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The effects of 2,2′-bipyridine and other metal chelators on the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in rice

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The effects of 2,2’-bipyridine and other metal chelators on the conversion of

l-aminocyclopropane-l-carboxylic

acid to ethylene in rice

Fei-Yi Tsai & Chiang Huei Kao*

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China *Author for correspondence

Received 1 August 1993; accepted 14 September 1993

Key words: l-aminocyclopropane-1-carboxylic acid, l-aminocyclopropane-1-carboxylic acid oxidase, 2,2’-

bipyridine, ethylene, 8-hydroxyquinoline, Oryza sativa, 1, IO-phenanthroline Abstract

Effects of metal chelators, 2,2’-bipyridine, 8-hydroxyquinoline and 1 ,lO-phenanthroline, on the conversion of l-aminocyclopropane-I-carboxylic acid (ACC) to ethylene in detached leaves of light-grown rice (Oryza

sativa) seedlings and detached shoots of etiolated rice seedlings were investigated. Metal chelators strongly

inhibited the in vivo ACC oxidase activity in detached leaves and detached etiolated shoots. This inhibition could be partially recovered by Fe2+. Our results support the notion that Fe2+ is an essential cofactor for the conversion of ACC to ethylene in vivo.

Abbreviations: ACC = l-aminocyclopropane-l-carboxylic acid; BP = 2,2’-bypyridine; HQ = 8-hydroxyl-

quinoline; MJ = methyl jasmonate; PA = 1, IO-phenanthroline; Put = putrescine.

1. Introduction

The final step in the biosynthesis of ethylene is catalyzed by ACC oxidase [12]. The strong inhibition of the conversion of ACC to ethylene by several chelators in intact tissues [l] indicates the involvement of a metal in the activity of ACC oxidase operating in vivo. The participation of a transition metal for the conversion of ACC to ethylene has been suggested [2, 3, 10, 121. Recent work indicates that in vitro and in vivo ACC oxidase activity requires divalent iron as a cofactor [4, 8, 111.

Unlike many other plant systems, ethylene biosynthesis in detached rice leaves was found to be promoted by polyamines [7] and inhibited by water stress [5]. Thus, rice leaves seem to be an interesting and unusual system to study ethylene biosynthesis. In the present study detached leaves of light-grown rice seedlings and detached shoots of etiolated rice seedlings were used to examine the effects of BP and other metal chelators (HQ

and PA) on the conversion of ACC to ethylene

in vivo.

2. Materials and methods

Seedlings of rice (Oryza sativa cv. Taichung Native 1) were grown in hydroponic culture as described previously [6]. The apical 3cm of the third leaves of 12-day-old light-grown seedlings were used for the experiments. For those experiments in which etiolated shoots of seedlings were used, the seeds, after sterilization, were allowed to germinate in Petri dishes containing filter papers moistened with Tris buffer (pH 7.0) at 27°C in the dark. After 3 days of germination, seedlings were transferred to a second Petri dish containing Tris buffer or 2 mM metal chelators in Tris buffer and incubated under dark conditions for a further 3 days. Etiolated shoots were then excised and used for the experiments.

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20 15 i c b E - t 10 0 2 G 5 0 I Contro: PA HQ BP Treatments T

Fig. 1. Effects of BP, HQ and PA on in vivo ACC oxidase activity in detached rice leaves. Detached rice leaves were pretreated with ACC (10mM) for 2 h and then treated wtih Tris buffer

(IOmM, pH 7.0) or Tris buffer containing metal chelators (5 m&I’) in darkness. Ethylene production was assayed after 6 h of treatment. Bars indicate standard error (n = 4).

For the determination of in viva ACC oxidase activity, leaf segments or excised shoots were pretreated with 10mM ACC for 2 h, then washed, blotted dry and transferred to test tubes (12.4 ml) containing treatment solution (1 ml). The test tubes were capped immediately and incubated at 27 “C in the dark. Ethylene was measured by gas chromatography at the times indicated as described previously [9].

3. Results

All metal chelators tested significantly inhibited the conversion of ACC to ethylene in detached rice leaves (Figure 1). However, PA was less effective than HQ and BP in inhibiting in vivo ACC oxidase activity. Inhibitory effect on in viva ACC oxidase activity caused by BP was detected within 2 h of application (Figure 2).

