Phytochrome B regulates reactive oxygen signaling during abiotic and biotic stress in plants.

Reactive oxygen species (ROS) and the photoreceptor protein phytochrome B (phyB) play a key role in plant acclimation to stress. However, how phyB that primarily functions in the nuclei impacts ROS signaling mediated by respiratory burst oxidase homolog (RBOH) proteins that reside on the plasma membrane, during stress, is unknown. Arabidopsis thaliana and Oryza sativa mutants, RNA-Seq, bioinformatics, biochemistry, molecular biology, and whole-plant ROS imaging were used to address this question. Here, we reveal that phyB and RBOHs function as part of a key regulatory module that controls apoplastic ROS production, stress-response transcript expression, and plant acclimation in response to excess light stress. We further show that phyB can regulate ROS production during stress even if it is restricted to the cytosol and that phyB, respiratory burst oxidase protein D (RBOHD), and respiratory burst oxidase protein F (RBOHF) coregulate thousands of transcripts in response to light stress. Surprisingly, we found that phyB is also required for ROS accumulation in response to heat, wounding, cold, and bacterial infection. Our findings reveal that phyB plays a canonical role in plant responses to biotic and abiotic stresses, regulating apoplastic ROS production, possibly while at the cytosol, and that phyB and RBOHD/RBOHF function in the same regulatory pathway.

Phytochrome B Is Required for Systemic Stomatal Responses and Reactive Oxygen Species Signaling during Light Stress.

Perception of a change in light intensity leads to the activation of multiple physiological, metabolic, and molecular responses in plants. These responses allow acclimation to fluctuating light conditions, e.g. sunflecks in field grown plants, preventing cellular damage associated with excess light stress. Perception of light stress by a single Arabidopsis (Arabidopsis thaliana) leaf was recently shown to activate different local and systemic responses that include rapid changes in stomatal aperture size; these were found to be coordinated by a systemic process of reactive oxygen species (ROS)-derived ROS production (i.e. the ROS wave). How light intensity is perceived, and how long the ROS wave stays "on" during this process are, however, unknown. Here we show that triggering of the ROS wave by a local excess light stress treatment results in the induction and maintenance of high levels of systemic ROS for up to 6 h. Despite these high systemic ROS levels, stomatal aperture size returns to control size within 3 h, and the systemic stomatal response can be retriggered within 6 h. These findings suggest that the ROS wave triggers a systemic stress memory mechanism that lasts for 3 to 6 h, but that within 3 h of its activation, stomata become insensitive to ROS and open. We further show that the excess light stress-triggered ROS wave, as well as the excess light stress-triggered local and systemic stomatal aperture closure responses, are dependent on phytochrome B function. Our findings reveal a delicate interplay between excess light stress, phytochrome B, ROS production, and rapid systemic stomatal responses.

The continuing arc toward phototropic enlightenment.

Phototropism represents a simple physiological mechanism-differential growth across the growing organ of a plant-to respond to gradients of light and maximize photosynthetic light capture (in aerial tissues) and water/nutrient acquisition (in roots). The phototropin blue light receptors, phot1 and phot2, have been identified as the essential sensors for phototropism. Additionally, several downstream signal/response components have been identified, including the phot-interacting proteins NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) and PHYTOCHROME SUBSTRATE 4 (PKS4). While the structural and photochemical properties of the phots are quite well understood, much less is known about how the phots signal through downstream regulators. Recent advances have, however, provided some intriguing clues. It appears that inactive receptor phot1 is found dispersed in a monomeric form at the plasma membrane in darkness. Upon light absorption dimerizes and clusters in sterol-rich microdomains where it is signal active. Additional studies showed that the phot-regulated phosphorylation status of both NPH3 and PKS4 is linked to phototropic responsiveness. While PKS4 can function as both a positive (in low light) and a negative (in high light) regulator of phototropism, NPH3 appears to function solely as a key positive regulator. Ultimately, it is the subcellular localization of NPH3 that appears crucial, an aspect regulated by its phosphorylation status. While phot1 activation promotes dephosphorylation of NPH3 and its movement from the plasma membrane to cytoplasmic foci, phot2 appears to modulate relocalization back to the plasma membrane. Together these findings are beginning to illuminate the complex biochemical and cellular events, involved in adaptively modifying phototropic responsiveness under a wide varying range of light conditions.

Phototropism: growing towards an understanding of plant movement.

