The asymmetric expression of plasma membrane H+-ATPase family genes in response to pulvinus-driven leaf phototropism movement in Vitis vinifera.

Phototropism movement is crucial for plants to adapt to various environmental changes. Plant P-type H+-ATPase (HA) plays diverse roles in signal transduction during cell expansion, regulation of cellular osmotic potential and stomatal opening, and circadian movement. Despite numerous studies on the genome-wide analysis of Vitis vinifera, no research has been done on the P-type H+-ATPase family genes, especially concerning pulvinus-driven leaf movement. In this study, 55 VvHAs were identified and classified into nine distinct subgroups (1 to 9). Gene members within the same subgroups exhibit similar features in motif, intron/exon, and protein tertiary structures. Furthermore, four pairs of genes were derived by segmental duplication in grapes. Cis-acting element analysis identified numerous light/circadian-related elements in the promoters of VvHAs. qRT-PCR analysis showed that several genes of subgroup 7 were highly expressed in leaves and pulvinus during leaf movement, especially VvHA14, VvHA15, VvHA16, VvHA19, VvHA51, VvHA52, and VvHA54. Additionally, we also found that the VvHAs genes were asymmetrically expressed on both sides of the extensor and flexor cell of the motor organ, the pulvinus. The expression of VvHAs family genes in extensor cells was significantly higher than that in flexor cells. Overall, this study serves as a foundation for further investigations into the functions of VvHAs and contributes to the complex mechanisms underlying grapevine pulvinus growth and development.

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.

A high concentration of abscisic acid inhibits hypocotyl phototropism in Gossypium arboreum by reducing accumulation and asymmetric distribution of auxin.

Hypocotyl phototropism is mediated by the phototropins and plays a critical role in seedling morphogenesis by optimizing growth orientation. However, the mechanisms by which phototropism influences morphogenesis require additional study, especially for polyploid crops such as cotton. Here, we found that hypocotyl phototropism was weaker in Gossypium arboreum than in G. raimondii (two diploid cotton species), and LC-MS analysis indicated that G. arboreum hypocotyls had a higher content of abscisic acid (ABA) and a lower content of indole-3-acetic acid (IAA) and bioactive gibberellins (GAs). Consistently, the expression of ABA2, AAO3, and GA2OX1 was higher in G. arboreum than in G. raimondii, and that of GA3OX was lower; these changes promoted ABA synthesis and the transformation of active GA to inactive GA. Higher concentrations of ABA inhibited the asymmetric distribution of IAA across the hypocotyl and blocked the phototropic curvature of G. raimondii. Application of IAA or GA3 to the shaded and illuminated sides of the hypocotyl enhanced and inhibited phototropic curvature, respectively, in G. arboreum. The application of IAA, but not GA, to one side of the hypocotyl caused hypocotyl curvature in the dark. These results indicate that the asymmetric distribution of IAA promotes phototropic growth, and the weakened phototropic curvature of G. arboreum may be attributed to its higher ABA concentrations that inhibit the action of auxin, which is regulated by GA signaling.

The JA-pathway MYC transcription factors regulate photomorphogenic responses by targeting HY5 gene expression.

Jasmonates are key regulators of the balance between defence and growth in plants. However, the molecular mechanisms by which activation of defence reduces growth are not yet fully understood. Here, we analyze the role of MYC transcription factors (TFs) and jasmonic acid (JA) in photomorphogenic growth. We found that multiple myc mutants share light-associated phenotypes with mutants of the phytochrome B photoreceptor, such as delayed seed germination in the dark and long hypocotyl growth. Overexpression of MYC2 in a phyB background partially suppressed its long hypocotyl phenotype. Transcriptomic analysis of multiple myc mutants confirmed that MYCs are required for full expression of red (R) light-regulated genes, including the master regulator HY5. ChIP-seq analyses revealed that MYC2 and MYC3 bind directly to the promoter of HY5 and that HY5 gene expression and protein levels are compromised in multiple myc mutants. Altogether, our results pinpoint MYCs as photomorphogenic TFs that control phytochrome responses by activating HY5 expression. This has important implications in understanding the trade-off between growth and defence as the same TFs that activate defence responses are photomorphogenic growth regulators.

Determination of Phototropism by UV-B Radiation.

UV-B phototropism in etiolated Arabidopsis seedlings has only been shown recently and needs further exploration. Here we elaborate on how to generate a customized setup with a unilateral UV-B light source, the required plant materials, different growth substrates, and a framework for data analysis.

