35

American Journal of Botany 100(1): 3546. 2013.

American Journal of Botany 100(1): 3546, 2013; http://www.amjbot.org/ 2013 Botanical Society of America

Plants depend on sunlight for photosynthesis and can adjust their growth according to the changes in their surrounding light environment. A good example of such a primary adaptive response is phototropic reorientation toward light ( Holland et al., 2009 ). Stems generally exhibit positive phototropism (growth toward the light), whereas roots show negative phototropism (growth away from the light). Phototropism is of particular adaptive signifi cance for germinating seedlings that must grow toward the light to survive, but has also been shown to contribute to plastic increases in root growth effi ciency under drought con-ditions ( Galen et al., 2004 , 2007a , b ). In his seminal work, The Power of Movement in Plants , Charles Darwin commented, No one can look at plants growing on a bank or on the borders of a thick wood, and doubt that the young stems and leaves place themselves so that the leaves may be well illuminated ( Darwin 1880 , p. 449). These observations led Darwin and his son Francis to dedicate a major portion of The Power of Movement in Plants to experimental analysis of the growth response of plant stems toward a light source, which they referred to as heliotropism. The term phototropism was later introduced to describe this light-driven growth phenomenon. Rather, heliotropism is now used to describe the motion of plant fl owers or leaves in response to the direction of the sun, a process that

does not depend on growth. Here, we discuss key developments since Darwins early work that contribute to our present understanding of the initial events that trigger higher plant phototropism. Because the subject of root phototropism has been described in detail very recently ( Kutschera and Briggs, 2012 ), this review will focus on the primary mechanisms underlying shoot phototropism. Readers are also directed to several excellent, in-depth reviews that cover various aspects of phototropism research ( Briggs, 2006 , 2010 ; Whippo and Hangarter, 2006 ; Holland et al., 2009 ; Sakai and Haga, 2012 ).

From Darwin to auxin For his phototropism studies, Darwin used primarily dark-grown (etiolated) seedlings of oat ( Avena sativa ) and canary grass ( Phalaris canariensis ) ( Darwin, 1880 ). The primary leaf of monocotyledonous grasses (monocots) is covered by the coleoptile, a hollow, cylindrical sheath that stops growing once the seedling has emerged from the soil and be-comes pierced by the leaf. Darwin found that etiolated grass coleoptiles enclosing the primary leaf (herein referred to as the coleoptile for simplicity) were phototropic when irradiated with unilateral light under laboratory conditions. Moreover, he dem-onstrated that coleoptiles were no longer phototropic when their tips were covered or removed ( Fig. 1A ). However, coleoptiles were still phototropic when their lower portions were covered leading Darwin to conclude the following: light is detected at the tip of the coleoptile and the middle portion of the coleoptile is where the majority of the curvature occurs. Charles and Francis Darwin also examined the universality of this phenomenon by studying phototropic bending in red cabbage ( Brassica olera-cea ) and other dicotyledonous seedlings (dicots). They con-cluded that the same principles applied to monocots and dicots. However, the response was more diffi cult to quantify in dicots due to a limited time frame in which to perform the bending measurements compared to grasses and the interference pre-sented by bulky cotyledons. Based on these and other experiments,

1 Manuscript received 4 July 2012; revision accepted 16 August 2012.

We thank The Royal Society, the Biotechnology and Biological Sciences Research Council, the National Science Foundation, and the U.S. Department of Energy for fi nancial support. We are grateful to Stuart Sullivan for providing the data for Fig. 3B and Stephen Barratt for Fig. 6. We also thank Wendy Peer, Stuart Sullivan, and Stephen Barratt for critically reading the manuscript.

4 E-mail: john.christie@glasgow.ac.uk, phone: +44 141 330 2392,

fax: +44 141 330 4447

5 E-mail: asmurphy@umd.edu, phone: 301 405 6244, fax: 301 314 9308

doi:10.3732/ajb.1200340

SHOOT PHOTOTROPISM IN HIGHER PLANTS: NEW LIGHT THROUGH OLD CONCEPTS 1 JOHN M. CHRISTIE 2,4 AND ANGUS S. MURPHY 3,5

2 Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences,

University of Glasgow, G12 8QQ, UK; and 3 Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland 20742 USA

Light is a key environmental factor that drives many aspects of plant growth and development. Phototropism, the reorientation of growth toward or away from light, represents one of these important adaptive processes. Modern studies of phototropism began with experiments conducted by Charles Darwin demonstrating that light perception at the shoot apex of grass coleoptiles induces differential elongation in the lower epidermal cells. This led to the discovery of the plant growth hormone auxin and the Cholod-nyWent hypothesis attributing differential tropic bending to lateral auxin relocalization. In the past two decades, molecular-ge-netic analyses in the model fl owering plant Arabidopsis thaliana has identifi ed the principal photoreceptors for phototropism and their mechanism of activation. In addition, several protein families of auxin transporters have been identifi ed. Despite extensive efforts, however, it still remains unclear as to how photoreceptor activation regulates lateral auxin transport to establish phototropic growth. This review aims to summarize major developments from over the last century and how these advances shape our current understanding of higher plant phototropism. Recent progress in phototropism research and the way in which this research is shed-ding new light on old concepts, including the CholodnyWent hypothesis, is also highlighted.

Key words: auxin; blue light; Darwin; CholodnyWent; lateral transport; photoreceptor; phototropism.

36 AMERICAN JOURNAL OF BOTANY [Vol. 100

coleoptile ( Fig. 1C ). This simple but effective agar-stimulated bending assay was subsequently used to identify the chemical nature of the growth promoting substance from human urine ( Kogl and Haagen-Smits, 1931 ), which was named auxin (from the Greek auxein, meaning to increase or grow). Later studies showed conclusively that indole-3-acetic acid (IAA) produced by the fungus Rhizopus suinus grown on tryptophan was identi-cal to the primary plant hormone ( Thimann and Went, 1934 ).

