Phototropic Responses of Vaucheria geminata to Intermittent Blue Light Stimuli

Phototropic responses of a tip-growing coenocytic alga Vaucheria geminata to intermittent blue light pulses were analyzed. Curvatures caused by repeated light pulses separated by dark intervals of various lengths were as large as those obtained by continuous light of the same incident energy, unless the length of the dark interval exceeded a critical value of about 30 to 40 seconds. If the dark interval was longer than the critical length, bending no longer took place. Another response to intermittent irradiation was found. In a narrow range of light pulse and dark interval lengths, i.e. light pulses longer than 10 milliseconds and dark intervals shorter than a value ranging from 15 to 150 milliseconds, intermittent irradiation was more effective than continuous irradiation of the same total energy. In this region, the maximum response to repeated pulses was equal to the curvature caused by a continuous irradiation of the same total elapsed time. It is evident that in this condition the dark interval is not perceived and the alga responds as if it were in light. The results suggest that two different dark reactions may be involved in the phototropic response of this algal cell.     

Adaptation to High-Intensity, Low-Wavelength Light among Surface Blooms of the Cyanobacterium Microcystis aeruginosa

Natural populations of the nuisance bloom cyanobacterium Microcystis aeruginosa obtained from the eutrophic Neuse River, N.C., revealed optimal chlorophyll a-normalized photosynthetic rates and resistance to photoinhibition at surface photosynthetically active radiation (PAR) intensities. At saturating PAR levels these populations exhibited higher photosynthetic rates in quartz than in Pyrex vessels. Eucaryotic algal populations obtained from the same river failed to counteract photoinhibition. At saturating PAR levels, such populations generally yielded lower photosynthetic rates in quartz containers than they did in Pyrex containers. Cultivation of natural Microcystis populations under laboratory conditions led to physiologically distinct populations which had photoinhibitory characteristics similar to those of other cultured cyanobacterial and eucaryotic algae. Our findings indicate that (i) photosynthetic production among natural surface populations is best characterized and quantified in quartz rather than Pyrex incubation vessels; (ii) extrapolation of natural photoinhibitory trends from laboratory populations is highly subjective to culture and PAR histories and may yield contradictory results; and (iii) buoyant surface-dwelling populations, rather than exhibiting senescence, are poised at optimizing PAR utilization, thereby maintaining numerical dominance in eutrophic waters when physico-chemical conditions favor bloom formation.     

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.     

Phototropic Response to Vectorial Light in Leaves of Lavatera cretica L

The mechanism by which the mature leaf of certain plants reorients its lamina to face the sun throughout the day was studied in Lavatera cretica L. The photoreceptor for this response differs fundamentally from the one involved in the phototropic growth response, by sensing light as a vector, rather than as a difference in luminous flux. The photoreceptor is located in the veins, which radiate in the plane of the lamina from the pulvinus situated at the junction between the lamina and petiole. The integrated response to the messages from the different veins takes place by differential turgor changes in a motor tissue surrounding the central vascular cylinder of the pulvinus, in which the veins coalesce. The differential turgor in the different segments of the motor tissue determines the orientation of the lamina. The photoreceptor reacts only to a parallel light beam striking the vein obliquely (from above). When half of the lamina is shaded, the leaf does not reorient in response to perpendicular illumination and its reorientation in response to an oblique beam is slower and partial, to a greater extend when the half-leaf is centrifugally illuminated than when it is centripetally illuminated. Application of 2,3,5 tri-iodobenzoic acid to the base of the veins in the shaded half-leaf eliminated all restrictions from the response to centrifugal illumination and totally inhibited the response to centripetal illumination. The results are consistent with a hypothesis that centrifugally illuminated veins generate turgor in their associated motor tissue in the pulvinus by activating K⁺ uptake, while centripetally illuminated veins cause loss of turgor in their associated motor tissue by deactivating K⁺ uptake, which leads to passive leakage of K⁺. When the entire lamina is exposed to oblique illumination, the centrifugally illuminated half and the centripetally illuminated half cooperate in the full response. Shaded parts of the lamina apparently interfere with the response by supplying their associated motor tissue with auxin, which presumably causes in it an active export of protons and concomitant uptake of K⁺, thereby establishing a static “dark turgor” in it.     

