Rainer Hertel (Prof. em.)
rainer.hertel@biologie.uni-freiburg.de

Research Report

Color Vision, Blue Photoreception, Auxin, Philosophy of Science

A. Red/green-blind men can help to understand the evolution of color vision.

Two puzzling neurobiological problems: (1) How can red/green-blind persons use their “red” and “green” percepts in a meaningful way? (2) How can color vision acquire a new degree of complexity and resolution between the primitive monkeys and higher primates, including man, by simply differentiating/duplicating a cone opsin gene?

Color vision of protanopes and deuteranopes was studied by Wachtler, Dohrmann, Hertel (2004), and is now further investigated in collaboration with Christian Garbers from Stefan Rotter’s group (BCCN Universität Freiburg), with Thomas Wachtler (now LMU München).
Our results confirmed earlier findings that red/green-blind subjects possess and apply “red” and “green” percepts. Our earlier model (Wachtler et al 2004) for the processing of photoreceptor signals in dichromats showed that, under physiologically plausible assumptions on color processing, a pseudo-trichromatic representation of dichromatic receptor signals can be achieved.
We hypothesise that the same scheme was also operating in our ancestors. In the dichromatic New World monkeys the two color perceptual axes, “red-green” as well as “blue-yellow”, may have existed before retinal trichromacy. A third, new receptor – evolving by diversification and duplication - can “immediately” be used for better wavelength discrimination (trichromacy).

A new model, with plausible neural correlates and with rod-input to color percepts, is presently under study. E.g. in protanopes, the output from M-cones bifurcates into parallel on- and off-pathways, where surrounding cones via horizontals are sending antagonistic signals. Additional input to the M-cone path comes from the rods, always in the same sense as the center cone.
We postulate that MON signals towards “green” and MOFF towards “red”.


B. Flavin binding proteins in plant membranes

Searching for biochemical elements and functions on the plant cell surface that have a role in sensory / developmental processes, we hope to find new types of signal perception / transduction mechanisms.

Some blue-light photoreceptors responsible for tropisms and for certain phototactic responses are presumably flavoproteins "fixed" in the plasmalemma. We started from the working hypothesis that the photoreceptor consists of a membrane protein with a reversibly bound flavin. We found large amounts of such flavin-binding in membranes from higher plants and from Phycomyces.
In presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution into (oxidizing) buffer, the adduct was resolved, and free flavin reappeared with a half time of ~ 20 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in presence of an electron donor (Lorenz et al 2003).
The adduct is probably a flavin–C4a-S-protein complex as shown e.g. by difference spectra with a peak of the adduct at 414 nm (Kunkel, Lorenz, Hertel, unpubl.). In plants, the natural ligand seems to be FMN (Lorenz, Dohrmann, Polaino-Orts, unpubl.).
In collaboration with Enrique Cerdà-Olmedo (Genética, Universidad de Sevilla), Volker Fries and myself are searching for a corresponding homologous or analogous protein in the lower fungus Phycomyces.
Several criteria – localization in the plasma membrane, high abundance, affinity to roseoflavin, photochemistry – argue for a role of the membrane-associated flavin-binding protein as photoreceptor. Its relation to the phototropin flavoproteins, certainly involved in plant phototropism, studied by the group of Winslow Briggs (e.g. Christie et al Science 282:1698-1701, 1998), remains to be analyzed.


C. Auxin transport and action: mechanisms revisited and the search for co-factors

Our plant hormone research has been motivated by the hope to find new types of signalling in the plasma membrane of higher plants.