The CholodnyWent hypothesis and auxin transport While Went worked on phototropism, Nicolai Cholodny (1928) had independently developed a similar model to explain how oat roots responded to gravitropic stimulation. A decade later, Went integrated Cholodnys work with his own to produce what is now referred to as the CholodnyWent hypothesis ( Went and Thimann, 1937 ). By this time, the central function of the organ tip in establishing tropic responses was apparently so well established that it was not mentioned in the publication ( Went and Thimann, 1937 ; Firn et al., 2000 ).

Since its introduction, the CholodnyWent hypothesis has received wide acceptance and experimental support ( Whippo and Hangarter, 2006 ; Holland et al., 2009 ). For phototropism, the hypothesis predicts that asymmetric light is perceived at the coleoptile tip and causes auxin to move from the irradiated to the shaded side. Auxin then moves down the coleoptile so that the lower region develops an asymmetry of auxin. The higher auxin concentration on the shaded side then promotes differen-tial elongation of epidermal cells and growth toward the light. A competing model, fi rst advanced by Anton Hendricks Blaauw (1919) , proposed that phototropic bending is brought about by light-mediated growth inhibition at the irradiated side and has continued to receive some experimental support ( Yamada et al., 2000 ).

An essential concept embedded within the CholodnyWent hypothesis is that of polar auxin transport. Indeed, Darwins proposal of some infl uence moving from the coleoptile tip is probably one of the fi rst descriptions of this phenomenon. In the shoot, auxin is synthesized mainly in the apex and young leaves and is directed into developmentally important polar streams by several transport mechanisms that are facilitated by chemioso-motic gradients ( Peer et al., 2011 ). Although most of the trans-port components have been identifi ed, the exact process by which lateral auxin redirection occurs in phototropism still remains unclear.

The fi rst efforts to quantify lateral auxin movement involved redefi nition of the coleoptile model system for phototropic experimentation. In 1957, Winslow Briggs and colleagues ( Briggs et al., 1957 ) inserted a physical barrier (a thin glass cover slip) between the irradiated and shaded sides of maize ( Zea mays ) coleoptile tips before measuring the amount of auxin diffusing into an agar block below. No redistribution of auxin was observed between the two sides when the barrier split the tip into two halves ( Fig. 1D ). However, partial splitting leaving the apex intact resulted in an accumulation of auxin on the shaded side, despite no change in the amount measured in the agar block. These fi ndings implied that neither the synthesis nor the break-down of auxin was infl uenced by unilateral light treatment. Instead, they were consistent with a model whereby auxin asymmetry arises from a lateral transport from the irradiated to the shaded side ( Briggs, 1963 ).

Further experiments using radiolabelled IAA applied to the coleoptile tip later confi rmed that phototropic stimulation results in an asymmetric redistribution of auxin ( Pickard and Thimann,

Darwin proposed that some infl uence was transmitted from the tip of the coleoptile to the regions below to promote pho-totropic bending.

Peter Boysen-Jensen (1911) was one of several scientists to extend Darwins observations by removing the tip of the co-leoptile and reattaching it with a piece of gelatin at the base ( Fig. 1B ). In doing so, he showed that Darwins infl uence could diffuse from the tip through the gelatin interface to promote phototropic curvature in the region below. Boysen-Jensen (1913) also found that inserting a thin sheet of impervious mica across the tip was suffi cient to prevent phototropism ( Fig. 1B ). Similarly, partial insertion of the mica at the shaded side and not the irradiated side inhibited phototropism implying that dif-fusion of Darwins signal occurred at the shaded side to pro-mote the differential growth. Frits Went (1926, 1928) later built upon these fi ndings by aiming to isolate the diffusible signal. Went used gelatin (later agar) blocks to absorb the diffusible signal from the coleoptile tip and found that these blocks could stimulate bending when placed at the one side of a decapitated

Fig. 1. Stylized representation of major historical advances that led to the discovery of auxin and its role in phototropism. (A) Darwins experi-ments demonstrating the importance of the coleoptile tip in phototropism. Phototropic bending to unilateral light is observed even when the lower region of the coleoptile is covered, whereas covering or removing the tip results in a loss of curvature. (B) Boysen-Jensens experiments showing that Darwins proposed infl uence could diffuse through an agar block inserted below the coleoptile tip. Inserting a thin piece of mica at that shaded side and not the irradiated side prevents curvature suggesting that diffusion of Darwins infl uence occurs at the shaded side. (C) Wents bioassay for auxin showing that Darwins growth-promoting infl uence can diffuse into an agar block and promote bending when placed unequally on top of a decapitated coleoptile. (D) Briggs experiment showing that a lateral accumulation of auxin occurs in response to unilateral light. Inser-tion of a thin glass barrier between the irradiated and shaded side causes equal amounts of auxin to diffuse into the agar block below. Lowering the barrier at the tip results in a net increase of auxin at the shaded side.

