Inhibition of root growth by spermidine is not due to enhanced production of ethylene

ELSEVIER

Plant Science

108 (1995) 121-124

Inhibition of root growth by spermidine is not due to enhanced production of ethylene Elvira Reaa, Giovanna Di Montea, Marina de Agazio*b aIstituto per la Nutrizione delle Piante, via della Navicella, 2 00184 Rome, Italy bIstituto di Biochimica ed EcofSologia Vegetale. Area aWa Ricerca, via Salaria Km 29 ooO16 Monterotondo Scala, Rome, Italy

Received 2 December 1994;revision received 31 March 1995;accepted 3 April 1995

Pretreatment of 3-day-old maize seedlings with spermidine induces an enhancement of ethylene production from roots compared to control tissues with no effect on l-aminocyclopropane-l-carboxylate oxidase activity and laminocyclopropane-1-carboxylic acid level, accompanied by a strong inhibition of root growth. Inhibitors of ethylene synthesis, cobalt chloride and aminoethoxy-vinylglycine, markedly inhibited ethylene production in both control and spermidine-pretreated roots. The decrease in ethylene production did not result in reversing the inhibition of root growth induced by spermidine. Results suggest that the stimulation of ethylene by spermidine, probably due to more available S-adenosylmethionine, is not responsible for the inhibitory effect of spermidine on root growth. This statement is substantiated by the ineffectiveness of silver thiosulfate, a known inhibitor of ethylene action, to remove spermidine-mediated inhibition of root growth. Keywords: Zea mays L.; Ethylene; Inhibitors;

Roots; Spermidine

1. Introduction Spermidine applied to 3-day-old maize seedlings through roots, induces several changes in the metabolism and structure of the primary root such as enhancement of K+ uptake and efflux [1,2], increase of putrescine titer [3] and a strong inhibition of root growth rate [4]. In this paper we focusAbbreviations: ACC,

I-aminocyclopropane-Icarboxylic

acid; ACCO, l-aminocyclopropane-l-carboxylate oxidase; AdoMet, Sadenosylmethionine; AVG, aminoethoxy-vinylglycine; Spd, spermidine; STS, silver thiosulfate. l Corresponding author.

ed our attention on the latter effect suggesting a possible implication of ethylene in the inhibition of root growth rate induced by Spd. Three different considerations prompted us to formulate this hypothesis: (1) Spd stimulates ethylene production in a few systems as reported by Pennazzio and Roggero [5], Chen et al. [6] and Botha and Whitehead [7]; (2) exogenous ethylene at high concentrations suppresses root elongation and increases radial expansion in maize [8]; (3) ethylene and Spd both share AdoMet as a biosynthetic intermediate and they are mutually inhibitory with respect to each other’s biosynthesis [9, lo]. The ad-

0 1995 Elsevier Science Ireland Ltd. All rights reserved 0168-9452/95/$09.50 SSDI 0168-9452(95)04134-G

122

E. Rea et al i Plans Science IOH i !995]

dition of exogenous Spd to maize roots could decrease the biosynthesis of endogenous Spd making AdoMet more available for ethylene biosynthesis; the increased ethylene production could then inhibit root growth rate. On the other hand, there is no convincing evidence that ethylene produced endogenously is involved in the restraining of root extension as suggested by lack of stimulation observed in the presence of ethylene-synthesis inhibitors [I 1,121. This paper examines the effect of exogenous spermidine on ethylene biosynthesis, ACCO activity and ACC level in the absence and presence of several inhibitors of ethylene biosynthesis in maize roots. 2. Materials and methods 2.1. Plant material Maize seeds (Zea mays L., hybrid line Plenus V516), supplied by the Dekalb Centre (Chiarano, Italy) were continuously rinsed with tap water for 8 h and then germinated in the dark at 27°C in a controlled growth chamber over 3 layers of filter paper wetted with a 0.5 mM CaS04 solution. 3day-old seedlings, selected on the basis of their root length (2.5 cm), were incubated for 3, 6 and 18 h, in the dark at 27”C, in plastic trays containing 0.5 mM CaS04 with or without Spd (1 mM), CoCl, (0.1 mM), STS (1 mM), AVG (0.01 mM). alone or in combinations as indicated. After incubation, 5 roots for each sample were excised and collected in order to measure ethylene production, level of free ACC and ACCO activity. AVG, ACC and Spd were purchased from Sigma. STS was prepared by mixing equal volumes of 2 mM AgNO, and 8 mM Na2S203. Increase in root length was usually measured 24 h after each treatment. 2.2. Ethylene measurements Five roots were gently introduced in lo-ml vials, flushed with ethylene-free air, sealed with rubber stoppers and incubated in the dark at 27°C. After 1 h, a 1 ml gas sample was withdrawn with a syringe and injected into a Carlo Erba Mega 2 series gas chromatograph fitted with a flame ionization detector and with a (2 m x 3.2 mm) column packed with 80-100 mesh Poropack-Q. Helium was

