Journal of Experimental Botany, Vol. 51, No. 349, pp. 1471-1473,
August 2000
© 2000 Oxford University Press
Short Communications |
Non-invasive photoacoustic spectroscopic determination of relative endogenous nitric oxide and ethylene content stoichiometry during the ripening of strawberries Fragaria anannasa (Duch.) and avocados Persea americana (Mill.)1
Faculty of Life Sciences, Jacob Vainstein Chair of Biblical Botany, Bar-Ilan University, Ramat Gan 52900, Israel
Received 31 January 2000; Accepted 4 April 2000
Abstract
Employing non-invasive photoacoustic spectrometry, emissions of nitric oxide (NO) and ethylene in post-harvest strawberries and avocados were monitored. A clear-cut stoichiometric relationship was found between the two gases: unripe fruit manifesting high NO and low ethylene levels—the converse in ripe fruit. Findings are discussed in the light of putative control of ethylene-promoted fruit senescence by endogenous NO.
Key words: Strawberries, avocados, photoacoustic spectroscopy, nitric oxide, ethylene.
Introduction
In the 1880s the first reports of the photoacoustic effect—the production of sound waves as a consequence of light absorption—were published practically concurrently by Bell in the USA, Tyndall in Britain and Röntgen in Germany (Bell, 1880
; Tyndall, 1881; Röntgen, 1881). A reverse effect based on the same physical principle, where ‘the people saw the voices’, is described even earlier in Exodus XX:15 upon the issuing of the Ten Commandments to Moses on Mt. Sinai.
Criticism has been expressed of the hypothesis that in fruit maturation an endogenous stoichiometric NO/C2H4 relationship exists and that, concomitantly, NO may be employed as a post-harvest senescence retarding agent (Leshem and Wills, 1998; Leshem et al., 1998). This criticism particularly pertains to endogenous plant NO emissions which, in the above reports, were partially based on data obtained from an invasive technique whereby an NO-sensitive microprobe was inserted into the fruit tissue and therefore that the wounding caused, albeit minor, NO emissions which ordinarily would not occur. The present research addresses this contention by using the recently renewed non-invasive and rapid PAS technique as described in the Materials and methods.
Materials and methods
Plant material
Hydroponically grown green and consumer ripe red strawberries (Fragaria anannasa cv. Malach) used for the initial experiment were obtained from the B'sor Regional Agricultural Experimental Farm in the northern Negev region of Israel. The fruits were air-shipped to the Netherlands, the time duration from dispatch to arrival in Nijmegen being c. 5 h. Subsequent experiments were performed on strawberries on fruits purchased at the Nijmegen wholesale fruit market. Firm and ripening avocados (Persea americana cv. Carmel) were also obtained from the market. Each experiment outlined in the next section was repeated five times.
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Photoacoustic spectroscopy
Alexander Graham Bell (Bell, 1880
) stated that ‘thin disks of very many substances emitted sounds when exposed to the action of a rapidly interrupted beam of sunlight’. The photoacoustic effect is based on acoustic wave generation due to light absorption. A gas molecule enters a higher ro-vibrational state as a result of absorption of infrared photon excitation. Collisions transfer the ro-vibrational energy to translational energy, i.e. heat. Light intensity modulation (‘chopping’) causes sample temperature periodically to increase and decrease. For a gas sample in a closed volume, this temperature variation is accompanied by pressure variation which creates an audible sound wave that can be detected with a sensitive microphone.
Present day development of the 120-year-old PAS phenomenon was made possible by the advent of high light intensities obtained by laser illumination combined with even more recently advanced hearing-aid technology. Figure 1
is a schematic diagram of the essentials of the system.
The PAS facility at Nijmegen, the Netherlands, very recently established by the European Community, has quantified to date 22 biological gases at hitherto unattainably low detection levels, e.g. 5.0 and 1.0 pptv, respectively, for C2H4 and NO (Harren and Reuss, 1997; Harren, 1998). In plant tissues, this PAS facility or its earlier model has been used for C2H4, CO2 and/or aldehyde production in Rumex under anoxic conditions (Voesenek et al., 1990), in red pepper Capsicum annuum maturation (Zuckermann et al., 1997) and in the abscission behaviour of climacteric fruits (de Vries et al., 1996). It is also of interest that a variation of this technique—that of using an acoustic optic fibre—and essentially based on the same physical principle as PAS, was efficiently used for the determination of certain stress-invoked photochemical responses in plants (Richter and Lichtenthaler, 1996).
All experiments were performed with a CO2 laser and previously weighed fruit samples—c. 25 strawberries or 1 avocado fruit—which were placed in the transparent sample cuvette at an ambient room temperature of 22 °C in fluorescent light at 150 µM s-1 m-2 PAR intensity. In order to prevent NO
NO2 conversion which in air may occur within 7 s (Snyder, 1992), when NO was measured, the sample cuvette was filled with N2 gas; when ethylene was measured, the cuvette contained air. Flow rate of the carrier gases was 1.0 l h-1. Each experiment was repeated five times, each of which produced essentially similar results. The data presented in Figs 2 and 3 are of a typical trial, one of the five repeats.
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Results and discussion
Figures 2 and 3 present data which indicate that upon ripening in both types of fruit—the non-climacteric strawberry and the climacteric avocado—the maturation process is clearly accompanied by a marked decrease of NO concomitant with an increase of C2H4. That exogenously applied NO can increase the shelf life of strawberries strongly suggests a regulative role of NO in senescing fruit, a claim also borne out by a similar ripening pattern revealed in many other species, as reported previously (Leshem et al., 1998; Leshem and Wills, 1998).
Note that the avocado induces higher gas emission than the strawberry since, as stated in the caption to Fig. 3, the relative basis of comparison on the NO/C2H4 ordinates was 10x more in the avocado than in the strawberry. This is in keeping with the well–documented phenomenon that ethylene emission in the ripening climacteric avocado fruit is higher than in most other edible fruits.
The above results substantiate the authors' previous results. In relation to the critique of their previous NO experimental data outlined in the Introduction, the authors quote John Tyndall, who in his paper on the photoacoustic effect, read before the Royal Society of London on the 10 January 1881, stated: ‘They thus ignore verifications, both general and special, which are to us of conclusive force’.
Acknowledgments
The authors wish to thank Dr Frans Harren, Director of the Trace Gas Facility, Department of Molecular and Laser Physics, which was established in the European Union TMR framework at the Nijmegen University, The Netherlands, for enabling us access and use of the Facility's laboratory and services. Special thanks are due Dr F Harren, Dr LJ Laarhoven and Dr S te Lintel Hekkert of the Facility for their counsel and expert aid in the experimental work. Invaluable advice and aid were also offered by Professor Zvy Dubinsky of the Bar–Ilan University's Life Sciences Faculty. Agronomist Yehoram Leshem of the B'sor Agricultural Experimental Station, Israel, kindly supplied some of the fruits for trials. This work was also financed in part by grants from the EU Community, the Jacob Vainstein Chair of Biblical Botany and the Bar–Ilan University Research Authority.
Notes
1 This research was carried out in part at the Life Science Trace Gas Exchange Facility, Department of Molecular and Laser Physics, University of Nijmegen, The Netherlands. 
2 To whom correspondence should be addressed. Fax: +972 3 5351824. E-mail: goldra{at}mail.biu.ac.il
Abbreviations
C2H4, ethylene; NO, nitric oxide; PAS, photoacoustic spectrometry..
References
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