Prof. R. P. Sharma
School of Life Sciences
University of Hyderabad
Hyderabad-500046
 

Introduction:

The life on our planet is sustained by continuous input of energy in biosphere in the form of the sunlight, which is harvested by plants, via a process called photosynthesis. Not only the plants use sunlight as a source of energy, to drive the process of photosynthesis, they have also evolved several specialized mechanisms, where they use the sunlight as the source of the information. Plants have developed mechanisms to sense the impending seasonal changes in the climate, to detect the shading and competition from the neighboring plants, to follow the path of the Sun etc. The bending of the plants towards the source of light, such as orientation of an indoor plant towards window of the room, is one of such well known phenomena. Each of these processes uses the light as a source of information and integrates this information with growth and development of plants. The processes involved in the detection of the light, transduction of light information signal to a chemical signal, to final execution of the photoresponse, consists of multiple steps and involves a coordinated process between different tissues and organs. The detailed steps involved in these processes are still not fully deciphered and are under intensive investigation. In the present talk I have summarized the current knowledge about this phenomena.

Historical Perspective

Though the orientation of plants towards the source of the sunlight is a well known phenomenon even to a layman, the first scientist to critically examine this process was none other than Charles Darwin. During his long voyage on the famous ship Beagle, Darwin kept the birds captured during his journey. He fed these birds with seedlings raised out of canary (Phalaris sp) grass, which he grew in small containers in his cabin in the ship. Being a keen naturalist he observed that the seedlings grown in his cabin were oriented towards the window, which was the only source of light in the dark cabin. He followed these observations with the experiments, which he eventually along with his son as a co-author published as a monograph namely "The power of the movement in the plants".  Darwin showed that in the absence of light, a germinating seed's coleoptile grew vertically. The coleoptile of the same seedlings on exposure to unidirectional light bent towards the light source. However, if the coleoptile tip was removed or covered by an opaque cap, coleoptiles did not bend towards light. Whereas, the bending toward the light source occurred when the coleoptiletip was covered by a transparent cap or the region where bending occurred was covered by an opaque material.  These experiments of Darwin laid the foundation for the modern plant hormonal physiology, which eventually lead to discovery that the coleoptile tip was the site of auxin synthesis. However, we first like to learn how would the coleoptile tip or a plant sense that the light is unidirectional in nature?

Plants see the light using specialized photoreceptors

Plants can see the light, the very same way, which we do, by using specific macromolecules called as photoreceptors that can detect the light. In case of mammals rhodospin is the visual pigment of the eye. Plants too have similar type of the photoreceptor molecules to gather information about its light environment. Plants obtain information about its light environment by several ways using one of the properties of the light. The way plants elicit this information is illustrated in the table. To detect an unidirectional light the plants have to make a two point measurement to detect a light gradient within the plant body. In the natural environment plants use different photoreceptors to collect the information about light gradient, duration, photoperiod etc.  For detecting each type of information, plants have evolved multiple photoreceptors to perform these tasks. There are enough physiological evidences for existence of different photoreceptors, however for most photoreceptors  their biochemical nature is yet to be identified. The known and yet to be identified photoreceptors of plants are listed in table.
 

How does one identify the photoreceptor?

The task of a photobiologist, once he identifies the photoresponse, is to identify the photoreceptor causing the response. Most often it is a job similar to finding a needle in the haystack, however the photobiologists are added in this task by a technique called action spectroscopy. The action spectra of photoresponses are derived by a careful study of the photoresponse at different wavelengths. First, the given plant material is exposed to different doses of light at a given wavelength and a dose response curve for the photoresponse is plotted. Similar dose responses curves are then obtained for the other wavelengths too, by increasing wavelength interval by 10 or 20 nm. Second, using these dose response curves, the number of photons required to elicit a fixed degree of the photoresponse at different wavelengths is determined. It is naturally expected the least amount of quanta would be required for eliciting the photoresponse at the most effective wavelength. The reciprocal of quanta required to elicit the response are then plotted against the wavelength and called as action spectra. The action spectra of a given photoresponse are then compared with the absorption spectra of known photochromic molecules. It is implicit in these comparative studies that the action spectra so obtained closely represent the absorption spectra of photoreceptor causing the photoresponse. In past the action spectroscopy has been successfully used to identify and isolate the plant photoreceptor phytochrome, which plays a major role in photoperiodic regulation of flowering in higher plants.

Action spectroscopy revealed that plant bending is caused by blue light

The action spectroscopy revealed that the effective spectral region triggering phototropism is in between 350-500 nm i.e. the blue region of the spectrum. The action spectra of oat coleoptile phototropism showed two prominent peaks at 475 and 450 nm with a shoulder at 420 nm. Interestingly the action spectrum for phototropic curvature in sporangiophore of the fungus Phycomyces also shows features similar to oat coleoptile with peak at 460 nm. It is of interest to note that the Nobel Laureate Max. Delbrück, after his pioneering work on bacteriophage genetics, for which he got the Nobel award, shifted his research area to study of phototropism in Phycomyces. These similarities highlight that photoreceptor mediating phototropism in higher plants and fungus may share few common elements.

