOH COOH Cyanoalanine

c Cysteine

ETHYLENE

Fig. 1. Regulation of ethylene biosynthesis: Hatched block, this is normally suppressed and is the rate-limiting step in the pathway; Large black arrow, induction of synthesis of ACC synthase and ACC oxidase; Large open arrow, inhibition of the reaction. ACC, AdoMet, AOA, AVG, DAdoMet, MACC, MET, MTA and MTR stand for 1-aminocyclopropane-I-carboxylic acid, S-adenosylmethionine, aminooxyacetic acid, aminoethoxyvinylglycine, decarboxylated AdoMet, l-(malonylamino)cycIopropane-1-carboxylic acid, methionine, 5’-methylthioadenosine and S-methylthioribose, respectively (adapted and modified from Yang, 1985; Miyazaki and Yang, 1987; Abeles et al., 1992).

serves as a signaling mechanism with profound physiological consequences (Yang and Hoffman, 1984; Abeles et al., 1992). The physiological role of wound-induced ethylene is not fully understood, but it is thought to be related to the defense mechanism of wounded plant tissue (Imaseki, 1991). The stimulation of ethylene production by wound stress typically occurs within a lag period of lo-30 min and

Rh4. Mathooko / Postharvest Biology and Technology 7 (1996) l-26

5

subsides after reaching a peak within several hours (Yang and Hoffman, 1984). The induction by wounding of ethylene production has been studied in a number of plant tissues (Boller and Kende, 1980; Yu and Yang, 1980; Kao and Yang, 1982; Cheverry et al., 1988; Mathooko, 1995). The induction of ACC synthase has been regarded as a key step in wound-induced ethylene biosynthesis (Boller and Kende, 1980; Yu and Yang, 1980; Hyodo, 1991) and is synthesized de novo following wounding (Yu and Yang, 1980; Mattoo and White, 1991; Abeles et al., 1992) but its mRNAs accumulate in the absence of protein synthesis (Huang et al., 1991; Liang et al., 1992; Lincoln et al., 1993; Zarembinski and Theologis, 1993) suggesting that their induction is a primary response to the inducer. The role of elevated CO;? levels in the regulation of wound-induced ethylene biosynthesis is still obscure. CO2 promotes ethylene production in excised leaves (Grodzinski et al., 1982; Kao and Yang, 1982) and inhibits ethylene production in excised apple (Chaves and Tomas, 1984; Cheverry et al., 1988), tomato and winter squash (Mathooko et al., 1993a), kiwifruit (Rothan and Nicolas, 1994) and in strawberry and pear (Rosen and Kader, 1989). ACC oxidase

It has been demonstrated that in excised tissues CO2 promotes ACC oxidase activity (Kao and Yang, 1982; Chaves and Tomas, 1984; Preger and Gepstein, 1984; Bufler, 1986; Philosoph-Hadas et al., 1986; Mathooko et al., 1993a; Rothan and Nicolas, 1994) which may involve de novo protein synthesis (Zacarias et al., 1990; Tian et al., 1994) or enhancement of its turnover (net synthesis and degradation) (Philosoph-Hadas et al., 1986). However, since the modulation of ACC conversion to ethylene by CO2 was rapid and reversible, Kao and Yang (1982) reasoned that CO2 regulates the activity, but not the synthesis of ACC oxidase. Recently, Tian et al. (1994) reported that CO2 induces ACC oxidase synthesis in pear and apple fruit discs since its stimulation by CO2 was inhibited by cycloheximide, an inhibitor of protein synthesis. Tian et al. (1994) further indicated that the stimulatory effect of CO2 on ACC oxidase activity cannot be caused by pretreatment of tissue with CO2, but for maximum expression of ACC oxidase both exogenous ACC and CO2 are required. This is contrary to previous observations which indicated that pretreatment of excised tobacco leaves (Philosoph-Hadas et al., 1986), tomato fruit pericarp tissue (Mathooko et al., 1993a) and apple and avocado fruit discs (Cheverry et al., 1988) with CO2 stimulated ACC oxidase activity. CO2 does not seem to stimulate wound-induced ACC oxidase activity in all tissues since it inhibits activity of the enzyme in apple (Chaves and Tomas, 1984) and in winter squash fruit (Mathooko et al., 1993a; Kubo et al., 1995; Mathooko, 1995; Fig. 2), suggesting that CO2 may regulate its activity in a tissue-specific manner. On the contrary, Grodzinski et al. (1982) proposed that CO2 promotes ethylene production in excised leaves indirectly by inhibiting ethylene retention or breakdown, thereby resulting in more ethylene being released. The inhibition of wound-induced ethylene production depends on the CO2 concentration (Zacarias et al., 1990; Mattoo and White, 1991; Mathooko et al., 1993a; Rothan and Nicolas, 1994).

EM. Mathooko I Postharvest Biology and Technology 7 (1996) l-26

0

6

12 18 Time after cutting (h)

24

Fig. 2. Changes in ACC oxidase activities in excised pericarp tissue of tomato (A) and mesocarp of winter squash (B) fruits during treatment with CO2. Vertical bars represent &SE (adapted Mathooko et al., 1993a).

tissue from

ACC synthase and ACC malonylation

Elevated CO2 inhibits wound-induced ACC synthase activity in tomato pericarp and winter squash mesocarp tissues (Mathooko et al., 1993a). However, despite its action in a concentration-dependent manner, CO2 does not seem to significantly influence the endogenous ACC levels in various excised tissues (Kao and Yang, 1982; Cheverry et al., 1988; Mathooko et al., 1993a; Rothan and Nicolas, 1994) irrespective of the concentration. On the other hand, Zamponi et al. (1990) indicated that 20% CO2 does not affect ACC synthase activity in tomato fruit discs. A CO2 level above 10% inhibited wound-induced conjugation of ACC into MACC in mesocarp tissue of winter squash fruit (Mathooko et al., 1993a). Since 10% and 60% CO2 inhibited ACC synthase activity to different extents, but maintained more or less similar levels of ACC while 10% had little effect on MACC accumulation, Mathooko et al. (1993a) proposed that in the presence of 10% COz, ACC that is not converted to ethylene is at once converted to MACC such that free ACC does not accumulate in the tissue. Little published work is available on the mode of regulation of wound-induced ACC malonylation by CO2. Studies on CO2 effects on the activity of ACC malonyltransferase, the enzyme that catalyzes the conversion of ACC to MACC, should no doubt help us in understanding how elevated CO2 regulates wound-induced ACC malonylation. At present it is not clear whether

l34. Mathooko I Postharvest Biology and Technology 7 (1996) 1-26

7

CO2 regulates wound-induced ACC synthase activity to such an extent that the net synthesis of ACC and its conversion to ethylene and MACC balance so that there is no net increase in ACC content, or through other mechanisms. Possible mode of CO2 action