37January 2013] CHRISTIE AND MURPHYSHOOT PHOTOTROPISM IN HIGHER PLANTS

later detected in maize and oat membranes ( Hager et al., 1993 ; Palmer et al., 1993b ; Salomon et al., 1997a ), as well as Arabi-dopsis where its size is approximately 120 kDa ( Fig. 3A ). The phosphoprotein has several attributes that correlate with those of phototropism ( Briggs et al., 2001b ). Highest levels of phos-phorylation were detected in the coleoptile tip, which is the most phototropically sensitive ( Hager et al., 1993 ; Palmer et al., 1993a ; Salomon et al., 1997b ). Light-induced phosphorylation obeyed the Busen-Roscoe law of reciprocity ( Short and Briggs, 1990 ), correlating with fi rst-positive phototropism. Moreover, action spectra for the phosphorylation reaction concurred with those for phototropism ( Hager et al., 1993 ; Palmer et al., 1993a ), implying a tight coupling of these processes. However, fl uence-response curves measured for the phosphorylation reaction both in vitro and in vivo were found to be an order of magnitude less sensitive than those measured for fi rst-positive phototropism ( Briggs et al., 2001b ). To date, the reason for this discrepancy has yet to be explained.

Further experiments demonstrated that unilateral blue light establishes a phosphorylation gradient across the oat coleoptile ( Salomon et al., 1997c ). A detailed study of phosphorylation

1963 ). Equivalent radiotracer experiments at increasingly small scales have also been used to measure changes in polar auxin transport associated with phototropism ( Haga et al., 2005 ; Esmon et al., 2006 ; Christie et al., 2011 ). In addition, lateral auxin redistributions can be monitored indirectly by using the syn-thetic auxin-responsive promoter DR5 to drive expression of a reporter gene such as green fl uorescent protein ( Christie et al., 2011 ; Ding et al., 2011 ). More recently, the DII-Venus reporter, which consists of the degron domain of the AUX-IAA auxin coreceptor fused to yellow fl uorescent protein, has been used to demonstrate differential auxin accumulations in gravistimu-lated roots ( Brunoud et al., 2012 ). This reporter responds much more dynamically to altered auxin levels and therefore holds great promise for use in phototropism studies. In the following sections, we summarize how the photoreceptor for phototro-pism was identifi ed before returning to recent research aimed at understanding how asymmetric auxin redistribution is achieved.

Photophysiology of phototropism The search for the iden-tity of the photoreceptor responsible for phototropism had already begun before the publication of Darwins work ( Briggs, 2006 ). For instance, Julius von Sachs (1864) was one of several researchers that used colored glass and solutions to illuminate plants with different wavelengths of light. Such studies there-fore measured the relative effectiveness of particular wavelengths to stimulate phototropism ( Briggs, 2006 ). Sachs found that blue wavelengths (390500 nm) were most effective. Several research-ers later produced more detailed action spectra indicating that the photoreceptor responsible absorbs ultraviolet-A (UV-A) wavelengths in addition to blue ( Fig. 2A ). Subsequent fl uence-response measurements revealed that the bending response showed a surprising degree of complexity ( Iino, 2001 ). A typical fl uence-response curve for phototropism can be divided into three phases depending on the fl uence and time requirements ( Fig. 2B ). The initial bell-shaped curve is referred to as fi rst-positive phototropism and is followed by a neutral zone where little or no curvature is detected. First-positive phototropism is only associated with bending of the coleoptile tip. Curvature rises once again at higher light intensities to produce second-positive phototropism, which occurs within the lower region of the coleoptile. This complex fl uence-response relationship for phototropism is not restricted to monocots, but is well docu-mented in dicots ( Iino, 2001 ), including Arabidopsis thaliana ( Konjevic et al., 1989 ), confi rming that the underlying mechanisms associated with phototropism are conserved in angiosperms.

Much of the studies involving grass coleoptiles have cen-tered on fi rst-positive phototropism, which obeys the Busen-Roscoe law of reciprocity ( Busen and Roscoe, 1855 ). This rule states that the magnitude of the response is directly proportional to the total energy dose supplied irrespective of the time admin-istered. Thus, an equivalent curvature response is expected if seedlings are exposed to bright light for a short duration or dim light for an extended time, as long at the total fl uence adminis-tered is the same. Second-positive phototropism occurs in re-sponse to prolonged irradiation in a time-dependent manner and does not obey the law of reciprocity ( Iino, 2001 ).

Light-induced phosphorylation and phototropism The fi rst step to identifying the biochemical nature of the photore-ceptor came again from Briggs and colleagues who discovered a protein in the plasma membrane of etiolated pea ( Pisum sati-vum ) epicotyls that is rapidly phosphorylated upon exposure to blue light ( Gallagher et al., 1988 ). This phosphoprotein was

Fig. 2. Photobiological characterization of phototropism. (A) Schematic representation of a typical action spectrum for phototropism ( Fig. 2A ). (B) Fluence-response curve for phototropism. Increasing fl uence results in two curvature responses referred to as fi rst- and second-positive phototropism, separated by a region of no curvature (neutral zone). A schematic represen-tation of the model proposed by Salomon et al. (1997b) to account for the different curvature responses that depend on the formation of a lateral gradient of protein phosphorylation across the coleoptile is also shown.

38 AMERICAN JOURNAL OF BOTANY [Vol. 100

altered in phototropism to low fl uence rates of unilateral blue light ( Khurana and Poff, 1989 ; Khurana et al., 1989 ). Three of these mutants were impaired specifi cally in hypocotyl phototro-pism (JK218, JK224 and JK229). JK218 showed no curvature to light, whereas JK224 and JK229 were altered in fi rst-positive phototropism, but were unaffected in second-positive photot-ropism. Notably, JK224 provided an important connection between phototropism and the phosphoprotein identifi ed by Briggs. Light-dependent phosphorylation of the 120-kDa plasma membrane protein was severely reduced in JK224 ( Reymond et al., 1992 ).

