Ullas V. Pedmale

Linking photoreceptor excitation to changes in plant architecture

Plants sense neighbor proximity as a decrease in the ratio of red to far-red light, which triggers a series of developmental responses. In Arabidopsis, phytochrome B (PHYB) is the major sensor of shade, but PHYB excitation has not been linked directly to a growth response. We show that the basic helix-loop-helix (bHLH) transcription factor PIF7 (phytochrome-interacting factor 7), an interactor of PHYB, accumulates in its dephosphorylated form in shade, allowing it to bind auxin biosynthetic genes and increase their expression. New auxin synthesized through a PIF7-regulated pathway is required for shade-induced growth, linking directly the perception of a light quality signal to a rapid growth response.

Cryptochrome 1 and phytochrome B control shade-avoidance responses in Arabidopsis via partially independent hormonal cascades.

Plants respond to a reduction in the red/far-red ratio (R:FR) of light, caused by the proximity of other plants, by initiating morphological changes that improve light capture. In Arabidopsis, this response (shade avoidance syndrome, SAS) is controlled by phytochromes (particularly phyB), and is dependent on the TAA1 pathway of auxin biosynthesis. However, when grown in real canopies, we found that phyB mutants and mutants deficient in TAAI (sav3) still display robust SAS responses to increased planting density and leaf shading. The SAS morphology (leaf hyponasty and reduced lamina/petiole ratio) could be phenocopied by exposing plants to blue light attenuation. These responses to blue light attenuation required the UV-A/blue light photoreceptor cry1. Moreover, they were mediated through mechanisms that showed only limited overlap with the pathways recruited by phyB inactivation. In particular, pathways for polar auxin transport, auxin biosynthesis and gibberellin signaling that are involved in SAS responses to low R:FR were not required for the SAS responses to blue light depletion. By contrast, the brassinosteroid response appeared to be required for the full expression of the SAS phenotype under low blue light. The phyB and cry1 inactivation pathways appeared to converge in their requirement for the basic/helix-loop-helix (bHLH) transcription factors PHYTOCHROME INTERACTING FACTORs 4 and 5 (PIF4 and PIF5) to elicit the SAS phenotype. Our results suggest that blue light is an important control of SAS responses, and that PIF4 and PIF5 are critical hubs for a diverse array of signaling routes that control plant architecture in canopies.

Regulation of Phototropic Signaling in Arabidopsis via Phosphorylation State Changes in the Phototropin 1-interacting Protein NPH3

Phototropism, or the directional growth (curvature) of various organs toward or away from incident light, represents a ubiquitous adaptive response within the plant kingdom. This response is initiated through the sensing of directional blue light (BL) by a small family of photoreceptors known as the phototropins. Of the two phototropins present in the model plant Arabidopsis thaliana, phot1 (phototropin 1) is the dominant receptor controlling phototropism. Absorption of BL by the sensory portion of phot1 leads, as in other plant phototropins, to activation of a C-terminal serine/threonine protein kinase domain, which is tightly coupled with phototropic responsiveness. Of the five phot1-interacting proteins identified to date, only one, NPH3 (non-phototropic hypocotyl 3), is essential for all phot1-dependent phototropic responses, yet little is known about how phot1 signals through NPH3. Here, we show that, in dark-grown seedlings, NPH3 exists as a phosphorylated protein and that BL stimulates its dephosphorylation. phot1 is necessary for this response and appears to regulate the activity of a type 1 protein phosphatase that catalyzes the reaction. The abrogation of both BL-dependent dephosphorylation of NPH3 and development of phototropic curvatures by protein phosphatase inhibitors further suggests that this post-translational modification represents a crucial event in phot1-dependent phototropism. Given that NPH3 may represent a core component of a CUL3-based ubiquitin-protein ligase (E3), we hypothesize that the phosphorylation state of NPH3 determines the functional status of such an E3 and that differential regulation of this E3 is required for normal phototropic responsiveness.

Cryptochromes Interact Directly with PIFs to Control Plant Growth in Limiting Blue Light

Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome wide and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions.

Modulation of Phototropic Responsiveness in Arabidopsis through Ubiquitination of Phototropin 1 by the CUL3-Ring E3 Ubiquitin Ligase CRL3 NPH3

This work demonstrates that the NPH3 protein of Arabidopsis represents a core component of a CULLIN3-based E3 ubiquitin ligase that targets the phototropin1 (phot1) photoreceptor for blue light–stimulated mono/multi- and polyubiquitination. In addition, it was shown that phot1 ubiquitination by this E3 complex is necessary for normal phototropic responsiveness. Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3NPH3. Under low-intensity BL, CRL3NPH3 mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi- and polyubiquitinated by CRL3NPH3, with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.

