Ethylene production in rice bronzing leaves induced by ferrous iron

Plant and Soil 149: 227-234, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands. PLSO 9712

Ethylene production in rice bronzing leaves induced by ferrous iron

X.X. PENG and M. YAMAUCHI International Rice Research Institute, P.O. Box 933, Manila, Philippines

Received 28 July 1992. Accepted in revised form 11 December 1992

Key words: bronzing, iron, ethylene, peroxidase, rice

Abstract

Bronzing, a nutritional disorder of rice plants which is widely distributed in tropical lowlands, was induced by dipping the cut end of rice leaves into FeSO 4 solution (pH 3.5). Ethylene production; the activities of peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase; and the effects of Co 2*, aminoethoxyvinylglycine, Ag +, cycloheximide, and 1-aminocyclopropane-l-carboxylate, were investigated in the course of bronzing development. It was found that ethylene production could be stimulated up to about 20 times that of the control by Fe 2÷, and a peak could be reached at about 24 h after incubation. The Fe2+-treated leaves also had 10-fold higher peroxidase activity than the control, whereas in vitro enzyme activity was inhibited by Fe 2*. Cycloheximide retarded in vivo stimulation of peroxidase, indicating that in vivo stimulation resulted from inducing de novo synthesis of the enzyme. No changes in the activities of phenylalanine ammonia-lyase and polyphenol oxidase were observed. The results, obtained from the incubation of leaves with Co 2÷, aminoethoxyvinylglycine, Ag+, cycloheximide, or 1-aminocyclopropane-l-carboxylate, showed that ethylene production was the effect of Fe 2+ stress and that it was not involved in the process of bronzing development, which is probably an acclimation process to enable plants to cope with stress. The accelerated peroxidase activity may be associated with bronzing development. Abbreviations: ACC - 1-aminocyclopropane-l-carboxylic acid, AVG - aminoethoxyvinylglycine, EFE - ethylene forming enzyme, P A L - phenylalanine ammonia-lyase, P O D - peroxidase, P P O - polyphenol oxidase, S E - standard error

Introduction

Bronzing, a nutritional disorder of rice plants, is widely distributed in tropical lowlands and is caused by iron toxicity (Ponnamperuma et al., 1955; Tanaka and Yoshida, 1970). The symptoms generally include development of brown spots and yellowing.

It is understood that higher plants have two mechanisms of mobilizing and taking up iron from the soil (Marschner et al., 1986; R6mheld, 1987; Takagi et al., 1984). Dicots and nongrass monocots mobilize iron by acidification and ferric reduction, while it is normally absorbed as ferric chelate in grasses (including rice). Since

ferrous iron is easily taken up by plants, the uptake is not controlled by such mechanisms when Fe 2+ level is high (Bienfait, 1989). Thus, excessive Fe 2+ uptake is eventually the main cause of iron toxicity, which arises not only from the high iron concentration of the soil solution, but also from soil status (i.e., highly reduced conditions and low pH of the soil solution) (Tanaka et al., 1966; Yamauchi, 1989). In addi- tion, nutrient deficiency may also weaken the tolerance of plants for excessive iron and aggra- vate the incidence of iron toxicity (Ottow et al., 1982; Yamauchi, 1989). When soluble iron concentration in the soil exceeds a critical level, plants may suffer from iron toxicity. But the

228 Peng and Yamauchi

critical levels of Fe concentration vary greatly, ranging from ten to several hundred ppm (Tanaka et al., 1966). This is probally due to the different varieties and experimental conditions used.

The increase in POD activity in rice seedlings suffering from iron toxicity had been reported (Inada, 1965; Ota and Yamada, 1962). The products of reaction between iron and oxidized polyphenols are probably the cause of bronzing as has been proposed by Yamauchi (1989). Polyphenol is oxidized by POD (Ke and Saltveit Jr, 1988).