Studies using other inhibitors of ethylene action such as 2,5-norbornadiene (NBD; Hyodo et al., 1993) and diazocyclopentadiene (DACP; Kubo et al., 1995; Mathooko, 1995) have indicated that wound-induced ACC synthase is regulated through a negative feedback control mechanism as indicated by the model in Fig. 3. Therefore, if CO2 regulates wound-induced ACC synthase by antagonizing the effects of ethylene, based on this model, it is expected that treatment of wounded tissues with CO2 should enhance ACC synthase activity. However, this has not been the case in a number of studies (Mathooko et al., 1993a; Kubo et al., 1995; Mathooko, 1995) and thus does not support this model which was proposed by Hyodo et al. (1993). Studies with rice seedlings (Sanders et al., 1990) have shown that CO2 did not block the ethylene binding site, but that NBD did. Moreover, in situations such as rice coleoptile growth (Sanders et al., 1990) and cocklebur seed germination (Ishizawa et al., 1988), where ethylene has stimulatory rather than inhibitory effects, CO2 enhances rather than counteracts ethylene action. Sisler and Wood (1988) stated that CO2 may act synergistically with ethylene rather than counteracting its effects. Such contradictory effects of CO2 compared to other inhibitors of ethylene action and the absence of conclusive data showing that CO2 alters ethylene binding suggest that CO;? effects on wound-induced ACC synthase is not modulated via ethylene receptor(s) and may be through other yet unknown mechanisms which need further investigation. Moreover, unlike other ethylene action inhibitors, CO2 is a product of the oxidative deamination of ACC to ethylene (Miyazaki and Yang, 1987) and, therefore, CO2 may act through a mass action effect in the regulation of wound-induced ethylene biosynthesis. Further, since treatment of wounded tissue with exogenous ethylene promotes ACC oxidase activity (Hyodo et al., 1993) as well as accumulation of its mRNA transcripts (Kim and Yang, 1994), one may argue that, in cases where CO2 inhibits wound-induced ACC oxidase activity, it may do so by counteracting the effects of ethylene. The inhibition of wound-induced ethylene production by elevated CO2 indicates that this treatment can be used to control hastened ripening and hence senescence that is associated with ethylene production resulting from bruising during harvesting and transportation of horticultural commodities over bumpy and uneven surfaces.

Methionine

,AdoMet *cc

s;!

Fig. 3. Model for the induction of ACC synthase and ACC oxidase wound ethylene (adapted from Hyodo et al., 1993).

by wounding

and the regulation

by

8

FM. Mathooko / Postharvest Biology and Technology 7 (1996) 1-26

It may also find practical application in the transport, storage or marketing of minimally processed fruits and vegetables in which cutting/wounding results in considerable increase in respiration and ethylene production. 4. Regulation

of ethylene biosynthesis

in ripening fruits

Climacteric fruits are characterized by a surge of ethylene production and respiration at the onset of ripening and it is recognized that ethylene plays an essential role in the ripening process (Yang and Hoffman, 1984; Yang, 1987; Theologis, 1992; Kende, 1993). There is now accumulated evidence that ethylene is the key regulatory molecule for fruit ripening and senescence, not a by-product of the ripening process (Theologis, 1992). Because large losses of fruits and vegetables are incurred worldwide annually due to ethylene’s effects on plant senescence, the significance of a means to regulate ethylene biosynthesis and/or action and hence control the ripening process and prevent spoilage is clear (Theologis, 1992). To retard fruit ripening, it has long been a commercial practice to store fruits under controlled atmospheres of reduced 02 and/or elevated CO2 levels which inhibit ethylene biosynthesis and/or action (Yang, 1985, 1987; Kader, 1986). The mechanism(s) by which elevated CO;! levels regulate ethylene biosynthesis during fruit ripening is still not fully understood and contradictory information has indeed been reported for the same commodity. Various hypotheses have been suggested including interference with ethylene metabolism through a mass action effect (Chaves and Tomas, 1984; Yang and Hoffman, 1984) besides displacing ethylene from its receptor site (Yang and Hoffman, 1984). Nevertheless, it has been demonstrated that elevated CO2 (5-20%) inhibits ethylene production in climacteric fruits by inhibiting ACC synthase activity (Bufler, 1984; Chavez-Franc0 and Kader, 1993; Mathooko et al., 1995b) and also ACC oxidase activity (Chavez-Franc0 and Kader, 1993; Mathooko et al., 1995b). Since ACC synthase has a rapid turnover (Kende and Boller, 1981), Mathooko et al. (1995b) proposed that the loss in ACC synthase activity during CO2 treatment could result from depletion of ACC synthase protein due to rapid degradation and/or inhibition of synthesis, inactivation of ACC synthase in vivo, or a combination of both processes. It has indeed been observed that treatment of pear fruit with elevated CO2 causes a decrease in ethylene production as well as extractable protein (Kerbel et al., 1988). However, data on actual rates of synthesis and turnover are required to confirm the relationship between exposure to elevated CO2 and suppression of protein synthesis. The inhibitory effect of CO2 on autocatalytic ethylene production in climacteric fruits could also be due to a competition for the active site (Burg and Burg, 1967). Based on results from treatment of fruits with exogenous ethylene, it is possible that the inhibition by CO2 of ethylene production (Mathooko et al., 1995b; Fig. 4) ACC synthase activity (Bufler, 1984; Table 1) and ACC oxidase activity (Cheverry et al., 1988) is, at least in part, due to its antagonistic effects on endogenous ethylene action. Treatment of preclimacteric apple fruit with exogenous ethylene also increases both in vivo and in vitro ACC oxidase activity and accumulation of its mRNA transcripts (Dong et al., 1992). Tonutti et al. (1993) indicated that

EM. Mathooko / Postharvest Biology and Technology 7 (1996) l-26

0

0.1

1

10

C2H4 concentration

50

100

($1 .I-‘)

Fig. 4. Ethylene production by tomato fruit after pretreatment at 25°C for 24 h with various concentrations of ethylene in the presence or absence of 20% CO2. Vertical bars represent +SE (adapted from Mathooko et al., 1995b).

