PHOTOTROPIN1 lysine 526 functions to enhance phototropism in Arabidopsis.

MAIN CONCLUSION: After blue-light exposure, ubiquitination of PHOTOTROPIN1 lysine 526 enhances phototropic responses. Arabidopsis blue-light photoreceptor, PHOTOTROPIN1 (PHOT1) mediates a series of blue-light responses that function to optimize photosynthesis efficiency. Blue-light sensing through the N-terminal sensory domain activates the C-terminal kinase activity of PHOT1, resulting in autophosphorylation. In addition to phosphorylation, PHOT1 lysine residue 526 (Lys526), after blue-light exposure, was found to carry a double glycine attachment, indicative of a possible ubiquitination modification. The functionality of PHOT1 Lys526 was investigated by reverse genetic approaches. Arginine replacements of PHOT1 Lys526, together with Lys527, complemented phot1-5 phot2-1 double mutant with attenuated phototropic bending, while blue-light responses: leaf expansion and stomatal opening, were restored to wild type levels. Transgenic seedlings were not different in protein levels of phot1 Lys526 527Arg than the wild type control, suggesting the reduced phototropic responses was not caused by reduction in protein levels. Treating the transformants with proteosome inhibitor, MG132, did not restore phototropic sensitivity. Both transgenic protein and wild type PHOT1 also had similar dark recovery of kinase activity, suggesting that phot1 Lys526 527Arg replacement did not affect the protein stability to cause the phenotype. Together, our results indicate that blocking Lys526 ubiquitination by arginine substitution may have caused the reduced phototropic phenotype. Therefore, the putative ubiquitination on Lys526 functions to enhance PHOT1-mediated phototropism, rather than targeting PHOT1 for proteolysis.

A phytochrome/phototropin chimeric photoreceptor promotes growth of fern gametophytes under limited light conditions.

Many ferns thrive even in low-light niches such as under an angiosperm forest canopy. However, the shade adaptation strategy of ferns is not well understood. Phytochrome 3/neochrome (phy3/neo) is an unconventional photoreceptor, found in the fern Adiantum capillus-veneris, that controls both red and blue light-dependent phototropism and chloroplast photorelocation, which are considered to improve photosynthetic efficiency in ferns. Here we show that phy3/neo localizes not only at the plasma membrane but also in the nucleus. Since both phototropism and chloroplast photorelocation are mediated by membrane-associated phototropin photoreceptors, we speculated that nucleus-localized phy3/neo possesses a previously undescribed biological function. We reveal that phy3/neo directly interacts with Adiantum cryptochrome 3 (cry3) in the nucleus. Plant cryptochromes are blue light receptors that transcriptionally regulate photomorphogenesis; therefore, phy3/neo may function via cry3 to synchronize light-mediated development with phototropism and chloroplast photorelocation to promote fern growth under low-light conditions. Furthermore, we demonstrate that phy3/neo regulates the expression of the Cyclin-like gene AcCyc1 and promotes prothallium expansion growth. These findings provide insight into the shade adaptation strategy of ferns and suggest that phy3/neo plays a substantial role in the survival and growth of ferns during the tiny gametophytic stage under low-light conditions, such as those on the forest floor.

Air spaces bend light in plant stems.

Intercellular air spaces are necessary for phototropism in Arabidopsis thaliana.

Air channels create a directional light signal to regulate hypocotyl phototropism.

In plants, light direction is perceived by the phototropin photoreceptors, which trigger directional growth responses known as phototropism. The formation of a phototropin activation gradient across a photosensitive organ initiates this response. However, the optical tissue properties that functionally contribute to phototropism remain unclear. In this work, we show that intercellular air channels limit light transmittance through various organs in several species. Air channels enhance light scattering in Arabidopsis hypocotyls, thereby steepening the light gradient. This is required for an efficient phototropic response in Arabidopsis and Brassica. We identified an embryonically expressed ABC transporter required for the presence of air channels in seedlings and a structure surrounding them. Our work provides insights into intercellular air space development or maintenance and identifies a mechanism of directional light sensing in plants.

Multiple light signaling pathways control solar tracking in sunflowers.

