discs ( 1 989) Vertical bars indicate standard erro r
7.3 TH E EFFECT OF CARBON DIOXI D E ON EFE ACTIVITY
Carbon dioxide might stimulate EFE activity directly by binding to the EFE binding site, or indirectly by interaction with the System I ethylene receptor which regulates EFE activity.
7.3.1 The d i rect stimulato ry effect of carbon dioxide on EFE activity The main stimulatory effect of C02 on EFE activity was caused by directly binding of C02 to th� EFE binding site in discs of Granny Smith apple at all stag
�
tested and�
nripe Hosui.apple fruits. (A) C2H4 or C3H6 effect on respiration (B) C2H4 production C2H4 o r C3H5 effect on C2H4 production (C) Endogenous C2H4 level
(D)
ACC ACC synthase (E) EFE Non-climacteric Hosui (unripe) C02 i while C2H4 or C3Hs present Low No effect Low Very low Very low Low EFE after low tmperature storagei C02 effect on EFE activity
i n short storage ii
after long storage No effect
C02 effect on EFE biosynthesis No effect (F) C2H4 receptor System I Climacteric apple (unripe) (ripening)
Permanent rise No effect
Low High
i · No effect
Low High
Very low High
Low High Absent High i ii i i i i No effect
Bengochea et al. (1 980a,b), and Thomas and Yang (1 987) indicated that enzyme preparations such as IAA oxidase, peroxidase, lipoxygenase, pea ste m homogenates and microsomal preparations were able to convert ACC to ethylene although these enzymes display high Km values for ACC, from 3 to 389 mM. The present results showed that C02 directly stimulated E FE activity and this stimulatory effect of C02 on EFE activity was
dependent on exogenous ACC when the EFE activity assay was carried out in the presence of CHI (which inhibited possible EFE biosynthesis during incubation) and high (1 -5 m M) ACC concentrations. Thus it was necessary to test whether C02 stimulated ethylene production through other pathways. The eo++ ion which is known to act as an EFE activity inhibitor (Yu and Yang, 1 979), completely blocked the stimulatory effect of co2. Enzyme kinetics results showed that C02 significantly increased the maximum reaction rate of EFE without changing the Km for ACC. Th�s suggests that C02 stimulated EFE activity through the formation of an co2-EFE-ACC complex and/or EFE-ACC-C02 complex in Hosui and Granny Smith fruits (See 'Chapter 5').
7.3.2 Carbon dioxide may sti mulate EFE located at plasma membrane
Recently, Pech et a/.(1 989) and Bouzayen et a/.(1 990) demonstrated in vitro cultured cells of Vitis vinifera L. cv. Muscat and in leaf tissues e:f. �-"'
Hordeum vulgare L. and Triticum aestivum L. that there were probably two sites of ethylene production: (1 ) An external site located at the plasma membrane, capable of conve rting apoplastic ACC to ethylene, which was very sensitive to high osmotica (0.8 M mannitol); and (2) An intracellular site, possibly located in the tonoplast, which was capable of converting internal ACC to ethylene and which remained unaffected even under severe
plasmolysis. In other grape cells cultured in vitro ( Vitis vinifera L. cv. Gamay) and in pea leaves
(
Pisum sativum L.), only the intracellular site was thought to operate as ethylene production was almost unaffected by plasmo�
sis. Thus they concluded that in cells sensitive to osmotica, ethylene forming activity is mainly located at the plasmalemma with a small activity inside the cell probably at the tonoplast. In osmoticum insensitive cells, the bulk of ethylene production is intracellular.In this work, mannitol which cannot, or can only slowly, penetrate into the cell (Berliner and Martindale, 1 981 ; Evans and Ting, 1 973; Ruesink,
1 978), stimulated EFE activity in Hosui and Granny Smith fruit discs when treated at an isotonic concentration (0.4 M). This suggests that mannitol stimulated EFE located at the plasma membrane. I n discs of both fruit, EFE activity was higher in 0.8 M mannitol solution than in 0.4 M, but the tissue lost its capacity to respond to C02 at the high mannitol concentration. It could mean that activity of EFE, located at plasma membrane, is o nly stim ulated by C02 when in an isotonic solution. Pech et al. (1 989) and Bouzayen et al. (1 990) concluded that the EFE located in the plasma membrane was dependent on ACC present in the apoplast. Because discs from Granny Smith produced ethylene without the addition of exogenous ACC, it is possible that in this fruit another form of EFE is located at the tonoplast, which mainly converted endogenous ACC (stored in the vacuole) to ethylene (Pech et al., 1 989).
