Joe Ortymski,2 Geir Johnsen, and Egil Sakshaug. I'rondheim Biological Station, ..... son between species without having to consider packaging, light scattering ...
./. f‘hycol. 33, 408-414 (1997)
THE SIGNIFICANCE OF INTRACELLULAR SELF-SHADING O N THE BIOOPTICAL PROPERTIES OF BROWN, RED, AND GREEN MACROALGAE’ Joe Ortymski,2 GeirJohnsen, and Egil Sakshaug I‘rondheim Biological Station, Norwegian University of Science and Technology, Bynesveien 46, N-7018 Trondheirn, Noiway
structural complexity and the varying absorption characteristics of the different pigment groups of macroalgae make measurements of light absorption and utilization under different environmental and physiological conditions difficult. Several approaches have been commonly applied to absorption analyses using macroalgae. Spectral absorption characteristics of thalli have been analyzed using in vitro absorption of the various pigments extracted in organic solvents (Haxo and Blinks 1950, Jeffrey 1980, Haxo 1985). Pigment extraction is relatively quick and easy and eliminates scattering. Lipophilic pigments (i.e. chlorophylls and carotenoids) , however, undergo spectral shifts ranging from 6 to 90 nm toward the blue when the pigment-protein complexes are detached (Britton 1984, Johnsen et al. 1994). These shifts make comparisons between in uiuo and in uitro absorption approximate. Smith and Alberte (1994) used in 7)iuo absorption spectra and fourth-derivative analyses to compare absorption in Chlorophytes, Phaeophytes, and Rhodophytes and to resolve differences in spectra due to changing environmental conditions. Mathematical analyses of absorption spectra cannot be used to assess pigment functionality. Moreover, the utility of mathematical analyses is compromised due to pigment packaging and scattering, which are significant in thick and/or heavily pigmented thalli. This was emphasized in recent work documenting in uivo light absorption and the potential package effect in macroalgae (Mercado et al. 1996). Therefore, traditional spectrophotometric techniques require pigment information and will provide only a relative comparison. Regardless of methodology, the structure of macroalgae has caused problems in quantifying the in viuo photophysiological state of a given species. Recent microalgal studies on in vivo light absorption in whole cells of phytoplankton have focused on the relationship between whole-cell light absorption and the concentration and composition of different pigment-protein complexes (Hiller et al. 1993, Iglesias-Prieto et al. 1993,Johnsen et al. 1994, Jovine et al. 1995) and on quantifying intracellular self-shading (i.e. the package effect) either experimentally (Kirk 1983, Geider and Osborne 1987, Berner et al. 1989, Sosik and Mitchell 1991, Johnsen et al. 1994) or mathematically (Morel and Bricaud 1981). The “package effect” describes the reduction in absorption of pigmented particles relative to the same quantity of pigment in solution, that is, macroalgal thalli represent packed absorption, and
ABSTRACT
The spectral light absorption and fluorescence excitation spectra from thalli (in vivo) and thylakoid micelles (no intracellular self-shading)from the macroalgae Laminaria digitata {I..) Edmonson, Palmaria palmata (L.) Kuntze, and Ulva sp. were examined. I n all the examined species, the intracellular self-shading (i.e. the package effect) was highly signijicant to the extent that absorption peaks related to individual pigments wfere signi3cantly suppressed. Thus, jor studie,r related to taxonomy and physiology, “unpacked ’’ spectra may be more infmative. Thylakoid micelk preparations offer opportunities to examine more closely species-specgic d@rences in the packagng effect, light hamesting and utilization, and p i ~ e n t a t i o nAs . well, the Lhylakoid micelles, when analyzed with biochemical assays orfluoromty, may povide insight into comflonents of the photosynthetic m a c h i n q that are speciJic to diferent classes of mncroalgne. We present the Chl a-speciJic absoqtion coe/ficients and the corresponding Jluorescence excitation .spectra oj‘macroalgal thylakoid micellesfor the three species. Kty index words: biuoptical taxonomy; fluorescence; macroalgae; package qfect; Pigments; spectral absorption The use of noninvasive biooptical techniques (spectral light absorption and fluorescence) to study photophysiology on relevant spatial and temporal scales is increasing, as instrument sensitivity, durability, and cost-effectiveness is improved. Although past research has dealt mostly with phytoplankton in the open ocean, recent efforts have focused on applying these new methods to coastal zones and estuarine environments. However, few studies have included macroalgae (Haxo and Blinks 1950, Neori et al. 1986). One of the most important parameters in bioop tical models is the spectrally weighted absorption coefficient used in mechanistic models for the estimation of photosynthetic efficiency and quantum yield (ap,.; cf, Morel 1991, Bidigare et al. 1992). This measurement is fiindamental in primary productivity work; however, it is extremely difficult to measure accurately in macroalgae. The varying thickness of the macroalgal thalli and the nonuniformly shaped and unevenly spaced cells cause multiple scattering. This prevents an accurate calculation of the lightpath length and the light-scattering coefficient. The
’ Received 90 Septemher 1996. Accepted 17 January 1997.
* Present address and author for reprint requests: Institute of Marine and Coastal Sciences, Rutgers Ilniversity, P.O. Box 231, New Brutiswick, NJ 089030231; e-mail address: joegQahah.rutgers.edu.
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THE PACKAGE EFFECT IN MACROALGAE
dispersed thylakoid micelles represent unpacked absorption. Studies of the package effect have resulted in a better understanding of the relationship between in vivo absorption, the corresponding package effect, and the light-harvesting efficiency of aigae. The objective of this study was to refine techniques for measurement of absorption in macroalgae. Our main goal was to eliminate the package effect in order to allow direct comparisons of the biooptical characteristics of different macroalgae. We describe a new procedure for studying spectral light absorption and fluorescence properties that may improve the utility of biooptical techniques in studies of macroalgal taxonomy and physiological ecology. MATERIALS AND METHODS
Three species of macroalgae were harvested in mid-August 1995, by scuba diving in Trondheimsfjord, Norway (63" N): Laminaria digatata (Phaeophyta), Palmana palmata (Rhodophyta), and Ulva sp. (Chlorophyta). They were harvested between 3 and 5 m depth and were virtually free of epiphytes. They were stored in seawater at 4" C under low light and used in experiments
