Search

During long-term storage of apples, detection of low O2 stress is used to optimise low O2 storage regimes for dynamic controlled atmosphere (DCA) storage. Monitoring respiratory characteristics, specifically the respiratory quotient (RQ), provides a tool to achieve this. The objectives of this study were to evaluate protocols to monitor response of apple consignments to low O2 using a respiration chamber, the SafePod, designed for use in commercial stores and research laboratories, and to compare the RQ response to changes in chlorophyll fluorescence (CF) yield from the fruit skin as used in DCA-CF. Protocols to identify the lowest oxygen limit (LOL), the O2 concentration below which RQ rises, were tested using ‘Braeburn’ (sensitive to low O2) and ‘Gala’ (less sensitive to low O2). A protocol that allows fruit to acclimatise at each O2 concentration takes several weeks and is therefore not practical for commercial use. A rapid profile without fruit acclimatisation can be completed in 2–3 days. Although this underestimates RQ values, and results in an increase in RQ at a higher O2 concentration than observed for acclimatised fruit, the rapid RQ protocol provides a practical method to compare response of apple consignments between cultivars, orchards and seasons. By the rapid protocol, the LOL of ‘Braeburn’ consignments was near 0.6 kPa and of ‘Gala’ consignments was near 0.2 kPa, consistent with detection of alcoholic taints below the LOL in each case. The RQ response using the SafePod was consistent with increase in CF yield using HarvestWatch™. Fruit respiration rates change through the storage season, including a substantial decrease over the first 2 months after harvest. As RQ response is affected by respiration rate, accurate comparison of consignments depends on profiles being measured at the same stage in the storage season. It is more difficult to determine the LOL by RQ profiling later in the season when respiration rates are lower.

Chlorophyll fluorescence has been tested by many researchers as a non-invasive method to assess physiological damage in chlorophyll containing fruits and vegetables. The characteristic Fv/Fm is most commonly considered. One draw-back of this method is that the fluorescence signal is affected primarily by the state of photosystem II, which is one of the components most sensitive to stresses, including heat, chilling and oxidative stress, so that the Fv/Fm may be more sensitive than the overall tissue. A more reliable strategy may be to look at the ability of the tissues to resynthesise photosystem II after damage, by looking at the rate of Fv/Fm recovery. This strategy shows promising results in predicting chilling injury in peppers and cucumbers, and heat stress in asparagus. Trials are also reported that attempt to predict low temperature injury in apples, but the results are not definitive.

Alternative methods for disinfestation of asparagus against thrips (Thrips tabaci) need to be developed for the Thai export trade, ensuring efficient thrips’ mortality, while maintaining quality and environmental safety. Hot air/water using temperatures of 40-60°C and high CO2 treatments were studied at laboratory level. An appropriate treatment involving hot water at 45°C for 15 min, with or without high CO2 treatment, followed by hydro-cooling with water at ambient temperature (~20°C) was identified. Methods to assess quality, such as objective tests (% mass loss, force measurement, colour measurement) and subjective tests (appearance, decay, off-odour, snap-test for texture) gave no indication of significant quality deterioration. Chlorophyll fluorescence and ion leakage measurements were used to assess physiological stress. The Fv/Fm ratio which indicates the intactness of photosystem II was shown to decrease immediately after all heat treatments tested, but recovered over 24h for non-damaging heat treatments (42.5 and 45°C). A rise in % K+ leakage was observed that increased with treatment temperature but recovered over 24h following treatment at 45°C. High CO2 showed neither significant detrimental effect nor indication of physiological stress, and could kill thrips although not at a sufficiently high rate. Confirmatory trials to demonstrate the efficacy of these treatments in Thailand using naturally infested asparagus were carried out. The mortality rate of thrips was as high as when this method was tested in the UK. Adoption of the selected disinfestation method into existing process lines in Thailand would not be too complicated.

A comparison has been made between the changes in absorption spectra and chlorophyll fluorescence emission occurring upon the induction of non-photochemical dissipation of excitation energy (qE) in isolated thylakoids and those accompanying the aggregation of detergent-solubilised spinach light-harvesting complex (LHCII). In support of a recent hypothesis for the mechanism of qE (Horton et al. (1991) FEBS Lett. 292, 1–4), it was found that absorbence changes at 530 nm were associated with qE and LHCII aggregation. Antimycin A inhibited these changes and prevented LHCII aggregation, as indicated by the electrophoretic mobility of the complex and its low-temperature fluorescence spectrum. An antimycin-insensitive partial aggregation of LHCII was associated with an absorbance change at 505 nm. Low concentration of detergent caused disaggregation of LHCII and the reversal of qE. These data are discussed in terms of the relationship between structural change in LHCII and the mechanism of non-photochemical quenching of chlorophyll fluorescence in thylakoids.