The fact that stress promotes ethylene production in plant tissues is universally accepted (Wang et al., 1990). It could thus be presumed that iron toxicity might cause stress ethylene to evolve, which then serves as a "second mes- senger" to induce other metabolic processes. Besides, it could be presumed that some enzymes-e.g., PAL, PPO, and POD, might be activated because they may be responsible for the formation of brown spots by increasing the production of polyphenols and their oxidation (Ke and Saltveit Jr, 1988). This paper aims to determine the relationship between ethylene production and bronzing development.

Materials and methods

Rice cultivation

Seeds of rice cultivar IR50, obtained from Inter- national Rice Germplasm Center, International Rice Research Institute, were sown in 4-liter pots containing Maahas clay loam soil with fertilizer (1 g N, 0.2 g P and 1 g K). Two seedlings per pot were grown until the reproductive phase.

Development of bronzing in excised rice leaves

The first and second leaves from the top were detached and placed in a test tube (2.5 × 20 cm), and the lower cut end dipped in solution with varying composition (pH 3.5, each treatment with 3 replications). Incubations were done in a growth chamber (26°C, 70% RH, dark), or

under normal room condition (24-27°C, ca. 10 h room light per day).

Determination of ethylene production

The treated detached leaf was enclosed in a test tube (1.5 x 30cm) with 1 mL of water. A vial containing 0.6 mL 40% KOH was placed in the tube to absorb CO 2 produced by the leaf. One mL of gas was withdrawn after 2h of dark incubation at 30°C. Ethylene concentration was determined by injecting 1 mL of gas samples onto a Alumina (Shimadzu) packed column maintained at 60°C in a Simadzu GC-8A gas chromatography equipped with a flame-ioniza- tion detector maintained at 120°C.

Extraction and assay of the enzymes

About 0.5 g of rice leaves was ground in 10 mL of 50 mM phosphate buffer (KH2PO4-KOH, pH 7.0). The homogenate was centrifuged at 10,000 g for 10 min. The pellet was washed once. The combined supernatants were used for assay of soluble POD and PPO activity. The pellet was suspended in 0.5 M KCI (dissolved in 50mM phosphate buffer, pH 7.0), and stirred at 25°C for 2 h. The solution was centrifuged at 10,000 g for 10 min. The supernatant was used for assay of ionically bound POD activity. The POD activity was determined using the method of Miller et al. (1990) with some modifications. The reaction mixture consisted of 1 mL of 50 mM phosphate buffer (pH 6.0), 1 mL of 0.3% H20 2, and l m L of 0.1% guaiacol. To initiate the reaction, 0.1 mL of enzyme was added. The increase in OD470 over time was observed at 30°C. One unit was defined as the amount of POD which increased 100 units of OD value in this reaction system. PPO activity was determined according to Miller et al. (1990), using 0.1M catechol (1,2-benzenediol) as substrate. PAL was extracted and assayed following the method of Hyodo and Yang (1971).

Measurement of chlorophyll content

Chlorophyll content was determined by a chlorophyll meter (SPAD-502, Minoruta, Tokyo). Each measurement was made on one side of the

midrib in the middle o f a leaf. Each t r ea tment had 3 replicat ions, and 4 leaves were used per repl icat ion.

Results

Induction of bronzing

Bronz ing was induced by dipping the cut end of rice leaves into F e S O 4 solution. In this case, Fe

Fe uptake (rag g-I FW) 4

3

O v r ~ . , ' ' - - i

0 80

7

20 4 0 60

Incubation time (h) Fig. 1. Fe uptake in detached leaves. F e S O 4 (0.89-5.39 mM) was applied through the cut end of a detached leaf.

Ethylene production in rice bronzing leaves 229

may be taken up th rough t ranspirat ion. Mea- surement of Fe up take f rom the solut ion showed that it increased linearly with increasing incubation t ime and Fe concent ra t ion in solut ion (Fig. 1), which suggests that Fe uptake is p redomin- antly a physical process. The small, dark b rown spots which appeared at the tip of the leaves as early as 6 h after incubat ion ex tended gradual ly but were generally confined to the upper por t ion of the leaf. W h e n incubat ion t ime was pro longed, the leaf became discolored (yel low with spots of brown). A prel iminary study noted that the symp tom which deve loped in the growth chamber was similar to that seen under normal room condit ion. We repor t here the results obta ined f rom exper iments conduc ted under normal room condit ion.

