Deetiolation Enhances Phototropism by Modulating NON-PHOTOTROPIC HYPOCOTYL3 Phosphorylation Status.

Phototropin (phot) receptor kinases play important roles in promoting plant growth by controlling light-capturing processes, such as phototropism. Phototropism is mediated through the action of NON-PHOTOTROPIC HYPOCOTYL3 (NPH3), which is dephosphorylated following phot activation. However, the functional significance of this early signaling event remains unclear. Here, we show that the onset of phototropism in dark-grown (etiolated) seedlings of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) is enhanced by greening (deetiolation). Red and blue light were equally effective in promoting phototropism in Arabidopsis, consistent with our observations that deetiolation by phytochrome or cryptochrome was sufficient to enhance phototropism. Increased responsiveness did not result from an enhanced sensitivity to the phytohormone auxin, nor does it involve the phot-interacting protein, ROOT PHOTOTROPISM2. Instead, deetiolated seedlings showed attenuated levels of NPH3 dephosphorylation and diminished relocalization of NPH3 from the plasma membrane during phototropism. Likewise, etiolated seedlings that lack the PHYTOCHROME-INTERACTING FACTORS (PIFs) PIF1, PIF3, PIF4, and PIF5 displayed reduced NPH3 dephosphorylation and enhanced phototropism, consistent with their constitutive photomorphogenic phenotype in darkness. Phototropic enhancement could also be achieved in etiolated seedlings by lowering the light intensity to diminish NPH3 dephosphorylation. Thus, phototropism is enhanced following deetiolation through the modulation of a phosphorylation rheostat, which in turn sustains the activity of NPH3. We propose that this dynamic mode of regulation enables young seedlings to maximize their establishment under changing light conditions, depending on their photoautotrophic capacity.

PIF4 and PIF5 transcription factors link blue light and auxin to regulate the phototropic response in Arabidopsis.

Both blue light (BL) and auxin are essential for phototropism in Arabidopsis thaliana. However, the mechanisms by which light is molecularly linked to auxin during phototropism remain elusive. Here, we report that phytochrome interacting factoR4 (PIF4) and PIF5 act downstream of the BL sensor phototropin1 (PHOT1) to negatively modulate phototropism in Arabidopsis. We also reveal that PIF4 and PIF5 negatively regulate auxin signaling. Furthermore, we demonstrate that PIF4 directly activates the expression of the auxin/indole-3-acetic acid (IAA) genes IAA19 and IAA29 by binding to the G-box (CACGTG) motifs in their promoters. Our genetic assays demonstrate that IAA19 and IAA29, which physically interact with auxin response factor7 (ARF7), are sufficient for PIF4 to negatively regulate auxin signaling and phototropism. This study identifies a key step of phototropic signaling in Arabidopsis by showing that PIF4 and PIF5 link light and auxin.

Mutants of phospholipase A (pPLA-I) have a red light and auxin phenotype.

pPLA-I is the evolutionarily oldest patatin-related phospholipase A (pPLA) in plants, which have previously been implicated to function in auxin and defence signalling. Molecular and physiological analysis of two allelic null mutants for pPLA-I [ppla-I-1 in Wassilewskija (Ws) and ppla-I-3 in Columbia (Col) ] revealed pPLA-I functions in auxin and light signalling. The enzyme is localized in the cytosol and to membranes. After auxin application expression of early auxin-induced genes is significantly slower compared with wild type and both alleles show a slower gravitropic response of hypocotyls, indicating compromised auxin signalling. Additionally, phytochrome-modulated responses like abrogation of gravitropism, enhancement of phototropism and growth in far red-enriched light are decreased in both alleles. While early flowering, root coils and delayed phototropism are only observed in the Ws mutant devoid of phyD, the light-related phenotypes observed in both alleles point to an involvement of pPLA-I in phytochrome signalling.

Arabidopsis thaliana life without phytochromes.

Plants use light as a source of energy for photosynthesis and as a source of environmental information perceived by photoreceptors. Testing whether plants can complete their cycle if light provides energy but no information about the environment requires a plant devoid of phytochromes because all photosynthetically active wavelengths activate phytochromes. Producing such a quintuple mutant of Arabidopsis thaliana has been challenging, but we were able to obtain it in the flowering locus T (ft) mutant background. The quintuple phytochrome mutant does not germinate in the FT background, but it germinates to some extent in the ft background. If germination problems are bypassed by the addition of gibberellins, the seedlings of the quintuple phytochrome mutant exposed to red light produce chlorophyll, indicating that phytochromes are not the sole red-light photoreceptors, but they become developmentally arrested shortly after the cotyledon stage. Blue light bypasses this blockage, rejecting the long-standing idea that the blue-light receptors cryptochromes cannot operate without phytochromes. After growth under white light, returning the quintuple phytochrome mutant to red light resulted in rapid senescence of already expanded leaves and severely impaired expansion of new leaves. We conclude that Arabidopsis development is stalled at several points in the presence of light suitable for photosynthesis but providing no photomorphogenic signal.