The isotonic potentials varied with species. The isotonic potentials of mannitol solution was shown to be 0.35-0.4 M for Granny Smith apple flesh tissues (with no significant changes occurring during ripening) (Harker,
1 986), 0.7 M for Kiwifruit (Joh n et al., 1 989) and 0.7-0.9 M for Delicious apple (Mattoo and Lieberman, 1 977). The effects of high osmotic potentials o n EFE activity also varied by either inhibiting or not affecting EFE activity; this effect would depend on whether EFE was located in plasma membrane o r tonoplast (Pech et al. , 1 989). John et al. (1 989) reported that the
m aximum EFE activity in kiwifruit membranes was observed when sucrose, mannitol and sorbitol were present in the medium at 0.7 M or at higher concentrations. Indeed their data showed that the maximum EFE activity was obtained at 1 .0 M mannitol. They also reported that the EFE activity was com pletely lost when the osmotic support was removed.
Although the mechanism by which high osmotica stimulate E FE activity is unknown (John et al. , 1 989), the explanation could be that some sugars, including mannitol, can stimulate EFE activity (see 'Chapter 3'). Thus the higher apparent EFE activity obtained in 0.8 M mannitol might be the consequence of the stimulatory effect of 0.8 M mannitol on EFE activity being greater in discs of both Hosui and G ranny Smith fruits than the
7.3.3 Carbon dioxide may stimulate EFE activity through altering the natu re of the ethylene receptor.
Libbenga and Mennes (1 987) reviewed hormone binding and its role in hormone action. They indicated that in hormonal systems, cells of different tissues and organs not only transmit signals, but they are also capable of detecting signals which they receive from other parts of the system, and responding to those signals in their own characteristic way. Receptor
activation triggers a molecular program which is simply a direct activation of a distinct set of enzymes. The normal experience with animal hormones is that receptors are C£_nfined to specific target organ. lt might be anticipated that receptors for plant hormones would be localized in hormone-responsive tissues, hence governing tissue sensitivity (Venis, 1 985). Libbenga and Mennes ( 1 987) also indicated that, i n general, chemical signals ultimately influence target calls, either by altering the activities or rates of synthesis of e.xisting proteins or by altering the synthesis of new ones. If the set of
responsive e nzymes is different in another type of target cell, then the same signal elicits a different response via a similar receptor-and-transduction
chain.
In plant/fruit systems, plant hormones (the present work, ethyrene) could bind to their receptors (in this case, the System I receptor) governing tissue responses (in this case, ethylene biosynthesis). Chemical signals (in this case, C02) could influence target cells (in Hosui and Granny Smith fruits) either by altering the activity or rates of synthesis of existing proteins or by altering the synthesis of new ones (in this case, EFE).
Veen (1 986) presented a theoretical model to explain the anti ethylene effects of silver thiosulphate and 2,5-norbornadiene. This model suggested how the receptors regulate ripening e nzyme activity and possible mechanisms of ethyle ne-action inhibitors, but it did not give any explanation for distinguishing between System I and System 11 ethylene receptors.
Silver ions and NDE are ethylene action inhibitors which inhibit ethylene action by binding at the ethylene receptor. In this work, using the nonclimacteric fruit, Hosui (in which only the System I ethylene receptor is thought to occur),- EFE lost the capacity to respond to C02 in the presence of Ag+. This suggested that Ag+ ions in some way inhibits the System I receptor which in turn regulates EFE activity.
lt has been reported that NDE is a competitive inhibitor of
autocatalytic ethylene synthesis (Sisler and Yang, 1 984) and ethylene action (Yang, 1 985; Yang and Hoffman, 1 984). Cycloheximide, a protein synthesis i nhibitor, inhibited the conversion of ACC to ethylene in mung bean
hypocotyls (Yu et al., 1 979), in preclimacteric cantaloupe tissue (Hoffman and Yang, 1 982) and in flavedo and albedo tissues of citrus fruits (Riov and Yang, 1 982b). In discs of Hosui and preclimacteric Granny Smith fruit, the combination of N DE and CHI inhibited EFE activity, and its inhibitory effect could be partially reve rsed by C02. In Granny Smith, when fruit was ripe, C02 could no longer reverse NDE inhibitory effect on EFE activity. Because E FE synthesis was inhibited by CH I, these experiments only measured the effect of C02 on EFE activity. NDE, an ethylene action inhibitor, inhibited EFE activity by binding to the System I receptor at the ethylene binding site, suggesting that at this more advanced ripening stage, the System I ethylene receptor regulates EFE activity. lt has been reported that C02 might
competitively bind to ethylene receptor with NDE at the ethylene binding site (Sisler and Yang, 1 984). A combination of NDE and C02 reversed the i nhibitory effect of NDE on EFE activity suggesting that C02 might reverse the inhibitory effect of NDE on EFE activity by binding to System I ethylene receptor at the ethylene binding site. When Granny Smith was ripe C02 still directly stimulated EFE activity, but lost its capability to stimulate EFE i ndirectly by binding to the System I receptor.