The application of K2SO 4 at pH3.5 to the leaves nei ther p roduced bronzing nor increased ethylene product ion and P O D activity (Table 1). The result negates the possibility that SO~ in FeSO 4 or its acidity cont r ibuted to bronz ing deve lopment .

Stimulation of ethylene production by ferrous iron

As shown in Figures 2 and 3, e thylene product ion was significantly s t imulated by Fe 2+. The product ion was increased as the incubat ion t ime proceeded , reached a peak at about 2 4 h , and thereaf ter decreased. W h e n the concen t ra t ion was increased, e thylene produc t ion was nearly

Table 1. Comparison of the effects of some chemicals on bronzing, ethylene production and POD activity

Treatment POD activity C2H 4 production Bronzing score

(unitsg 'min t) (%) (nLg t h t) (%)

Control 3.67 100 3.70 100 0 F e S O 4 19.21 523 57.75 1559 7.0 K2SO 4 3.78 103 2.52 68 0 ACC 3.78 103 63.36 1771 0 C o 2~ + F e S O 4 11.41 302 6.56 177 7.0 AVG + FeSO 4 11.52 314 5.33 144 7.3 Ag ~ + AVG + F e S O 4 - - 7.59 205 7.8 CH + F e S O 4 1.83 50 0.33 9 2.8

The leaves were pretreated with H20 [for treatments with FESO4(3.57 mM), K2SO 4 (3.57mM), and ACC (2mM)], C o C l 2

(2 mM), AVG (0.05 raM), cyeloheximide (CH, 0.5 mM), or AgNO 3 (0.14 mM)+ AVG for 8 h, then transferred to a solution with FeSO 4 (3.57 raM) + corresponding inhibitor, K2SO4, and ACC. For the control H20 was supplied throughout. POD activity and ethylene production were determined 24 h after pretreatment. Bronzing was scored 72 h after pretreatment according to the International Rice Testing Program's method (1980).


Ethylene production (nLg -1FW h -1 )

40

30

20

10

0

5 . 5 7 m M Fe

I I I

0 20 40 60 80

Incubation time (h) Fig. 2. Ethylene production in detached leaves treated with ferrous iron.

Ethylene production (nLg "1FW h "1) 80

• •

6 0

4 0

2O

0 q I I 0 1 2 5 4 5 6

Fe concentration (mM) Fig. 3. Effect of Fe concentrat ion on ethylene production. The leaves were supplied with FeSO 4 at various concentrat ions for 24 h.

proportionally increased at low concentration (0-1 .79mM), reached maximum, and then slightly decreased.

Induction of POD activity by ferrous iron

It was found that the Fe-treated leaves had higher soluble and bound POD activity than the

control (Fig. 4). The activities were continuously increased as incubation time was extended. The extent of enhancement could reach up to more than 10 times. This enhancement was eliminated when cycloheximide was applied (Fig. 5). In vitro POD was inhibited by Fe 2÷ at the concentration of 17.8-71.6/zM (Fig. 6). These results suggest that the increase in POD activity is attributed to the induced de novo synthesis of the enzyme and that the interaction of inorganic ions with enzymes in vitro may be different from the interaction in vivo (Mittal and Dubey, 1991).

Interrelationship between bronzing, ethylene production, and POD activity

The relationship between bronzing and ethylene and some enzyme activities was investigated (Table 1, Fig. 7). When the leaves were treated with ACC, the precursor of ethylene synthesis (Gepstein and Thimann, 1981), ethylene production was increased 17-fold at 24 h after incubation, but no bronzing symptom was induced. Co 2÷ and AVG, the inhibitors of ACC synthet- ase and EFE, respectively (Gepstein and Thimann, 1981), retarded the Fe2÷-induced ethylene production, but bronzing was not alle- viated. Even when Ag ÷, an antagonist of ethylene binding to the acceptor (Gepstein and Thimann, 1981), was added together with AVG, there was still no inhibitory effect on bronzing development, ruling out the possibility that ethylene is involved in bronzing development.