Molecular nature of the blue light photoreceptor's chromophore

The similarity between the action spectra and absorption spectra of known biological pigments has been used to predict the likely molecular nature of the photoreceptor, or at least the nature of chromophore associated with the photoreceptor. Based on the similarity between action spectra of phototropism and absorption spectra, it has been predicted that a carotenoid, or a flavin, or a pterin act as putative chromophore for the photoreceptor. Arguments and evidences for and against each of these molecules have been advanced in the past. In recent years most people have favored flavin and pterin as potential candidates for the chromophore, however, still few people support carotenoid as chromophore molecule. A great deal of the controversy regarding the molecular nature of the chromophore has been generated due to lack of identification of the photoreceptor molecules per se. However, in recent years the remarkable progress has been made for identification for at least few likely photoreceptor candidates, which is described below.

Dose response curve of phototropism has two peaks

 Normally the dose responses curves for most photoresponses are linear in shape till it reaches a plateau. However the dose response curves for the phototropism in higher plants have a very distinct shape, consisting of three zones. The initial rising part of the curve and the first peak is called as first-positive curvature, the subsequent decline to a minimum is called as first-negative curvature, and the second rising part is called as the second-positive curvature. This unusual shape of the dose response curve for the phototropic curvature has added to the complexities of the identification of the photoreceptors. The views for and against the multiple photoreceptors have been advanced on the basis of the physiological consideration. The recent results on identification of the photoreceptors have not still clearly resolved the phenomena. The evidence is still incomplete to say firmly if one or more than one photoreceptor sense phototropic blue light.

One of the earliest responses in the phototropism is phosphorylation of a membrane bound protein

 The etiolated seedlings of higher plants are hypersensitive to blue light, even a brief exposure of one or two second of weak blue light is sufficient to elicit phototropic curvature. The work carried out by the Winslow Briggs group revealed that exposure to blue light for such a short duration causes phosphorylation of a 120 kD protein likely associated with the membranes. Both in vivo irradiation of etiolated plants or in vitro irradiation of the homogenates could elicit the protein phosphorylation. Moreover, the phosphorylation was observed in most of the higher plants examined including both dicots and monocots. The physiological studies conducted showed a close correlation between the phototropic responses and blue light mediated phosphorylation. It was speculated that this phosphorylation might represent one of the initial events in the phototropic signal transduction pathway.

Mutants have aided the identification of photoreceptors

 The physiological studies during the past century though aided to characterization of the phototropic response, the molecular identity of the photoreceptor could not be ascertained. However during past decade a significant progress was made to identify the photoreceptors by isolating mutants from Arabidopsis, also known as "Botanical Drosophila". Jiten Khurana while in Ken Poff’s lab was first to actively screen Arabidopsis for the phototropic mutants. He isolated several mutants defective in phototropism, which were eventually found to be defective in the photoreceptor and also in the components of the signal transduction chain. A proof that the blue light mediated phosphorylation was indeed the part of phototropic response was obtained using those mutants. The mutant JK224 which was defective in the phototropism was also defective in phosphorylation. Winslow Briggs supplemented to the studied of Khurana by isolating few more mutants and  rechristened JK mutants as nph (non-phototropic hypocotyl) mutants in Arabidopsis. These mutants were found to be defective in the signal transduction and photoreceptor per se. The molecular analysis of these mutants particularly of nph1 mutant lead to the identification and isolation of first putative photoreceptor for the phototropism.

NPH1 gene encodes a kinase and has a flavin binding site

 The NPH1 gene was cloned using the technique of Amplified Fragment Length Polymorphism in 1997. The perusal of the NPH1 gene sequence and its comparison with other gene sequences in the data bases showed that it encodes a novel protein with significant homology with proteins with LOV domain found in bacteria and higher organism. The gene sequence also indicated that the protein encodes a serine-threonine protein kinase. The protein has a putative flavin-binding site too, confirming for the first time that flavin rather than carotenoid act as chromophore for phototropism. The confirmation that NPH1 protein is the photoreceptor for the phototropism rather than an intermediate in the signal pathway came for its expression studies. The NPH1 protein can be expressed in the insect's cells and does undergo autophosphorylation on exposure to the blue light.  Recombinant NPH1 binds FMN noncovalently and its fluorescence excitation spectrum is similar to action spectrum of the phototropism. Since mutant defective in the NPH1 protein lacks  phototropic responses, it is believed that NPH1 is the photoreceptor eliciting phototropism. The NPH1 protein has been renamed phototropin, to indicate  its biochemical function. Studies on structure and function of phototropin are currently being done by Briggs laboratory to understand  how this protein mediates phototropic signaling.