Table 1 Effect of CO2 on ethylene-enhanced Treatment 2 2 2 2 200 200 200 200

~1 I-’ ,Ul I-’ WI 1-l WI I-’ ~1 1-l ~1 I-’ WI I-’ WI 1-l

Adapted

ACC synthase Relative

CZH4 C2H4 C2H4 C2H4 C2H4 CrH4 C2H4 C2H4

+ 1% CO2 + 5% CO2 + 10% CO2 + 1% CO2 + 5% CO2 + 10% Co2

activity ACC synthase

activity

(%)

100 61 55 33 100 100 100 55

from Bufler (1984).

treatment of kiwifruit with exogenous ethylene stimulated ACC accumulation and ACC oxidase activity and that these effects of ethylene were counteracted by the presence of 5% CO2. These observations support the view that the autocatalytic signal associated with ethylene action during fruit ripening stimulates the activities of both enzymes. Therefore, the effects of elevated CO2 on ethylene biosynthesis at the levels of ACC synthesis and its oxidation to ethylene may, at least in part, contribute to the extension of postharvest storage life of fruits in addition to its action as an antagonist of ethylene. Although in most instances the inhibition of ethylene production by CO;? is through inhibition of ACC synthase activity, which will ultimately affect ACC content in the tissue, the effect of CO2 on the level of ACC appears controversial in the literature, even within the same commodity. For instance, elevated CO2 did not cause any change in ACC content in ‘Granny Smith’ apple (Cheverry et

10

EM. Mathooko IPosthantest Biology and Technology 7 (1996) l-26

al., 1988) in ‘Ben Davis’ apple (Li et al., 1983) and in ‘Golden Delicious’ apple (Gorny and Kader, 1994), but another report indicated an increase in ACC content in ‘Granny Smith’ apple (Chaves and Tomas, 1984). These differing results could be related to the gas composition used in the various experiments, particularly the 02 level, since the conversion of ACC to ethylene is entirely dependent on 02 concentration. However, CO;! reduced ACC content in ‘Jonathan’ apple fruit even at reduced 02 tension which tended to stimulate its accumulation (Levin et al., 1993). Elevated CO;! also inhibits ACC accumulation in tomato (Mathooko et al., 1995b) and in pear (Chavez-Franc0 and Kader, 1993) fruits. The reason for these differential effects of CO2 on ACC content in ripening fruits is not known and could depend on many factors, among them the commodity and its stage of ripeness, COz-02 combinations, temperature, duration of exposure and probably different cultural practices. It has generally been accepted since McMurchie et al. (1972) that ethylene biosynthesis is regulated by at least two receptors commonly referred to as System I and System II. Fruit tissues which are preclimacteric may demonstrate an autoinhibitory response to ethylene because only System I receptor is functional. With the appearance or developing functionality of the System II for yet unknown reasons, the endogenous ethylene is capable of inducing autocatalytic production; of course there is likely to be an overlap of these two systems. It is possible that this could, at least in part, explain why CO;! has differing or reversed effects at various developmental stages as it might react in alternative ways with each of the two receptors. CO2 may also regulate ethylene biosynthesis during fruit ripening by inhibiting the conjugation of ACC into MACC (Mathooko et al., 1995b). This could be through its effect on ACC malonyltransferase activity. Results from experiments with other inhibitors of ethylene action have indicated that MACC formation and the capability to convert ACC to MACC would also decline when autocatalysis is blocked (Liu et al., 1985). However, experimental evidence on the regulation of ACC malonyltransferase activity by CO2 during fruit ripening is lacking and, therefore, further studies should be undertaken to examine how CO2 regulates its activity. Although CO:! inhibits ethylene biosynthesis in several fruits and vegetables, it does not permanently damage the fruit tissues’ capability to synthesize ethylene, thereby indicating that its effects are not as a result of general toxicity to the plant tissue, but tissue response to the stimulus. This is because ethylene production in peach (Kubo et al., 1990), tomato (Kubo et al., 1990; Mathooko et al., 1995b), apple (Chaves and Tomas, 1984), avocado (Marcellin and Chaves, 1983) and strawberry (Li and Kader, 1989) and the activities of ACC synthase and ACC oxidase in tomato fruit (Mathooko et al., 1995b) increase to the control level upon withdrawal of CO2 gas from the storage atmosphere. Since the recovery is gradual, it is evident that the inhibition of ethylene biosynthesis by CO2 cannot be due to just a simple mass action effect contrary to some hypotheses regarding the mode of action of CO2. This suggests that the messengers coding for ACC synthase and ACC oxidase are suppressed by CO2 and are gradually reactivated after CO2 is withdrawn and is indicative of the phenomenon Li and Kader (1989) termed as the ‘residual effect’ of controlled atmosphere storage. This response is important in postharvest handling