Integration of Light and Photoperiodic Signaling in Transcriptional Nuclear Foci

Light regulates major plant developmental transitions by orchestrating a series of nuclear events. This study uncovers the molecular function of the natural variant, TZP (Tandem Zinc-finger-Plus3), as a signal integrator of light and photoperiodic pathways in transcriptional nuclear foci. We report that TZP acts as a positive regulator of photoperiodic flowering via physical interactions with the red-light receptor phytochrome B (phyB). We demonstrate that TZP localizes in dynamic nuclear domains regulated by light quality and photoperiod. This study shows that phyB is indispensable not only for localizing TZP to transcriptionally active nuclear photobodies, but also for recruiting TZP on the promoter of the floral inducer FLOWERING LOCUS T (FT). Our findings signify a unique transcriptional regulatory role to the highly enigmatic plant nuclear photobodies, where TZP directly activates FT gene expression and promotes flowering.

Phototropism: mechanism and outcomes.

Plants have evolved a wide variety of responses that allow them to adapt to the variable environmental conditions in which they find themselves growing. One such response is the phototropic response - the bending of a plant organ toward (stems and leaves) or away from (roots) a directional blue light source. Phototropism is one of several photoresponses of plants that afford mechanisms to alter their growth and development to changes in light intensity, quality and direction. Over recent decades much has been learned about the genetic, molecular and cell biological components involved in sensing and responding to phototropic stimuli. Many of these advances have been made through the utilization of Arabidopsis as a model for phototropic studies. Here we discuss such advances, as well as studies in other plant species where appropriate to the discussion of work in Arabidopsis.

PIL1 Participates in a Negative Feedback Loop that Regulates Its Own Gene Expression in Response to Shade