Cycloheximide, an inhibitor of protein synthesis, inhibited not only the induction of POD activity by Fe 2÷ but also delayed bronzing development (Table 1, Fig. 7). Chlorophyll degradation caused by Fe 2÷ was also restrained by cycloheximide (Fig. 7). These results, however, are not enough to conclude that POD is responsible for bronzing development because cycloheximide may block many of the physiological processes.

Discussion

Rice roots have the iron excluding power which prevents excess Fe 2+ from penetrating into plants (Yoshida, 1981). When the power is not


P O D a c t i v i t y ( u n i t s g-1 F W min -1)

6 0 S o l u b l e 6 Bound

3.572 mM Fe 4O 4

2O 2

O ~ W v i = ~ O, I I t 0 20 40 60 80 100 0 20 40 60 80

Incubation t i m e ( h )

Fig. 4. POD activity in detached leaves treated with ferrous iron. Incubation time indicates the time after the start of FeSO~ (3.57 mM) application.

Soluble POD activity (%) 120

E

10

0 2 4

f i f / f J f i f J f J f J f J f f

, - - f J

°~ f I

e- / / " 0 f i ¢~ J J ~ J J

E / /

tO / ' J

÷

LU f i J J

"Z

4 8

Soluble POD activity (units g-1FW mir~ 1) 20

Incubation time (h)

Fig. 5. Effect of cycloheximide on the induction of POD activity by ferrous iron.

s t r ong e n o u g h , Fe z+ en te r s in to roo t t issue and t hen t r a n s p o r t e d to the shoot . O x y g e n and h y d r o x y l - f r e e rad ica l s a re f o r m e d when Fe 2+ is ox id i zed , and then m a y incur the p e r o x i d a t i o n of

lOOq

80

60

40

20

O 0 20 4 0 6 0 80

Fe concentration (pM) Fig. 6. Effect of ferrous iron on in vitro POD activity. POD was isolated as described in "Materials and methods". F e S O 4

was directly added to the reaction mixture of POD assay (see "Materials and methods"), then activity was measured spec- trophotometrically at OD470.

m e m b r a n e , nick D N A , o r inac t iva te e n z y m e s (Bienfa i t , 1989). A l t h o u g h the ac t ion of the radicals and the i r effect on p lan t m e t a b o l i s m a re

Chlorophyll content (%) 120

80

Fe + 0.5mM.= c,~cloheximide

4 0 -


Bronzing score

8

6

4

2

0 i I O' o 4 0 8o 12o o loo

Fe B

20 4 0 60 80

Incubation time (h) Fig. 7. Effect of cycloheximide on bronzing and chlorophyll degradation. Changes in chlorophyll content were determined with chlorophyll meter. Bronzing was scored according to the International Rice Testing Program's method (1980).

not well known at the cellular level, the induction of bronzing and POD activity and stimulation of ethylene production reported here might directly or indirectly be elicited by those radicals.

That various kinds of stresses including certain chemicals such as Ag, SO2, and Cu can stimulate ethylene production has been well documented (Wang et al., 1990). Our findings revealed that Fe is another effective stimulant to ethylene production. Inasmuch as bronzing appears to be a complex process of deposition of brown pig- ments (russet) and degradation of chlorophyll associated with senescence, ethylene related to these two aspects should be considered.

In recent years, ethylene has received increasing attention as a potential regulator of senescence in vegetative tissues. But the argument still exists because differing patterns of ethylene production have been reported in senescing leaf tissues, and the discrepancy between the large acceleration of ethylene production and the much smaller acceleration of senescence rate in response to ACC suggests some limit on the senescence-accelerating effect of elevated rate of ethylene production (Goldthwaite, 1987). It was reported that ethylene was able to hasten the degradation of chlorophyll so as to promote

senescence (Aharoni and Lieberman, 1979; Gepstein and Thimann, 1981).

