Respiration and ethylene in harvested asparagus spears during aging at 20°C

Scientia Horticulturae, 43 (1990) 189-195 Elsevier Science Publishers B.V., Amsterdam

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Respiration and ethylene in harvested asparagus spears during aging at 20 °C S. Hennion and C. Hartmann* Laboratoire de Physiologic du Vieillissement et de la Sbnescence des Vbgbtaux Supbrieurs, Universitb d'Orlbans, F 45067 Orlbans Cbdex 2 (France) (Accepted for publication 21 December 1989)

ABSTRACT Hennion, S. and Hartmann, C., 1990. Respiration and ethylene in harvested asparagus spears during aging at 20 ° C. Scientia Hortic., 43:189-195. Respiration, ethylene (C2H4) production and concentrations of l-aminocyclopropane-l-carboxylic acid (ACC) and its malonyl conjugate (MACC) were followed during aging of harvested white asparagus spears (Asparagus officinalis L., Institut National de la Recherche Agronomique (INRA) F~ male hybrid). Respiration increased slightly during the first hours of aging, followed by a steady decrease concurrent with increasing age. C2H4 production did not vary greatly during the first days of aging, but increased after 10 days. ACC steadily increased in the tip of the spear after 1 day and exhibited a maximum at Day 10, then decreased subsequently and exhibited a final rise. The tip as well as the butt accumulated large quantities of MACC at the end of storage. Spears treated with silver thiosulphate (STS) exhibited a large increase in C2H4production. Concomitantly, ACC and MACC levels rose markedly, while respiration was not significantlymodified. Keywords:-aging; 1-aminocyclopropane-l-carboxylicacid; asparagus; Asparagus officinalis; ethylene; 1 - (malonylamino)cyclopropane- 1-carboxylic acid; post-harvest physiology. Abbreviations: ACC: 1-aminocyclopropane-l-carboxylicacid; C2H4: ethylene; INRA: Institut National de la Recherche Agronomique; MACC: 1-(malonylamino)cyclopropane- 1-carboxylic acid; STS: silver thiosulphate.

INTRODUCTION

For a long time, ethylene was mostly associated with fruit ripening. It is now well recognized that this hormone regulates many aspects of plant physiology, from germination to senescence (Yang and Hoffman, 1984). A great majority of papers concern fruits, flowers and leaves, while the literature dealing with non-fruit vegetables is relatively scarce. Concerning asparagus, Sal*Author to whom correspondence should be addressed.

0304-4238/90/$03.50 © 1990 - - Elsevier Science Publishers B.V.

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veit and Kasmire ( 1985 ) measured respiration rates during storage at 2.5 °C, King et al. ( 1988 ) described respiration, flavour, protein and carbohydrates changes, and Haard et al. (1974) followed C2H4 production by freshly harvested spears (from 45 to 165 min) in relation to isoperoxidase changes. Changes occur in asparagus after harvest, including a progressive toughening due to an increasing crude fibre content, which is a major factor in the post-harvest loss of quality. Post-harvest lignification has been studied by Haard et al. (1974) and Chen et al. (1980). C2H4 is known to stimulate the synthesis of phenolic compounds and to be implicated in the control of lignification (Rhodes et al., 1976). To better understand the lignification processes, we followed respiration, C2H4 production and changes of 1-aminocyclopropane-l-carboxylic acid (ACC) (its immediate precursor) and 1(malonylamino)cyclopropane-l-carboxylic acid (MACC) during the aging of harvested asparagus spears. The effect of silver thiosulphate (STS), an inhibitor of ethylene, was also studied. MATERIALSAND METHODS P l a n t material. - White (blanched) asparagus spears ( A s p a r a g u s officinalis

L., Institut National de la Recherche Agronomique (INRA) F~ male hybrid No. 156 ) were cut during April and May 1988 in an experimental field situated at Soings-en-Sologne (41-France) at around 9 a.m. and transported to the laboratory. Spears were washed with fresh water, trimmed to 17 cm in length, weighed, and stored in the dark at 20 °C and a relative humidity of ~ 50%. Time 0 was chosen as the end of a 2-h equilibration period. There were three treatments: stored dry, with the cut end (spear) in water and with the cut end in a freshly prepared solution of 2 m M STS complex (Veen, 1983 ). In the two last treatments, the butt section was recur and water or STS solution renewed each 2 days. Aging was stopped when visible decay or fungal attack appeared. C 0 2 a n d C2H4m e a s u r e m e n t s . - Two samples of 10 spears were used for each experiment and followed throughout storage. All measurements were made in duplicate. CO2 output in a continuous air flow was measured with an IR gas analyser (Hartmann and Brown, Uras 7N, flow rate 20 1 h -1, relative humidity 50%, plant material 10 spears; i.e. from 300 to 400 g fresh weight ( F W ) ) . C2H4 production was followed in closed jars (relative humidity 50%, plant material 300-400 g FW, volume 1.51) by gas chromatography (column of activated alumina, T = 65 °C) on a sample from the head space. Each experimental point was calculated from at least two samples and three measures (time 30, 60 and 90 rain). C2H4 output was linear during at least 150 min. The confinement time was < 120 min. E x t r a c t i o n a n d d e t e r m i n a t i o n o f A C C . - After different lengths of aging, spears