Several years later, Liscum and Briggs (1995) isolated eight additional phototropism mutants that mapped to four genetic loci designated N ON- P HOTOTROPIC H YPOCOTYL 14 ( NPH1 NPH4 ). Physiological characterization demonstrated that phototropism was specifi cally altered in nph1 , nph2 , and nph3 , whereas nph4 has defects in both phototropism and grav-itropism. Both nph1 and nph3 lack fi rst- and second-positive phototropism, and all nph1 mutant alleles have impaired light-induced phosphorylation of the 120-kDa plasma membrane protein. Indeed, JK224 is allelic to nph1-2 , which has severely reduced levels of the 120-kDa phosphoprotein ( Fig. 3A ). Liscum and Briggs (1995) proposed that the NPH1 phosphoprotein was closely linked to the photoreception process mediating all phototropic responses in Arabidopsis. By contrast, nph2 , nph3 (allelic to JK218), and nph4 retain normal levels of the light-induced phosphorylation activity and were concluded to represent lesions in downstream signaling events. During this time, Kiyotaka Okada and colleagues isolated R OOT P HOTO T ROPISM 1-3 ( rpt1-3 ) mutants of Arabidopsis ( Okada and Shimura, 1992 ). Later studies revealed that rpt1 is allelic to nph1 ( Sakai et al., 2000 ), consistent with the observation that nph1 mutants are defective in both hypocotyl and root phototropism ( Liscum and Briggs, 1995 ). Similarly, rpt3 (allelic to nph3/ JK218) and rpt2 are also defective in both shoot and root phototropism ( Sakai et al., 2000 ). NPH4 encodes an auxin-regulated transcrip-tion factor that is required for normal phototropism and gravit-ropism ( Stowe-Evans et al., 1998 ). Mutants lacking NPH4 and other related family members therefore highlight the need for auxin-regulated gene expression in establishing phototropic curvatures. The molecular identity of the NPH2 locus still re-mains unresolved. However, nph2 may be allelic to rpt2 since these mutants exhibit similar hypocotyl and root phototropism phenotypes ( Liscum and Briggs, 1996 ; Sakai et al., 2000 ).

Phototropin receptor kinases Cloning of the NPH1 locus and subsequent photochemical/biochemical characterization of the NPH1 fl avoprotein kinase fi nally resolved the molecular identity of the photoreceptor for phototropism ( Huala et al., 1997 ; Christie et al., 1998 ). Recombinant expression in insect cells established that NPH1 was a photoreceptor kinase that autophos-phorylates in response to blue light irradiation ( Christie et al., 1998 ). Briggs and colleagues therefore renamed the protein pho-totropin 1 (phot1) after its role in phototropism ( Christie et al., 1999 ; Briggs et al., 2001a ). Lower-case usage (phot1) is used typically by plant photobiologists to defi ne the photoreceptor holoprotein complete with its chromophore following the nomenclature system introduced to defi ne members of the phyto-chrome family of red/far-red light absorbing photoreceptors ( Quail et al., 1994 ). A summary of past and present terminology used to describe the most studied Arabidopsis mutants isolated from genetic screens is provided in Table 1 for convenience.

rates between the tip and the base of the coleoptile led to one possible model that could explain the underlying complexity of the fl uence-response curve. At low fl uences, a lateral gradient in phosphorylation occurs across the tip to produce the bending response associated with fi rst-positive phototropism ( Fig. 2B ). At higher fl uences, phosphorylation between the irradiated and shaded sides becomes saturated and consequently negates the curvature response. Continued light exposure results in a phos-phorylation gradient at the base of the coleoptile to bring about second-positive phototropism. While this model provides some explanation for the complexity of the fl uence response curve for phototropsim, it does require further validation, especially when it does not consider potential deactivation and regenera-tion of the photoreceptor system. Nonetheless, identifi cation of the light-induced phosphorylation reaction and its correlation with phototropism represented a major advance. Subsequent genetic analysis using Arabidopsis fi nally confi rmed the iden-tity of the phosphoprotein as the photoreceptor for phototro-pism. Events leading to this pivotal discovery are summarized in the following section.

Arabidopsis mutants altered in phototropism Kenneth Poff and coworkers were the fi rst to describe mutants of Arabidopsis

Fig. 3. Phototropin phosphorylation and protein abundance. (A) Auto-radiograph showing light-activated autophosphorylation of a 120-kDa protein later identifi ed as phot1 (formerly nph1) in extracts prepared from wild-type Arabidopsis seedlings (WT) or from phot1-2 ( nph1-2/ JK224) and phot1-7 ( nph1-7 ) mutants. Membrane proteins were prepared under dim red light and given a mock irradiation (D for dark) or a pulse of light (L) prior to the addition of radiolabelled ATP. Proteins were separated on a polyacrylamide gel and exposed to autoradiography. The immunoblot of the phot1 protein levels is below the autoradiograph. (B) Autoradiographs of phot1 and phot2 protein levels in etiolated Arabidopsis seedlings. Seed-lings were grown in the dark for 3 d prior to the exposure of blue light (20 molm 2 s 1 ).

39January 2013] CHRISTIE AND MURPHYSHOOT PHOTOTROPISM IN HIGHER PLANTS

Whereas phot2 overlaps in function with phot1 to mediate sec-ond-positive phototropism at high fl uence rates of blue light (>1 molm 2 s 1 ), phot1 alone is responsible for mediating fi rst- and second-positive phototropism to low fl uence rates of blue light (1 molm 2 s 1 ), implying the presence of a second phototropic receptor. This protein was originally named NPH1-Like 1 (NPL1) ( Sakai et al., 2001 ) and later designated phot2 in accordance with the phototropin nomen-clature introduced by Briggs et al. (2001a) . Like phot1, phot2 is present in monocots ( Kanegae et al., 2000 ), contains two LOV domains, a kinase domain and undergoes light-activated au-tophosphorylation ( Christie et al., 2002 ; Sakai et al., 2001 ).