The pH dependence of maximum chlorophyll fluorescence yield (Fm) was examined in spinach thylakoids in the presence of nigericin to dissipate the transthylakoid pH gradient. 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) was present to eliminate photochemical quenching. Thylakoids were prepared from dark adapted leaves ('dark' thylakoids) or preilluminated leaves ('light' thylakoids). In the latter there had been approximately 50% conversion of the xanthophyll violaxanthin to zeaxanthin, while no conversion had occurred in the former. In the presence of a reductant such as ascorbate, antimycin A sensitive quenching was observed (half maximal quenching at 5 μM), whose pH dependence differed between the two types of thylakoid. Preillumination of leaves resulted in more quenching at pH values where very little quenching was observed in 'dark' thylakoids (pH 5-7.6). This was similar to activation of high-energy-state quenching (qE) observed previously (Rees D, Young A, Noctor G, Britton G and Horton P (1989) FEBS Lett 256: 85-90). Thylakoids isolated from preilluminated DTT treated leaves, that contained no zeaxanthin, behaved like dark thylakoids. A second form of quenching was observed in the presence of ferricyanide, that could be reversed by the addition of ascorbate. This was not antimycin A sensitive and showed the same pH dependence in both types of thylakoid. The former type of quenching, but not the latter, showed similar low temperature fluorescence emission spectra to qE, and was considered to occur by the same mechanism.

A new hypothesis is presented to explain the major molecular process that regulates the efficiency of light harvesting by chloroplast membranes. It is proposed that in excess light the decrease in the thylakoid lumen pH causes an increase in aggregation of the light harvesting complexes of photosystem II resulting in formation of an efficient pathway for non-radiative dissipation of excitation energy. The aggregation is potentiated by the conversion of violaxanthin to zeaxanthin. This hypothesis is based upon (i) similarity between the spectroscopic changes associated with energy dissipation and those observed upon aggregation of isolated light harvesting complex; and (ii) the link between changes in light scattering and increased energy dissipation.

Thylakoids isolated from light-treated spinach leaves (‘light’ thylakoids) which contain significant zeaxanthin levels show amplification of high-energy-state chlorophyll fluorescence quenching (qE) with respect to the transthylakoid pH gradient, when compared to thylakoids from dark-adapted leaves (‘dark’ thylakoids) which contain no zeaxanthin. In 77 K fluorescence emission spectra q E in ‘dark’ thylakoids shows maximal quenching with respect to fluorescence yield near 680 nm, whereas in ‘light’ thylakoids additional quenching occurs near 700 nm, which is associated with the appearance of a chlorophyll species absorbing at long wavelengths. It is suggested that this long-wavelength species is associated with an aggregated state of LHC II.

The quenching of variable fluorescence yield (qN) and the quenching of dark level fluorescence yield (q0) directly atributable to high-energy-state fluorescence quenching (qE) was studied to distinguish between energy dissipation in the antenna and light harvesting complexes (antenna quenching) and energy dissipation at the reaction centres (reaction centre quenching). A consistent relationship was obtained between qN and q0 in barley leaves, the green alga Dunaliella C9AA and in pea thylakoids with 2,3,5,6-tetramethyl-p-phenylene diamine (DAD) as mediator of cyclic electron flow around PS 1. This correlated well with the relationship obtained using m-dinitrobenzene (DNB), a chemical model for antenna quenching, to quench fluorescence in Dunaliella C9AA or pea thylakoids. The results also correlated reasonably well with theoretical predictions by the Butler model for antenna quenching, but did not correlate with the predictions for reaction centre quenching. It is postulated that qE quenching therefore occures in the antenna and light harvesting complexes, and that the small deviation from the Butler prediction is due to PS 2 heterogeneity.