Dear Editor, Plants grown in close proximity experience a change in light quality, and respond by reallocating energy resources from storage organs to stem-like organs. This adaptive response, called the shade-avoidance syndrome (SAS), allows the shaded plant to grow and compete effectively against its neighbors. SAS is initiated upon detection by the phytochrome photoreceptors of a lowering of the ratio of red to far-red light (R/FR), leading to the synthesis of plant hormones and a transcriptional cascade that targets genes involved in growth. Among these genes is PIL1 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 1), which encodes a bHLH transcription factor, whose expression is induced by up to 100-fold within 30min of exposure to shade (Salter et al., 2003); yet, PIL1’s precise role in shade avoidance is unknown. The Salter paper concluded that PIL1 worked with TOC1 to restrict growth to a particular time of day, and that PIL1 is necessary for the normal display of the rapid elongation response to shade (Salter et al., 2003). Later, Roig-Villanova and colleagues (2006) showed that PIL1 is a negative regulator of the SAS with only phenotype of pil1-4 and pil1-4phyB without any mechanism. To further understand the function of PIL1 in transducing phytochrome signals during the shade-avoidance response, we examined phenotypes of PIL1 loss- and gain-of-function mutants in simulated shade and proposed three possible modes of PIL1 action based on its protein stability and interaction with DNA and PIFs to regulate gene expression. We first obtained two Arabidopsis mutants with T-DNA insertions (Salk_043937C termed pil1-4 and Salk_025598C termed pil1-6) in the PIL1 coding region and also generated plants that stably overexpressed a PIL1–YFP fusion protein under the CaMV 35S promoter (35S::PIL1–YFP #17 and #13). Consistently with a previous report (Roig-Villanova et al., 2006), pil1-4 and pil1-6 mutant seedlings had slightly longer hypocotyls under shade conditions (Figure 1A). Furthermore, hypocotyls of PIL1-overexpressing lines were ~50% shorter than wild-type under shade (Figure 1A). This observation suggests that PIL1 plays a role as a decelerator of growth during early shade avoidance. Figure 1 PIL1 Participates in a Negative Feedback Loop that Regulates Its Own Gene Expression in Response to Shade. Although shade-induced accumulation of PIL1 transcripts is well documented, the regulation of PIL1 protein levels or activity has not been reported. Seedlings of line 35S::PIL1–YFP #17 were grown under continuous white light for 3 d, and then PIL1 protein levels were monitored over time from 0 to 24 h following transfer of seedlings from white light to shade. PIL1 protein gradually accumulated after seedlings were transferred to shade when compared with white light (Figure 1B). We then pretreated PIL1ox seedlings with 26S proteasome inhibitor MG132 or mock-treated with solvent control DMSO and then subjected the seedlings to shade or white-light conditions. MG132 treatment led to accumulation of PIL1 in white light. This indicates a white-light-dependent proteasome degradation of PIL1 protein (Figure 1B) which may explain why pil1 mutant and overexpression have no obvious phenotypes under white-light (high R/FR) conditions. To further examine light-mediated control of PIL1 stability, we measured protein accumulation in etiolated seedlings upon transfer to R or FR light. As shown in the Supplementary Data, long-term FR light treatment slightly stabilized PIL1 whereas R light had the opposite effect. Previous studies have shown that the atypical HLH transcription factors HFR1, PAR1, and PAR2 are negative regulators of the shade-avoidance response (Hornitschek et al., 2009; Galstyan et al., 2011). These proteins do not directly bind DNA. Instead, they function through modulating the activity of other DNA-binding bHLHs, such as PIF4 and PIF5, by interactions through the HLH domain (Hornitschek et al., 2009; Galstyan et al., 2011; Hao et al., 2012). PIL1 is predicted to be a typical bHLH protein with H/K9–E13–R17 DNA-binding domain. We tested whether PIL1 can bind DNA by employing a previously described in vitro DNA-binding assay (Vert and Chory, 2006). 3xHA–PIL1 and 3xHA–HFR1 were synthesized in cell-free extracts to test binding to biotin-labeled dsDNA probes. We chose the G-box-containing region from CCA1 promoter (–301/–266) and PIL1 promoter (–1412/–1375) as probe. PIL1 pelleted readily with the G-box containing dsDNA probes (Figure 1C, lanes 3 and 7). This binding was effectively competed by an unlabeled DNA probe (Figure 1C, lanes 4, 5, and 8), indicating that PIL1 can directly bind to DNA similarly to PIFs whereas HFR1 (Figure 1C, lane 6) cannot. In addition, we tested whether PIL1 is able to bind with PIFs. In yeast two-hybrid assays, the β-Gal reporter was activated when PIL1 was co-expressed with PIL1, PIF7, PIF4, and PIF5 (Figure 1D and Supplementary Data). A glutathione S-transferase (GST) pull-down assay was performed using GST–PIL1 purified from Escherichia coli which could pellet PIF4 and PIF7 from the extraction of seedlings overexpressing Flash-tagged (9Myc–6His–3Flag) PIF4 and PIF7 (Figure 1D and Supplementary Data). BiFC (Bimolecular Fluorescence Complementation) also confirmed the interaction between PIL1 and PIF7/PIF4, indicating that PIL1 is able to form a homodimer or heterodimers with PIFs in vivo. To better understand how PIL1 gene expression is controlled and how it affects the shade-regulated transcription network, we constructed a transgenic line that fuses the PIL1 promoter to the firefly luciferase (pPIL1::LUC) reporter. The PIL1 from this transgenic line reported similar expression levels and responses to shade as the endogenous PIL1 locus. And the shade induction of LUC gene expression and activity in pil1-4 background was similar to that in wide-type (Supplementary Data). In contrast, PIL1–CFP overexpression in the pPIL1::LUC line reduced LUC activity and suppressed hypocotyl elongation. These phenotypes were associated with PIL1 overexpression, as they were only manifested in three independent lines (lines 2, 4, and 5) which succeeded in overexpression but not in the other five lines which failed to express the transgene (Figure 1E). To confirm the self-regulation directly, we conducted a transactivation assay in tobacco. We used the LUC reporter gene under the control of the 1.5-Kb region upstream from the translation initiation site of PIL1 as a reporter, and PIL1, PIF5, and control (GUS) were used as effectors under the control of a 2xCaMV 35S promoter. Lastly, a Renilla LUC gene was driven by the CaMV 35S promoter and used as a control for transformation efficiency (Figure 1F). Agrobacterium tumefaciens harboring this construct were infiltrated into tobacco and the ratio of Luc/Ren activity was measured in leaf punches for determining any effects on the PIL1 promoter. PIF5 has been shown as an activator of PIL1 transcription (Hornitschek et al., 2009) and, consistently, expression of PIF5 strongly increased the ratio of Luc/Ren, whereas expression of PIL1 reduced the ratio of Luc/Ren compared to the control samples (Figure 1F). When PIL1 and PIF5 were co-expressed at the same time, the ratio of Luc/Ren is between that from only PIL1 and only PIF5, and lower than co-expression of PIF5 and GUS. Besides PIL1 itself, we examined the expression level of YUCCA8, IAA5, and IAA29 by qRT–PCR in Col-0 and PIL1 overexpression lines treated by 1h of shade (Supplementary Data). Except a higher level of PIL1 in the PIL1 overexpression line, the expression of YUCCA8, IAA5, and IAA29 were lower than that in Col-0, which may explain the short hypocotyl length of PIL1 overexpression under shade. Compared to other negative regulators of SAS, unlike HFR1 and PAR1/2, PIL1 can bind DNA and regulate gene expression (Figure 1E). On the other hand, similarly to HFR1, PIL1 could form heterodimers with PIFs. The attenuation of reporter by introducing PIL1 expression (Figure 1F) could be caused by either a homo-dimerization of PIL1 itself or hetero-dimerization of PIL1 and other PIF, namely PIF5. Thus, two modes of PIL1 action are possible: (1) PIL1 may outcompete PIFs for binding DNA or PIL1/PIF heterodimers may reduce the growth promoting function of PIFs. (2) PIL1 may directly regulate gene expression in a PIF-independent manner through binding to different sites in promoters of downstream genes. Another difference with HFR1, PIL1 contains an Active Phytochrome B-binding (APB) domain which is required for phyB-specific binding (Khanna et al., 2004). Despite the lack of evidence for full-length PIL1 interacting with phyB in vitro, the PIL1 APB motif has been shown to bind phyB Pfr in a photo-reversible manner (Khanna et al., 2004). The APB domain is required for PIF turnover (Khanna et al., 2004; Al-Sady et al., 2006; Lorrain et al., 2008; Bu et al., 2011). It is not sure whether APB of PIL1 affects the protein stability, while PIL1 is degraded in the light (Figure 1B) with similar kinetics to PIFs, which raises the third possibility that PIL1 may outcompete PIFs for binding to phyB. How do these negative regulators cooperate during shade? HFR1 is induced by up to 4 d of shade treatment, whereas PIL1 is rapidly induced by 1 h of shade treatment and is self-limited. PIL1 accumulates rapidly and transiently in response to shade, which might be an early signal for Arabidopsis to ‘pause growth’, thereby allowing the plant to determine whether prolonged shading is imminent. This would slow down a commitment to the shade-avoidance lifestyle if it were unnecessary. Finally, a more robust and long-lasting negative feedback loop involves other negative regulators: HFR1, PAR1, and PAR2 (Hornitschek et al., 2009; Galstyan et al., 2011), which ensures that plants sense sustained shade conditions and make a ‘self-confident’ decision.