Russet can also be induced by application of ethylene to lettuce leaves (Ke and Saltveit Jr, 1988). They found that 1 -3ppm of ethylene induced the formation of brown spots on the midrib of the leaf, which, in their opinion, resulted from the activation of POD, PPO, PAL, and IAA oxidase by ethylene. In this study, however, we found that the stimulated ethylene production was the effect of iron toxicity and not in any way involved with the process of bronzing development. This conclusion is based on the following evidences (Table 1): (1) the quantities of ethylene accumulation by applying ACC to the leaves failed to induce any symptom of bronzing; (2) the inhibition of ethylene production by Co 2÷ and AVG in Fe2+-treated leaves did not alleviate bronzing development; and (3) PAL and PPO activities in treated and nontreated leaves were too small to be detected (data not shown).

Although POD activity was significantly increased in Fe2+-treated leaves, it seems not to be related to ethylene action because the large acceleration of ethylene production by ACC failed to stimulate POD activity and it still

increased even when Fe2+-induced ethylene production was inhibited (Table 1). It remains to be studied whether or not Fe2+-induced ethylene is directly produced by the increased POD activity. According to Gasper et al. (1985), the stimulus (stress) may first activate basic POD, which then serves as ACC oxidase and IAA oxidase. The resulting ACC and IAA oxidase-mediated changes in ethylene production would further induce other metabolic processes (Gasper et al., 1985; Kevers et al., 1984; Osswald et al., 1989).

Since the production of stress ethylene is often concomitant with visible leaf injuries, as well as latent lesions or cellular disturbance (Wang et al., 1990), it has been suggested as an indicator of the extent of injury from the environmental stresses. Whether or not the Fe-stressed ethylene production can be successfully used for diagnosing or screening rice varieties tolerant to iron toxicity is being investigated.

On the other hand, Fe-stressed ethylene is probably part of the acclimation process that plants develop to cope with the stress conditions. This presumption is based on the fact that ethylene can stimulate the formation of aerenchyma in plant tissues, and aerenchyma aids in the passive transfer of 0 2 from leaves to roots, which subsequently diffuses into the rhizosphere according to the prevailing 0 2 concentration gradient (Jackson, 1985; Smirnoff and Crawford, 1983), so as to increase the iron excluding power. In some other stresses, responsive ethylene is formed to alleviate salinity, higher temperature, and osmotic stresses during germina- tion and plant establishment (Khan et al., 1987).

We found that POD activity was stimulated by treating the leaves with Fe 2+ (Fig. 5). It has been reported that POD can also be stimulated by other different kinds of stresses (e.g., low temperature, SO 2 stress, salinity, water stress, etc.) such that it is often called a "stress enzyme" (Edreva et al., 1989; Mittal and Dubey, 1991; Wang et al., 1990). Accordingly, POD has been accepted as a responsively defensive factor, probably by preventing incipiently the formation of stress-induced free radicals, and has already been applied as a biochemical marker for diagnosing cryoresistance in cereals (Mittal and Dubey, 1991; Savich and Peruanskii). On the other hand, others opine that the accelerated


POD activity may also indicate the severity of damage, probably resulting from lipid peroxida- tion of cell membranes (Edreva et al., 1989). Now it can be easily detected that the case of stress POD is very similar to that of stress ethylene. More interestingly, there are publi- cations reporting that both ethylene and POD can stimulate each other (Abeles et al., 1989; Gasper et al., 1985; Osswald et al., 1989).

Acknowledgements

This work is part of a collaborative project between the Government of Japan and I R R I - the development of stabilization technology for rice double cropping in the t ropics- sponsored by the Government of Japan. The authors thank Dr. S Peng for his technical assistance in chlorophyll measurement.

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Ethylene production in rice bronzing leaves induced by ferrous iron