The auxin efflux carrier – originally ”marked” by NPA-binding - is complex and, according to our studies, should possess 5 different binding sites: two different, interacting recognition sites for phytotropins, a site for 2,3,5-TIBA, an IAA/NAA translocating site, and an IAA-"regulatory" site. Several laboratories try to isolate the genes and proteins related to this interesting “machine”.
Auxin transmembrane translocation is thought to occur by cooperation of two processses in the plasma membrane: (1) influx by passive diffusion of IAAH and/ or via a saturable and specific H+/IAA- symport, and (2) efflux via a basally placed IAA-anion carrier protein.
Contrary to this chemiosmotic theory, we assume that the auxin exporter is a primary ATP-driven pump. Suggestive indications were found by Godbolé, Gräber, Hertel (2001, Plant Growth Regul 32:151-155), and hard molecular evidence for an involvement of ABCB (mdr-pgp) transporters was provided by Noh, Murphy and Spalding (2001, Plant Cell 13:2441-2454).

Action on elongation and the transport of the plant hormone auxin have been studied extensively in numerous species using many substances structurally related to indoleacetic acid (IAA). The resulting specificity patterns show striking similarities. Following classical work of Åberg, we (e.g. Hössel, Schmeiser and Hertel 2005) examined and compared polar transport of 2-naphthoxyacetic acid in different species. For both elongation as well as for polar transport, 2-NOA was much more active in dicots and in Allium than it was in Zea mays.
This finding supports the hypothesis that a single, common protein mediates auxin efflux as well as primary auxin action on elongation. Transporters may have a dual function as pumps and as signaling elements. Important in this context is the finding by Schenck, Christian, Jones, Lüthen (2010, Plant Physiol 152:1183-1185) that the “soluble” TIR1/AFB-receptors, important for gene expression (Dharmasiri and Estelle, 2004, TiPS 9:302-308), do not play the major role in triggering the rapid phase of cell elongation.
Our results question the roles of the extensively studied auxin binding protein 1 (ABP1) as well as that of the proposed efflux carrier PIN proteins. Both proteins undoubtedly perform important, essential functions: some control of plasma membrane functions by ABP1, and at least a structural association with cellular polarity in case of PIN. However, neither auxin action on growth, nor auxin efflux are carried out by these proteins.

The question of specificity of auxin transport can also be asked in a different way: in the plant tissue, is there any substance, other than IAA, that might be transported in a polar way like auxin? Such a substance - if synergistic with IAA – might also explain some of the paradoxes of gravitropism left open by the Cholodny-Went theory. Working on the transport aspect in the laboratory of M. Iino (Osaka, in fall 2001), we found indications of a co-auxin.
From segments of lower zones of maize coleoptiles, but not from the tips, a substance – about 20-30% the amount of the endogenous indoleacetic acid (IAA) – was exported in a polar, basipetal manner; its export was inhibited by NPA. We are trying to identify the material; molecular weight and HPLC elution time indicate that it was not IAA, but a “similar” UV-absorbing organic acid.
In the context of published results on elongation, we propose a model with two differrent, but similar receptor subunits, for IAA and for the co-auxin, respectively, where both have to be occupied by hormone in order to signal.

In a collaborative research with a group at Yonsei University Seoul, Korea, we found an auxin/ethylene-synergism on elongation in the semiaquatic Ranunculus sceleratus (Park, Kang, Hertel 2010).


D. Some philosophical aspects: mind/body, “complexification”, physics <-> biology

In addition to experimental research, I am studying problems somewhat beyond biology, e.g. the “old” mind/body problem ("Not being a dualist, how should the biologist think (talk) about his mind?" in Hertel R 2000 Wie soll der Biologe von seiner Seele reden? S.119-140. In: Hosp I Hrsg. Naturforschung und Aufklärung. Eur Akad Bolzano).

Plain biologists would neither imagine a "ghost in the machine", nor should they talk about "Nothing but a bunch of neurons". The mainstream biologist can learn by analogy: how biological processes are explained by molecular processes. A “slim” system theory might be appropriate, with some of John Dewey’s cautioning slogans: "Process, not underlying substance!" and "A complex process is not less real than a simple one!"
Complementarity does not help to cope with the mind/body problem. Terminology must be adequate to the level of organization, and the essential contribution to mental processes by cultural traditions and institutions (Popper's world 3) should be appreciated by biologists, as stimulus patterns interacting with the central nervous system.

