METHODS AND PLATFORMS 



The in situ photoprotective effect of xanthophyll interconversions will be estimated from 

 sampling vertically through the euphoric zone, by triggering collection bottles containing cycle 

 inhibitors. Deckboard analysis of net photosynthesis (as 2 evolution) and gross photosynthesis 

 (from pulse fluorometry) will be performed at irradiances at, above and below those determined 

 at the depth of sampling. Comparison with the performance of uninhibited phytoplankton 

 samples should provide a meaningful assessment of any protective role for de-epoxidized 

 xanthophyll cycle components (diatoxanthin and zeaxanthin). It will also assess any disadvantage 

 accruing from the presence of these quenchers at low iiradiance. 



STRENGTHS AND LIMITATIONS OF PROPOSED RESEARCH 



The use of selective inhibitors of the deepoxidase such as dithiothreitol, or of ionophores that 

 collapse the transmembrane pH gradient, have provided considerable insight into the relationships 

 between xanthophyll cycling, energy-dependent quenching, and photosynthesis in higher plants, 

 and to a limited extent in phytoplankton. However, a convenient chemical inhibitor for the 

 epoxidase is not presently available. Zeaxanthin (or diatoxanthin) is converted to violaxanthin 

 (diadinoxanthin) in the light as well as in the dark by an enzyme that uses NADPH and 

 molecular oxygen, and thus, can be blocked by anaerobiosis. The use of chemical inhibitors 

 would be more advantageous than anaerobicity in blocking epoxidation, since the local 

 environment of the thylakoid membrane is inevitably exposed to photosynthetically-derived 2 . 

 However, to be effective in providing meaningful information on the role of xanthophyll cycling 

 in ocean margins, the selected inhibitors must be equally potent in a wide variety of species 

 across varying taxonomic lines. 



STATUS OF RESEARCH 



Two classes of antimycotic agents, known to block the epoxidation of squalene in fungi and 

 mammalian tissues, have been tested for their ability to inhibit zeaxanthin epoxidase in barley 

 chloroplasts. Epoxidation was monitored by illuminating samples to accumulate zeaxanthin then 

 inhibiting deepoxidation by simultaneous darkening and addition of the ionophore nigericin. 

 Analysis of the magnitude and kinetics of the slow absorbance decrease at 505 nm, indicative of 

 violaxanthin formation from zeaxanthin, demonstrated that the allylamines terbinafine and 

 naftifine, and the thiocarbamate tolnaftate are potent inhibitors of zeaxanthin epoxidation. The 

 concentrations yielding 50% inhibition of initial epoxidation rates were 4.6 5M tolnaftate, 6.0 5M 

 naftifine, and 10.6 5M terbinafine. The 505 nm absorbance increase during actinic illumination, 

 which reflects net accumulation of zeaxanthin, was apparently enhanced in the presence of these 

 inhibitors, and an initial lag phase was abolished. Ascorbate-loading of chloroplasts in the 

 presence of 20 5M tolnaftate yielded the highest observed rate of absorbance increase at 505 nm 

 during actinic illumination. The dynamic interaction between epoxidation and deepoxidation in 

 intact chloroplasts is remarkable in that epoxidation seems to compete effectively when 



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