TABLE 1. Mutants of Arabidopsis isolated with altered hypocotyl phototropism

Mutant DesignationOld/other Current Protein Name Function Phenotype Source

nph1 / rpt1 / JK224 phot1 phot1 Photoreceptor Aphototropic to low blue light, but phototropic to high blue light (owing to the presence of phot2)

Khurana and Poff, 1989 ; Okada and Shimura, 1992 ; Liscum and Briggs, 1995 ; Sakai et al., 2000

npl1 phot2 phot2 Photoreceptor Phototropic to low and high blue light (owing to the presence of phot1)

Sakai et al., 2001

rpt3 / JK218 NPH3 NPH3 Phototropic signaling Aphototropic to low and high blue light Khurana and Poff, 1989 ; Okada and Shimura, 1992 ; Motchoulski and Liscum, 1999

rpt2 RPT2 Phototropic signaling Reduced phototropism under low and high blue light

Sakai et al., 2000

arf7 nph4 NPH4 Auxin-response transcription factor

Reduced phototropism under low and high blue light

Stowe-Evans et al., 1998

40 AMERICAN JOURNAL OF BOTANY [Vol. 100

auxin. NPH3 is vital for this process because nph3 mutants of rice ( Oryza sativa ) and Arabidopsis ( Liscum and Briggs, 1995 ) are aphototropic owing to a lack of lateral auxin translocation ( Haga et al., 2005 ). NPH3 is expressed predominantly at the coleoptile tip ( Matsuda et al., 2011 ) and has been shown to in-teract with phot1 both in vitro ( Motchoulski and Liscum, 1999 ) and in vivo ( Lariguet et al., 2006 ). NPH3 is a member of a large plant-specifi c gene family consisting of 32 members in Arabi-dopsis ( Celaya and Liscum, 2005 ) and at least 24 in rice ( Kimura and Kagawa. 2006 ). NPH3 is hydrophilic in nature, but like phot1, is associated with the plasma membrane ( Motchoulski and Liscum, 1999 ) and is thought to function in component as-sembly of a photoreceptor complex. RPT2 is closely related to NPH3 and also interacts directly with phot1 ( Inada et al., 2004 ; Sullivan et al., 2009 ). However, rpt2 mutants exhibit moderate defects in hypocotyl phototropism ( Sakai et al., 2000 ) indicat-ing that RPT2 most likely functions to have a modulatory role in establishing hypocotyl curvature in Arabidopsis .

Biological roles for other members of the N PH3/ R PT2- l ike (NRL) family have also been reported. A subset of NRL pro-teins known as N aked p ins in y ucca (NPY) function redun-dantly in regulating auxin movements required for organogenesis ( Cheng et al., 2007 ; Furutani et al., 2007 ). These and other fi nd-ings suggest that NRL family members act collectively to regu-late various aspects of auxin traffi cking and signaling ( Li et al., 2011 ). Intriguingly, NPY proteins appear to function in concert with AGC (protein kinase A /cyclic G MP-dependent protein kinase/protein kinase C ) kinase family members ( Cheng et al., 2008 ). NRL proteins might therefore share a common mecha-nism of action given that phototropins are also AGC kinases ( Bogre et al., 2003 ).

Assigning a biochemical activity for NPH3 will be key to un-derstanding its role in orchestrating auxin transport regulation. Photoactivation of phot1 leads to dephosphorylation of NPH3 in etiolated Arabidopsis seedlings, a signaling process that has been linked to the onset of phototropic curvature ( Pedmale and Liscum, 2007 ). Yet, mutation of potential NPH3 phosphoryla-tion sites does not appear to affect its function ( Tsuchida-Mayama et al., 2008 ), raising questions as to the biological signifi cance of NPH3 dephosphorylation. Yeast-three hybrid screening using phot1 and NPH3 as a bait complex has recently identifi ed as a second interacting target for NPH3. E n h anced b ending 1 (EHB1) is a potential calcium-binding protein that appears to function as a negative regulator of tropic responsive-ness ( Knauer et al., 2011 ). Consequently, ebh1 mutants show enhanced phototropic and gravitropic responses. However, it is not yet known whether EBH1 directly or indirectly affects lateral auxin redistribution to modulate tropic responsiveness.

More recent work indicates that NPH3 functions as part of a Cullin 3-based E3 ubiquitin ligase to target proteins for ubiquit-ination, and in turn, facilitates their subcellular relocalization or degradation ( Wan et al., 2008 ; Roberts et al., 2011 ). One such target is phot1 ( Roberts et al., 2011 ), which has been shown to partially relocalize away from the plasma membrane upon pho-toactivation ( Sakamoto and Briggs, 2002 ; Kaiserli et al., 2009 ). The biological signifi cance of phot1 internalization is presently not known, but may represent some form of receptor desensiti-zation. Alternatively, internalization could function to recruit active phot1 to a specifi c subcellular region. New evidence showing that NPH3 can infl uence the subcellular localization of proteins known to be involved in auxin transport ( Wan et al., 2012 ) is particularly noteworthy and provides additional support

step in phototropic signaling ( Inoue et al., 2008 ). A functional assessment of the equivalent serine residue in Arabidopsis phot2 also indicates that phosphorylation of the kinase activa-tion loop is important for function ( Inoue et al., 2011 ). While both LOV domains have been shown to undergo a unique mode of photochemical activity ( Fig. 4C ), LOV2 is the predominant light sensor controlling phototropin kinase activity ( Christie et al., 2002 ; Cho et al., 2007 ). To date, the biological role for LOV1 is still not known, but this domain may be involved in receptor dimerization ( Salomon et al., 2004 ; Nakasako et al., 2008 ) upon light exposure ( Kaiserli et al., 2009 ).