Attractiveness of light-emitting diodes (LEDs) of different wavelengths to the South American rice water weevil / Atratividade de diodos emissores de luz (LEDs) de diferentes comprimentos de onda aos adultos da bicheira-da-raiz

The South American rice water weevil Oryzophagus oryzae (Costa Lima) (Coleoptera: Curculionidae) is a key pest of irrigated rice in Brazil. Light traps could be used as an integrated pest management (IPM) strategy since these weevils have strong phototropism. Thus, the aim of this study was to search the attractiveness of LEDs of different wavelengths to O. oryzae to define the best light-emitting diodes (LED) arrangement to build the lamp of photovoltaic light traps. LEDs of different wavelengths were tested in a dark room with a hexagonal arena with exits to light sources. Groups of 200 insects were released in the center of the arena and captured at the end of exposure time in plastic bags placed in the exits. The LEDs in the short-wave band of the light spectrum (365 and 460 nm) and white LEDs, as well as the light mix of 365 with 460 nm or white LEDs, were attractive to O. oryzae. Beyond the LEDs with ultraviolet (UV) spectrum, the LEDs above 380 nm were the most attractive and, this way, might be used to build LED lamps of photovoltaic light traps.(AU)

Uma das pragas-chave do arroz irrigado é a bicheira-da-raiz, denominação comum atribuída às larvas do gorgulho aquático Oryzophagus oryzae (Costa Lima) (Coleoptera: Curculionidae). A utilização de armadilhas luminosas poderia ser uma alternativa viável no manejo integrado, pois esse gorgulho apresenta elevado fototropismo positivo. Assim, objetivou-se determinar a atratividade de diodos emissores de luz (LEDs) de diferentes comprimentos de onda ao O. oryzae e definir o seu melhor arranjo para compor a lâmpada de armadilhas luminosas fotovoltaicas. LEDs de diferentes comprimentos de onda foram testados em sala escura, em uma arena hexagonal com saídas para as fontes de luz. Grupos de 200 indivíduos foram liberados no centro da arena e capturados em sacos plásticos nas saídas ao fim do tempo de exposição. Os LEDs da faixa do espectro luminoso de ondas curtas (365 e 460 nm) e LEDs brancos, bem como as misturas de luzes de LEDs 365 com 460 nm ou branco, foram atrativos ao O. oryzae. Entre os LEDs com espectro ultravioleta, os mais atrativos foram aqueles acima de 380 nm, devendo estes compor, prioritariamente, lâmpadas de LED para uso nas armadilhas luminosas fotovoltaicas.(AU)

Developmental reprogramming by UV-B radiation in plants.

Plants are extremely plastic organisms with the ability to adapt and respond to the changing environmental conditions surrounding them. Sunlight is one of the main resources for plants, both as a primary energy source for photosynthesis and as a stimulus that regulates different aspects of their growth and development. UV-B comprises wavelengths that correspond to a high energy region of the solar spectrum capable of reaching the biosphere, influencing plant growth. It is currently believed that plants are able to acclimate when growing under the influence of this radiation and perceive it as a signal, without stress signs. Nonetheless, many UV-B induced changes are elicited after DNA damage occurs as a consequence of exposure. In this review we focus on the influence of UV-B on leaf, flower and root development and emphasize the limited understanding of the molecular mechanisms for most of this developmental processes affected by UV-B documented over the years of research in this area.

Arabidopsis phot1 and phot2 phosphorylate BLUS1 kinase with different efficiencies in stomatal opening.

In Arabidopsis thaliana, phototropins (phot1 and phot2), light-activated receptor kinases, redundantly regulate various photoresponses such as phototropism, chloroplast photorelocation movement, stomatal opening, and leaf flattening. However, it is still unclear how phot1 and phot2 signals are integrated into a common target and regulate physiological responses. In the present study, we provide evidence that phot1 and phot2 phosphorylate BLUE LIGHT SIGNALING1 (BLUS1) kinase as a common substrate in stomatal opening. Biochemical analysis revealed that the recombinant phot2 protein directly phosphorylated BLUS1 in vitro in a blue light-dependent manner, as reported for phot1. BLUS1 phosphorylation was observed in both phot1 and phot2 mutants, and phot2 mutant exhibited higher phosphorylation of BLUS1 than did phot1 mutant. Transgenic plants expressing phot1-GFP (P1G) and phot2-GFP (P2G) at a similar level under the PHOT2 promoter demonstrated that P1G initiated higher phosphorylation of BLUS1 than P2G, suggesting that phot1 phosphorylates BLUS1 more efficiently. Similarly, P1G mediated a higher activation of the plasma membrane H(+)-ATPase and stomatal opening than P2G, indicating that the phosphorylation status of BLUS1 is a key determinant of physiological response. Together, these findings provide insights into the signal integration and different properties of phot1 and phot2 signaling.

A phytochrome/phototropin chimeric photoreceptor of fern functions as a blue/far-red light-dependent photoreceptor for phototropism in Arabidopsis.

