Phototropism: Some History, Some Puzzles, and a Look Ahead

Though few and far between, phototropism studies through 1937 established a number of important principles. (1) Blue light is the active spectral region. (2) The phototropic stimulus is perceived by the coleoptile tip, and the consequences of the stimulation progress down into the growing region. (3) Lateral transport of auxin mediates the curvature response. (4) The reciprocity law holds for first positive curvature, whereas second positive curvature is time dependent. (5) Red light treatment had a major effect on phototropic sensitivity. Later studies established the following. (6) Seven blue light receptors (cryptochromes, phototropins, and three F-box proteins) were identified and characterized. (7) A flavin was established as the photoreceptor chromophore for all seven. (8) The chromophore domain, designated the LOV domain (for light, oxygen, or voltage), carries out a unique photochemistry. (9) LOV domains must be truly ancient chromophore domains. There remain some puzzles. The fluence-response threshold level for first positive curvature is far below that for phototropin photochemistry. Likewise, the fluence-response threshold level for the red light effect on coleoptile phototropism is far below those for phytochrome phototransformation. Cytological effects of red light are also very insensitive compared with the physiological effects of red light. What is the mechanism allowing for this extraordinary photosensitivity? How is phototropin specificity controlled? What are the functions of the phytochrome kinase substrate proteins in both phytochrome and phototropin responses? What mechanism leads to lateral auxin transport? Finally, are LOV domain proteins true photoreceptors in all of the bacteria in which they occur? If so, what is their biological function? Even in the ancient world, astute observers noted that plants could turn to face the sunlight. What was originally designated heliotropism for plants that followed the sun eventually became divided into two distinct response categories: solar tracking (the real heliotropism), a repetitive and completely reversible turgor-driven process; and phototropism, an irreversible directional growth response determined by light direction. Over the past 200 years, a large number of brilliant biologists, including Julius Sachs (1864), Charles Darwin (1881), Frits Went (1928), and Kenneth Thimann (Went and Thimann, 1937) have applied their talents to examining and elucidating the mechanisms accounting for both of these responses. The entire history of phototropism and solar tracking parallels and is intertwined with that for a number of other blue light responses, found not just in higher plants but in bryophytes, ferns, algae, fungi, and, most recently bacteria. For a detailed account of this history, see Briggs (2006). Progress in research on blue light-activated processes over the last half century was severely hampered by a lack of knowledge of the relevant blue light receptors. By contrast, the discovery and initial characterization of a red/far-red-reversible phytochrome (Butler et al., 1959) nurtured an enormous and sophisticated body of knowledge about these photoreceptors: their structure, their chromophores, their photophysics and photochemistry, and the extraordinary signal transduction networks that they rule. Meanwhile, although a huge and scattered body of knowledge on blue light responses in plants and fungi accumulated, there was scarcely a clue to what the photoreceptor(s) might be, and competing hypotheses abounded. Attention focused on the physical and physiological characterization of responses to blue light and, eventually, biochemical investigations of intriguing in vitro photochemistry. It was only with the advent of modern molecular genetics and its extraordinary capabilities that research on blue light photobiology finally began to catch up with phytochrome photobiology 20 years ago and the first blue light receptor, cryptochrome1 (cry1), became identified (Ahmad and Cashmore, 1993). This review will go back to some of the puzzles of earlier years, a few over 100 years old, and provide a glimpse of the status of these puzzles today. They arose from studies of response kinetics, action spectroscopy, interactions between blue and red regions of the visible spectrum, and discrepancies between in vivo and in vitro results. Although the focus will be on higher plant phototropism, several other blue light responses will contribute to the discussion. No effort will be made to be comprehensive. Any effort here would be redundant with that of three recent excellent reviews on phototropism (Sakai and Haga, 2012; Christie and Murphy, 2013; Hohm et al., 2013) and on LOV (for light, oxygen, or voltage) domain photochemistry (Losi and Gärtner, 2011, 2012). The article concludes with a look at the unexpected role of LOV domains in prokaryotes. Over a century ago, Fröschel (1908) and Blaauw (1909) first reported that