Retromer Contributes to Immunity-Associated Cell Death in Arabidopsis

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.     

A Kinetic Model for Adaptation and the Light Responses of Phycomyces

A kinetic model is described consisting of two sequential first order processes connected by two parallel reaction pathways, one of which is light-catalyzed. A change in light flux changes the rate constant of the light-dependent process, whereupon the levels of two chemical intermediaries readjust. The model's output duplicates all the main features of the cell's light-growth and dark-growth responses except their latent periods. An asymmetric modification of the model reproduces the two types of phototropic inversion discovered by Reichardt and Varjú and by Dennison. Simple exponential equations describe these responses of the model, as well as the theoretical course of its light and dark adaptation. It is concluded that adaptation in Phycomyces consist in the photocatalytic adjustment of the level of a metabolic reservoir.     

Phenotypic Plasticity Contributes to Maize Adaptation and Heterosis

Plant phenotypic plasticity describes altered phenotypic performance of an individual when grown in different environments. Exploring genetic architecture underlying plant plasticity variation may help mitigate the detrimental effects of a rapidly changing climate on agriculture, but little research has been done in this area to date. In the present study, we established a population of 976 maize F₁ hybrids by crossing 488 diverse inbred lines with two elite testers. Genome-wide association study identified hundreds of quantitative trait loci associated with phenotypic plasticity variation across diverse F₁ hybrids, the majority of which contributed very little variance, in accordance with the polygenic nature of these traits. We identified several quantitative trait locus regions that may have been selected during the tropical-temperate adaptation process. We also observed heterosis in terms of phenotypic plasticity, in addition to the traditional genetic value differences measured between hybrid and inbred lines, and the pattern of which was affected by genetic background. Our results demonstrate a landscape of phenotypic plasticity in maize, which will aid in the understanding of its genetic architecture, its contribution to adaptation and heterosis, and how it may be exploited for future maize breeding in a rapidly changing environment.     

Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves

Attaining defined steady-state carotenoid levels requires balancing of the rates governing their synthesis and metabolism. Phytoene formation mediated by phytoene synthase (PSY) is rate limiting in the biosynthesis of carotenoids, whereas carotenoid catabolism involves a multitude of nonenzymatic and enzymatic processes. We investigated carotenoid and apocarotenoid formation in Arabidopsis (Arabidopsis thaliana) in response to enhanced pathway flux upon PSY overexpression. This resulted in a dramatic accumulation of mainly β-carotene in roots and nongreen calli, whereas carotenoids remained unchanged in leaves. We show that, in chloroplasts, surplus PSY was partially soluble, localized in the stroma and, therefore, inactive, whereas the membrane-bound portion mediated a doubling of phytoene synthesis rates. Increased pathway flux was not compensated by enhanced generation of long-chain apocarotenals but resulted in higher levels of C₁₃ apocarotenoid glycosides (AGs). Using mutant lines deficient in carotenoid cleavage dioxygenases (CCDs), we identified CCD4 as being mainly responsible for the majority of AGs formed. Moreover, changed AG patterns in the carotene hydroxylase mutants lutein deficient1 (lut1) and lut5 exhibiting altered leaf carotenoids allowed us to define specific xanthophyll species as precursors for the apocarotenoid aglycons detected. In contrast to leaves, carotenoid hyperaccumulating roots contained higher levels of β-carotene-derived apocarotenals, whereas AGs were absent. These contrasting responses are associated with tissue-specific capacities to synthesize xanthophylls, which thus determine the modes of carotenoid accumulation and apocarotenoid formation.In plants, the synthesis of carotenoids is plastid localized, with the plastid type determining their function (Ruiz-Sola and Rodríguez-Concepción, 2012; Nisar et al., 2015). In nonphotosynthetic chromoplasts, carotenoids and their volatile derivatives attract pollinating insects or zoochoric animals. Here, carotenoids are sequestered in diverse suborganellar structures, which can be tubulous, globulous, membranous, or crystalline (Sitte et al., 1980; Egea et al., 2010). In chloroplasts, carotenoids are present in light-harvesting complex proteins and photosynthetic reaction centers. They extend the light spectrum used for photosynthetic energy transformation and act photoprotectively because of their ability to quench excitation energy from singlet- or triplet-state chlorophylls, thereby decreasing the risk that singlet oxygen forms (Niyogi, 1999; Demmig-Adams and Adams, 2002). Furthermore, the regulated epoxidation and deepoxidation of zeaxanthin in the xanthophyll cycle contribute to the nonphotochemical quenching of energy (Niyogi, 1999; Ballottari et al., 2014). In contrast to these processes, which maintain carotenoid integrity, carotenoids are also capable of chemically quenching singlet oxygen by their own oxidation, which is accompanied by the release of various carotenoid degradation products (Ramel et al., 2012a, 2013).The various functions of carotenoids require their dynamic qualitative and quantitative tuning in response to environmental conditions to attain and maintain adequate steady-state concentrations. These include both the regulation of their synthesis and the formation, release, or disposal of their breakdown products. The synthesis of carotenoids is initiated by the condensation of two molecules of geranylgeranyl diphosphate to form phytoene catalyzed by the enzyme phytoene synthase (PSY), which is considered as the rate-limiting enzyme (von Lintig et al., 1997; Li et al., 2008; Rodríguez-Villalón et al., 2009; Welsch et al., 2010; Zhou et al., 2015). In plants, two desaturases, phytoene desaturase and ζ-carotene desaturase, and two carotene cis-trans-isomerases convert the colorless phytoene into the red-colored all-trans-lycopene (Isaacson et al., 2002; Park et al., 2002; Chen et al., 2010; Yu et al., 2011). Two lycopene cyclases introduce either β- or ε-ionone rings, yielding α-(ε,β-)-carotene and β-(β,β)-carotene. In Arabidopsis (Arabidopsis thaliana), four enzymes hydroxylate carotenes with partially overlapping substrate specificity (Kim et al., 2009). Two nonheme iron-dependent β-carotene hydroxylases (BCH), BCH1 and BCH2, convert β-carotene into zeaxanthin. The second set of hydroxylases, cytochrome P450 (CYP)97A3 and CYP97C1, prefers α-carotene and produces zeinoxanthin and lutein, respectively. Absence of each cytochrome P450 hydroxylase constitutes a distinct phenotype, named lutein deficient5 (lut5) for CYP97A3 deficiency and lut1 for CYP97C1 deficiency, characterized by altered pigment compositions and the accumulation of monohydroxylated intermediates, whereas deficiency in BCH1 and BCH2 does not affect the pigment composition.In green tissues, photooxidative destruction seemingly predominates and consumes carotenoids (Simkin et al., 2003). Moreover, ¹⁴CO₂ pulse-chase experiments with Arabidopsis leaves identified α- and β-carotene as the main targets for photooxidation, whereas xanthophylls were less affected (Beisel et al., 2010). Oxidation assays with β-carotene showed epoxy- and peroxy-derivatives as the main primary products, which however, undergo additional reactions, yielding more stable degradation products that are, in part, the same apocarotenals/ones as those being produced enzymatically (Ramel et al., 2012a, 2013).In Arabidopsis, genes coding for carotenoid cleaving enzymes (carotenoid cleavage dioxygenases [CCDs]) form a small gene family comprising nine members, five of which are attributed to the synthesis of abscisic acid (ABA; nine-cis-epoxycarotenoid cleavage dioxygenases [NCEDs]; AtNCED2, AtNCED3, AtNCED5, AtNCED6, and AtNCED9; Iuchi et al., 2001; Tan et al., 2003), whereas two are committed to strigolactone biosynthesis (CCD7/MORE AXILLARY GROWTH3 [MAX3] and CCD8/MAX4; Alder et al., 2012; Bruno et al., 2014). Orthologs of CCD1 are involved in the generation of volatile apocarotenoids contributing to flower scents and aroma production (e.g. saffron [Crocus sativus; Rubio et al., 2008; Frusciante et al., 2014] and tomato [Solanum lycopersicum; Simkin et al., 2004]), whereas CCD4 enzymes are involved in citrus peel and chrysanthemum (Chrysanthemum morifolium) petal coloration (Ohmiya et al., 2006; Rodrigo et al., 2013). Recent analysis of Arabidopsis mutants revealed a major function of CCD4 in regulating seed carotenoid content with only a minor contribution of CCD1 (Gonzalez-Jorge et al., 2013). Moreover, CCD4 activity was required for the synthesis of an apocarotenoid-derived signaling molecule involved in leaf development and retrograde gene expression (Avendaño-Vázquez et al., 2014).Elevated carotenoid pathway flux caused by PSY overexpression increases carotenoid accumulation in various nongreen tissues, such as tomato fruits, canola (Brassica napus) seeds, cassava (Manihot esculenta) roots, and rice (Oryza sativa) endosperm (Shewmaker et al., 1999; Ye et al., 2000; Fraser et al., 2002; Welsch et al., 2010). Similarly, the constitutive overexpression of PSY in Arabidopsis results in dramatically increased carotenoid amounts accumulating as crystals in nongreen tissues, such as roots and callus, yielding β-carotene as the main product (Maass et al., 2009). However, leaves of the very same plants do not show altered pigment composition, and phytoene or other pathway intermediates are not detected. Similarly, increased levels of active PSY protein achieved though overexpression of the ORANGE protein exclusively affect carotenoid amounts in roots but not in leaves (Zhou et al., 2015). Leaves from constitutively PSY-overexpressing tomato and tobacco (Nicotiana tabacum) plants are also reported to show only slightly increased carotenoid levels compared with the wild-type control (Fray et al., 1995; Busch et al., 2002). These contrasting responses of leaves versus nongreen tissues to elevated pathway flux suggest fundamental differences in the modes of carotenoid formation and/or degradation.In this work, we identified xanthophyll-derived apocarotenoid glycosides (AGs) in Arabidopsis leaves that increase upon higher pathway flux. This suggests that apocarotenoid glycosylation functions as a valve regulating carotenoid steady-state levels in leaves. The analysis of Arabidopsis mutants enabled us to conclude on potential precursor carotenoids and assess the contribution of carotenoid cleavage enzymes on their formation. Moreover, apocarotenoids but not the identified glycosides were increased in carotenoid-hyperaccumulating roots, indicating tissue-specific different modes of carotenoid turnover regulation.     