KM. MathookolPostharvest Biology and Technology 7 (1996) 1-26

11

of climacteric fruits since practical postharvest benefits during retail marketing may be derived from such a ‘cosmetic’ response to elevated COz. The effect of elevated CO2 on ACC oxidase activity in ripening fruits seems to depend on its concentration, commodity and probably the method of assay, that is, whether it is assayed in vivo or in vitro. Based on in vitro assay, 20% CO2 inhibits ACC oxidase activity in pear (Chavez-Franc0 and Kader, 1993) and tomato (Mathooko et al., 1995b). CO2 also inhibits in vivo ACC oxidase activity in apple (Chaves and Tomas, 1984; Cheverry et al., 1988; Gorny and Kader, 1994) avocado (Cheverry et al., 1988) kiwifruit (Rothan and Nicolas, 1994) and pear (ChavezFranc0 and Kader, 1993). Levin et al. (1993) reported that CO2 concentrations up to 10% stimulated in vivo ACC oxidase activity and 20% had an inhibitory effect. Similarly, in pear, both in vivo and in vitro ACC oxidase activities were stimulated by 1% CO;! and inhibited by 20% (Chavez-Franc0 and Kader, 1993; Fig. 5C). Thus, at low concentrations (about 1%) CO2 may promote ethylene production in climacteric fruits (Bufler, 1986; Chavez-Franc0 and Kader, 1993; Fig. 5A) while at the same time inhibiting and stimulating the activities of ACC synthase and ACC oxidase, respectively (Chavez-Franc0 and Kader, 1993; Fig. 5B, C). The measurements by Chavez-Franc0 and Kader (1993) were made in pear fruit discs after wound-induced ethylene production had subsided and, therefore, represent ripening-associated ethylene rather than wound-induced ethylene biosynthesis. It is plausible, therefore, that at low concentrations CO:! may regulate ethylene production in ripening fruits by modulating ACC oxidase rather than ACC synthase. If not so, the stimulator-y effect of CO2 at this low level could be a balance between its stimulatory effect on ACC oxidase activity and its inhibitory effect on ACC synthase activity with the contribution by the former being more significant. Gorny and Kader (1994) however, indicated that 20% CO2 inhibited in vivo ACC oxidase activity in apple and had no effect on in vitro ACC oxidase activity, while the same CO1 level inhibited in vitro ACC oxidase activity in pear fruit (Chavez-Franc0 and Kader, 1993). On the other hand, Rothan and Nicolas (1994) indicated that the stimulation or inhibition of ethylene production by CO2 depends on the level of ACC in the tissue, promoting it when ACC concentration is beyond a certain threshold (in this particular case 55 PM) and inhibiting it when the ACC concentration is low, that is, at an in vivo concentration. It is not clear, however, how ACC concentration influences stimulation or inhibition of ethylene production by COz. It has been proposed that cytosolic pH changes induced by CO2 may reduce the in vivo catalytic capacity of ACC oxidase, and hence significantly reduce ethylene biosynthesis in some fruit tissues (Gorny and Kader, 1994; Rothan and Nicolas, 1994). Similar sentiments were expressed by Mizutani et al. (1994). Although the cytoplasmic pH of plant cells appears fairly constant despite metabolic processes which generate or consume Hf, and despite wide variation in external pH (Smith and Raven, 1979) data are available to indicate that exposure of lettuce tissue to 20% CO2 causes a decrease in cytoplasmic pH from 6.7 to 6.3 (Siriphanich and Kader, 1986). Recently, Philosoph-Hadas et al. (1993) threw some light onto a possible ethylene-independent action of CO2. These workers observed that exposure of

12

EM. Mathooko I Postharvest Biology and Technology 7 (1996) l-26

0

40

80

120

160

200

Hours at 20°C Fig. 5. Ethylene production rates (A), ACC synthase activities (B) and in vivo ACC oxidase activities (C) in ‘Bartlett’ pear fruit discs kept in air and in air + 1% or 20% CO2 at 20°C. Vertical bars represent *SD (adapted from Chavez-Franc0 and Kader, 1993).

chervil leaves to 10% CO2 caused an increase in the cell sap pH by one unit and this was related to the maintenance of the original levels of polyamines which are nitrogenous bases. The high levels of polyamines could, therefore, exhibit a possible pH-stat process for regulating internal pH. Irving and Honnor (1994) also reported that besides delaying a burst in ethylene production, treatment of carnations with CO2 caused a pH increase of one unit as measured in extracts from the petals. It is not known whether this pH increase is just a consequence of CO2 effects on normal metabolism, or is a direct reaction by plants to counteract the acidic effects of CO2. It has been suggested (Irving and Honnor, 1994) that upon removal of tissue from

EM. Mathooko /Postharvest Biology and Technology 7 (1996) 1-26

13

a COz-enriched atmosphere, the rapid diffusion of CO2 into the air and inefficient buffering capacity of the cytoplasm and vacuole to cope with the resulting proton consumption (OH- production) may result in an increase in the vacuolar pH. Experimental evidence is still needed, however, to relate the effect of elevated CO? on intracellular pH and polyamine levels to their regulation of ethylene biosynthesis in plant tissues. Thus, while all elevated levels of CO2 inhibit ACC synthase activity, ACC oxidase activity is differentially regulated by CO2, being stimulated at low COz levels and inhibited at high CO2 levels. 5. Carbon dioxide stress-induced

ethylene biosynthesis

Elevated levels of CO2 can have mild to serious toxic effects on plant tissues (Abeles et al., 1992). Therefore, subjecting a cultivar of a given commodity to CO2 levels above its tolerance limits at a specific temperature-time combination may result in stress to the living plant tissue which is manifested in various symptoms, such as increased ethylene production, irregular ripening, inhibition and/or aggravation of certain physiological disorders and increased susceptibility to decay (Kader, 1986; Zagory and Kader, 1988). Thus, although CO2 may induce ethylene biosynthesis in a number of horticultural commodities (Kader and Morris, 1977; Kader, 1986; Kubo et al., 1990), it is only recently that the mode of induction has been characterized. Pesis et al. (1994) proposed that enhancement of ethylene production by CO2 could result from either activation of the enzymes involved in ethylene biosynthesis, or a response to stress. Using cucumber fruit at horticultural maturity, Mathooko et al. (1995a) demonstrated that CO2 stimulates ethylene production (Fig. 6A) in cucumber fruit by inducing both ACC synthase (Fig. 6B) and ACC oxidase activities. The induction of ACC synthase led to accumulation of ACC. A commodity’s tolerance to elevated levels of CO2 depends on its physiological condition, maturity, the CO2 concentration within the tissue, the duration of exposure, the internal 02 concentration and temperature, among other variables (Zagory and Kader, 1988). Therefore, since cucumber fruit at horticultural maturity are physiologically immature, the observed response of the tissue to elevated CO2 level was related to CO2 stress. The induction of ethylene production by CO? is inhibited by treatment of the tissue with aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to AdoMet (Mathooko et al., 1995a), indicating that CO2 indeed induces ACC synthase. CO2 stress-induced ethylene production and ACC synthase activity were further inhibited by cycloheximide, an inhibitor of protein synthesis. This indicates that the induction of ACC synthase by CO2 is not a consequence of the activation of pre-existing inactive enzyme, but is due to de novo synthesis of the enzyme. Therefore, the ethylene producing system induced by CO2 is rapidly turning over. These observations indicate that the CO2 stress-induced ACC synthase is similar, if not identical, to ACC synthase induced by other stimuli. The induction of ethylene biosynthesis by CO;? requires continuous presence of the gas (Mathooko, 1995) and may resemble the ethylene producing system induced by fungal elicitor in which continuous presence of the stimulus is required. Ethylene production

14

EM. Mathooko / Postharvest Biology and Technology 7 (1996) l-26 _ 7 -c ; ‘y -E 8 ‘= i