Sunflowers are famous for their ability to track the sun throughout the day and then reorient at night to face east the following morning. This occurs by differential growth patterns, with the east sides of stems growing more during the day and the west sides of stems growing more at night. This process, termed heliotropism, is generally believed to be a specialized form of phototropism; however, the underlying mechanism is unknown. To better understand heliotropism, we compared gene expression patterns in plants undergoing phototropism in a controlled environment and in plants initiating and maintaining heliotropic growth in the field. We found the expected transcriptome signatures of phototropin-mediated phototropism in sunflower stems bending towards monochromatic blue light. Surprisingly, the expression patterns of these phototropism-regulated genes are quite different in heliotropic plants. Most genes rapidly induced during phototropism display only minor differences in expression across solar tracking stems. However, some genes that are both rapidly induced during phototropism and are implicated in growth responses to foliar shade are rapidly induced on the west sides of stems at the onset of heliotropism, suggesting a possible role for red light photoreceptors in solar tracking. To test the involvement of different photoreceptor signaling pathways in heliotropism, we modulated the light environment of plants initiating solar tracking. We found that depletion of either red and far-red light or blue light did not hinder the initiation or maintenance of heliotropism in the field. Together, our results suggest that the transcriptional regulation of heliotropism is distinct from phototropin-mediated phototropism and likely involves inputs from multiple light signaling pathways.

Negative phototropism of the shoots helps temperate liana Hedera helix L. to locate host trees under habitat conditions.

Lianas employ a variety of searching mechanisms to find support; however, it is not clear to what extent environmental signals are used to help direct the search. Several adventitious root climbers have been shown to bend away from light and grow toward darker areas or objects, in one case including actual tree trunks. In the literature, this negative phototropism (NP) has also been informally and inconsistently reported from a temperate root climber Hedera helix L. (common ivy). In this study, rigorous laboratory tests have confirmed the occurrence of NP in both seedlings and prostrate shoots of H. helix. Furthermore, a field experiment with potted ivy seedlings placed around tree trunks demonstrated their ability to remotely locate trees. This finding was corroborated by a survey of growth directions in wild-growing prostrate ivy shoots in two woodland habitats. An additional outdoor experiment showed that the ability to locate support is expressed in shade but supressed by full sun conditions. These results show that H. helix uses NP to locate support and indicate that this ability is a component of the species' shade escape strategy.

MIZU-KUSSEI1 (MIZ1) and GNOM/MIZ2 control not only positive hydrotropism but also phototropism in Arabidopsis roots.

In response to unilateral blue light illumination, roots of some plant species such as Arabidopsis thaliana exhibit negative phototropism (bending away from light), which is important for light avoidance in nature. MIZU-KUSSEI1 (MIZ1) and GNOM/MIZ2 are essential for positive hydrotropism (i.e. in the presence of a moisture gradient, root bending towards greater water availability). Intriguingly, mutations in these genes also cause a substantial reduction in phototropism. Here, we examined whether the same tissue-specific sites of expression required for MIZ1- and GNOM/MIZ2-regulated hydrotropism in Arabidopsis roots are also required for phototropism. The attenuated phototropic response of miz1 roots was completely restored when a functional MIZ1-green fluorescent protein (GFP) fusion was expressed in the cortex of the root elongation zone but not in other tissues such as root cap, meristem, epidermis, or endodermis. The hydrotropic defect and reduced phototropism of miz2 roots were restored by GNOM/MIZ2 expression in either the epidermis, cortex, or stele, but not in the root cap or endodermis. Thus, the sites in root tissues that are involved in the regulation of MIZ1- and GNOM/MIZ2-dependent hydrotropism also regulate phototropism. These results suggest that MIZ1- and GNOM/MIZ2-mediated pathways are, at least in part, shared by hydrotropic and phototropic responses in Arabidopsis roots.

PIN-FORMED is required for shoot phototropism/gravitropism and facilitates meristem formation in Marchantia polymorpha.

PIN-FORMED auxin efflux transporters, a subclass of which is plasma membrane-localised, mediate a variety of land-plant developmental processes via their polar localisation and subsequent directional auxin transport. We provide the first characterisation of PIN proteins in liverworts using Marchantia polymorpha as a model system. Marchantia polymorpha possesses a single PIN-FORMED gene, whose protein product is predicted to be plasma membrane-localised, MpPIN1. To characterise MpPIN1, we created loss-of-function alleles and produced complementation lines in both M. polymorpha and Arabidopsis. In M. polymorpha, gene expression and protein localisation were tracked using an MpPIN1 transgene encoding a translationally fused fluorescent protein. Overexpression of MpPIN1 can partially complement loss of an orthologous gene, PIN-FORMED1, in Arabidopsis. In M. polymorpha, MpPIN1 influences development in numerous ways throughout its life cycle. Most notably, MpPIN1 is required to establish gemmaling dorsiventral polarity and for orthotropic growth of gametangiophore stalks, where MpPIN1 is basally polarised. PIN activity is largely conserved within land plants, with PIN-mediated auxin flow providing a flexible mechanism to organise growth. Specifically, PIN is fundamentally linked to orthotropism and to the establishment of de novo meristems, the latter potentially involving the formation of both auxin biosynthesis maxima and auxin-signalling minima.