the phototropic responses of Lepidium spp. seedlings and Avena spp. coleoptiles, respectively, obeyed the reciprocity law of Bunsen and Roscoe (1862). The law states that as long as the light dose (fluence: intensity × time) is held constant, a given light response, in these two cases phototropism, will remain the same over a broad range of intensity × time combinations. In both cases, the authors were investigating threshold responses, something that later investigators tended to forget. Meanwhile, du Buy and Nuernbergk (1934) collected data from a number of authors and published the fluence-response curve shown in Figure 1 for the phototropic responses of Avena spp. coleoptiles over an immense range of fluences (8 orders of magnitude). The first maximum was designated first positive curvature, the immediately subsequent minimum first negative curvature, the second maximum second positive curvature, the next minimum a “zone of indifference,” and the final ascent third positive curvature. First positive curvature was usually located only a short distance below the coleoptile tip, whereas second positive curvature extended somewhat farther down the coleoptiles. For some reason, reciprocity tests were never carried out at any fluences above the threshold assays used by Fröschel (1908) and Blaauw (1909), although their conclusions, unfortunately, became accepted and applied to higher plant phototropism in general. All of this early work was with white light from a variety of sources, and the unit of fluence at the time was the meter-candle second (MCS). Fluence-response curve for the phototropic responses of Avena spp. coleoptiles assembled from the literature by du Buy and Nuernbergk (1934). From Briggs (1960). Over 50 years ago, Briggs (1960) finally reinvestigated the reciprocity relationships of phototropism for maize (Zea mays) and oat (Avena sativa) coleoptiles over a wide range of fluences and demonstrated that the reciprocity law was valid only for first positive curvature but not for second. In all cases, for fluences above 10,000 MCS, maximum phototropic curvature for both species was attained after about 20 min of unilateral light stimulus irrespective of fluence. Indeed, a flashing-light experiment with a total fluence of just over 30,000 MCS showed that with increasing dark periods but constant flash duration, maximum curvature for maize coleoptiles was only achieved when the total exposure time, dark plus light, was 20 min or more. Zimmerman and Briggs (1963a, a for the phototropic responses of oat coleoptiles, on a detailed of fluence-response a of to the The for first positive phototropism an initial photoreceptor and its subsequent a The they for second positive curvature the of an photoreceptor followed by a dark that to the curvature response. The a and time for the response, accounting for the lack of It also a second that the first By the early was established that first positive curvature is to the response is with to the of the number of photoreceptor first positive curvature, blue light of also the reciprocity law both in vivo and in vitro et al., with this The of the response curve the first positive maximum is to a in the between of the photoreceptor on of the coleoptiles. fluences are of the photoreceptor on both of the coleoptiles all eventually and the This not the from Zimmerman and Briggs for first positive of photoreceptor on the could the photoreceptor their the for second positive curvature. exposure time the phototropin are to to their dark and be there is a light the is on the on the of the coleoptiles and a to second positive curvature, time on the for the the of Zimmerman and Briggs is not but the of photoreceptors on the the of the could the they for of oat coleoptiles et al., the that a light leads to a biochemical in an both for first and second positive curvature. of in between and by blue light, from light-activated phototropin et al., this shown to be for phototropism et al., the time for of phototropin to its dark could account for the time of second positive curvature. Indeed, the of to light-activated in vivo over a of a light et al., is that is to all of the photoreceptor at on both and of the a that to curvature, the first minimum in the fluence-response curve for phototropism see Briggs et al., The of phototropic after a the same time of the du Buy and Nuernbergk (1934) fluence-response curve remain first negative curvature and third positive curvature. First negative curvature only demonstrated for oat coleoptiles, is for maize coleoptiles, and never reported for It a although Zimmerman and Briggs a to that for first positive curvature, that a to the positive curvature might be by the that the data to this response in fluence and exposure time light could have to in response to an positive The exposure to these fluences as the light Indeed, there for a third positive curvature in any species first and second positive to be coleoptiles and seedlings Zimmerman and Briggs and and et al., including