1.6 1.4 1.2 1

0.6 0.8

g 0.4 -?z 2 0.2 G 0

01 “““‘I’I’I’I 0

6

12

18

24

30

36

42

Time (h) Fig. 6. Time in cucumber COz-enriched

courses of CO2 stress-induced ethylene production (A) and ACC synthase activity (B) fruit held at 25°C. The arrows indicate the time of transfer of the fruit from the atmosphere to air. Vertical bars represent fSE (adapted from Mathooko et al., 1995a).

induced by CO;! in podded pea and lettuce (Kubo et al., 1990) sweet pepper (Wang, 1977) and in cucumber fruit (Mathooko et al., 1995a) returned to control level upon withdrawal of CO2 gas. In other instances, the induction of ethylene production is not manifested in the presence of the gas but upon withdrawal of the gas. This latter trend has been demonstrated in spinach and eggplant (Kubo et al., 1990) preclimacteric ‘Golden Delicious’ apple (Pesis et al., 1994) and in figs (Mathooko et al., 1993~). It has been proposed that this burst in ethylene production immediately after removal from the stress treatment may result from a build up of ACC during CO2 treatment (Pesis et al., 1994). CO2 stress-induced ethylene production is also related to the time of exposure to the gas. Buescher (1979) observed that, while exposure of tomato fruit to 40% and 60% CO2 for four days inhibited ethylene production, further exposure induced CO2 stress-related ethylene production to levels two to three times that of the fruit held in air. The response of tissue to CO2 may also depend on the level of CO2 available within the cell. The contrasts between external CO2 concentration and the amount available within the cell is determined largely by the resistance of the plant organ to gas diffusion which varies among various plants, plant cultivars, plant organs and stage of maturity (Kader and Morris, 1977; Zagory and Kader, 1988). Indeed, these anatomical features responsible for differing diffusion resistance rather than alterations in biochemical pathways among various fruits and vegetables, may be

Eh4. Mathooko I Postharvest Biology and Technology 7 (I 996) 1-26

15

largely responsible for contrasting tolerances to reduced 02 and/or elevated CO2 levels (Kader and Morris, 1977). 6. Regulation of ethylene biosynthesis

at the molecular level

The role of ACC synthase as the rate-limiting enzyme in ethylene biosynthesis and the multitude of factors which govern its activity have led to considerable interest in the molecular mechanisms underlying ACC synthase gene expression. It has been indicated that during increased ethylene production induced by various stimuli, expression of multiple ACC synthase genes is activated (Van Der Straeten et al., 1990; Huang et al., 1991; Rottman et al., 1991; Yip et al., 1992; Lincoln et al., 1993) and increase in ACC synthase activity is related to the enhanced level of its mRNA (Nakajima et al., 1988; Olson et al., 1991; Kende, 1993; Lincoln et al., 1993) as shown by northern blotting or RNAase protection assay. There is evidence that the ACC synthase genes are regulated differentially and induced under different circumstances (Nakajima et al., 1990; Van Der Straeten et al., 1990; Nakagawa et al., 1991; Olson et al., 1991; Kende, 1993) and the regulation may be tissue-specific (Huang et al., 1991). Thus, ACC synthase has been the major subject of investigation concerning the regulation of ethylene biosynthesis and hence characterization of the regulation of this enzyme by CO2 at the molecular level is of both fundamental and applied importance. A fundamental question arises as to how CO2 regulates ethylene biosynthesis at the molecular level. For example, given that the enzyme is encoded by a highly divergent multigene family whose members are differentially regulated by more than one inducer, does CO2 regulate these ACC synthase genes in a similar manner or differentially? CO2 itself could also act as an inducer of ACC synthase depending on the commodity (Mathooko et al., 1995a). It has been demonstrated that although the wound-induced ACC synthase and the am&r-induced ACC synthase enzymes are immunochemically different (Nakagawa et al., 1988) and are encoded in two genes that are expressed differentially (Nakagawa et al., 1991) CO2 regulates woundinduced (Mathooko et al., 1993a) and auxin-induced (Mathooko et al., 1993d) ethylene biosynthesis specifically through suppression of ACC synthase activity. This may indicate that CO2 regulates ACC synthase in a similar manner irrespective of the mode of induction. Northern blot analysis using the respective cDNAs as probes has demonstrated that the increase in ACC synthase activity following wounding of winter squash (Nakajima et al., 1988, 1990; Kende, 1993), zucchini (Sato and Theologis, 1989; Huang et al., 1991) and tomato (Olson et al., 1991; Li et al., 1992; Yip et al., 1992; Lincoln et al., 1993; Mattoo et al., 1993) is accompanied by increases in its mRNA level. Recently, Kubo et al. (1995) and Mathooko (1995) studied the molecular basis for the CO2 regulation of wound-induced ethylene biosynthesis in mesocarp tissue of winter squash fruit. Using a cDNA probe prepared with a Hind III fragment from pCMW33, a plasmid clone containing the cDNA (an insert of 1.8 kb) for a woundinducible ACC synthase gene (Nakajima et al., 1990) these workers demonstrated that CO2 inhibits ACC synthase activity (Fig. 7B) by inhibiting the accumulation of

16

EM. Mathooko / Postharvest Biology and Technology 7 (1996) 1-26

1

2

0

3

2

(*) 4

5

4 6 8 Time after cutting (h)

6

7

10

12

Fig. 7. Time course of the appearance of mRNA encoding ACC synthase gene (A) and ACC synthase activity (B) in mesocarp tissue of winter squash fruit after wound induction and treatment at 25°C with COz or DACP Excised tissue was treated with air or air + DACP for 10 h or with 60% COz for 6 h and then transferred to air (arrow) for an additional 4 h. Total RNA was extracted at time 0 h (lane 7), and from excised tissue treated with air (lane 6), DACP (lane 5) or CO:! (lane 4) after 6 h or air (lane 3), DACP (lane 2) after 10 h or 4 h after transfer from COz-enriched atmosphere to air (lane 1). Total RNA (20 pg per lane) was hybridized with 32P-labeled cDNA inserts of pCMW33 (Nakajima et al., 1990) (adapted from Mathooko, 1995).

its mRNA transcripts (Fig. 7A). The inhibition of ACC synthase activity and the accumulation of its mRNA transcripts, however, required continuous presence of CO2 (Mathooko, 1995). Comparison of the effect of CO2 with that of another inhibitor of ethylene action, viz. DACP, indicated that DACP promotes wound-induced ACC synthase activity and the accumulation of its mRNA, further supporting the view that woundinduced ACC synthase is regulated by a negative feedback control mechanism as shown in the model in Fig. 3. When various CO2 concentrations were tested, the expression of the wound-inducible ACC synthase gene always reflected the total ACC synthase activity (Kubo et al., 1995; Mathooko, 1995; Fig. 8A, B). The increased ACC synthase activity and its mRNA accumulation at CO2 levels above 20% could be related to stress effects of the gas. These observations further support the hypothesis that CO2 effect on wound-induced ACC synthase activity and gene expression could be through another mechanism other than inhibition of