Phototropism, or the differential cell elongation exhibited by a plant organ in response to directional blue light, provides the plant with a means to optimize photosynthetic light capture in the aerial portion and water and nutrient acquisition in the roots. Tremendous advances have been made in our understanding of the molecular, biochemical, and cellular bases of phototropism in recent years. Six photoreceptors and their associated signaling pathways have been linked to phototropic responses under various conditions. Primary detection of directional light occurs at the plasma membrane, whereas secondary modulatory photoreception occurs in the cytoplasm and nucleus. Intracellular responses to light cues are processed to regulate cell-to-cell movement of auxin to allow establishment of a trans-organ gradient of the hormone. Photosignaling also impinges on the transcriptional regulation response established as a result of changes in local auxin concentrations. Three additional phytohormone signaling pathways have also been shown to influence phototropic responsiveness, and these pathways are influenced by the photoreceptor signaling as well. Here, we will discuss this complex dance of intra- and intercellular responses that are regulated by these many systems to give rise to a rapid and robust adaptation response observed as organ bending.

Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-Ring E3 ubiquitin ligase CRL3(NPH3).

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, CRL3(NPH3). Under low-intensity BL, CRL3(NPH3) 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 CRL3(NPH3), 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.

Understanding phototropism: from Darwin to today.

Few individuals have had the lasting impact on such a breadth of science as Charles Darwin. While his writings about time aboard the HMS Beagle, his study of the Galapagos islands (geology, fauna, and flora), and his theories on evolution are well known, less appreciated are his studies on plant growth responses to a variety of environmental stimuli. In fact, Darwin, together with the help of his botanist son Francis, left us an entire book, 'The power of movements in plants', describing his many, varied, and insightful observations on this topic. Darwin's findings have provided an impetus for an entire field of study, the study of plant tropic responses, or differential growth (curvature) of plant organs in response to directional stimuli. One tropic response that has received a great deal of attention is the phototropic response, or curvature response to directional light. This review summarizes many of the most significant advancements that have been made in our understanding of this response and place these recent findings in the context of Darwin's initial observations.

A Method for Investigating the Pseudomonas syringae-Arabidopsis thaliana Pathosystem Under Various Light Environments.

Arabidopsis thaliana and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) comprise an effective model pathosystem for resolving mechanisms behind numerous aspects of plant innate immunity. Following the characterization of key molecular components over the past decades, we may begin investigating defense signaling under various environmental conditions to gain a more holistic understanding of the underlying processes. As a critical regulator of growth and development, exploration into the influence of light on pathogenesis is a logical step toward a systems-level understanding of innate immunity. Based on methods described previously, here we describe a method for investigating plant immune responses under various light environments.

Plant tropisms: providing the power of movement to a sessile organism.

In an attempt to compensate for their sessile nature, plants have developed growth responses to deal with the copious and rapid changes in their environment. These responses are known as tropisms and they are marked by a directional growth response that is the result of differential cellular growth and development in response to an external stimulation such as light, gravity or touch. While the mechanics of tropic growth and subsequent development have been the topic of debate for more than a hundred years, only recently have researchers been able to make strides in understanding how plants perceive and respond to tropic stimulations, thanks in large part to mutant analysis and recent advances in genomics. This paper focuses on the recent advances in four of the best-understood tropic responses and how each affects plant growth and development: phototropism, gravitropism, thigmotropism and hydrotropism. While progress has been made in deciphering the events between tropic stimulation signal perception and each characteristic growth response, there are many areas that remain unclear, some of which will be discussed herein. As has become evident, each tropic response pathway exhibits distinguishing characteristics. However, these pathways of tropic perception and response also have overlapping components - a fact that is certainly related to the necessity for pathway integration given the ever-changing environment that surrounds every plant.

TWISTED DWARF 1 Associates with BRASSINOSTEROID-INSENSITIVE 1 to Regulate Early Events of the Brassinosteroid Signaling Pathway.

A genome-wide screen for mutants showing altered brassinosteroid (BR) sensitivity or bri1-like phenotypes resulted in the identification of two new mutant alleles of TWISTED DWARF 1 (TWD1), twd1-4, and twd1-5. Previous studies indicated that TWD1, also named as ULTRACURVATA 2 or FKBP42, associates with auxin efflux transporters and is essential for their biological functions. Although earlier reports showed that BR signaling is downregulated in twd1, how TWD1 is integrated in BR signaling has not been elucidated. Here, we provide genetic and biochemical evidence demonstrating that TWD1 interacts with the BR receptor BRI1 in vivo in a BR-independent manner. Further analyses indicated that TWD1 modulates the BR signal transduction not by altering ER quality control or protein abundance of BRI1; instead, TWD1 appears to be critical in BR-induced interaction of BRI1 and its co-receptor BAK1, as well as BR-induced phosphorylation of these two proteins. These results provide better understanding of the early events of the BR signaling pathway.

Phototropins and associated signaling: providing the power of movement in higher plants.