et al., Fluence-response curve for the phototropic responses of maize coleoptiles. From Briggs (1960). There is a in to light-activated phototropin in vivo with the threshold and fluences for blue light of first positive curvature between 1 and orders of below those for et al., The physiological response curve is not with the response This is that blue light phototropic curvature and red light not see a of the early phototropism studies on red light as a noted for the first time that fluences of red light the phototropic of oat coleoptiles in the first positive curvature range by 1 of Zimmerman and Briggs (1963a, this effect both for first positive curvature for first negative of oat coleoptiles and demonstrated in that red light had the effect on second positive its by a of and Briggs reported with maize coleoptiles. and Briggs also demonstrated that the maize response to red light was reversible and to be a phytochrome response. There is a with a of the on with maize coleoptiles to a Briggs and fluence-response relationships for the red light effect on first positive phototropism and compared with those relationships for phytochrome in vivo for maize coleoptiles. phototropism was by fluences of red light orders of to of phytochrome This parallels the in blue light phototropic curvature at fluences far below those that phototropin in vitro or in vivo This is all the in that the red light effect on phototropism is reversible the light are the same in phototropic that red responses were later designated responses and used This is also Fluence-response curve for the phototropic curvature of maize coleoptiles and for phytochrome in vivo From Briggs and The in is somewhat Red light both first and second positive curvature. and first showed that red light treatment phototropic curvature in the first positive and shown to be by et al., et showed that red light the between the of blue light and the of curvature, an and demonstrated that the role of was and that and could also the of phototropism to higher blue light fluences (for see et al., there is for effects of red light on first and second positive curvature in effect of red light on coleoptiles might be to red in phototropic sensitivity. Briggs found that red light treatment of maize coleoptiles the of auxin that could be collected in to of that from dark The was a level after about a dark after a red light the auxin to the dark over a of about The time for these auxin were to the red in phototropic and its subsequent in the dark studies with with mechanism for the red in phototropic found in and Briggs first noted that blue light the of from the into the a response in by et The response to min to go to et reported a of from the in et found that red light given to phototropic completely the blue of The photoreceptor with the that lateral auxin transport is to be with of a blue light red light treatment could account for the of phototropism in this red light effect could only be in the growing of the the above mechanism be valid for the red for be the red light coleoptiles in the first positive curvature first positive curvature second positive curvature the threshold for this red light effect in coleoptiles is far below that reported by et for the in in and the of phototropin at the is not the For the third time, an between and photochemistry and Briggs reported that red light treatment the between the 1 of oat coleoptiles by about to a in a the The began about after the of red light and to the dark level after about min that time, a of light after about to a in of about followed by dark over several A second red light had effect there had not an light are phytochrome and some of action at the some years earlier had with the red/far-red-reversible effects on that could only be by phytochrome in to the et have demonstrated an of phytochrome with phototropin at the in of the They also used a to an of phytochrome with phototropin when they were both into although they to for an in a recently a review on by the early history as as recent on the and their signal transduction in recent years focused on their effects on and and et al., 2012). It is that is to of at the Red in between the 1 of an oat coleoptile and a in a the and Briggs In et reported an important the by blue light but both in vivo and in vitro with and and is also was shown to phototropism and et al., A second was to both and two responses et al., Finally, et showed that some is for a phototropic response. the two proteins provide the between phytochrome and a that are with the large between and photochemistry, of is that the discrepancies between the of phytochrome and both phototropic and phototropin could be by some mechanism at the an mechanism This mechanism could be when only a very few photoreceptor are a mechanism might be to the a very few are to in a Frits Went originally that the phototropism of coleoptiles was by lateral transport of a on of auxin from and of coleoptile the total auxin was that from dark of higher