Mutants of Arabidopsis thaliana with Decreased Amplitude in Their Phototropic Response

Two mutants of Arabidopsis thaliana have been identified with decreased phototropism to 450-nanometer light. Fluence-response relationships for these strains (ZR8 and ZR19) to single and multiple flashes of light show thresholds, curve shapes, and fluence for maximum curvature in `first positive' phototropism which are the same as those of the wild type. Similarly, there is no alteration from the wild type in the kinetics of curvature or in the optimum dark period separating sequential flashes in a multiple flash regimen. In addition, in both strains, gravitropism is decreased compared to the wild type by an amount which is comparable to the decrease in phototropism. Based on reciprocal backcrosses, it appears that the alteration is due to a recessive nuclear mutation. It is suggested that ZR8 and ZR19 represent alterations in some step analogous to an amplifier, downstream of the photoreceptor pigment, and common to both phototropism and gravitropism.     

ROP3 GTPase Contributes to Polar Auxin Transport and Auxin Responses and Is Important for Embryogenesis and Seedling Growth in Arabidopsis

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.     

miR824-Regulated AGAMOUS-LIKE16 Contributes to Flowering Time Repression in Arabidopsis

The timing of flowering is pivotal for maximizing reproductive success under fluctuating environmental conditions. Flowering time is tightly controlled by complex genetic networks that integrate endogenous and exogenous cues, such as light, temperature, photoperiod, and hormones. Here, we show that AGAMOUS-LIKE16 (AGL16) and its negative regulator microRNA824 (miR824) control flowering time in Arabidopsis thaliana. Knockout of AGL16 effectively accelerates flowering in nonvernalized Col-FRI, in which the floral inhibitor FLOWERING LOCUS C (FLC) is strongly expressed, but shows no effect if plants are vernalized or grown in short days. Alteration of AGL16 expression levels by manipulating miR824 abundance influences the timing of flowering quantitatively, depending on the expression level and number of functional FLC alleles. The effect of AGL16 is fully dependent on the presence of FLOWERING LOCUS T (FT). Further experiments show that AGL16 can interact directly with SHORT VEGETATIVE PHASE and indirectly with FLC, two proteins that form a complex to repress expression of FT. Our data reveal that miR824 and AGL16 modulate the extent of flowering time repression in a long-day photoperiod.     