The involvement of a protein kinase in phototaxis and gravitaxis of Euglena gracilis.

The unicellular flagellate Euglena gracilis shows positive phototaxis at low-light intensities (<10 W/m(2)) and a negative one at higher irradiances (>10 W/m(2)). Phototaxis is based on blue light-activated adenylyl cyclases, which produce cAMP upon irradiation. In the absence of light the cells swim upward in the water column (negative gravitaxis). The results of sounding rocket campaigns and of a large number of ground experiments led to the following model of signal perception and transduction in gravitaxis of E. gracilis: The body of the cell is heavier than the surrounding medium, sediments and thereby exerts a force onto the lower membrane. Upon deviation from a vertical swimming path mechano-sensitive ion channels are activated. Calcium is gated inwards which leads to an increase in the intracellular calcium concentration and causes a change of the membrane potential. After influx, calcium activates one of several calmodulins found in Euglena, which in turn activates an adenylyl cyclase (different from the one involved in phototaxis) to produce cAMP from ATP. One further element in the sensory transduction chain of both phototaxis and gravitaxis is a specific protein kinase A. We found five different protein kinases A in E. gracilis. The blockage of only one of these (PK.4, accession No. EU935859) by means of RNAi inhibited both phototaxis and gravitaxis, while inhibition of the other four affected neither phototaxis nor gravitaxis. It is assumed that cAMP directly activates this protein kinase A which may in turn phosphorylate a protein involved in the flagellar beating mechanism.

The ABC subfamily B auxin transporter AtABCB19 is involved in the inhibitory effects of N-1-naphthyphthalamic acid on the phototropic and gravitropic responses of Arabidopsis hypocotyls.

N-1-Naphthylphthalamic acid (NPA) causes the abnormal growth and development of plants by suppressing polar auxin transport. The mechanisms underlying this inhibition, however, have remained elusive. In Arabidopsis, we show that a defect in the ABC subfamily B auxin transporter AtABCB19 suppresses the inhibitory effects of NPA on hypocotyl phototropism and gravitropism, but not on hypocotyl elongation. Expression analysis using the auxin reporter gene DR5:GUS further suggests that NPA partially inhibits the asymmetric distribution of auxin in an AtABCB19-dependent manner. These data thus suggest that AtABCB19 plays an important role in the inhibitory effects of NPA on hypocotyl tropism induced by auxin.

Induction of beta-glucosidase activity in maize coleoptiles by blue light illumination.

The role of beta-glucosidase during the phototropic response in maize (Zea mays) coleoptiles was investigated. Unilateral blue light illumination abruptly up-regulated the activity of beta-glucosidase in the illuminated halves, 10 min after the onset of illumination, peaking after 30 min and decreasing thereafter. The level of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which is released from DIMBOA glucoside (DIMBOA-Glc) by beta-glucosidase, and its degradation compound 6-methoxy-benzoxazolinone (MBOA) were elevated within 30 min in the illuminated halves as compare to the shaded halves, prior to the phototropic curvature. Furthermore, beta-glucosidase inhibitor treatment significantly decreased the phototropic curvature and decreased growth suppression in the illuminated sides. These results suggest that blue light induces the activity of beta-glucosidase in the illuminated halves of coleoptiles causing an increase in DIMBOA biosynthesis and the growth inhibition that leads to a phototropic curvature.

Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize.

Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

Phototrophic cultivation of a thermo-tolerant Desmodesmus sp. for lutein production: effects of nitrate concentration, light intensity and fed-batch operation.

Four indigenous thermo-tolerant Desmodesmus sp. strains were examined for their ability to produce lutein. Among them, Desmodesmus sp. F51 was the best strain for this purpose. The medium composition, nitrate concentration and light intensity were manipulated to improve the phototrophic growth and lutein production of Desmodesmus sp. F51. It was found that a nitrogen-sufficient condition was required for lutein accumulation, while a high light intensity enhanced cell growth but caused a decrease in the lutein content. The best cell growth and lutein production occurred when the light intensity and initial nitrate concentration were 600 µmol/m(2)/s and 8.8 mM, respectively. The fed-batch cultivation strategy was shown to further improve lutein production. The highest lutein productivity (3.56±0.10 mg/L/d) and content (5.05±0.20 mg/g) were obtained when pulse-feeding of 2.2 mM nitrate was employed. This study demonstrated the potential of using Desmodesmus sp. F51 as a lutein producer in practical applications.

Glutamatergic elements in an excitability and circumnutation mechanism.