Phototropism requires NPH3 It is well accepted that for-mation of the biochemical gradient of phot1 autophosphoryla-tion ( Salomon et al., 1997b ) across the shoot underlies the directionality of the phototropic response. While much prog-ress was made in uncovering the molecular basis of the photo-receptors involved ( Christie, 2007 ; Demarsy and Fankhauser, 2009 ), less information is available on how asymmetric stimu-lation of the photoreceptor initiates a lateral redistribution of

Fig. 4. Structural and photochemical features of phot1. (A) Cartoon illustrating the domain structure of phot1. Relative positions of the light, oxygen or voltage (LOV) domains and the serine/threonine kinase domain are indicated. Relative positions of in vivo phosphorylation sites identifi ed for Arabidopsis phot1, including serine residue 851 are shown with arrows. (B) Absorption spectrum of the LOV domain expressed and purifi ed from E. coli . (C) Photochemical reactivity of the LOV domain. Purifi ed LOV domains bind fl avin mononucleotide (FMN) as chromophore and form a covalent adduct between the FMN and a conserved cysteine residue within the LOV domain upon irradiation, which is reversible in darkness.

41January 2013] CHRISTIE AND MURPHYSHOOT PHOTOTROPISM IN HIGHER PLANTS

Arabidopsis pin3 seedlings were found to exhibit reduced tropic bending, and PIN3 was found to relocalize dynamically to the side walls of auxin conducting cells at the root apex after gravi-stimulation ( Friml et al., 2002 ). The simultaneous discovery that plant dynamic cellular traffi cking mechanisms associated with A DP- r ibosylation f actor (ARF) GTPases and that the ARF- g uanidine e xchange f actor (ARF-GEF) GNOM directs PIN1 and PIN2 vesicular cycling ( Geldner et al., 2001 ; 2003 ), further strengthened the case for dynamic redirection of PIN-mediated auxin fl ows in tropic growth. More recently, reduced hypocotyl bending has been described in gnom mutants ( Ding et al., 2011 ), although it is not clear whether this is a constitu-tive effect of diminished PIN function or a direct effect on pho-totropic responses. Phototropins could also impact such a mode of transporter recycling given that they have been shown to in-teract with ARF proteins in yeast two-hybrid studies ( Sullivan et al., 2009 ).

Initially, the mechanisms that direct changes in PIN abun-dance at the plasma membrane appeared to be too slow to func-tion in phototropic responses. However, two recent studies from the groups of Zhenbiao Yang and Jiri Friml ( Chen et al., 2012 ; Lin et al., 2012 ) demonstrated that PIN endocytosis is regulated by the ROP ( R ho guanidine triphosphate hydrolases o f p lants) family of Rho-like GTPases and their associated RICs ( R OP i nteractive C RIB motif-containing proteins), which function in stabilization of actin cytoskeletal fi laments. These studies re-ported nearly instantaneous effects on PIN2 abundance at the plasma membrane in gravistimulated roots and showed that the response is dependent on the ROP/RIC mechanism. Although the initial redirection of auxin in the graviresponding root does not appear to be directed by this mechanism, the speed of the response (within 30 s) ( Xu et al., 2010 ) indicates that such a role for PINs in redirecting auxin during phototropism is possible.

for a role of NPH3 in regulating subcellular protein traffi cking. These transporter proteins and their involvement in phototro-pism are discussed in the follow sections.

Phototropism and PIN auxin transporters Genetic analy-sis in Arabidopsis has been instrumental in defi ning the identity of several protein families that function to transport the plant growth hormone auxin ( Vanneste and Friml, 2009 ; Peer et al., 2011 ). Members of the Pin -formed (PIN) family, named after the pin-like infl orescence phenotype of the pin1 mutant, are the primary mediators of directional auxin fl uxes that regulate plant development ( Krecek et al., 2009 ). Arabidopsis contains eight PINs that are distantly related to fungal transport proteins com-prising 911 transmembrane spanning helices ( Fig. 5A ). PIN1PIN4 and PIN7 encode full-length transporter proteins that function in directing auxin effl ux at the plasma membrane, while PIN5 , PIN6 , and PIN8 encode short PINs that appear to mediate intracellular auxin movement ( Mravec et al., 2009 ; Vanneste and Friml, 2009 ; Zadnikova et al., 2010 ; Ding et al., 2011 ; Peer et al., 2011 ).

The directionality of polar auxin transport is facilitated by the polar subcellular distributions of PIN proteins. For example, PIN1 is localized to the basal side of cells within the central vasculature of shoots ( Fig. 5B ) consistent with its role in polar auxin transport. PIN2 is localized to the upper plasma mem-brane in root epidermal cells that conduct auxin from the shoot apex to the root elongations zone ( Vanneste and Friml, 2009 ). Hence, the longitudinal asymmetric distribution of PIN1 con-tributes to establishing the polarity of auxin fl ow from the shoot apex to the base. From the outset, the polar cellular localiza-tions of PIN proteins suggested that they are the most likely mediators of the lateral auxin fl uxes required for phototropism. This hypothesis gained a great deal of support when etiolated

Fig. 5. Auxin transport activities associated with Arabidopsis phototropism. (A) Cartoon illustrating the topologies of PIN1 and ABCB19 auxin effl ux carriers. White cylinders depict membrane-spanning regions and the cytosolic ATP-binding loops of ABCB19 are indicated. (B) Schematic representation of a longitudinal section through the hypocotyl elongation zone of etiolated seedlings. Relative positions of the epidermis (epi), cortex (cor), endodermis (end) and vasculature (vas) are indicated. Red lines represent main avenues of polar auxin transport. Relative positioning of PIN1 in cells of the vasculature is shown. (C) Relative positioning of PIN3 on the irradiated side of etiolated seedlings. (D) Proposed auxin traffi cking in de-etiolated seedlings following unilateral blue light irradiation. Initially, phot1 phosphorylates the effl ux activity of ABCB19 to promote auxin accumulation in the hypocotyl apex. This build up of auxin is growth inhibitory and stops vertical elongation of the hypocotyl. Unknown transporters then promote a lateral accumulation of auxin to the shaded side, which is then channeled to the elongation zone below to promote differential cell elongation across the hypocotyl.