The reversal of the inhibition of the in vivo ACC oxidase activity in detached leaves caused by BP

-i 40 c 7n z 30 g" 20 Q) 5 10 ii 0 O-BP . +BP o-1 2-3 4-5 6-7 Time, h

Fig. 2. Changes with times in in viva ACC oxidase activity in detached rice leaves treated with BP. Detached rice leaves were pretreated with ACC (lOmA4) for 2 h and then treated with BP (SmM) in darkness. Rates of ethylene production were determined at the times indicated. Bars indicate standard errors

(n = 4).

was studied using a range of divalent cations (Figure 3). Inhibition caused by BP could be partially reversed by Fe2+ and Cu2+. However, Zn2+ and Mn2+ had no effect.

Our previous work showed that MJ and putescine (Put) were effective in promoting the conversion of ACC to ethylene 15, 71. If in vivo

ACC oxidase requires a metal as cofactor, then MJ or Put would be unable to promote in vivo

ACC oxidase activity in BP-pretreated detached

Control Fez+ Cu” Zn”

Treatments

1

Mn*+

Fig. 3. Reversal of the inhibitory effect on in viva ACC oxidase activity of BP by various metals in detached rice leaves. Detached rice leaves were pretreated with BP (2 mM) for 2 h and then treated wtih sulfate salts of various metals (1 mM) for 3 h in the dark. Detached rice leaves were then fed with ACC (10mM) and their ethylene production was determined after 2 h in the dark. Bars indicate standard errors (n = 4). Ethylene production in excised leaves pretreated with water and treated in the absence of metals was 23.3 l 1.6 nl g-’ h-l_

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0

Cl:-BP Fg:+Bp

Control MJ Put

Treatments

Fig. 4. Effect of BA on MJ- and Put-enhanced in vivo ACC oxidase activity in detached rice leaves. Detached rice leaves were pretreated with or without BP (2 mM) for 2 h and then treated with MJ (45 PM) or Put (5 mM) for 3 h in the dark. Detached rice leaves were fed with ACC (10mM) and their ethylene production was determined after 2 h in the dark. Bars indicate standard errors (n = 4).

leaves. As indicated in Figure 4, MJ and Put were indeed unable to promote the conversion of ACC to ethylene in BP-pretreated detached rice leaves.

To understand whether the effects of metal chelators on in vivo ACC oxidase activity are specific for green leaf tissue, the effects of metal chelator on in vivo ACC oxidase activity in etiolated shoots from 6-day-old dark-grown seedlings were also studied. As found for light- grown leaves, all metal chelators tested were effective in inhibiting the in vivo ACC oxidase activity in etiolated shoots (Figure 5).

Metal deficiency in shoots can be achieved by growing rice seedlings in the presence of metal chelators. With these shoots were excised and their in vivo ACC oxidase activity was measured, ACC oxidase activity was found to be significantly less than that of shoots excised from seedlings growing in the absence of metal chelators (Figure 6). In vivo ACC oxidase activity in shoots starved for metals could be partially recovered by Fe2+ or Cu2+ (Figure 7). Both Fe2+ and Cu2+ were able to recover in vivo ACC oxidase activity in shoots from seedlings grown

r

40 -i 30 ?n ‘: f 20 0) ‘; 5 W 10 0 -

II

Control i HQ Treatments T

1L

BP

in PA. However, in vivo ACC oxidase activity in shoots excised from seedlings grown in BP or HQ could only be partially recovered by Fe2+.

1

Fig. 5. Effect of BP, HQ and PA on in vivo ACC oxidase activity in detached shoot of rice seedlings germinated in darkness. Etiolated shoots were excised from 6-day-old rice seedlings growing in Tris buffer (IOmM, pH 7.0). Detached shoots were pretreated with ACC (10 mM) and then treated with Tris buffer (IOmM, pH 7.0) or Tris buffer containing metal chelators (5 mM) in darkness. Ethylene production was determined after 2 h of treatement. Bars indicate standard errors (n = 4).

bnrol PA HO BP

Fig. 6. In vivo ACC oxidase activity in detached shoots starved for metals. Starvation of metals in shoots was achieved by growing seedlings in metal chelators (2mM) in darkness. Detached shoots were treated with ACC (1OmM) and their ethylene production was measured after 2 h in darkness. Bars indicate standard errors (n = 4).