The Arabidopsis rcn1-1 Mutation Impairs Dephosphorylation of Phot2, Resulting in Enhanced Blue Light Responses

Phototropins (phot) sense blue light through the two N-terminal chromophore binding LOV domains and activate the C-terminal kinase domain. The resulting phototropin autophosphorylation is essential for biological activity. We identified the A1 subunit of Ser/Thr protein phosphatase 2A (PP2A) as interacting with full-length phot2 in yeast and also interacting with phot2 in an in vitro protein binding assay. Phenotypic characterizations of a phot1-5 rcn1-1 (for root curling in n-naphthylphthalamic acid1) double mutant, in which phot2 is the only functional phototropin and PP2A activity is reduced, showed enhanced phototropic sensitivity and enhanced blue light–induced stomatal opening, suggesting that PP2A activity is involved in regulating phot2 function. When treated with cantharidin, a chemical inhibitor of PP2A, the phot1-5 mutant exhibited enhanced phot2-mediated phototropic responses like those of the phot1-5 rcn1-1 double mutant. Immunoblot analysis to examine phot2 endogenous phosphorylation levels and in vitro phosphorylation assays of phot2 extracted from plants during dark recovery from blue light exposure confirmed that phot2 is more slowly dephosphorylated in the reduced PP2A activity background than in the wild-type PP2A background, suggesting that phosphorylated phot2 is a substrate of PP2A activity. While reduced PP2A activity enhanced the activity of phot2, it did not enhance either phot1 dephosphorylation or the activity of phot1 in mediating phototropism or stomatal opening.     

The Effects of Paraffin Oil on Phototropic and Geotropic Responses in Avena Coleoptiles

Experiments are described which were designed to test the significanceof the coleoptile tip as the site of reception of light stimulusleading to negative photo-tropic response under paraffin oil.The results show clearly that the tip is of paramount importancein this respect. Further experiments in which the coleoptiletips were bisected in a plane at right angles to the light rayslend support to the hypothesis that negative phototropism underoil is related tolateral transport of material in the tip. Lastly,experiments which show that the geotropic response of coleoptilesis not reversed by immersion in oil are described. These findingsare discussed in relation to certain hypotheses concerning themechanism of negative phototropism under oil.     

Adaptation of the Cyanobacterium Microcystis aeruginosa to Light Intensity

Light intensity adaptation (20 to 565 microeinsteins per square meter per second) of Microcystis aeruginosa (UV-027) was examined in turbidostat culture. Chlorophyll a and phycocyanin concentrations decreased with increasing light intensity while carotenoid, cellular carbon, and nitrogen contents did not vary. Variation in the number but not the size of photosynthetic units per cell, based on chlorophyll a/P₇₀₀ ratios, occurred on light intensity adaptation. Changes in the numbers of photosynthetic units partially dampened the effects of changes in light intensity on growth rates.     

ROOT HAIR DEFECTIVE 2 vesicular delivery to the apical plasma membrane domain during Arabidopsis root hair development

Arabidopsis (Arabidopsis thaliana) root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species generated by A. thaliana nicotinamide adenine dinucleotide phosphate (NADPH) oxidase respiratory burst oxidase homolog protein C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2). Loss-of-function root hair defective 2 (rhd2) mutants have short root hairs that are unable to elongate by tip growth, and this phenotype is fully complemented by GREEN FLUORESCENT PROTEIN (GFP)-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent molecular marker mCherry-VTI12 as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which corresponds with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we revealed that structural sterols might be involved in the accumulation, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs. These results help in clarifying the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth.

Structural sterols might participate in delivering GFP-RHD2 to the apical plasma membrane of developing root hairs.