42 AMERICAN JOURNAL OF BOTANY [Vol. 100

moderately reduced in pin7 mutants, suggesting that this trans-porter may contribute to establishing the lateral auxin gradients involved ( Ding et al., 2011 ). PIN7 is relatively abundant in the epidermis and cortex of the hypocotyl elongation zone ( Zadnikova et al., 2010 ; Christie et al., 2011 ) and may play a role in mobi-lizing auxin to this region.

Phosphorylation of PINs could certainly regulate the direc-tional auxin fl ows mediated by these proteins. The phototropin-related AGC kinase PINOID (PID) phosphorylates members of the PIN family to control their subcellular traffi cking ( Dhonukshe et al., 2010 ). Yet, there is no evidence to date to suggest that PINs are direct substrate targets for phototropin kinases ( Ding et al., 2011 ). Changes in the subcellular traffi cking of PIN2 have been proposed recently to be involved in generating negative phototropic curvature in Arabidopsis roots ( Wan et al., 2012 ), but PIN2 functions only in mobilizing auxin from its site of relocalization at the root tip to the root elongation zone, not in the relocalization processes itself ( Peer et al., 2011 ). However, these light-dependent changes in PIN2 localization are dependent on phot1 and NPH3 ( Wan et al., 2012 ) implying that phot1/NPH3 may exert their effects by altering the subcel-lular cycling of auxin transport proteins. Whether this involves substrate phosphorylation of specifi c PINs by phot1 or some other related AGC kinase(s) requires further study.

ATP-binding cassette proteins and phosphorylation New evidence has shown that phot1 can phosphorylate and regulate the activity of another class of auxin transporters known as A TP- b inding c assette B (ABCB) proteins ( Christie et al., 2011 ). ABCBs belong to a large transporter family in Arabidopsis comprising 21 members. Like PINs, ABCBs are integral mem-brane proteins with multiple membrane spanning regions ( Fig. 4A ). To date, ATP-driven auxin effl ux activities have been shown for ABCB1, ABCB4, and ABCB19 in plant cells and in heterologous systems ( Peer et al., 2011 ). ABCB19 is pre-dominantly localized apolarly at the plasma membrane and co-localizes, as well as functions coordinately, with PIN1 in tissues within the shoot apex ( Blakeslee et al., 2007 ) to mediate polar auxin transport ( Noh et al., 2003 ).

Arabidopsis mutants lacking ABCB19 exhibit enhanced phototropic curvature, which has been attributed to an enhanced accumulation of auxin in the upper hypocotyl as a conse-quence of decreased PIN1-B19-mediated polar transport ( Noh et al., 2003 ; Nagashima et al., 2008 ; Christie et al., 2011 ). ABCB19 physically interacts with phot1 in Arabidop-sis and is phosphorylated by phot1 in vitro ( Christie et al., 2011 ). Moreover, coexpression studies in HeLa cells indicate that ABCB19-mediated auxin effl ux is inhibited by phot1 kinase activity ( Christie et al., 2011 ). Since auxin is growth inhibiting

An impact of ROP GTPases on the cellular distribution of other PIN family members has also been demonstrated ( Nagawa et al., 2012 ).

PIN3 is expressed in the hypocotyl endodermis, central vas-culature, and epidermis. In a recent study, Friml and coworkers have reported important changes in PIN3 localization that oc-cur within endodermal cells at the hypocotyl elongation zone following phototropic stimulation ( Ding et al., 2011 ). On the irradiated side of the hypocotyl, PIN3 becomes more localized at the inner side of the cell ( Fig. 5C ). These changes in PIN3 localization are dependent on phot1 and are proposed to divert auxin back into the vasculature, thereby reducing auxin move-ment to the epidermis at the irradiated side. However, given that phototropic impairment in etiolated pin3 seedlings is mod-erate and changes in PIN3 abundance do not correlate tempo-rally with reduced bending in the pin3 mutant ( Christie et al., 2011 ; Ding et al., 2011 ), it seems likely that lateral auxin trans-port is coordinated by the action of multiple transporter pro-teins. This redundancy might explain why no auxin transporter has been identifi ed to date in genetic screens for Arabidopsis mutants impaired in phototropism. Determining whether redun-dancy between PIN family members contributes to phototropic responsiveness will be challenging given that multiple pin mu-tants display general growth defects ( Ding et al., 2011 ; Friml et al., 2003 ). Indeed, it is likely that altering auxin homeostasis would indirectly impact tropic responsiveness. However, in a very recent report, Haga and Sakai (2012) suggested more spe-cifi c roles for PIN isofroms in Arabidopsis phototropism. De-tailed phototropic analysis revealed that pin1 pin3 pin7 triple mutants are severely impaired in pulse-induced, fi rst-positive phototropism but are unaffected in continuous-light-induced, second-positive phototropism. Furthermore, lateral auxin trans-port mediated by PIN3 appears to participate in fi rst- but not second-positive phototropism. These fi ndings strongly suggest the existence of functionally separable mechanisms in estab-lishing fi rst- and second-positive phototropic curvatures.