1,7/- 124

used as carrier gas at a flow rate of 30 ml min-‘, and injector, column and detector temperatures were 21O”C, 100°C and 210°C respectively. The ethylene measurements were made in triplicate for each treatment and the experiments were repeated at least twice. 2.3. Determination of ACC content Extraction and determination of free ACC in the tissue were carried out according to the method of Lizada and Yang [ 131 with some minor modifications. Ten segments (about 0.25 g) were homogenized with 2 volumes of 5% (w/v) sulphosalicylic acid. After centrifugation the precipitate was extracted twice more with 5% sulphosalicylic acid. Suflicient 70% (w/v) KzCOl was then added to bring the final pH to 4-5. An aliquot (0.1 ml) was placed in lo-ml vials to which 0.25 ml of 10 mM HgC12 was added and the volume was brought up to 1 ml. The vials were sealed with a rubber serum stopper and 0.2 ml of a cold mixture of 5% of NaOCl and saturated NaOH (2:l v/v) was injected through the stoppers by means of a l-ml syringe. Reaction mixtures were agitated on a vortex mixer for a period of 5 s, immediately after the hypochlorite addition and incubated in ice for 2.5 min. The mixtures were then agitated for another 5 s prior to gas sampling. The released ethylene was determined as above. The efficiency of conversion of standard ACC to ethylene was about 75%. 2.4. ACCO activity ACCO activity was measured by determining ethylene production in the presence of a saturating concentration of ACC ( 10 mM). Roots from control and 6 h Spd pretreated seedlings were incubated in sealed vials at 27°C in the dark in the presence of 10 mM ACC and the released ethylene was determined as above. 3. Results and discussion 3-day-old maize seedlings were treated with Spd applied through roots at concentrations ranging from l-5 mM. At different time intervals primary roots were cut and collected for measuring ethylene production. Data, reported in Table 11indicate

E. Rea et al. /Plant Science 108 (1995) 121-124

123

Table I Effect of Spd pretreatment as a function of concentration on ethylene evolution from primary roots Pretreatment

Time of pretreatment 3h

6h

9h

12 h

24 h

Ethylene (pmol/Wg J wt.)

Control Spd (I mM) Spd (I.5 mM) Spd (2 mM) Spd (5 mM)

178.30 f 298.50 zt 310.20 + 336.50 + 330.70 l

17.2 40.2 33.2 25.1 30.3

160.70 f 335.50 f 300.30 zt 394.70 f 315.00 l

13.0 35.0 30.2 16.3 34.5

Data are means k S.D. from a representative experiment run

a stimulation of ethylene production induced by Spd pretreatment with a maximum after 9 h for all concentrations except for 1 mM, which showed a maximum after 6 h. The values successively decreased reaching those of control seedlings. The most effective concentration is 1.5 mM. However, we chose 1 mM for the following experiments because of its shorter time of action. Table 2 shows no differences in endogenous free ACC levels at all time intervals tested between control and Spd pretreated roots. Similarly, no effect of Spd on ACCO activity, determined by measuring ethylene production in the presence of saturating ACC levels, obtained with 10 mM external concentration, was observed. In fact after 6 h of Spd pretreatment the ACCO activity was identical in both control and pretreated tissues (0.50 and 0.47 nmol/h/g f. wt. of ethylene, respectively). Inhibitors of ethylene biosynthesis, Co*+ and AVG, and of ethylene action, STS, were used alone or in combination

Table 2 Effects of Spd on free ACC level. Maize seedlings were pretreated for 3, 6, I8 h with Spd (I mM) before determining the ACC amounts in primary roots Pretreatment

Time of pretreatment 3h

6h

I8 h

1.36 * 0.06 1.58 f 0.20

1.54 + 0.06 I.11 f 0.10

ACC (nmoUgj: wt.)

Control Spd

1.39 f 0.20 1.62 f 0.07

Data are means & SD. from a representative experiment run in triplicate.

196.00 f 338.30 l 616.70 f 540.30 + 703.50 f

3.00 50.3 53.2 52.4 40.1

105.50 f 304.30 f 464.70 zt 482.20 zt 337.40 zt

3.6 37.0 25.0 IO.1 48.2

133.50 f 24.7 117.20 f 10.3 155.50 f 15.5 178.00 zk 9.8 273.30 f 20.2

in triplicate.

with 1 mM Spd for 6 h following which ethylene production was measured. Data reported in Table 3 show a strong inhibition of ethylene production by Co*+ and AVG in both control and Spd pretreated roots; however a 100% stimulation was still observed in ethylene production when Spd was present. On the contrary STS, a known inhibitor of ethyiene action, gave a stimulatory effect on ethylene production, which is additive with the stimulatory effect induced by Spd. The effect of inhibitors was also followed by measuring growth rate of primary root in seedlings grown in their presence with or without Spd. As shown in Table 3, neither inhibition of ethylene synthesis by Co*+ and AVG, nor inhibition of ethylene binding by STS, influence during 24 h growth rate in both control and Spd treated tissue. Our assumption that a Spd pretreatment of maize seedlings can stimulate ethylene production from roots, has been confirmed by experimental results. In fact, roots from Spd pretreated seedlings produced 2-fold more in ethylene as compared with control roots. Co*+ and AVG, inhibitors of ethylene biosynthesis, strongly inhibited both basal- and Spd-stimulated ethylene, indicating that the latter is produced also by the classical pathway of biosynthesis. The fact that Spd pretreatment is ineffective in stimulating ACCO activity, together with only small variation in ACC level, seems to be consistent with our hypothesis that more AdoMet is available for the increase in ethylene production. However, it does not seem that the effect of Spd on growth rate of primary root occurred via ethylene. In fact, the in-