Recent developments in phototropin biology have provided exciting new findings on the roles of these photoreceptor proteins in plants. Much of the recent work has focused on phototropin photochemistry and the structural alterations in both the chromophoric and peptide components of the molecule associated with light perception. In this review, specific aspects of phototropin action in higher plants will be discussed in the context of these new findings. Although, as their name suggests, phototropins play a key role in phototropic responses in plants, increasing evidence shows they have many other functions in plants. In this review, the roles of phototropins in additional plant "movement" responses will be addressed; in particular their roles in stomatal aperture control and chloroplast movements. In discussing these various movement responses special attention will be given to identified and hypothesized downstream signaling partners or events that enable the phototropins to selectively participate in any one or more of these responses in a given light condition.

An experimental test of the adaptive evolution of phototropins: blue-light photoreceptors controlling phototropism in Arabidopsis thaliana.

Phototropins are blue-light photoreceptor molecules mediating the capacity for phototropism or bending toward or away from directional light. Like the red-light sensing phytochromes that control shade avoidance, phototropins modulate developmental plasticity in plant architecture. Yet, unlike phytochromes, the adaptive significance of phototropins has been largely a topic of conjecture. In Arabidopsis thaliana, phototropism of seedling and plant stems is under the control of two paralogous genes, PHOT1 and PHOT2, that encode different phototropins with partially redundant light response qualities. The PHOT1 gene product interacts with the NPH3 gene product to cause phototropic bending over a broad range of light intensity, from very weak light in the soil to stronger light in the aerial environment. The PHOT2 gene product modulates shoot bending in response to light of higher intensity only. We compared the fitness of wild-type, phot1, phot2, and nph3 genotypes over a range of light conditions in the field. Seeds were sown in the field on the soil surface and left bare or covered with either gravel or bark mulch chips. Plantings were made under full sun and dense canopy cover. Rates of seedling emergence, survival to flowering, and total seed set were measured. All mutant genotypes had significantly reduced lifetime fitness compared to wild-type. Consistent with their different fluence rate sensitivities, phot1 and phot2 signaling pathways affected fitness at discrete life-cycle stages. Fitness costs of phot1 and nph3 were expressed mainly during seedling emergence from the soil whereas that of phot2 was expressed solely after emergence. Surprisingly, the only significant genotype-by-environment interaction for fitness occurred during emergence: genotypes blind to dim blue light (phot1 and nph3) had poor emergence in the open, but not in the shade. Possibly, the loss of negative phototropism in seedling roots of mutant genotypes reduced establishment success in open (dry soil) conditions. Results show that phototropin-modulated pathways are adaptive and that their evolution has involved functional specialization. However, mechanism(s) of selection on these pathways remain a mystery.

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.

Disruptions in AUX1-dependent auxin influx alter hypocotyl phototropism in Arabidopsis.

Phototropism represents a differential growth response by which plant organs can respond adaptively to changes in the direction of incident light to optimize leaf/stem positioning for photosynthetic light capture and root growth orientation for water/nutrient acquisition. Studies over the past few years have identified a number of components in the signaling pathway(s) leading to development of phototropic curvatures in hypocotyls. These include the phototropin photoreceptors (phot1 and phot2) that perceive directional blue-light (BL) cues and then stimulate signaling, leading to relocalization of the plant hormone auxin, as well as the auxin response factor NPH4/ARF7 that responds to changes in local auxin concentrations to directly mediate expression of genes likely encoding proteins necessary for development of phototropic curvatures. While null mutations in NPH4/ARF7 condition an aphototropic response to unidirectional BL, seedlings carrying the same mutations recover BL-dependent phototropic responsiveness if co-irradiated with red light (RL) or pre-treated with either ethylene. In the present study, we identify second-site enhancer mutations in the nph4 background that abrogate these recovery responses. One of these mutations--map1 (modifier of arf7 phenotypes 1)--was found to represent a missense allele of AUX1--a gene encoding a high-affinity auxin influx carrier previously associated with a number of root responses. Pharmacological studies and analyses of additional aux1 mutants confirmed that AUX1 functions as a modulator of hypocotyl phototropism. Moreover, we have found that the strength of dependence of hypocotyl phototropism on AUX1-mediated auxin influx is directly related to the auxin responsiveness of the seedling in question.

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.

Functional ecology of a blue light photoreceptor: effects of phototropin-1 on root growth enhance drought tolerance in Arabidopsis thaliana.