The yield of photosynthetic O2 evolution was measured in cultures of Dunaliella C9AA over a range of light intensities, and a range of low temperatures at constant light intensity. Changes in the rate of charge separation at Photosystem I (PS I) and Photosystem II (PS II) were estimated by the parameters ΦPS I and ΦPS II . ΦPS I is calculated on the basis of the proportion of centres in the correct redox state for charge separation to occur, as measured spectrophotometrically. ΦPS II is calculated using chlorophyll fluorescence to estimate the proportion of centres in the correct redox state, and also to estimate limitations in excitation delivery to reaction centres. With both increasing light intensity and decreasing temperature it was found that O2 evolution decreased more than predicted by either ΦPS I or ΦPS II. The results are interpreted as evidence of non-assimilatory electron flow; either linear whole chain, or cyclic around each photosystem.

Light-dependent quenching of room-temperature chlorophyll fluorescence associated with the “energisation” of the thylakoid membrane is usually referred to as qE; improvements in the analysis of chlorophyll fluorescence yield (1,2) allow its quantification. qE is generally taken to reflect an increase in energy dissipation by non-radiative pathways (heat) at Photosystem 2. This increased dissipation has been implicated in the control of electron transport through PS2 (3,4) and in protection of PS2 reaction centres against photoinhibition (5). However, the mechanistic links between the onset of ΔpH and the increased energy dissipation remain obscure.

The thylakoid membranes of higher plants possess several mechanisms that control both the distribution and rate of dissipation of absorbed light. These mechanisms, which allow regulation of photosynthetic electron transport in response to alteration in external and internal factors, can be observed as the various processes that quench chlorophyll fluorescence. By using the 'light-doubling techniques’, together with analysis of quenching relaxation, it is possible to assess quantitatively the extents of these regulatory processes and to allow their interrelations to be studied. These techniques can be applied to in vitro systems or to leaves, and can be particularly useful when applied with electron-transport measurements and when models are used to aid interpretation. Results of quenching analysis at different light intensities in isolated thylakoids, intact chloroplasts, protoplasts, algae and leaves of a variety of species are presented.

The extent of energy-dependent quenching of chlorophyll fluorescence in broken spinach chloroplasts has been quantitatively related to the size of the thylakoid proton gradient as measured by the quenching of 9-aminoacridine fluorescence by titration at constant irradiance with the uncoupler nigericin or by change in irradiance. It was found that chloroplasts prepared from leaves that had been pre-illuminated with strong light for 30 min showed energy-dependent quenching at a lower proton gradient than chloroplasts prepared from dark-adapted leaves. Measurement of the carotenoid composition of the thylakoids showed that light treatment raised the ratio of zeaxanthin: violaxanthin. The possible dependence of energy-dependent quenching on xanthophyll composition and the physiological implications of this light-activation process to the regulation of photosynthetic electron transport are discussed.

The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH4Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN3 inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.

The quantitative relationship between energy-dependent quenching of chlorophyll fluorescence (qE) and trans-thylakoid pH difference (ΔpH, estimated with 9-aminoacridine) was compared in chloroplasts from leaves preilluminated at low oxygen in the absence of CO2 with chloroplasts from leaves darkened under the same conditions. The extent of both phenomena was varied by changes in actinic light intensity. Chloroplasts from preilluminated leaves contained high levels of zeaxanthin (up to 15% of total carotenoid) and were capable of forming qE at lower ΔpH values than chloroplasts from dark-adapted leaves, which lacked zeaxanthin. Infusion of dithiothreitol into leaves prior to preillumination prevented the light-induced formation of zeaxanthin; chloroplasts prepared from these leaves showed the same relationship between qE and ΔpH observed in chloroplasts fom dark-adapted leaves. The rate of appearance and disappearance of the change in relationship between qE and ΔpH upon leaf preillumination and darkening was closely matched by the kinetics of synthesis and degradation of zeaxanthin. The quantitative relationship between qE and quenching of the dark-level of fluorescence (F0) was very similar in chloroplasts from preilluminated or dark-adapted leaves, containing disparate amounts of zeaxanthin. In both sets of chloroplasts, qE was inhibited by antimycin A. These latter observations suggest that the formation of qE involves mechanistically similar features in chloroplasts containing or lacking zeaxanthin. Overall, the data are discussed in terms of action of zeaxanthin as a ‘quenching amplifier’, functioning physiologically to allow qE formation in the absence of high ΔpH potentially prohibitive to high rates of CO2 fixation.