RM. Mathooko / Postharvest Biology and Technology 7 (1996) 1-26

17

(4 1

2

3

4

5

6

7

8

I

I

_

I

DACP

( 80

I

I

40 20 CO, concentntion

IO (7%)

5 ) Wounded Conlml

Intact contml

Treatment Fig. 8. Effects of various CO2 concentrations and DACP on the expression of wound-induced ACC synthase gene (A) and ACC synthase activity (B) in mesocarp tissue of winter squash fruit held at 25°C for 8 h. Total RNA (20 kg per lane) from unwounded control (lane 8), wounded control (lane 7), wounded tissue treated with 5% (lane 6), 10% (lane S), 20% (lane 4), 40% (lane 3), 80% CO2 (lane 2) or DACP (lane 1) was hybridized with 32P-labeled cDNA inserts of pCMW33 (Nakajima et al., 1990) (adapted from Kubo et al., 1995).

ethylene action. Wound-induced ACC synthase gene expression in winter squash fruit was found to be repressed in the presence of ethylene and enhanced under conditions when ethylene action was inhibited by NBD (Nakajima et al., 1990). On the contrary, Mattoo et al. (1993) reported that the accumulation of a 1.8 kb wound-induced ACC synthase transcript in tomato fruit is ethylene-inducible and the presence of NBD inhibited wound-induced ethylene production, ACC synthase activity and the accumulation of its mRNA transcripts. These workers proposed that NBD might inactivate the ethylene receptor site. Whether CO2 functions in a similar manner remains to be elucidated. Since ACC synthase is encoded by a multigene family, these inhibitors of ethylene action may regulate different genes. CO2 also inhibits wound-induced ACC oxidase activity in mesocarp tissue of winter squash fruit (Mathooko et al., 1993a; Kubo et al., 1995; Mathooko, 1995). Expression of a wound-induced ACC oxidase gene pEAT1 from Arubidopsis thaliuna was enhanced by treatment with ethrel (an ethylene-releasing compound) (Gomez-Lim et al., 1993) further supporting the view that wound-induced ACC oxidase is under a positive feedback control mechanism. Recently, Kim and Yang (1994) reported that the increase in wound-induced ACC oxidase activity in mung bean hypocotyl is accompanied by increase in its mRNA transcripts and this accumulation of ACC oxidase mRNA transcripts was further enhanced by treatment of the excised tissue with ethylene while treatment with NBD suppressed completely the increase in the

18

EM. Mathooko 1 Postharvest Biology and Technology 7 (1996) l-26

enzyme transcripts. This observation further supports the view that wound-induced ACC oxidase is under a positive feedback control mechanism. CO2 may thus act in a similar manner in its inhibition of wound-induced ACC oxidase activity. 7. Modulation of ACC oxidase by CO2 A number of studies have previously demonstrated that CO2 stimulates ACCdependent ethylene production in leaf tissues (Kao and Yang, 1982; Yang and Hoffman, 1984). However, since the effect of CO2 was rapid and reversible, Kao and Yang (1982) proposed that CO2 exerts its effect by directly activating ACC oxidase in vivo rather than stimulating its synthesis. This enzyme had previously been thought to require tissue and membrane integrity and possibly a transmembrane ion gradient and, at least part of ACC oxidase, might be localized in the tonoplast (Yang and Hoffman, 1984; Abeles et al., 1992; Kende, 1993). However, in vitro activity of the enzyme was demonstrated (Ververidis and John, 1991) leading to its purification and characterization (Dong et al., 1992; Dilley et al., 1993; Fernandez-Maculet et al., 1993; Hyodo et al., 1993; Mathooko et al., 1993b; Poneleit and Dilley, 1993; Smith and John, 1993; Finlayson and Reid, 1994). These reports have indicated that ACC oxidase is activated by CO2 and requires both ascorbate and Fe2+ as co-factors, suggesting that it is a dioxygenase that belongs to the superfamily of Fe2+/ascorbate oxidases (McGarvey and Christoffersen, 1992). Dong et al. (1992) established the following stoichiometry for the C02-activated conversion of ACC to ethylene: ACC + ascorbate + 02

Fe2f,CPZ C2Hd + HCN + 2H20 + dehydroascorbate

+ CO2

Although CO2 activates ACC oxidase, it increases the V,, of the reaction (Fernandez-Maculet et al., 1993; Poneleit and Dilley, 1993; Finlayson and Reid, 1994; Rothan and Nicolas, 1994; Tian et al., 1994) and decreases the affinity of the enzyme towards its substrate, ACC (Fernandez-Maculet et al., 1993; Hyodo et al., 1993; Mathooko et al., 1993b; Poneleit and Dilley, 1993; Smith and John, 1993; Finlayson and Reid, 1994; Rothan and Nicolas, 1994). However, Tian et al. (1994) indicated that CO2 does not affect the apparent K, value of the enzyme towards its substrate, ACC, and suggested that in such a situation CO2 may act as a noncompetitive antagonist. Although CO2 lowers the affinity of the enzyme towards ACC, the rate of reaction is always greater in the presence of additional CO2 than in air (Mathooko et al., 1993b; Smith and John, 1993). The enzyme from melon (Smith et al., 1992), avocado (McGarvey and Christoffersen, 1992), winter squash (Mathooko et al., 1993b) and apple (Poneleit and Dilley, 1993) exhibits a non-linear time course during incubation which is not altered by the presence of COz, perhaps due to substrate inactivation of ACC oxidase (Mathooko et al., 1993b; Mathooko, 1995) or due to inactivation by the entire reaction mixture (Smith et al., 1994) during the enzyme catalytic action. This non-linearity is still not yet well understood and further research is needed in this direction. The HCO, also activates ACC