In the fern Adiantum capillus-veneris, the phototropic response of the protonemal cells is induced by blue light and partially inhibited by subsequent irradiation with far-red light. This observation strongly suggests the existence of a phytochrome that mediates this blue/far-red reversible response; however, the phytochrome responsible for this response has not been identified. PHY3/NEO1, one of the three phytochrome genes identified in Adiantum, encodes a chimeric photoreceptor composed of both a phytochrome and a phototropin domain. It was demonstrated that phy3 mediates the red light-dependent phototropic response of Adiantum, and that phy3 potentially functions as a phototropin. These findings suggest that phy3 is the phytochrome that mediates the blue/far-red response in Adiantum protonemata. In the present study, we expressed Adiantum phy3 in a phot1 phot2 phototropin-deficient Arabidopsis line, and investigated the ability of phy3 to induce phototropic responses under various light conditions. Blue light irradiation clearly induced a phototropic response in the phy3-expressing transgenic seedlings, and this effect was fully inhibited by simultaneous irradiation with far-red light. In addition, experiments using amino acid-substituted phy3 indicated that FMN-cysteinyl adduct formation in the light, oxygen, voltage (LOV) domain was not necessary for the induction of blue light-dependent phototropism by phy3. We thus demonstrate that phy3 is the phytochrome that mediates the blue/far-red reversible phototropic response in Adiantum. Furthermore, our results imply that phy3 can function as a phototropin, but that it acts principally as a phytochrome that mediates both the red/far-red and blue/far-red light responses.

Plasma membrane H⁺ -ATPase regulation is required for auxin gradient formation preceding phototropic growth.

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H⁺ -ATPases that are required to control apoplastic pH. Our results show that H⁺ -ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H⁺ -ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH.

A mechanism for reorientation of cortical microtubule arrays driven by microtubule severing.

Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

Analysis of phenotypic evolution in Dictyostelia highlights developmental plasticity as a likely consequence of colonial multicellularity.

Colony formation was the first step towards evolution of multicellularity in many macroscopic organisms. Dictyostelid social amoebas have used this strategy for over 600 Myr to form fruiting structures of increasing complexity. To understand in which order multicellular complexity evolved, we measured 24 phenotypic characters over 99 dictyostelid species. Using phylogenetic comparative methods, we show that the last common ancestor (LCA) of Dictyostelia probably erected small fruiting structures directly from aggregates. It secreted cAMP to coordinate fruiting body morphogenesis, and another compound to mediate aggregation. This phenotype persisted up to the LCAs of three of the four major groups of Dictyostelia. The group 4 LCA co-opted cAMP for aggregation and evolved much larger fruiting structures. However, it lost encystation, the survival strategy of solitary amoebas that is retained by many species in groups 1-3. Large structures, phototropism and a migrating intermediate 'slug' stage coevolved as evolutionary novelties within most groups. Overall, dictyostelids show considerable plasticity in the size and shape of multicellular structures, both within and between species. This probably reflects constraints placed by colonial life on developmental control mechanisms, which, depending on local cell density, need to direct from 10 to a million cells into forming a functional fructification.

Disruption of ROOT PHOTOTROPISM2 gene does not affect phototropin-mediated stomatal opening.

Phototropins (phot1 and phot2), blue light-receptor protein kinases in plants, mediate stomatal opening by activating the plasma membrane H(+)-ATPase in guard cells, but the signaling from phototropins to the H(+)-ATPase remains unknown. A recent study concluded that ROOT PHOTOTROPISM2 (RPT2) is involved in the primary step of this process. However, this conclusion is based solely on the determination of stomatal apertures in the epidermis. We investigated the role of RPT2 in blue light-dependent stomatal opening in more detail. We generated double mutants of rpt2 and phototropins (phot1 or phot2) in the Col ecotype background and obtained the typical phenotypes of rpt2 mutants, including the impairment in phototropism. In contrast, neither blue light-dependent H(+) pumping nor blue light-dependent H(+)-ATPase activation in guard cells was affected in the rpt2 mutants of rpt2, phot1 rpt2, and phot2 rpt2. Stomata in these rpt2 mutants opened widely by blue light in both epidermal peels and intact leaves, and no difference in the responses was found between the wild type and the mutants. From these results, we concluded that RPT2 gene disruption does not affect blue light-dependent stomatal opening.

Photosensitivity of kinase activation by blue light involves the lifetime of a cysteinyl-flavin adduct intermediate, S390, in the photoreaction cycle of the LOV2 domain in phototropin, a plant blue light receptor.