Single/multiple photoreceptors for phototropic response

 The unique dose response curve of the phototropic responses has been interpreted as a result of the operation of two independent photoreceptors. The isolation of nph1 mutants indicated that one single photoreceptor may be sufficient to trigger phototropism in plants. However blue light also mediates other responses in the Arabidopsis seedlings such as inhibition of hypocotyl elongation and promotion of cotyledon expansion using two other photoreceptors namely cryptochrome 1 and cryptochrome 2 respectively. Similarly to NPH1 the cryptochrome 1 and cryptochrome 2 have also been cloned from Arabidopsis, and found to have strong homology with microbial DNA photolyases. The cryptochromes are dual photochromic molecules having two chromophore binding sites one for a flavin and another for pterin. The mutants deficient in either cryptochrome 1 or cryptochrome 2 are normal in their phototropic behavior. However, a double mutant of cry1/cry2 lacks the first-positive curvature of phototropism. Since the double mutant shows normal blue light mediated phosphorylation of 120 kD protein, it has been assumed that NPH1 is present and functions normally in these mutants. It has been proposed that the first-positive curvature is mediated by cooperative action of cryptochrome 1 and cryptochrome 2 genes, whereas NPH1 may be a component of the signal transduction chain acting down stream of these photoreceptors. Since the above double mutants retains normal second-positive curvature, it can be assumed that either NPH1 or another yet unknown photoreceptor triggers this reaction. It is expected that in coming years the detailed analysis of mutants would clarify the relative roles of these photoreceptors in phototropism.
 

Additional photoreceptors may be involved in phototropism

 As outlined above, the current knowledge obtained from studies on Arabidopsis, indicates that there are at least three putative candidates for the blue light photoreceptor mediating phototropism namely, NPH1, cryptochrome 1 and cryptochrome 2. It is likely that there may additional photoreceptors in the plants, which may mediate this response.  The action spectra of phototropism have indicated that the wavelength higher than 500 nm are ineffective in triggering phototropic reaction. One of the major photoreceptor which sense the wavelength higher than 500 nm is phytochrome which detects red/far-red regions of the spectrum. It has been shown that the in blue light mediated responses phytochrome plays only a modulatory role. The etiolated seedlings if exposed to a brief pulse of red light about 1-2 hour prior to blue light exposure shows the increase in the amplitude of the first-positive curvature compared to control seedlings. However red light is ineffective in causing phototropic curvature. An evidence for a major role of phytochrome in phototropic responses has come from analysis of phototropism in phytochrome deficient mutants. These studies highlighted that in Arabidopsis both phytochrome A and B are required for manifestation of the phototropic responses. The double mutants of phytochrome A and B respond very sluggishly to blue light and show extremely delayed curvatures in Arabidopsis.

Differential growth is needed for the expression of the response

 Darwin identified tip as the site of perception for phototropism and subsequently Went showed that tip actively secreted plant hormone auxin. These two observation were then rationalized to explain the phototropic curvature of plants by formulating classical Went and Cholodny hypothesis. This hypothesis stated that the differential growth during phototropism occurred due to redistribution of auxin. Light caused a grater accumulation of auxin on the shaded side resulting in the faster elongation growth on that side, compared to illuminated side leading to curvature of the seedlings. There has been extensive debate for and against his hypothesis. Some support for this hypothesis has recently come from analysis of nph4 mutant of Arabidopsis, which is a signal transduction mutant. This mutant shows defect both in phototropism and geotropism and therefore a signal chain mutant. It also highlights that the phototropism and geotropism may share the components of signal chain, a prediction of Cholodny and Went hypothesis. The above mutant is defective in auxin metabolism and show that auxin is needed for displaying normal phototropic curvature in plants.

Conclusion

The research on plant phototropism has finally seen the light at the end of the tunnel. The combination of studies using genetics, molecular biology and physiology of plants and the newly generated mutants of plant development would greatly add to our understanding of these phenomena. It is expected that more elements in the perception of light and signal transmission and execution of the response would be identified. At the end I would like to mention that most research on phototropism has been carried out using etiolated seedlings and we have only little information on phototropism of green plants. It is expected that in future further studies would provide us with a comprehensive picture of the process of phototropism in plants.
 

References

Ahmad M, Jarillo JA, Smirnova O, Cashmore AR (1998) Cryptochrome blue-light photoreceptors of Arabidopsis implicated in phototropism. Nature 392:720-723

Christi JM, Reymond P, Powell GK, Bernasconi P, Raibekas AA, Liscum E, and Briggs WR (1998) Arabidopsis NPH1 has properties of a blue-light photoreceptor for phototropism. Science 282: 1698-1701

Huala E, Oeller PW, Liscum E, Han E-S, Larsen E, and Briggs WR (1997) Arabidopsis NPH1: A protein kinase with a putative redox-sensing domain. Science 278: 2120-2123

Liscum E and Briggs WR (1995) Mutations in the NPH1 locus of Arabidopsis disrupt the perception of phototropic stimuli. Plant Cell 7: 473-485

Stowe-Evans EL, Harper RM, Motchoulski AV, and Liscum E (1998) NPH4, a conditional modulator of auxin-dependent differential growth responses in Arabidopsis. Plant Physiol 118: 1265-1275