E. Rea et al. /Plant Science 108 (1995) 121-124

124

Table 3 Effect of ethylene inhibitors in the presence or absence of Spd on ethylene evolution and primary root growth rate in maize seedlings after 6 h and 24 h, respectively. Pretreatment

Ethylene (pmolih/g f. wt.)

Root elongation/ 24 h (cm)

% Inhibition of root elongation

Control Spd (1 mM) Coz+ (0.1 mM) Co2+ and Spd AVG (0.01 mM) AVG and Spd STS (I mM) STS and Spd

99.20 f 196.60 f 43.62 + 100.14 f 35.85 f 71.85 * 251.00 f 237.00 f

2.87 1.28 2.60 1.30

55.40 9.41 54.70 31.36 60.28 0.00 54.70

8.34 41.04 6.90 0.48 6.00 8.30 24.0 20.0

1.97 1.14 2.95 1.30

Data are means + S.D. from a representative experiment run in triplicate.

hibitory effect of Spd was not reversed in the presence of inhibition of ethylene biosynthesis and action. On the other hand, these results are in agreement with the lack of growth rate stimulation in roots treated with ethylene biosynthesis inhibitors, previously observed by others in lettuce 1111and also maize 1121.It has been supposed that these plants present a very low ‘basal’ ethylene which is insufficient to be concentration, physiologically active, or which is able to enhance rather than restrain root elongation [14].

151

161

171

Acknowledgements

181

The authors wish to thank Dr. Francesco Misiti for his collaboration during the preliminary phases of this research. Research partially supported by National Research Council of Italy, Special Project RAISA, Sub-project N. 2, Paper N. 2113.

PI

1101

References

[III

111M. de Agazio, S. Grego and F. De Cesare, Enhancement of KC uptake through the plasma membrane in freshly cut root segments from maize seedlings pretreated with spermidine. PIant Sci., 76 (1991) 85-89. 121M. de Agazio, E. Rea, F. De Cesare and S. Grego, Influence of exogenous spermidine on potassium balance in maize seedlings. J. Plant Nutr., 16 (1993) 2555-2562. [31 F. De Cesare, S. Grego and M. de Agazio, Metabolic changes of endogenous polyamines in roots from maize seedlings after spermidine treatment. Plant Physiol. (Life Sci. Adv.), 13 (1994) 5-11.

1121

[I31

[I41

M. de Agazio, R. Federico, R. Angelini, F. De Cesare and S. Grego, Spermidine pretreatment or root tip removal in maize seedlings: effects on K+ uptake and tissue modifications. I. Plant Physiol., 140 (1992) 741-746. S. Pennazio and C. Roggero, Exogenous polyamines stimulate ethylene synthesis by soybean leaf tissue. Ann. Bot., 65 (1990) 45-50. S.L. Chen, CT. Chen and C.H. Kao, Polyamines promote the biosynthesis of ethylene in detached rice leaves. Plant Cell Physiol., 32 (1991) 813-818. M.L. Botha and C.S. Whitehead, The effect of polyamines on ethylene synthesis during normal and pollination-induced senescence of Petunia hybrida L. flowers. Planta, 188 (1992) 478-483. MC. Whalen and L.J. Feldman, The effect of ethylene on root growth of Zea mays seedlings. Plant Physiol., 91 (1989) 310-314. Z. Ever&hen, A.K. Mattoo and R. Goren, Inhibition of ethylene biosynthesis by aminoethoxyvinylglicine and by polyamines shunts label from 3.4[14C]methionine into spermidine in aged orange peel discs. Plant Physiol., 69 (1982) 385-388. H. Kende, Ethylene biosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol., 44 (1993) 283-307. F.B. Abeles and S.G. Wydoski, Inhibitors of ethylene synthesis and action: a comparision of their activities in a lettuce root growth model system. J. Am. Sot. Hort. Sci., 112 (1987) 122-125. G.I. Moss, K.C. Hall and M.B. Jackson, Ethylene and responses of roots of maize (Zea mays L.) to physical impedance. New Phytol., 109 (1988) 303-311. M.C.C. Lizada and S.F. Yang, A simple and sensitive assay for I-aminocyclopropane-I-carboxylic-acid. Anal. B&hem., 100 (1979) 140-145. M.B. Jackson, Ethylene in root growth and development, in: A.K. Mattoo and J.C. Suttle (Eds.), The plant hormone ethylene, CRC Press Inc. Boca Raton, FL, 1991. pp. 159-181.