Chlorophyll Fluorescence and Whole Fruit Senescence in Golden Delicious Apple

1. Introduction

2. Materials and Methods

Vacuum infiltrated PSH acceptor, 2,6-dimethylbenzoquinone (DMQ), 500 uM and PSI acceptors, Methyl Viologen (MV), 1 mM and N,N-dimethyl-p-nitrosoaniline (PNDA), 200 uM were used to drain electrons from photosynthetic electron transport chain in whole fruit. The photosynthetic electron transport was blocked by vacuum infiltrating 3-(3,4-dichlorophenyl)- 1, 1-dimethylurea (DCMU), 20 uM into the fruit.

3. Results

3.1 Post-storage changes in Chl fluorescence parameters

The combined changes in Fo and Fm were reflected in the variable fluorescence pattern of the fruit. The Fv/Fm declined rapidly from day 4 through 9. Holding fruit beyond 9 days under ambient conditions did not influence the Fv/Fm values in apple.

3.2 Post storage changes in chloroplast activity.

3.3 Post-storage changes in Chlorophyll content

3.4 Effect of artificial electron acceptors on variable fluorescence quenching in whole fruit.

4. Discussion

Golden Delicious apples change color from green to yellow on maturation or in storage. The color transformation window is relatively wider in comparison to the varieties which change from green to red, like Red Delicious. This phenomenon helps one to have a close track of whole fruit senescence as well as chloroplast senescence during storage. For clarity, we mean by chloroplast senescence, fruit senescence-associated in vivo changes. It differs from chloroplast aging which on the other hand, indicates time dependent alterations in chloroplast activity under in vitro conditions.

In the photosynthetic apparatus light absorbing by the antenna pigments results in transfer of excitation energy to the reaction centers of two photosystems, PSII and PSI. Consequently, well known photochemical reactions are initiated to conserve this energy into various chemical forms. At low light, the yield of photochemical energy conversion in a photosynthetic system is much higher than in high light. (Bjorkman and Demmig, 1987). The amount of energy that is not conserved by the photosynthetic system is emitted as fluorescence, a reaction that results in deactivation of excited chlorophyll molecules.

During senescence the quality of apple fruit with regard to color, texture and flavor change dramatically. There is a general agreement that membrane and cell wall deterioration (Meir et al.,1992) associated with fruit softening (Dull, 1970) is a fundamental aspect of fruit senescence. While the chloroplast membranes retain their physical integrity until late in senescence, photosynthetic capacity declines from the earliest stages of senescence (Gepstein, 1988). In our experiments, we used chlorophyll a fluorescence parameters, Fo and Fm as an index of photosynthetic pigment complex (Fig. 1). Quantitative determinations of Chl over time were also made (Fig. 3). The fluorescence parameters, Fo and Fm declined rapidly during first two weeks of storage (Fig. 1). Since Fo and Fm represent the Chl a concentration in the fruit, a similar pattern as can be seen in Fo or Fm was observed in Chl a degradation (Fig. 3). The degradation of Chl b, by and large, followed Chl a almost closely maintaining a Chl a/Chl b ratio of 3.7 to 4 during entire period of storage (Fig. 3). This pattern is some what different from leaves where marked differences in degradation rates of Chl a and Chl b during senescence are observed (Grover and Mohanty, 1993). To test the physiological state of the chloroplasts during senescence, we used two approaches. Approach 1 consisted of measurements of Fv/Fm (Fig. 1) in whole fruit over time and approach 2 was quantification of photosynthetic O2 evolution due to photolysis of H20 at PSII (Fig. 2). The Fv/Fm ratio declined from 0.78 to 0.71 during air-storage. The O2 evolution due to CO2 fixation also declined over time, stopping completely in 3rd week of storage (Fig. 2). If the fluorescence quenching is only due to CO2 fixation then one would expect no fluorescence quenching (Fig. 4) once no net O2 evolution is seen from the apple chloroplasts (Fig. 2). However, It has been shown that the total electron generation as can be calculated (4 electrons for every O2 mol evolved) from net O2 may underestimate the total capacity of photosynthetic electron transport due to many potential pathways of O2 metabolism in biological systems, pseudocyclic photophosphorylation being the predominant one (Sultemeyer, 1993; Mir et al., 1995b). Interestingly, when DCMU which blocks electron transport at QA to QB was added to the senescent fruits, no quenching of variable fluorescence was observed (Fig. 4). These results confirm that photosynthetically generated electrons are also utilized in other processes (possibly in Mehler's reaction) other than CO2 reduction, resulting in no net evolution of O2 . Such a mechanism is known to exist in cyanobacteria (Mir et al., 1995b), green algae (Sultemeyer et al., 1993) and vascular plants (Schreiber et al, 1993). In an effort to understand the mechanism of fluorescence quenching, we isolated the thylakoids from apple peel and studied various components of electron Import chain (Mir et al., 1996, unpublished results). We have shown that thylakoids from apple peel have a great potential to reduce O2 in a Mehler type reaction and when this reaction was blocked by 1 mM KCN, the MV dependent O2 uptake increased 4-fold. These results further support our finding that in-vivo fluorescence quenching observed in apple is largely due to photoreduction of O2. When photo-reduction of O2 was blocked by DCMU infiltration into the whole fruit (Fig.4), quenching of variable fluorescence was prevented.

Refrences

Van Kooten, 0. and J. F. H. Snel. 1990. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynthesis Research 25:147-150.