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were cut into three segments of 5 cm each (tip, mid and butt), frozen in liquid nitrogen and immediately lyophilized. Results given in this paper concern tip and butt parts. The middle parts have not been analysed. Free ACC was extracted from lyophilized material as follows. One gram of dry tissue was pulverized in a Dangoumau type grinder and extracted in boiling methanol-water (80:20 v / v ) for 15 min. Extracts were centrifuged ( 12 000 ×g, 15 rain). Measurements were performed as described by Lizada and Yang (1979) except t h a t H g 2+ and NaOC1 concentrations were increased to 3.3 and 170 mM, respectively. Known amounts of pure A C C added to aliquots of the sample allowed the calculation of the regression line and the yield of conversion from ACC to C2H4. Each experimental point was calculated from six determinations. The correlation between C2H4 production and A C C concentration was larger than 0.9 and yields ranged from 75 to near 100%. Assay of total A C C was performed directly upon 1 g of tissue after incubating in 3 N HC1 at 100 °C for 3 h in order to hydrolyse MACC. ACC produced was measured subsequently as mentioned above except that Hg 2+ and NaOC1 concentrations were increased to 6.6 and 260 mM, respectively. Under these conditions, the yields were better than 80%. MACC was obtained by substracting ACC from total A C C . RESULTS

R e s p i r a t i o n . - Respiration increased slightly during the first hours of aging up

to a maximum and decreased subsequently; rapidly until Day 2, more slowly afterwards (Fig. 1 ). Dipping the cutting in water had no significant effect. STS slightly stimulated respiration during the first 2 days. 300

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Fig. 1. Respiration of asparagus spears during aging at + 20 ° C in the dark. Air: aging without water supply. Water: aging with water supply. STS: aging with STS. Vertical bars indicate 95% confidence intervals (when not represented, smaller than symbol).

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S. HENNION AND C. HARTMANN 5

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POSTHARVEST RESPIRATION AND ETHYLENE IN ASPARAGUS

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Fig. 4. Effect of STS on ACC and MACC levels in the apical and basal segment of asparagus spears during aging at 20 ° C. The cut ends of the spears were dipped in water and in STS solution, respectively. Vertical bars as in Fig. 1.

C 2 H 4 p r o d u c t i o n . - At the beginning of the storage in air treatment C2H4 output decreased from H o u r 0 to Hour 6, regained its initial rate at Hour 12, decreased again, then increased steadily until a final rise. With a water supply no significant rise in C2H4 production was observed during the first hours and the final rise was m u c h reduced (Fig. 2 ). STS strongly stimulated C2H4 production, which increased sharply after 7 h, reached a level of ~ 10 nl h - i g-1 FW at Day 1, remained constant until Day 5, then increased to > 25 nl at Day 10. Compared with the water sample, on average C2H4 production was multiplied by a factor of 20 (Fig. 2 ).

a n d M A C C . - During the first day of aging without water supply, ACC levels in the apical and basal parts of the spears did not exhibit great variations. A sharp increase then occurred up to a m a x i m u m at Day 10, followed by a second increase at the end of the aging period. At the same time, MACC steadily increased (Fig. 3). In the water treatment, ACC decreased from Hour 4 to Hour 20 in both