Phototropin (phot) is a light-regulated protein kinase that mediates a variety of photoresponses in plants, such as phototropism, chloroplast positioning, and stomata opening. Arabidopsis has two homologues, phot1 and phot2, that share physiological functions depending on light intensity. A phot molecule has two photoreceptive light oxygen voltage-sensing domains, LOV1 and LOV2, and a Ser/Thr kinase domain. The LOV domains undergo a photocycle upon blue light (BL) stimulation, including transient adduct formation between the chromophore and a conserved cysteine (S390 intermediate) that leads to activation of the kinase. To uncover the mechanism underlying the photoactivation of the kinase, we have introduced a kinase assay system composed of a phot1 LOV2-linker-kinase polypeptide as a light-regulated kinase and its N-terminal polypeptide as an artificial substrate (Okajima, K., Matsuoka, D., and Tokutomi, S. (2011) LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1. FEBS Lett. 585, 3391-3395). In the present study, we extended the assay system to phot2 and compared the photochemistry and kinase activation by BL between phot1 and phot2 to gain insight into the molecular basis for the different photosensitivities of phot1 and phot2. Photosensitivity of kinase activation by BL and the lifetime of S390 of phot1 were 10 times higher and longer, respectively, than those of phot2. This correlation was confirmed by an amino acid substitution experiment with phot1 to shorten the lifetime of S390. The present results demonstrated that the photosensitivity of kinase activation in phot involves the lifetime of S390 in LOV2, suggesting that the lifetime is one of the key factors for the different photosensitivities observed for phot1 and phot2.

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.

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.

Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling.

Musa (banana and plantain) is an important genus for the global export market and in local markets where it provides staple food for approximately 400 million people. Hybridization and polyploidization of several (sub)species, combined with vegetative propagation and human selection have produced a complex genetic history. We describe the application of the Ecotilling method for the discovery and characterization of nucleotide polymorphisms in diploid and polyploid accessions of Musa. We discovered over 800 novel alleles in 80 accessions. Sequencing and band evaluation shows Ecotilling to be a robust and accurate platform for the discovery of polymorphisms in homologous and homeologous gene targets. In the process of validating the method, we identified two single nucleotide polymorphisms that may be deleterious for the function of a gene putatively important for phototropism. Evaluation of heterozygous polymorphism and haplotype blocks revealed a high level of nucleotide diversity in Musa accessions. We further applied a strategy for the simultaneous discovery of heterozygous and homozygous polymorphisms in diploid accessions to rapidly evaluate nucleotide diversity in accessions of the same genome type. This strategy can be used to develop hypotheses for inheritance patterns of nucleotide polymorphisms within and between genome types. We conclude that Ecotilling is suitable for diversity studies in Musa, that it can be considered for functional genomics studies and as tool in selecting germplasm for traditional and mutation breeding approaches.

Impact of two different types of heat stress on chloroplast movement and fluorescence signal of tobacco leaves.

Although the chloroplast movement can be strongly affected by ambient temperature, the information about chloroplast movement especially related to high temperatures is scarce. For detailed investigation of the effects of heat stress (HS) on tobacco leaves (Nicotiana tabacum L. cv. Samsun), we used two different HS treatments in dark with wide range of elevated temperatures (25-45 degrees C). The leaf segments were either linearly heated in water bath at heating rate of 2 degrees C min(-1) from room temperature up to maximal temperature (T (m)) and then linearly cooled down to 25 degrees C or incubated for 5 min in water bath at the same T (m) followed by 5 min incubation at 25 degrees C (T-jump). The changes in light-induced chloroplast movement caused by the HS pretreatment were detected after the particular heating regime at 25 degrees C using a method of time-dependent collimated transmittance (CT) and compared with the chlorophyll O-J-I-P fluorescence rise (FLR) measurements. The inhibition of chloroplast movement started at about 40 degrees C while the fluorescence parameters responded generally at higher T (m). This difference in sensitivity of CT and FLR was higher for the T-jump than for the linear HS indicating importance of applied heating regime. A possible influence of chloroplast movement on the FLR measurement and a physiological role of the HS-impaired chloroplast movement are discussed.

An unexpected advantage of whiteness in horses: the most horsefly-proof horse has a depolarizing white coat.

White horses frequently suffer from malign skin cancer and visual deficiencies owing to their high sensitivity to the ultraviolet solar radiation. Furthermore, in the wild, white horses suffer a larger predation risk than dark individuals because they can more easily be detected. In spite of their greater vulnerability, white horses have been highly appreciated for centuries owing to their natural rarity. Here, we show that blood-sucking tabanid flies, known to transmit disease agents to mammals, are less attracted to white than dark horses. We also demonstrate that tabanids use reflected polarized light from the coat as a signal to find a host. The attraction of tabanids to mainly black and brown fur coats is explained by positive polarotaxis. As the host's colour determines its attractiveness to tabanids, this parameter has a strong influence on the parasite load of the host. Although we have studied only the tabanid-horse interaction, our results can probably be extrapolated to other host animals of polarotactic tabanids, as the reflection-polarization characteristics of the host's body surface are physically the same, and thus not species-dependent.