Establishing the initial directionality of lateral auxin trans-port could possibly occur through alterations in coordinated traffi cking of multiple transporter proteins. Of the other PIN proteins, PIN1 polar localization is the most dynamic and es-sential for normal development. Because PIN1 has been shown to relocalize during development of the shoot apex ( Heisler et al., 2005 ), it is tempting to speculate that dynamic PIN1 relocal-ization might motivate lateral auxin redistribution during pho-totropism. Arabidopsis mutants impaired in PIN1 localization apparently have altered hypocotyl phototropism ( Noh et al., 2003 ; Blakeslee et al., 2004 ). By contrast, the phototropic re-sponses of etiolated pin1 , pin2 , and pin4 seedlings are not im-paired ( Ding et al., 2011 ). Hypocotyl phototropism is, however,

Fig. 6. Phototropic responsiveness of de-etiolated wild type (WT) and phot1 phot2 double mutant Arabidopsis seedlings following dark acclimation.

43January 2013] CHRISTIE AND MURPHYSHOOT PHOTOTROPISM IN HIGHER PLANTS

Conclusions and outlook Although recent evidence has provided further support for the CholodnyWent hypothesis and its applicability to both monocots and dicot phototropism, the signaling mechanisms responsible for accumulating auxin on the shaded side of the stem remain poorly defi ned. Spatial and temporal mapping of auxin traffi cking processes within the shoot apex, combined with an improved understanding of the role of NPH3 in regulating these events, will be instrumental in gaining insights into the mechanisms involved. A major chal-lenge for future research will be to develop new strategies to dissect and identify the regulatory mechanisms by which lateral auxin gradients arise as a result of asymmetric photoreceptor activation across the stem. Molecular genetic advances in Arabi-dopsis have given much headway over the last decade, and the resolution of assays to visualize auxin levels in discrete tissues continues to improve ( Brunoud et al., 2012 ; Christie et al., 2011 ). Transitioning such tools to monocot systems will logi-cally extend this line of research with an aim of being able to help spatially defi ne the auxin transporter activities required to initiate and sustain phototropic bending and how these compare to dicots given their anatomical differences. With new strate-gies to study phototropism and auxin transport emerging, rapid progress is now anticipated to answer how these fundamen-tal aspects of light and hormone signaling coordinate to shape plant growth.

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at high concentrations, the transient accumulation at the shoot apex arising from an inhibition of ABCB19 activity by phot1 is hypothesized to halt vertical growth prior to the onset of phototropic curvature ( Fig. 5D ). This model agrees with sev-eral reports showing that an inhibition of polar auxin transport ( Shen-Miller et al., 1969 ), as well as light-mediated growth inhibition ( Folta and Spalding, 2001 ; Haga et al., 2005 ; Christie et al., 2011 ) precedes the onset of phototropic curvature. Fur-ther work has shown that ABCB-mediated auxin effl ux activity can also be regulated by the phot1-related AGC kinase PID ( Henrichs et al., 2012 ). These fi ndings, together with the evi-dence for PIN regulation by both AGC ( Ding et al., 2011 ) and D6 ( Zourelidou et al., 2009 ; Dhonukshe et al., 2010 ) protein kinases implicate a key role for phosphorylation in regulating auxin transporter activity.

CholodnyWent revisited Despite the considerable prog-ress made in phototropism research over the last century, it is still not clear as to how the lateral redistribution of auxin in the shoot is established. Recent efforts that adopted a different experimental strategy, however, have given new insights into the spatial initiation of auxin fl uxes in Arabidopsis ( Christie et al., 2011 ). As described throughout this review, phototro-pism is largely studied using etiolated seedlings as an experi-mental system. However, studies performed in this manner examine the combined effects of phototropism as well as seed-ling photomorphogenesis. Moreover, PIN and ABCB auxin transporters contribute to directing light-mediated develop-ment in Arabidopsis , including hypocotyl growth suppression ( Nagashima et al., 2008 ; Tsuchida-Mayama et al., 2008 ) and apical hook opening ( Zadnikova et al., 2010 ). This level of complexity makes it diffi cult to discriminate auxin transport processes associated with phototropism from those involved in photomorphogenesis.

In a recent report, Murphy and colleagues have shown that auxin fl uxes associated with phototropism can be monitored post-photomorphogenesis ( Fig. 6 ) by simply subjecting light-grown (de-etiolated) Arabidopsis seedlings to a period of dark-ness prior to phototropic stimulation ( Christie et al., 2011 ). Lateral auxin accumulation in these so-called dark-acclimated seedlings, is initiated at and above the hypocotyl apex ( Fig. 5D ) as visualized by monitoring the auxin-responsive transcrip-tional reporter DR5. The lateral auxin translocation system in dark-acclimated Arabidopsis seedlings therefore complies with the CholodnyWent hypothesis, and Darwins early work showing that the site of photoreception and the initiation of lat-eral auxin gradients occur above the site of elongation. Genera-tion of this auxin gradient does not appear to depend on PIN3 or ABCB19 ( Christie et al., 2011 ). Indeed, phototropic respon-siveness in this experimental system could not be ascribed to any of the well-characterized auxin transporters, as mutants lacking these proteins were still phototropic ( Christie et al., 2011 ). These observations are therefore consistent with a model in which coaction between multiple transporters is required to drive the lateral redirection of auxin necessary for phototropic growth. Furthermore, excision of the cotyledons prior to photo-stimulation of dark-acclimated Arabidopsis seedlings does not stop phototropism or lateral auxin accumulation from occur-ring, indicating that their contribution to phototropic bending is likely to be minimal. A detailed spatial assessment of cellular auxin levels will now be necessary to determine where the lat-eral transport of auxin is initiated from within the shoot apex of dicot seedlings.

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