on the on the was not as a mechanism to curvature. The auxin mechanism in coleoptiles Briggs et demonstrated that an between and of maize coleoptile any auxin auxin and Briggs and and Thimann used and extended these somewhat recent studies have demonstrated that the growth between and of is a in growth on the is by an in growth on the and Briggs this response in maize coleoptiles, and et with of coleoptiles as curvature demonstrated a growth in of All three of these studies are with a that lateral transport of auxin any or of the the response. is Darwin first demonstrated the of the coleoptile in the phototropic light The coleoptile had to be in for curvature of the regions of the coleoptile to curvature. and and the to a of 1 of the coleoptile demonstrated that physical between the and of the oat coleoptile was also in to phototropic curvature. the coleoptile and a of or a of into the to unilateral at to the curvature was If the was to the of light, curvature with the of the Finally, the and the two back any curvature. Briggs the experiment with maize coleoptiles a and a the was at to the light, second positive curvature was and first positive curvature was completely Briggs showed that with only of coleoptile unilateral light as a in auxin between and of coleoptiles as when the of the coleoptile was et showed that the 1 of maize coleoptiles showed the level of light-activated of what was identified as All of these for phototropism (1) that in the coleoptile is (2) that lateral between and is and (3) that a phototropin is the three to be the large between the fluence for threshold phototropism and the fluence for threshold blue phototropin something of a over the a phototropin in coleoptiles. The growth growth on and of established that lateral transport of auxin could be in as as In as et demonstrated a in between and of In the past a of about the several proteins in auxin transport (Sakai and Haga, 2012; Christie and Murphy, an is the of of the light signal in the as in coleoptiles, is perceived in the and as in the of Christie et used the a that to to this They found that unilateral blue light an of the of auxin from the into the and by an of Lateral of auxin into the above the and the auxin between on and down into the by the of curvature. Although blue of the auxin for the initial growth which auxin are in the lateral transport is not the over a century ago by Darwin for coleoptiles is also valid for and for other light above the growing and by lateral transport of an auxin that down into the growing the of the first what of light was for plant noted that the of light for a in to the at to the light or solar the first that there might be a photoreceptor that light for its and years demonstrated that true phototropism of seedlings was also in the blue of the followed with detailed studies but the of the century for and and Thimann and (1960) to the first action for first positive curvature of oat the range to and Thimann an action for second positive curvature that over the same In subsequent years, action for by blue light with for responses as as phototropism in the two fungi and in stimulation of by the fungi and in the of a of in and and several other and The of all of these action were a broad of in the a in the blue with a maximum and of of the and the with sativa) seedlings what the action by far for first positive It from into the of the to and for the first time a of to about as as the action with that by Thimann and (1960) for oat coleoptiles over the entire range by both for was that there was a blue light to all of these responses. The was the chromophore was a a or something It was only in that the first blue light was in (Ahmad and Cashmore, 1993). et identified a second The are to proteins in and both a flavin and a or a as were found to a flavin and most as the second chromophore et al., et al., an initial for the discovery of the of the photoreceptor chromophore for phototropism and that the an important of the for might be the chromophore for phototropism in maize coleoptiles. et found that the coleoptiles of maize seedlings on the of showed phototropism in the of any et that might be out the photoreceptor for et the responses to both blue light and phototropism in in and a a and the recently and and only the first positive phototropism in response to blue light and et al., the authors out of the and both for et also as the showed in the of a phototropic response. they that there must be a minimum of blue light receptors in this two and an photoreceptor that for but not The of this is The et al., et al., had not identified at the et had used a fluence in the studies that was to whereas the fluence they used for the phototropic studies was to (Sakai et al., they to phototropism but for the blue light out to be and not a completely is what the photoreceptor is for the by and It could be the of of the of the flavin chromophore of or both or of or in a photoreceptor It could also be the of the of a recently