In plants, an electrical potential and circumnutation disturbances are a part of a response to environmental and internal stimuli. Precise relations between electrical potential changes and circumnutation mechanisms are unclear. We have found recently that glutamate (Glu) injection into Helianthus annuus stem induced a series of action potentials (APs) and a transient decrease in circumnutation activity. A theoretical explanation for this finding is discussed here taking into considerations data about the ion mechanism of AP and circumnutation as well as about the metabolic and signaling pathways of glutamate and their possible interactions.

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.

A brassinosteroid-hypersensitive mutant of BAK1 indicates that a convergence of photomorphogenic and hormonal signaling modulates phototropism.

The phototropic response of Arabidopsis (Arabidopsis thaliana) is induced by the phototropin photoreceptors and modulated by the cryptochrome and phytochrome photoreceptors. Downstream of these photoreceptors, asymmetric lateral redistribution of auxin underlies the differential growth, which results in phototropism. Historical physiological evidence and recent analysis of hormone-induced gene expression demonstrate that auxin and brassinosteroid signaling function interdependently. Similarly, in this study we report evidence that interactions between brassinosteroids and auxin signaling modulate phototropic responsiveness. We found that elongated, a previously identified photomorphogenesis mutant, enhances high-light phototropism and represents a unique allele of BAK1/SERK3, a receptor kinase implicated in brassinosteroid perception. Altogether, our results support the hypothesis that phototropic responsiveness is modulated by inputs that influence control of auxin response factor-mediated transcription.

Characterization of the eyespot regions of "blind" Chlamydomonas mutants after restoration of photophobic responses.

Chlamydomonas reinhardtii exhibits photophobic and positive and negative phototactic responses that can be defined for cell populations using computerized cell tracking and motion analysis. Mutants CC-2359 and FN68 are pigment deficient mutants that are blocked in carotenoid synthesis and lack these photo responses. In particular, neither mutant exhibits flash-induced photophobic responses to visible light stimuli to which wild-type gametic cells exhibit a strong response, with several behavioral stages. Upon addition of all-trans retinal to these mutants, the photophobic responses are restored with minor quantitative differences from wild-type populations. Using both light and electron microscopy, we have compared the ultrastructural characteristics of wild-type C. reinhardtii to those of both mutants. As previously described, wild-type cells contain an eyespot consisting of 2-4 layers of pigmented granules encased within thylakoid membranes, located between the distal extremities of the flagellar root. This structure is also visible as an orange-red spot in light microscopy. The photoreceptor is thought to be concentrated in the plasma membrane above the eyespot. The mutant, CC-2359, lacks this eyespot as seen by both light and electron microscopy, even when the photophobic response has been restored. FN68-like mutants studied earlier by Morel-Laurens and Feinlieb and others contain an eyespot which can be seen only by electron microscopy. In FN-68, the eyespot generally has the same dimensions as in wt cells, differing mainly in pigment granule appearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of phototropic responsiveness in decapitated maize coleoptiles.

The literature indicates that the tip of maize (Zea mays L.) coleoptiles has the localized functions of producing auxin for growth and perceiving unilateral light stimuli and translocating auxin laterally for phototropism. There is evidence that the auxinproducing function of the tip is restored in decapitated coleoptiles. We examined whether the functions for phototropism are also restored by using blue-light conditions that induced a first pulse-induced positive phototropism (fPIPP) and a time-dependent phototropism (TDP). When the apical 5 mm, in which photosensing predominantly takes place, was removed, no detectable fPIPP occurred even if indole-3-acetic acid (lanolin mixture) was applied to the cut end. However, when the blue-light stimulation was delayed after decapitation, fPIPP became inducible in the coleoptile stumps supplied with indole-3-acetic-acid/lanolin (0.01 mg g-1), indicating that phototropic responsiveness was restored. This restoration progressed 1 to 2 h after decapitation, and the curvature response became comparable to that of intact coleoptiles. The results for TDP were qualitatively similar, but some quantitative differences were observed. It appeared that the overall TDP was based on a major photosensing mechanism specific to the tip and on at least one additional mechanism not specific to the tip, and that the tip-specific TDP was restored in decapitated coleoptiles with kinetics similar to that for fPIPP. It is suggested that the photoreceptor system, which accounts for fPIPP and a substantial part of TDP, is regenerated in decapitated coleoptiles, perhaps together with the mechanism for lateral auxin translocation.

Evidence that zeaxanthin is not the photoreceptor for phototropism in maize coleoptiles.