Protein S-acylation controls the subcellular localization and biological activity of PHYTOCHROME KINASE SUBSTRATE.

PHYTOCHROME KINASE SUBSTRATE (PKS) proteins are involved in light-modulated changes in growth orientation. They act downstream of phytochromes to control hypocotyl gravitropism in the light and act early in phototropin signaling. Despite their importance for plant development, little is known about their molecular mode of action, except that they belong to a protein complex comprising phototropins at the plasma membrane (PM). Identifying evolutionary conservation is one approach to revealing biologically important protein motifs. Here, we show that PKS sequences are restricted to seed plants and that these proteins share 6 motifs (A to F from the N to the C terminus). Motifs A and D are also present in BIG GRAIN, while the remaining 4 are specific to PKSs. We provide evidence that motif C is S-acylated on highly conserved cysteines, which mediates the association of PKS proteins with the PM. Motif C is also required for PKS4-mediated phototropism and light-regulated hypocotyl gravitropism. Finally, our data suggest that the mode of PKS4 association with the PM is important for its biological activity. Our work, therefore, identifies conserved cysteines contributing to PM association of PKS proteins and strongly suggests that this is their site of action to modulate environmentally regulated organ positioning.

Phototropin phosphorylation of ROOT PHOTOTROPISM 2 and its role in mediating phototropism, leaf positioning, and chloroplast accumulation movement in Arabidopsis.

Directional movements impact the ability of plants to respond and adjust their growth accordingly to the prevailing light environment. The plasma-membrane associated protein, ROOT PHOTOTROPISM 2 (RPT2) is a key signalling component involved in chloroplast accumulation movement, leaf positioning, and phototropism, all of which are regulated redundantly by the ultraviolet/blue light-activated AGC kinases phototropin 1 and 2 (phot1 and phot2). We recently demonstrated that members of the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)/RPT2-like (NRL) family in Arabidopsis thaliana, including RPT2, are directly phosphorylated by phot1. However, whether RPT2 is a substrate for phot2, and the biological significance of phot phosphorylation of RPT2 remains to be determined. Here, we show that RPT2 is phosphorylated by both phot1 and phot2 at a conserved serine residue (S591) within the C-terminal region of the protein. Blue light triggered the association of 14-3-3 proteins with RPT2 consistent with S591 acting as a 14-3-3 binding site. Mutation of S591 had no effect on the plasma membrane localization of RPT2 but reduced its functionality for leaf positioning and phototropism. Moreover, our findings indicate that S591 phosphorylation within the C-terminus of RPT2 is required for chloroplast accumulation movement to low level blue light. Taken together, these findings further highlight the importance of the C-terminal region of NRL proteins and how its phosphorylation contributes to phot receptor signalling in plants.

Chloroplasts in plant cells show active glassy behavior under low-light conditions.

Plants have developed intricate mechanisms to adapt to changing light conditions. Besides phototropism and heliotropism (differential growth toward light and diurnal motion with respect to sunlight, respectively), chloroplast motion acts as a fast mechanism to change the intracellular structure of leaf cells. While chloroplasts move toward the sides of the plant cell to avoid strong light, they accumulate and spread out into a layer on the bottom of the cell at low light to increase the light absorption efficiency. Although the motion of chloroplasts has been studied for over a century, the collective organelle motion leading to light-adapting self-organized structures remains elusive. Here, we study the active motion of chloroplasts under dim-light conditions, leading to an accumulation in a densely packed quasi-2D layer. We observe burst-like rearrangements and show that these dynamics resemble systems close to the glass transition by tracking individual chloroplasts. Furthermore, we provide a minimal mathematical model to uncover relevant system parameters controlling the stability of the dense configuration of chloroplasts. Our study suggests that the meta-stable caging close to the glass transition in the chloroplast monolayer serves a physiological relevance: Chloroplasts remain in a spread-out configuration to increase the light uptake but can easily fluidize when the activity is increased to efficiently rearrange the structure toward an avoidance state. Our research opens questions about the role that dynamical phase transitions could play in self-organized intracellular responses of plant cells toward environmental cues.

Photosensory adaptation mechanisms in hypocotyl phototropism: how plants recognize the direction of a light source.