EM. Mathooko IPostharvest Biology and Technology 7 (1996) 1-26

19

oxidase; however, experimental evidence is available to suggest that COZ rather than the HCO, is the species that activates ACC oxidase (Fernandez-Maculet et al., 1993; Smith and John, 1993; Vioque and Castellano, 1994). The level of CO2 required to maintain maximum ACC oxidase activity varies widely. A concentration of 4-5% is required for ACC oxidase from apple (Dong et al., 1992; Fernandez-Maculet et al., 1993), winter squash (Hyodo et al., 1993; Mathooko et al., 1993b) and more than 10% for ACC oxidase from pear (Vioque and Castellano, 1994) and melon (Smith and John, 1993). These differences in optimal CO2 concentrations could be related to the different buffers and pH used. Smith et al. (1992) reported that the nature of buffers affect ACC oxidase activity, presumably due to formation of different Fe2+-buffer complexes and this may affect the CO2 concentration required to maintain maximum ACC oxidase activity. On the other hand, Mizutani et al. (1994) indicated that there is a negative correlation between pH and the CO2 concentration required for maximum ACC oxidase activity. This effect by pH may also account for the variation in the apparent K, value for ACC reported in the presence and/or absence of COz, since K, value for ACC increases as the pH is decreased (Mizutani et al., 1994). The affinity of ACC oxidase for ACC has also been shown to decrease with increasing CO2 concentration (Rothan and Nicolas, 1994). In some cases, wound-induced ACC oxidase (Mathooko et al., 1993b) and ACC oxidase from ripe fruit (Tian et al., 1994) for instance, the presence of exogenous CO2 during assay has little effect on the stimulation of in vivo ACC oxidase activity. This is taken to indicate that the CO2 resulting from wound-induced respiration and as a result of fruit respiration is sufficient for the stimulation of ACC oxidase activity and as such, supplemental CO2 would not be expected to stimulate ACC oxidase much. Inhibition of ethylene production in leaves by light (Kao and Yang, 1982; Preger and Gepstein, 1983) may, therefore, be explained by photosynthesis lowering the endogenous CO2 level in the leaves. A number of hypotheses have been put forward to explain the mode of activation of ACC oxidase by CO 2. Dong et al. (1992) observed that the enzyme was reversibly activated by CO2, leading to the conclusion that this was the more likely mechanism rather than any direct effect on the substrate. Further work by Fernandez-Maculet et al. (1993) confirmed this, with evidence based on an altered pH profile for the enzyme following pre-incubation with COz, and they suggested the formation of a carbamate on one of the amino groups of the enzyme. This probably involves carbamylation of a specific lysine residue within the protein (Poneleit and Dilley, 1993; Wilson et al., 1993) as is known to occur in ribulose-l$bisphosphate carboxylase (rubisco), the only other plant enzyme known to be activated by CO2 (Lorimer and Miziorko, 1980). Carbamylation of Lys2O’ of rubisco has been proposed as the basis for CO;! activation since decarbamylation inactivates the enzyme (Poneleit and Dilley, 1993). Indeed, using lysine-specific reagents, sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (SAMA), 2,4,6_trinitrobenzene sulfonic acid and sulfo succimidyl acetate, Ververidis and Dilley (1994) demonstrated that these reagents inhibit ACC oxidase activity in a manner suggesting the involvement of a single lysyl residue as the target for CO2

20

Rh4. Mathooko I Postharvest Biology and Technology 7 (1996) l-26

carbamylation and thereby serving as the basis for CO2 activation of ACC oxidase. There is good evidence that a single lysyl residue among the 29 present in the protein is required for ACC oxidase activity and that the presence of CO2 protects the enzyme activity loss upon treatment with the lysyl reagent SAMA (D.R. Dilley, pers. commun., 1994). In an in vivo system, Tian et al. (1994) proposed that the mechanism of CO2 stimulation of ACC oxidase may be ‘direct’ probably through interaction with a non-substrate binding site on ACC oxidase and that CO2 might combine reversibly with an ACC oxidase-ACC complex to increase V,,,,, of the reaction. The mechanism through which CO2 activates ACC oxidase is still not yet quite clear and warrants further investigation. 8. Concluding remarks In essence, the present review has attempted to integrate the current knowledge regarding the biochemical and physiological bases for the regulation of ethylene biosynthesis by CO2 as well as the possible mode of action of CO2 in the activation of ACC oxidase. The available information indicates that the regulation involves ACC synthase, the rate-limiting enzyme in the ethylene biosynthetic pathway and to some extent ACC oxidase depending on the mode of induction of ethylene biosynthesis. Depending on the mode of induction of ethylene production, CO2 also has some regulatory influence through its antagonistic effects on ethylene action. It still remains to be elucidated how CO2 regulates wound-induced ACC oxidase activity in a commodity-specific manner and its exact mode of activation of ACC oxidase. Since there are at least three forms of ACC oxidase including ethl, eth2 and eth3 from tomato (Bouzayen et al., 1993) which could probably be induced and/or synthesized in different tissues and at different developmental stages, it is quite possible that CO2 can affect each of these forms of ACC oxidase differently. Besides, more research needs to be undertaken to elucidate the regulation of ACC malonylation, especially the effect of elevated CO2 on ACC malonyltransferase activity, since this enzyme is a major factor limiting the availability of ACC for ethylene production (Mattoo and White, 1991). Following isolation of cDNAs for ACC synthase induced by various stimuli and treatment of tissue with the respective cDNA as probes, it has become apparent that the increase in ACC synthase activity is accompanied by a concomitant accumulation of its mRNA transcripts. Since the biochemical and physiological bases for the effect of elevated CO2 on ethylene biosynthesis are clear, attention should now be focused on understanding the regulation by CO2 of ethylene biosynthesis at the molecular level, focusing not only on ACC synthase but also on ACC oxidase. It is now certain that CO2 inhibits wound-induced ethylene production by suppression of ACC synthase gene expression at the transcriptional level. However, it remains to be elucidated how the mode of action of elevated CO2 in the regulation of woundinduced and probably auxin-induced ACC synthase enzymes is different from that of the other known inhibitors of ethylene action. The cloning of the CO2 stress-induced ACC synthase cDNA has not yet been accomplished. Cloning, characterization and studies on the expression of the CO2 stress-induced ACC synthase gene should

EM. Mathooko / Postharvest Biology and Technology 7 (1996) 1-26

21

further advance our understanding on tissues’ response to elevated CO2 in relation to ethylene production. Cloning of this ACC synthase cDNA thus represents the first step towards understanding CO2 stress-induced ethylene biosynthesis at the molecular level and may thus open the possibility of an improved tolerance of plant tissues to elevated CO* levels. On the other hand, cloning of ACC synthase gene(s) and regulation of their expression by CO2 opens the possibility of improving the postharvest longevity and quality of horticultural commodities by controlling the expression of the endogenous gene(s) through appropriate genetic engineering approaches. Acknowledgments

I thank Professor D.R. Dilley, Michigan State University, for allowing me access to his unpublished results on the mode of activation of ACC oxidase by CO2. References Abeles, EB., Morgan, PW. and Saltveit, Academic Press, New York, 414 pp.