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25 20 15 10 5 0 BP

1

10 8 6 4 2 0 HQ 1

ltmm

H20 Fe2+ Cu2+ Zn2+ AIn*+ Mg2+ Fig. 7. Reversal of in vivo ACC oxidase activity by metals in etiolated shoots excised from seedlings grown in metal chelators (2mM). Detached shoots were pretreated with ACC (10mM) for 2 h and then treated with sulfate salts of various metals (1 mM) in the dark. Ethylene production was measured at 2 h after treatment. Bars indicate standard errors (n = 4). Ethylene production in etiolated shoots excised from seedlings grown in buffer solution and then treated in the absence of metals was 28.5 f 0.9nlg’ h-‘.

4. Discussion

Apelbaum et al. [l] reported that metal chelators strongly inhibited EFE activity. They concluded that the copper ion was involved in the conversion of ACC to ethylene. However, recent investigation by Bouzayen et al. [4] showed that Fe2+ was required for in vivo ACC oxidase activity. The present investigation demonstrated that metal chelators (BP, PA and HQ) tested were effective

in inhibiting in vivo ACC oxidase activity in both detached leaves and etiolated-shoots. Our results also showed that inhibition of ACC oxidase activity caused by metal chelators was partially recovered by Fe2+. The partial recovery

of in vivo ACC oxidase activity by adding CL?’

to BP-treated detached leaves and to shoots excised from seedlings grown in the presence of PA most likely resulted from the displacement of Fe” from PA or BP complex by Cu2+ as suggested by Bouzayen et al. [4]. It is concluded that Fe2+ is essential for the conversion of ACC to ethylene in rice.

Acknowledgements

This work was supported by a research grant from the National Science Council of the Republic of China (NSC82-0 115COO2- 1238B).

References

1. Apelbaum A, Burgoon AC, Anderson JD, Solomos T and Liberman M (1981) Some characteristics of the system converting l-aminocyclopropane-I-carboxylic acid to ethylene. Plant Physiol 67: 80-84

2. Baldwin JE, Jackson DA, Adlington RW and Rawlings BJ (1985) The stereochemistry of oxidation of l-aminocyclo- propane-1-carboxylic acid. J Chem Sot Chem Commun 1985: 206

3. Boller T, Herner RC and Kende H (1979) Assay for and enzymatic formation of an ethylene precursor, l-aminocyclopropane-I-carboxylic acid. Planta 145: 293-303

4. Bouzayen M, Felix G, Latche A, Pech JC and Boller T (1991) Iron: an essential cofactor for the conversion of I-aminocyclopropane-I-carboxylic acid to ethylene. Planta 184: 244-247

5. Chen CT and Kao CH (1990) Comparative study of the metabolism of l-aminocyclopropane-l-carboxylic acid and senescence of water-stressed and ABA-treated excised rice leaves. Plant Cell Physiol 31: 463-468

6. Chen CT, Chou IT and Kao CH (1990) Senescence of rice leaves XX. Changes of proton secretion during senescence. Plant Sci 66: 29-34

7. Chen SL, Chen CT and Kao CH (1991) Polyamines promote the biosynthesis of ethylene in detached rice leaves. Plant Cell Physiol 32: 813-817

8. Fernandez-Maculet JC and Yang SF (1992) Extraction and partial characterization of the ethylene-forming enzyme from apple fruit. Plant Physiol 99: 751-754 9. Kao CH and Yang SF (1982) Light inhibition of the

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ethylene in leaves is mediated through carbon dioxide. 11. Ververidis P and John P (1991) Complete recovery in viva of Planta 155: 261-266 ethylene-forming enzyme activity. Phytochemistry 30: 725-727 0. Pirung MC (1983) Ethylene biosynthesis. 2. Stereo- 12. Yang SF and Hoffman NE (1984) Ethylene biosynthesis chemistry of ripening, stress, and model reactions. J Am and its regulation in higher plants. Annu Rev Plant Physiol

數據

Fig. 2.  Changes  with  times  in  in  viva  ACC  oxidase  activity  in  detached  rice  leaves treated  with  BP
Fig.  5. Effect  of  BP,  HQ  and  PA  on  in  vivo  ACC  oxidase  activity  in  detached  shoot  of  rice  seedlings  germinated  in  darkness
Fig.  7.  Reversal  of  in  vivo  ACC  oxidase  activity  by  metals  in  etiolated  shoots excised from  seedlings  grown  in  metal  chelators  (2mM)

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