Plants recognize the direction of a light source and exhibit phototropic responses. Physiological studies have predicted that differences in the light intensity received by the cells on the irradiated and shaded sides of a coleoptile or hypocotyl cause differences in the amounts of photoproduct. This hypothetical photoproduct appears to regulate a signaling pathway that controls cell elongation in which cells under lower light intensity elongate more than those under higher light intensity. This results in a bending growth toward a light source and has been proposed as the photoproduct-gradient model of phototropism. In this review, we summarize recent findings on the photosensory adaptation mechanisms involving a blue-light photoreceptor, phototropin1 (phot1), ROOT PHOTOTROPISM2, NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and another photoreceptor family, the phytochromes. The current evidence demonstrates that, in addition to the transition of the phot1-NPH3 photoreceptor complexes to their active state, the presence of a certain population of the phot1-NPH3 complexes showing a steady state, even in a light environment, is essential for recognition of the light source direction in phototropism. This is consistent with the photoproduct-gradient model, and a dissociation state of the phot1-NPH3 complex would be considered an entity of the hypothetical photoproduct in this model.

Physiological Analysis of Phototropic Responses to Blue and Red Light in Arabidopsis.

Plants utilize light as sole energy source. To maximize light capture, they are able to detect the light direction and orient themselves toward the light source. This phototropic response is mediated by the plant blue-light photoreceptors phototropin1 and phototropin2 (phot1 and phot2). Although fully differentiated plants also exhibit this response, it can be best observed in etiolated seedlings. Differences in light between the illuminated and shaded site of a seedling stem lead to changes in the auxin distribution, resulting in cell elongation on the shaded site. Since phototropism connects light perception, signaling, and auxin transport, it is of great interest to analyze this response with a fast and simple method. Moreover, pre-exposure to red light enhances the phototropic response via phytochrome A (phyA) and phyB action. Here we describe a method to analyze the phototropic response of Arabidopsis seedlings to blue light and the enhanced response with a red-light pretreatment. With numerous mutants available, its fast germination, and its small size, Arabidopsis is well suited for this analysis. Different genotypes can be simultaneously probed in less than a week.

Spaceflight studies identify a gene encoding an intermediate filament involved in tropism pathways.

We performed a series of experiments to study the interaction between phototropism and gravitropism in Arabidopsis thaliana as part of the Seedling Growth Project on the International Space Station. Red-light-based and blue-light-based phototropism were examined in microgravity and at 1g, a control that was produced by an on-board centrifuge. At the end of the experiments, seedlings were frozen and brought back to Earth for gene profiling studies via RNASeq methods. In this paper, we focus on five genes identified in these space studies by their differential expression in space: one involved in auxin transport and four others encoding genes for: a methyltransferase subunit, a transmembrane protein, a transcription factor for endodermis formation, and a cytoskeletal element (an intermediate filament protein). Time course studies using mutant strains of these five genes were performed for blue-light and red-light phototropism studies as well as for gravitropism assays on ground. Interestingly, all five of the genes had some effects on all the tropisms under the conditions studied. In addition, RT-PCR analyses examined expression of the five genes in wild-type seedlings during blue-light-based phototropism. Previous studies have supported a role of both microfilaments and microtubules in tropism pathways. However, the most interesting finding of the present space studies is that NFL, a gene encoding an intermediate filament protein, plays a role in phototropism and gravitropism, which opens the possibility that this cytoskeletal element modulates signal transduction in plants.

The phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 affects phot2-dependent phototropism in Arabidopsis.

The blue light photoreceptors, phototropin 1 (phot1) and phot2, and their signal transducer, NONPHOTOTROPIC HYPOCOTYL3 (NPH3), are activators of the phototropic responses of Arabidopsis hypocotyls. In a recent study, we reported that the control of NPH3 phosphorylation at serine 7 (S7: or S5), S213, S223, S237, S467, S474 (or S476), and S722 (or S723) contributes to the photosensory adaptation of phot1 signaling during the phototropic response. Phosphomimetic NPH3SE mutant and unphosphorylatable NPH3SA mutant on those serine residues function efficiently under blue light conditions at fluence rates of 10-5 µmol m-2 s-1 and 10-3 µmol m-2 s-1 or more, respectively. We here demonstrate that phosphomimetic NPH3SE, but not unphosphorylatable NPH3SA, promotes phot2-dependent phototropism under blue light condition at 100 µmol m-2 s-1. This result suggests that phot1 negatively controls phot2 signaling through the dephosphorylation of NPH3 at those residues and that the hyperactivation of phot1- and phot2-NPH3 complexes does not occur at the same time under high intensity blue light. We hypothesize that the dephosphorylation of NPH3 on those serine residues suppresses both phot1 and phot2 signaling, which results in different impacts on phot1- and phot2-dependent hypocotyl phototropism due to the differences in the photosensitivity and activation levels of phot1 and phot2.