In the present work, EFE developed during coolstorage at 1 °± 1 oc in both Hosui and Granny Smith fruit. The response of EFE to C02 in Hosui and Granny Smith fruit discs were different; C02 stimulated EFE activity and its synthesis in preclimacteric Granny Smith fruit discs, but it only stimulated E FE activity in discs of more ripe Granny Smith and Hosui fruits. These results indicated that EFE was able to develop during storage in Hosui and G ranny S mith. The difference betwee n the nonclimacteric and climecteric fruits was their response to co2. The following hypothesis is prese nted to explain the above results:
EFE was measured in fruit tissue which consisted of very uniform cells in both Hosui and Granny Smith fruits. EFE is capable of developing in both fruits, but there is a difference in response to C02 between Hosui and preclimacteric G ranny Smith fruits, and betwee n Granny Smith fruit at different ripening stages. lt is suggested that EFE is regulated by the same recaptor (System I ethylene recaptor) but that ethylene recaptor has different functions which regulates EFE synthesis and EFE activity separately.
lt is suggested that there are two different forms of System I
receptors, System I-A1 ethylene recaptor which regulates E FE biosynthesis, and System I-A2 ethylene recaptor which regulates EFE activity, both of which are thought to be located in the same target cell. Unripe G ranny Smith fruit and Hosui fruit possess both System I-A1 and System 1-A2 receptors. Ethylene could bind with both of them to stimulate EFE synthesis and activity. C02 could combine with System I-A1 recaptor in preclimacteric Granny Smith fruit to form an activated recaptor which stimulates EFE synthesis. C02 can bind to System I-A2 recaptor in both preclimacteric Granny Smith and unripe Hosui fruits, but not from ripe Hosui and Gran ny smith fruits, to stimulate EFE activity (Table 7-1 ).
Table 7 -1 . Summary o f effects o f co2 o n EFE synthesis and its activity . Ho sui Effects of C02 Unripe Ripe Direct effect : + Indirect effect : System I -A1 System I -A2 + Type number of cell 1 2 Granny Smith P reclimacteric Ripe + + + 3 + 4
*Direct effect means that co2 directly affected EFE activity .
* * I ndirect effects mean that co2 regulated EFE activity or synthesis through binding with ethylene receptors , in which the System I -A1 regulates EFE synthesis , and the System I -A2 regulates EFE activity . * * * ' + ' and ' - ' represent C02 st�ulatory effect and no effect on EFE respectively .
Osborne (1 989) has indicated that there are numbe rs of target cells in plants where ethylene can induce specific effects. lt that even within uniform tissue, such as that found in the fruit there may be cells, or organs within cells, which may respond differently and selectively to ethylene, or treatments which influenced ethylene action. If it is suggested that there are 4 types of cells, or regions in cells, in fruit cortical tissue, then the above results could be explained according to their response to C02 and the mechanisms of the responses:
Type 1 cell: (to be found in unripe Hosui fruit).
C02 stim ulated EFE activity directly by interacting with the EFE binding site; C02 did not stimulate EFE biosynthesis indirectly through interacting with the System I-A1 ethylene receptor;
C02 stimulated EFE activity indirectly through interacting with the System I A2 ethylene receptor;
Type 2 cell: (to be found in ripe Hosui fruit).
C02 did not stimulate EFE activity directly by interacting with the EFE binding site;
C02 did not stimulate EFE biosynthesis indirectly through interacting with the System l-A1 ethylene receptor;
C02 did not stimulate EFE activity indirectly through interacting with the System l-A2 ethylene receptor;
Type 3 cell: (to be found in preclimacteric Granny Smith fruit).
C02 stim ulated EFE activity directly by interacting with the EFE bi nding site ; C02 stimulated EFE biosynthesis indirectly through interacting with the System I-A1 ethylene receptor;
C02 stimulated EFE activity indirectly through i nteracting with the System l A2 ethylene receptor;
Type 4 cell : (to be found in climacteric or in ripe Granny Smith fruit).
C02 stimulated EFE activity directly by interacting with the EFE binding site ; C02 did not stimulate EFE biosynthesis indirectly through interacting with the System l-A1 ethylene receptor;
C02 did not stimulate EFE activity indirectly through interacting with the System l-A2 ethylene receptor;
Hosui fruit may have Type 1 and 2 cells. When fruit develop
�
s from unripe to ripe, the type of cell changes from Type 1 to 2. Granny Smith fruit may have Type 3 and 4 cells. Fruit at the preclimacteric stage have Type 3 cells, while fruit at all climacteric stages have Type 4 cells.lt is possible that the types of cells described are morphologically distinct. However it is more likely that these types as described represent regions of the cells which change in their responsiveness to C02 with development of maturation and ripening.
7.4 A POSSIBLE MODEL OF THE REG U LATION OF EFE BY CAR BON