Signaling in Phototropism

Land plants cope with the same environmental challenges as animals but have the added complication of being fixed to the ground. Thus, adaptability to variable environmental circumstances is essential to plant survival and fitness. A consequence of this condition is the necessity of plants to possess sophisticated sensors to adjust to changes. Plants take the input from their myriad of physiological sensors and respond physiologically. Among these responses are the tropisms, or directional growth responses that are oriented relative to a directional stimulus. Phototropism is among one of the best-studied tropic responses where plant tissues perceive and grow directionally upon perception of a directional light stimulus - positively, or towards the light source, in the case of shoots and negatively, or away from the light source, in the case of roots. From a historical perspective, the phototropic phenomenon has been known for hundreds of years (Whippo and Hangarter in Plant Cell 18:1110–1119, 2006). Yet, only in the past few decades has the phenomenon been carefully studied to the extent that the basis of this response has become clearer. While recent analyses have yielded detailed biochemical mechanisms for some of the phototropic receptors, a great deal remains unknown. In this review of phototropism in plants, the focus is on the growth of our understanding of phototropism from the simple observations of plant growth, to the initial physiological experiments, to the most recent detailed molecular mechanisms. With the advances in genetic and molecular tools we are now in a position to understand the nature of phototropic signaling and its regulation in great detail. Over the past few years we have come to learn much about the complex interplay of molecules, including the photoreceptors, accessory proteins, transcription factors, and effector molecules necessary to perceive the light cues, modulate signaling, activate gene transcription, and elicit physiological change. While some of these players are known, undoubtedly a role for many others will emerge in future studies and such advances will provide new avenues of research.