For a long time the behavior of algae in response to light stimuli has attracted the attention of
scientists, who hoped that the simplicity of the material would make this phenomenon easy to
investigate. On the contrary, the undertaking turned out to be more difficult than expected and to
date a clear understanding of the phenomenon has still to be attained, while comprehension of
the visual process in humans is extensive. Initial ideas on the chemical nature of receptors for
photobehavioral responses were gained by measurements of their spectral sensitivity. Action spectroscopy
results were then screened and supported by other methods such as biochemistry, absorption
spectroscopy, electrophysiology, and molecular biology, which when integrated with different
investigative approaches shed more and more light on nature and functioning of photoreceptive
pigments.
In the following we will briefly describe the characteristics of rhodopsin-like proteins and flavoproteins
and compare spectroscopic and biochemical techniques that have been used to define the
light-absorbing properties of photoreceptive proteins either in vivo (single cell or cell population) or
in vitro (extracted material). To provide a clear perspective on the efficacy of different techniques as
tools for the study of photoreceptive structures, a discussion of the pros and cons, the advantages
and limitations of each technique are included.
Rhodopsin-Like Proteins
Rhodopsins are photoreceptor proteins, universally used from archeabacteria to humans, consisting
of a proteic part, the opsin, organized in seven transmembrane a-helices, and a light absorbing
group, the retinal (i.e., the chromophore). The retinal is located inside a pocket of the opsin,
approximately in its center.
Why these proteins are so special? First, retinal–opsin complex has an intense absorption band
whose maximum can be shifted into the visible region of the spectrum, over the entire range from
380 to 640 nm. Second, light isomerizes the retinal inside the protein very efficiently and rapidly.
This isomerization, that is, the event initiating the vision reaction cascade, can be triggered almost
exclusively by light; in the dark it occurs only about once in a thousand years. Third, remarkable
structural changes (movements of single a-helix) are produced by isomerization of retinal. Light is
converted into atomic motion of sufficient magnitude to trigger a signal reliably and reproducibly. |
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Fourth, the photocycle (the photoreceptive protein upon light excitation undergoes a series of conformational
changes which can be driven back to the original conformational state) is very fast,
hence the intracellular photoreceptive machinery is immediately reset for a new response. Fifth,
retinal is derived from β-carotene, a precursor with a widespread biological distribution.
About 1000 rhodopsin-like protein genes have been so far detected in the microbial world.
Genes were detected in
Anabaena (aka Nostoc) sp. (Cyanophyta),
Guillarda theta (Cryptophyta),
Pyrocystis lunula (Dynophyta), and
Chlamydomonas sp. (Chlorophyta). Biochemical and spectroscopical
evidences for a retinal-based photoreceptor were reported in one lineage of prokaryotes,
that is, in
Leptolyngbya sp., (Cyanophyta), and at least in four lineages of eukaryotic algae, that
is,
Ochromonas sp. and
Silvetia, sp. (Heterokonthophyta);
Euglena sp. (Euglenophyta);
Gymnodinium sp. (Dinophyta), and
Dunaliella sp.,
Spermatozopsis sp., and
Volvox sp. (Chlorophyta).
Flavoproteins
Flavoproteins, or yellow enzymes, are a diverse group of more than 70 oxidoreductases found in
animal, plants, and microorganisms, which have a flavin as a prosthetic group covalently attached to the protein. The proposition that these proteins could function as a near-UV-visible-light detector
dates back more than 40 years. Despite the ubiquity and ancient origins of flavoproteins, their role
in acquiring information from the radiation environment still remains a complex area of study.
Apart from difficulties in their identification, much of the reason for the lack of understanding
lies in their diversity of function. Typical absorption spectra of flavoproteins show a dominant
protein peak at 280 nm, and maior peaks at 380 and 460 nm. The overall similarity of many
blue-light action spectra with flavoprotein absorption spectra is one of the main reasons for the
belief that flavoproteins can function as blue-light photoreceptors. So far, the only biochemical
identification of flavin-based photoreceptor has been carried out in
Euglena gracilis. Despite this
paucity of evidence, the hypothesis of a flavin-based photoreceptor in algae has withstood time
and still remains an accepted working hypothesis.
Action Spectroscopy
Action spectroscopy still represents the classic way to investigate photopigments. By means of
action spectroscopy the photosensitivity of a cell at different wavelengths can be measured, thus
providing information on the nature of the pigments involved in photoreception. It is still a
common belief that this approach represents the only feasible way to study the photosensory pigments
of a large number of species. However, direct measure of an action spectrum is much more
difficult than that of an absorption spectrum. Moreover, action spectra may not be directly correlated
with the absorption peaks of the pigments involved in photoreception, as light scattering
can cause several errors. When many pigments with similar absorption characteristics in the
same visible range are present, action spectroscopy often fails to discriminate between them.
Even when there is only one predominant pigment, it is not always possible to identify it. To
obtain more reliable results, threshold action spectra should be preferred to eliminate adaptation
phenomena and screening modulation, limiting the utilization of action spectroscopy to the
study of changes or increases in the photosensitivity of a mutant cell, after the exogenous addition
of a presumptive photoreceptor pigment which the cell lacks. It may be hasty to indicate the nature
of a photoreceptor only on the basis of data obtained from action spectroscopy. This is especially
true in the case of photoreceptors such as rhodopsins, which have retinal as the chromophoric
group. Retinal absorption can be fine-tuned by amino acid charges of the retinal pocket, which
allows the entire spectrum between 380 and 640 nm to be covered. Moreover, the presence or
formation of photo-intermediates may shift the absorption maxima, and make the interpretation
of the action spectrum difficult.