photoreceptor in et al., will Finally, there have two of et reported very phototropic of maize It could be by the seedlings on a to curvature by Red light from above to unilateral red light completely the phototropic response, the authors to that the photoreceptor was a Likewise, et a phototropic response to unilateral red light from that had in They reported a in growth about min after the of red light and that the curvature was the of growth the Red light from above also the response, the authors to the same It that a chromophore functions for a photoreceptor for phototropism, although phototropism is its major of the finally became with the of that were in phototropism and et found that of the of and was severely in its and Briggs a phototropism designated that to to unilateral light in the first positive curvature range and the light-activated et used this to and the the that was the substrate for of a It two domains about 100 long with very which they designated LOV domains they were to domains in other proteins that were to light, oxygen, or was a kinase domain, a of the kinase et al., A Christie et the in and demonstrated (1) that a flavin and (2) that light its in the of any other plant authors that was a photoreceptor for Christie et demonstrated that the LOV domains were both for It was not long a second was identified et al., et al., there is a third of blue light receptors in the et al., et al., and et al., a role in phototropism, they are in and through proteins are all F-box proteins that the same unique LOV domain photochemistry for the and blue light receptors (Losi and Gärtner, 2012). both both phototropins, and all three F-box proteins a flavin as the flavin in of a blue light into blue light two a two phototropins, and three F-box There is a between the identified for signal transduction and For the is to but role in the of and The is for by but effect on by a at the to phototropism, or et al., 2012). mediates the of growth of seedlings and the response and are only by and although responses be by with on only of the two for a signal transduction for responses must at the level of the A for response is completely for response. For of these and several other see and LOV domains have a of for investigating chromophore and et demonstrated that they carried out a unique the of a between a and the of the to a et first reported that the LOV domain the of a flavin to et also reported and chromophore by light et showed next that LOV domain to about a in on et showed that to an of an just from the LOV domain, the to the kinase domain accounting for the reported by et A number of LOV domain have by the reviews by et and and for a of progress in this active Although progress made in the past two in plant blue light receptors and the processes they there remain a few puzzles that have to or have The huge between the of light that a physiological response and the that to the photoreceptor is Indeed, when a is about the and of a be a phototropin or a the there a phytochrome is the phototropin the and photochemistry by about orders of whereas the phototropin must only with a of between 1 and orders of How is phototropin specificity What a given response is by or both What a a role in but not or of phototropin a there other that How unilateral light the lateral transport of What auxin are plant for a long the discovery of LOV domains in et al., and of their unique photochemistry, light-activated of a et al., LOV domains have identified in proteins not only from fungi et al., but in proteins from over of all bacteria far (Losi and Gärtner, 2012; et al., 2012). The first photoreceptor to be was from et al., and of are from all major of fungi et al., including et al., the of which were the of of phototropism the and the of a number of in a blue The first reported to have a LOV domain was a from (Losi et al., LOV domains are found from a large number of the LOV domains and domains. There are also short LOV proteins with et al., from the but also from the in the of LOV domains on the of a a minimum of to be for and a of et al., 2012). There is a of proteins LOV domains in as as bacteria. to proteins LOV there are that or blue light domain Indeed, some bacteria two of photoreceptors and some three and to the LOV domain or the entire LOV they all LOV domain on a major what are the functions of these LOV domain a a in a number of recent cases, the have of a LOV domain in the its in a et al., the of a in the et al., a response through the in et al., A mediates by the plant et al., 2012). Likewise, a mediates light-activated for et al., A mediates and in et al., 2012). an important role in in through a a response that also a phytochrome et al., a and to the and This will on the signal transduction that these LOV proteins is is that there is a of wide for research on plant photoreceptors not only about plant photoreceptors but an for and for their review of the and for in and the of published also for the for this light, oxygen, or meter-candle second