M.E. Jr. (Editors),

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Adams, D.O. and Yang, S.F., 1979. Ethylene biosynthesis: Identification of l-aminocyclopropane-lcarboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA, 76: 170-174. Boller, 7: and Kende, Bouzayen,

H., 1980. Regulation

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Cheverry, J.L., Sy, M.O., Pouliqueen, J. and Marcellin, P., 1988. Regulation by CO2 of l-aminocyclopropane-1-carboxylic acid conversion to ethylene in climacteric fruits. Physiol. Plant., 72: 535540. Clijsters, H., De Proft, M., Marcelle, R. and Van Poucke, M. (Editors), 1989. Biochemical and Physiological Aspects Publishers, Dordrecht,

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Dilley, D.R., Kuai, J., Poneleit, L.S., Zhu, Y., Pekker, Y., Wilson, I.D., Burmeister, D.M., Gran, C. and Bowers, A., 1993. Purification and characterization of ACC oxidase and its expression during ripening Aspects

in apple fruit. In: J.C. Pech, A. Latche and C. Balague (Editors), Cellular and Molecular of the Plant Hormone Ethylene. Kluwer Academic Publishers, Dordrecht, pp. 46-52.

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Dong, J.G., Fernandez-Maculet, J.C. and Yang, SF., 1992. Purification and characterization of laminocyclopropane-1-carboxylate oxidase from apple fruit. Proc. Natl. Acad. Sci. USA, 89: 97899793. Fernandez-Maculet, J.C., Dong, J.G. and Yang, SF., 1993. Activation of l-aminocyclopropane-lcarboxylate oxidase by carbon dioxide. Biochem. Biophys. Res. Commun., 193: 1168-1173. Finlayson, S.A. and Reid, D.M., 1994. Influence of CO2 on ACC oxidase activity from roots of sunflower (Helianthus annuus) seedlings. Phytochemistry, 34: 847-851. Fuchs, Y. and Chalutz, E. (Editors), 1984. Ethylene: Biochemical, Physiological and Applied Aspects. Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, 348 pp. Gomez-Lim, M.A., Valdes-Lopez, V., Cruz-Hernandez, A. and Saucedo-Arias, L.J., 1993. Isolation and characterization of a gene involved in ethylene biosynthesis from Arabidopsis thafiana. Gene, 134: 217-221. Gorny, J.R. and Kader, A.A., 1994. The mode of CO;? action on ACC oxidase and its role in the inhibition of ethylene biosynthesis. HortScience, 29: 533 (Abstr.). Grodzinski, B., Boesel, I. and Horton, RF., 1982. Ethylene release from leaves of Xanthium strumarium L. and Zea map L. J. Exp. Bot., 33: 344-354. Hoffman, N.E., Yang, SF. and McKeon, ‘LA., 1982. Identification of l-(malonylamino)cyclopropane1-carboxylic acid as a major conjugate of 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor in higher plants. Biochem. Biophys. Res. Commun., 104: 765-770. Huang, PL., Parks, J.E., Rottmann, W.H. and Theologis, A., 1991. Bvo genes encoding l-aminocyclopropane-1-carboxylate synthase in zucchini (Cucurbita pepo) are clustered and similar but differentially regulated. Proc. Natl. Acad. Sci. USA, 88: 7021-7025. Hyodo, H., 1991. Stress/wound ethylene. In: A.K. Mattoo and J.C. Suttle (Editors), The Plant Hormone Ethylene. CRC Press, Boca Raton, FL, pp. 43-63. Hyodo, H., Hashimoto, C., Morozumi, S., Hu, W. and Tanaka, K., 1993. Characterization and induction of the activity of l-aminocyclopropane-1-carboxylate oxidase in the wounded mesocarp tissue of Cucurbita maxima. Plant Cell Physiol., 34: 667-671. Imaseki, H., 1991. The biochemistry of ethylene biosynthesis. In: A.K. Mattoo and J.C. Suttle (Editors), The Plant Hormone Ethylene. CRC Press, Boca Raton, FL, pp. l-20. Irving, D.E. and Honnor, L., 1994. Carnations: effects of high concentrations of carbon dioxide on flower physiology and longevity. Postharvest Biol. Technol., 4: 281-287. Ishizawa, K., Hoshima, M., Kawabe, K. and Esashi, Y., 1988. Effects of 2,5-norbornadiene on cocklebur seed germination and rice coleoptile elongation in response to carbon dioxide and ethylene. J. Plant Growth Regul., 7: 45-58. Kader, A.A., 1986. Biochemical and physiological basis for effect of controlled and modified atmospheres on fruits and vegetables. Food Technol., 40(5): 99-100, 102-104. Kader, A.A. and Morris, L.L., 1977. Relative tolerance of fruits and vegetables to elevated CO2 and reduced 02 levels. Mich. State Univ. Hortic. Rept., 28: 260-265. Kao, C.H. and Yang, SF., 1982. Light inhibition of the conversion of l-aminocyclopropane-1-carboxylic acid to ethylene is mediated through carbon dioxide. Planta, 155: 261-266. Kende, H., 1989. Enzymes of ethylene biosynthesis. Plant Physiol., 91: 1-4. Kende, H., 1993. Ethylene biosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol., 44: 283-307. Kende, H. and Boller, T, 1981. Wound ethylene and 1-aminocyclopropane-l-carboxylate synthase in ripening tomato fruit. Planta, 151: 476-481. Kerbel, E.L., Kader, A.A. and Roman, R.J., 1988. Effect of elevated CO2 concentrations on glycolysis in intact ‘Bartlett’ pear fruit. Plant Physiol., 86: 1205-1209. Kidd, F. and West, F., 1927. A relationship between the concentration of oxygen and carbon dioxide in the atmosphere, rate of respiration and length of storage in apples. G.B. Dept. Sci. Ind. Res. Rept. Food Investment Board, 1925, 1926, pp. 41-45. Kim, W.T. and Yang, SF., 1994. Structure and expression of cDNAs encoding l-aminocyclopropane1-carboxylate oxidase homologs isolated from excised mung bean hypocotyls. Planta, 194: 223229.

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