* The blue light photoreceptor phototropin-1 has been shown to enhance fitness in Arabidosis thaliana under field conditions. Here, we ask whether performance consequences of phototropin-1 reflect its impact on root growth and drought tolerance. * We used a PHOT1-GFP gene construct to test whether phototropin-1 abundance in roots is highest at shallow soil depths where light penetration is greatest. We then compared root growth efficiency and size at maturity between individuals with and without functional phototropin-1. Comparisons were made under wet and dry conditions to assess the impact of phototropin-1 on drought tolerance. * Phototropin-1 was most abundant in upper root regions and its impact on root growth efficiency decreased with soil depth. Roots of plants with functional phototropin-1 made fewer random turns and traveled further for a given length (higher efficiency) than roots of phot1 mutants. In dry (but not wet) soil, enhancement of root growth efficiency by phototropin-1 increased plant size at maturity. * Results indicate that phototropin-1 enhances performance under drought by mediating plastic increases in root growth efficiency near the soil surface.

Light-sensing in roots.

Light gradients in the soil have largely been overlooked in understanding plant responses to the environment. However, roots contain photoreceptors that may receive ambient light through the soil or piped light through the vascular cylinder. In recent experiments we demonstrated linkages between phototropin-1 photoreceptor production, root growth efficiency, and drought tolerance, suggesting that root plasticity in response to light signals contributes to the ecological niche of A. thaliana. However, the availability of light cues in natural soil environments is poorly understood, raising questions about the relevance of light-mediated root growth for fitness in nature. Additionally, photoreceptor expression is characterized by pleiotropy so unique functions cannot be clearly ascribed to root vs. shoot sensory mechanisms. These considerations show that challenges exist for resolving the contribution of light-sensing by roots to plant adaptation. We suggest that blue-light sensing in roots of A. thaliana provides a model system for addressing these challenges. By calibrating blue light gradients in soils of diverse A. thaliana habitats and comparing fitness of phot1 mutant and wild-type controls when grown in presence or absence of soil light cues, it should be possible to elucidate the ecological significance of light-mediated plasticity in roots.

A gradient of auxin and auxin-dependent transcription precedes tropic growth responses.

Plants, although sessile, can reorient growth axes in response to changing environmental conditions. Phototropism and gravitropism represent adaptive growth responses induced by changes in light direction and growth axis orientation relative to gravitational direction, respectively. The nearly 80-year-old Cholodny-Went theory [Went, F. W. & Thimann, K. V. (1937) Phytohormones (Macmillan, New York)] predicts that formation of a gradient of the plant morphogen auxin is central to the establishment of tropic curvature. Loss of tropic responses in seedling stems of Arabidopsis thaliana mutants lacking the auxin-regulated transcriptional activator NPH4/ARF7 has further suggested that a gradient of gene expression represents an essential output from the auxin gradient. Yet the molecular identities of such output components, which are likely to encode proteins directly involved in growth control, have remained elusive. Here we report the discovery of a suite of tropic stimulus-induced genes in Brassica oleracea that are responsive to an auxin gradient and exhibit morphologically graded expression concomitant with, or before, observable curvature responses. These results provide compelling molecular support for the Cholodny-Went theory and suggest that morphologically graded transcription represents an important mechanism for interpreting tropically stimulated gradients of auxin. Intriguingly, two of the tropic stimulus-induced genes, EXPA1 and EXPA8, encode enzymes involved in cell wall extension, a response prerequisite for differential growth leading to curvatures, and are up-regulated before curvature in the flank that will elongate. This observation suggests that morphologically graded transcription likely leads to the graded expression of proteins whose activities can directly regulate the establishment and modulation of tropic curvatures.

PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism.

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1-PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.

MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana.

We have isolated a dominant, auxin-insensitive mutant of Arabidopsis thaliana, massugu2 (msg2), that displays neither hypocotyl gravitropism nor phototropism, fails to maintain an apical hook as an etiolated seedling, and is defective in lateral root formation. Yet other aspects of growth and development of msg2 plants are almost normal. These characteristics of msg2 are similar to those of another auxin-insensitive mutant, non-phototropic hypocotyl4 (nph4), which is a loss-of-function mutant of AUXIN RESPONSE FACTOR7 (ARF7) (Harper et al., 2000). Map-based cloning of the MSG2 locus reveals that all four mutant alleles result in amino acid substitutions in the conserved domain II of an Auxin/Indole-3-Acetic Acid protein, IAA19. Interestingly, auxin inducibility of MSG2/IAA19 gene expression is reduced by 65% in nph4/arf7. Moreover, MSG2/IAA19 protein binds to the C-terminal domain of NPH4/ARF7 in a Saccharomyces cerevisiae (yeast) two-hybrid assay and to the whole latter protein in vitro by pull-down assay. These results suggest that MSG2/IAA19 and NPH4/ARF7 may constitute a negative feedback loop to regulate differential growth responses of hypocotyls and lateral root formation.