The issue of reductionism, e.g. the relation between physics and biology was addressed in a comment (2007) on Max Delbrück’s informal philosophy.
Bohr’s suggestion (Light and Life, 1933 Naturwiss 21:245-250) that life held some special secret like complementarity in quantum physics of light, may be defused by subdivi-ding the issue into four.
(1) Limited predictability, analyzability of living organisms:
It sounds trivial and has nothing to do with Heisenberg‘s uncertainty. Bohr (in solemn German): „damit aber ist der Analyse der Lebenserscheinungen mittels physikalischer Begriffe eine prinzipielle Grenze gesetzt durch das Absterben des Organismus bei dem Eingriff, welchen eine vom atomtheorischen Gesichtspunkt möglichst vollständige Beobachtung erfordert.“
Wordsworth (in English verse): “Sweet is the lore which Nature brings; / Our meddling intellect / Mis-shapes the beauteous forms of things / We murder to dissect.”
Biologists have to be aware that any testing interferes with future behavior.
(2) Old vitalism - extra factors, extra laws in living systems, which are not found in physics - was rejected by Bohr and by Delbrück. There is however a variant which is in agreement with “the laws of physics”: are there any deep (physical) laws that show up only in living systems? Delbrück’s answer, e.g. for replication was: No! “With the discovery of the double helix, the mystery of gene replication was revealed as a ludicrously simple trick, making those who had expected a deep solution feel silly.”
(3) Why is physics not enough to explain life?
There are two essentially equivalent ways to argue: from the (high) level of organi-zation or from the (long) evolutionary history. Processes in living systems, and of course mental processes, are so complex that it is “practically” impossible to reconstruct them – in their individual course especially – from elementary physics. At the time of Bohr, the chemical bond had been explained in terms of quantum physics, and yet the chemistry and biochemistry, e.g., of ligand-receptor binding, is still done without reconstructing every scheme from quantum physics, and biochemists have not yet lost their job to physicists.
Delbrück argues from evolution: cells, organisms as old, wise guys with a lot of wisdom picked up during a long history of mutation and selection. “.. a cell [is] more an historical than a physical event”; thus “biology is too difficult for physicists”.
(4) Are the profound structures of physics, covered by quantum field theory, string theory, by quantum information, relevant for biology?
80 years ago, Bohr had invoked “complementarity” to grasp the paradoxes of light, of particles and waves. One may call it an “excuse” for the difficulties to visualize the quantum phenomena with intuitive models. The great attempts of a “fundamentalization”, to model past/future, wave-clouds/particle-points, implicate/explicate order are very important not only for the science of physics, but also for our gehobenes Weltbild, for a sophisticated worldview. Efforts, however, to use such ideas for the problems of mind/body or of free will, are doomed.
The interesting arguments concerning the living cell, the soul or responsible action are not found on the atomic level, but on a higher level of organization, “grown” in the long, long history of evolution.

Another general problem under study concerns complexity, increasing in evolution (Hertel 2003). (The issue discussed above – how a new, third color receptor can be used immediately – is one example within this context.)
Evolutionary theory exists in two editions, down-to-earth, selectionist biology and grand evolutionary philosophy: one theory, orthodox Neodarwinism, staying inside biology, dealing with variation (mutation), selection, speciation; the other, partly philosophical approach (see e.g. Bresch, 1977, Zwischenstufe Leben. Piper München) embraces cosmology and cultural evolution as well as the history of life on earth; it deals with complexification. Hardly addressed by Neodarwinists, mechanisms that increase complexity are presently studied intensively by molecular and developmental biologists. The interesting processes happen between neighboring levels of organization. Systems are coming together and form new systems (nets), from one level to the next higher one: E - S. Fusions, duplications, multiplications and differentiation are considered key processes of anagenesis.

The distinction of the two types of evolutionary theory is also relevant for arguments concerning ethics. The relation of ethics and evolutionary thought was discussed (in German) in “Theory of biological Evolution, ethics and the fear of inequality” (Hertel 2010).

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Publications 2003 -2010:

A collaboration with the Hertel-Lab:

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[update May 2010]