ACC

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apical and basal segments, as did C 2 H 4 output, and increased subsequently. STS enhanced ACC and MACC concentrations in the apical as well as in the basal segment (Fig. 4). The ACC concentration was very m u c h enhanced, especially in the apex, whereas at Day 4 MACC production was apparently slowed down in the apical segment. DISCUSSION In the present work we observed spears aged from ~ 4 h after harvest (2 h transportation + 2 h equilibration) up to 16 days, except for ACC and MACC which were already measured at Hour - 2 . Therefore the immediate effects of harvest stress or the initial status before harvest are not known. Nevertheless, the results presented here indicate that the " w o u n d effect" has apparently relatively few consequences on respiration or C2H4 production. Haard et al. (1974) observed a rise in C2H4 output between 45 and 95 m i n after harvest, while King et al. ( 1988 ) described a rapid decline during the first 2 days. In our work, respiration was increased ~ 20% during the first 12 h, then decreased rapidly until Day 4. After a decline, a small m a x i m u m in C z H 4 production occurred in the air, but not in the water treatment. After a 48-h transitory period, the respiration rate decreased, while C2H4 output increased steadily. The end of storage was characterized by enhanced C2H4 production. MACC was accumulated, indicating that both ACC synthase and malonyltransferase were active. STS had a marked effect on development, probably by blocking C2H4 action. For instance, it stopped tomato fruit ripening (Hobson et al., 1984) and greatly increased the vase life of cut carnations (Reid et al., 1980). It markedly stimulated C2H4 production by asparagus spears as it did for the tomato (Atta-Aly et al., 1987 ) and the cherry fruit (Hartmann, 1989 ). The effectiveness of STS is generally attributed to a greater mobility of Ag +, but sulphur may also play a role, either directly or indirectly, by for instance control of bacteria. However in the tomato, Na2S203 failed to inhibit maturation and to enhance C2H4 production. Moreover, a control experiment was conducted with spears supplied with a Na28203 solution. Ethylene production was not significantly increased. It appeared, consequently, that the effect of STS on CzH 4 production can probably be attributed to the silver ion. A question arises: what is the mechanism of the effect of STS o n C z H 4 production? Tomato is a climacteric fruit and possesses a positive feedback system (autocatalysis--system II of McMurchie et al., 1972 ). In contrast, cherry (a non-climacteric fruit) and asparagus (a vegetative organ), are system II deficient. If the rate of C2H4 synthesis is governed by a negative feedback effect (Yang and Hoffman, 1984), the silver ion can act either by suppressing this effect or as a heavy metal by enhancing S-adenosylmethionine (SAM) conversion to C2H4 and stimulating CzH4 forming enzyme (EFE).

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ACKNOWLEDGEMENTS

We thank Madame Lucette Corriols and her coworkers (INRA, Versailles, France) for helpful assistance. Stephane Hennion was supported by a grant from the Conseil R6gional de la R6gion Centre.

REFERENCES Atta-Aly, M.A., Saltveit, M.E. and Hobson, G.E., 1987. Effect of silver ions on ethylene biosynthesis by tomato fruit tissue. Plant Physiol., 83: 44-48. Chen, S.C., Lin, C.H. and Hsue, J.C., 1980. On the study of postharvest lignification of white asparagus stalks. Natl. Sci. Counc. Monthly, 8:627-634 (in Chinese). Haard, N.F., Sharma, S.C., Wolfe, R. and Frankel, C., 1974. Ethylene induced isoperoxidase changes during fiber formation in postharvest asparagus. J. Food Sci., 39: 452-456. Hartmann, C., 1989. Ethylene and ripening of a non climacteric fruit: the cherry. Acta Hortic., 258: 89-96. Hobson, G.E., Nichols, R., Davies, J.N. and Atkey, P.T., 1984. The inhibition of tomato fruit ripening by silver. J. Plant Physiol., 116:21-29. King, A.G., Henderson, K.G., O'Donoghue, E.M., Martin, W. and Lill, R.E., 1988. Flavour and metabolic changes in asparagus during storage. Scientia Hortic., 36:183-190. Lizada, M.C.C. and Yang, S.F., 1979. A simple and sensitive assay for 1-aminocyclopropane 1carboxylic acid. Anal. Biochem., 100: 140-145. McMurchie, E.J., McGlasson, W.B. and Eaks, I.L., 1972. Treatment of fruit with propylene gives information about the biogenesis of ethylene. Nature (London), 237: 235-236. Reid, M.S., Paul, J.L., Farhoomand, M.B., Kofranek, A.M. and Staby, G.-L., 1980. Pulse treatments with the silver thiosulfate complex extend the vase life of cut carnations. J. Am. Hottic. Sci., 105: 25-27. Rhodes, M.J.C., Hill, A.C.R. and Wooltorton, L.S.C., 1976. Activity of enzymes involved in lignin biosynthesis in swede root disks. Phytochemistry, 15:707-710. Salveit, M.E. and Kasmire, R.F., 1985. Changes in respiration and composition of different length asparagus spears during storage. HortScience, 20:1114-1116. Veen, H., 1983. Silver thiosulfate: an experimental tool in plant science. Scientia Hortic., 20: 211-224. Yang, S.F. and Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol., 35:155-i 89.