Absorption and Fluorescence Microspectroscopy
These techniques do not disturb the integrity of the organism or subcellular components, and allow
the examination of an uninjured system with its physiological functions intact. The spectroscopic
overshadowing of one pigment by another is avoided because each pigment is packaged in a different
structure. Thus, cellular structure can be easily correlated with pigment type by direct observation.
It is possible to make exact quantitative determinations of various reactions at the time
of their occurrence in the sample, the progressive changes in these reactions, and their relationships
to different conditions in the external medium. Because of the fundamental connection between
optical parameters and properties of molecular structures, microspectroscopy allows assessments
of minute changes in the state of the molecules of various substances in the organism, the
degree of their aggregation, and the interconversions of various forms of pigments and other
important biochemical compounds with characteristic spectra. In many cases the lability and reversibility
of such changes make microspectroscopy the only possible method of investigation. There
is virtually no light scattering problem associated with microspectroscopic measurements, even if
the analyzed structure has a dimension of 1 µm. Obviously, the absorption spectrum cannot provide
adequate information about the photochemical action of photons as a function of their fundamental energy; however, the identification of the chromophores in the photoreceptive structures does provide information about possible mechanisms of energy transfer. The measurements are very difficult
when small changes in absorption have to be measured in the presence of a strong total signal
(luminous background), as photon noise is proportional to the square root of the intensity of the
incident light. Then, fluorescence spectroscopy is recommended: it can achieve more reliable
results compared with absorption spectroscopy, because the background emission is much
reduced. In this case the sensitivity of detection is not limited by the signal-to-noise ratio, but
rather by the presence, virtually unavoidable, of fluorescent contaminants.
Biochemical and Spectroscopic Study of Extracted Visual Pigments
Extraction of visual pigments (chromophore or protein), either by means of detergents such as digitonin
and TRITON or by organic solvents, could be the best method for providing large quantities
of photoreceptive pigments in an accessible in vitro form for subsequent detailed biochemical
analysis. Such samples allow a very accurate determination of spectroscopic parameters. Spectroscopy
of solubilized pigment may be complicated, however, by the simultaneous extraction of
several other pigments in the cell, which cause distortion of absolute spectra and necessitate
special procedures of purification. These problems can be solved by separating the different pigments
after extraction, for example, by high performance liquid chromatography (HPLC) and
final identification with gas chromatography – mass spectrography (GC–MS) for chromophores
or affinity chromatography for proteins. As pigment extraction permanently removes the identifying
link to a particular cell structure and may change the spectroscopic properties of a receptor
because native interactions are disrupted, these detrimental factors mandate a careful evaluation
of the results obtained by this method.
Electrophysiology
Light excitation of the photoreceptor generates a cascade of electrical events. Electrophysiology
was the elective method in the study of vertebrate photoreceptors. However, this technique has
been applied with less success in the algae because it is very difficult to locate the photoreceptors
in the cell body and, when this is possible, to produce a good sealing between the glass pipette and
the cell membrane.
Flash-induced transient depolarizing potentials using intracellular glass microelectrode were
first identified in Acetabularia crenulata. However, the first detailed analysis of photocurrents
were possible by employing a suction pipette technique (patch clamp technique) in Haematococcus
pluvialis and in the wall-free mutants of the unicellular green alga Chlamydomonas sp. In these
experiments whole cells were gently sucked into fire-polished pipettes, forming seals with resistances
up to 250 MW, allowing cell attached recordings from a relatively large membrane area,
though higher resistance seals were not achieved. Recently, Negel et al. (2002) demonstrated by
means of electrophysiology that the rhodopsin-like protein of
Chlamydomonas, expressed in
Xenopus laevis oocytes in the presence of all-trans retinal produces a light gated conductance
that shows characteristics of a channel selectively permeable for protons.
Molecular Biology Investigations
DNA hybridization is useful in attempting to determine phylogenetic interrelationship between
species. The rationale is that similarities between DNA structures correlate to interrelatedness.
It is used to detect and isolate specific sequences and to measure the extent of homology between
nucleic acids. It represents an alternative to the study of visual pigments at the protein level, as
the genes encoding these proteins can be identified, their sequences determined, and the comparative
genetic information assessed. Genomic Southern blot hybridization is used to probe the genomes of
a variety of species in a manner analogous to that reported for other protein families. The potential for using bovine rhodopsin opsin complementary DNA (cDNA) probe to identify homologous genes
in other species was demonstrated by Martin et al. (1986). These authors identified coding regions of
bovine opsin that are homologous with visual pigment genes of vertebrate, invertebrate, and phototactic
unicellular species. Successful application of this method requires closely homologous genes,
and in general additional criteria, such as protein sequence information, is desirable for eliminating
false positives on Southern blots. A molecular biology approach has been used also by Sineshchekov
et al. (2002) in Chlamydomonas. These authors identified gene fragments with homology to the
archaeal rhodopsin apoprotein genes in the expressed sequence-tag data bank of Chlamydomonas
reinhardtii. Two quite similar genes were identified having almost all the residues of bacteriorhodopsin
in the retinal binding site. The authors suspected that these genes were related to the putative
retinal-based pigments already suggested for Chlamydomonas.
However, to show that the pigments are a part of the genuine signaling system, ideally one
would like to delete each gene by using homologous recombination, but it is not easy to do such
gene knockouts in any algal species. The problem can be overcome partially by using RNA interference
(RNAi) technology to preferentially suppress the synthesis of the pigments to convincingly
show that the pigment is a genuine segment of the algal phototactic response.
Understanding the molecular mechanism used by algal cells to “see the light,” as we have tried
to explain, is a very difficult task. At least a century has been wasted without any success. It is discouraging
to think that even if the algae are not as intelligent as men are, they have “understood”
very well how to orientate themselves in their light environment, and do it very efficiently. Maybe
the compass mechanism they use is too simple for our complex brain.