A novel device to study altered gravity and light interactions in seedling tropisms.

Long-duration space missions will need to rely on the use of plants in bio-regenerative life support systems (BLSSs) because these systems can produce fresh food and oxygen, reduce carbon dioxide levels, recycle metabolic waste, and purify water. In this scenario, the need for new experiments on the effects of altered gravity conditions on plant biological processes is increasing, and significant efforts should be devoted to new ideas aimed at increasing the scientific output and lowering the experimental costs. Here, we report the design of an easy-to-produce and inexpensive device conceived to analyze the effect of interaction between gravity and light on root tropisms. Each unit consisted of a polystyrene multi-slot rack with light-emitting diodes (LEDs), capable of holding Petri dishes and assembled with a particular filter-paper folding. The device was successfully used for the ROOTROPS (for root tropisms) experiment performed in the Large Diameter Centrifuge (LDC) and Random Positioning Machine (RPM) at ESA's European Space Research and Technology centre (ESTEC). During the experiments, four light treatments and six gravity conditions were factorially combined to study their effects on root orientation of Brassica oleracea seedlings. Light treatments (red, blue, and white) and a dark condition were tested under four hypergravity levels (20 g, 15 g, 10 g, 5 g), a 1 g control, and a simulated microgravity (RPM) condition. Results of validation tests showed that after 24 h, the assembled system remained unaltered, no slipping or displacement of seedlings occurred at any hypergravity treatment or on the RPM, and seedlings exhibited robust growth. Overall, the device was effective and reliable in achieving scientific goals, suggesting that it can be used for ground-based research on phototropism-gravitropism interactions. Moreover, the concepts developed can be further expanded for use in future spaceflight experiments with plants.

Functional mapping of gravitropism and phototropism for a desert tree, Populus euphratica.

Background: Plants have evolved the dual capacity for maximizing light assimilation through stem growth (phototropism) and maximizing water and nutrient absorption through root growth (gravitropism). Previous studies have revealed the physiological and molecular mechanisms of these two processes, but the genetic basis for how gravitropism and phototropism interact and coordinate with one another to determine plant growth remains poorly understood. Methods: We designed a seed germination experiment using a full-sib F1 family of Populus euphratica to simultaneously monitor the gravitropic growth of the radicle and the phototropic growth of the plumule throughout seedling ontogeny. We implemented three functional mapping models to identify quantitative trait loci (QTLs) that regulate gravitropic and phototropic growth. Univariate functional mapping dissected each growth trait separately, bivariate functional mapping mapped two growth traits simultaneously, and composite functional mapping mapped the sum of gravitropic and phototropic growth as a main axis. Results: Bivariate model detected 8 QTLs for gravitropism and phototropism (QWRF, GLUR, F-box, PCFS4, UBQ, TAF12, BHLH95, TMN8), composite model detected 7 QTLs for growth of main axis (ATL8, NEFH, PCFS4, UBQ, SOT16, MOR1, PCMP-H), of which, PCFS4 and UBQ were pleiotropically detected with the both model. Many of these QTLs are situated within the genomic regions of candidate genes. Conclusions: The results from our models provide new insight into the mechanisms of genetic control of gravitropism and phototropism in a desert tree, and will stimulate our understanding of the relationships between gravity and light signal transduction pathways and tree adaptation to arid soil.

Regulation of plant phototropic growth by NPH3/RPT2-like substrate phosphorylation and 14-3-3 binding.

Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.

Light-triggered and phosphorylation-dependent 14-3-3 association with NON-PHOTOTROPIC HYPOCOTYL 3 is required for hypocotyl phototropism.

NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent plant phototropic growth response. We report that NPH3 directly binds polyacidic phospholipids, required for plasma membrane association in darkness. We further demonstrate that blue light induces an immediate phosphorylation of a C-terminal 14-3-3 binding motif in NPH3. Subsequent association of 14-3-3 proteins is causal for the light-induced release of NPH3 from the membrane and accompanied by NPH3 dephosphorylation. In the cytosol, NPH3 dynamically transitions into membraneless condensate-like structures. The dephosphorylated state of the 14-3-3 binding site and NPH3 membrane recruitment are recoverable in darkness. NPH3 variants that constitutively localize either to the membrane or to condensates are non-functional, revealing a fundamental role of the 14-3-3 mediated dynamic change in NPH3 localization for auxin-dependent phototropism. This regulatory mechanism might be of general nature, given that several members of the NPH3-like family interact with 14-3-3 via a C-terminal motif.