The photoreceptor that mediates blue-light-induced phototropism in dark-grown seedlings of higher plants has not been identified, although the carotenoid zeaxanthin has recently been proposed as the putative chromophore. In the experiments described in this paper, we analyzed phototropism and a blue-light-induced protein phosphorylation that has been genetically and physiologically implicated in phototropism in wild-type maize (Zea mays L.) seedlings and compared the results with those from seedlings that are either carotenoid deficient through a genetic lesion or have been chemically treated to block carotenoid biosynthesis. The blue-light-dependent phototropism and phosphorylation responses of seedlings deficient in carotenoids are the same as those of seedlings containing normal levels of carotenoids. These results and those in the literature make it unlikely that zeaxanthin or any other carotenoid is the chromophore of the blue-light photoreceptor for phototropism or the blue-light-induced phosphorylation related to phototropism.

Changes in blue-light-dependent protein phosphorylation during the early development of etiolated oat seedlings.

Blue light induced the phosphorylation of a 116-kDa plasma-membrane-associated protein in dark-grown seedlings from Avena sativa L. The response was restricted to the phototropically sensitive tissue of the coleoptile tip. Surprisingly, this protein showed different properties in membrane preparations from plants that were grown for 3 d than in those from 5-d-old seedlings. In contrast to the younger coleoptiles, in 5-d-old seedlings phosphorylation of the 116-kDa protein depended strictly on the addition of Triton X-100 or other non-ionic detergents and was not abolished when the membranes were pretreated with trypsin. These latter membranes were also characterized by the appearance of two additional blue-light-regulated phosphoproteins of slightly lower molecular masses, exhibiting properties similar to the 116-kDa protein from 3-d-old plants. The data, together with solubilization studies, indicate that the 116-kDa protein is strongly membrane-bound only at the very beginning of seedling development and becomes more loosely associated in the course of coleoptile growth. In addition, we demonstrate that the capacity of the light-activated photoreceptor to recover photosensitivity in the dark also can occur under in-vitro conditions.

Mutation in domain II of IAA1 confers diverse auxin-related phenotypes and represses auxin-activated expression of Aux/IAA genes in steroid regulator-inducible system.

Most of Aux/IAA genes are rapidly induced by auxin. The Aux/IAA proteins are short-lived nuclear proteins sharing the four conserved domains. Domain II is critical for rapid degradation of Aux/IAA proteins. Among these gene family members, IAA1 is one of the earliest auxin-inducible genes. We used a steroid hormone-inducible system to reveal putative roles and downstream signaling of IAA1 in auxin response. Arabidopsis transgenic plants were generated expressing fusion protein of IAA1 (IAA1-GR) or IAA1 with a mutation in domain II (iaa1-GR) and the glucocorticoid hormone-binding domain (GR). IAA1-GR transgenic plants did not exhibit any discernable phenotypic differences by DEX treatment that allows nuclear translocation of the fusion protein. In contrast, diverse auxin-related physiological processes including gravitropism and phototropism were impaired by DEX treatment in roots, hypocotyls, stems, and leaves in iaa1-GR transgenic plants. Auxin induction of seven Aux/IAA mRNAs including IAA1 itself was repressed by DEX treatment, suggesting that IAA1 functions in the nucleus by mediating auxin response and might act as a negative feedback regulator for the expression of Aux/IAA genes including IAA1 itself. Auxin induction of Aux/IAA genes in the presence of cycloheximide can be repressed by DEX treatment, showing that the repression of transcription of the Aux/IAAs by the iaa1 mutant protein is primary. Wild-type IAA1-GR could not suppress auxin induction of IAA1 and IAA2. These results indicate that inhibition of auxin-activated transcription of Aux/IAA genes by the iaa1 mutant protein might be responsible for alteration of various auxin responses.

Mutations in the huge Arabidopsis gene BIG affect a range of hormone and light responses.

In independent genetic screens, for shade-avoidance response and cytokinin sensitivity, we identified two Arabidopsis mutants, attenuated shade avoidance 1 (asa1) and umbrella1 (umb1), which have very similar pleiotropic phenotypes. asa1 and umb1 are allelic to tir3-1, and are caused by mutations in BIG, which is required for normal auxin efflux. They have a compact rosette, fewer lateral roots, delayed flowering, more secondary inflorescence, smaller seeds and, in the Laer-0 background, much shorter internodes between adjacent flowers, suggesting an interaction between BIG and ERECTA. These mutants have organ-specific defects in response to cytokinins, ethylene, N-1-naphthylphthalamic acid (NPA) and gibberellin (GA). The phenotype of the asa1 ga1-3 double mutant is consistent with defects in GA signalling. There are subtle effects in responses to auxins, abscisic acid and brassinolide. Elongation growth associated with shade avoidance in phyA phyB null mutants is suppressed by asa1 in all organs other than the hypocotyl. Therefore, we here provide evidence that BIG is a key player not just in auxin signalling, but in a multitude of light and hormone pathways.