Cryptochromes are the dominant photoreceptors mediating heliotropic responses of Arabidopsis inflorescences.

Inflorescence movements in response to natural gradients of sunlight are frequently observed in the plant kingdom and are suggested to contribute to reproductive success. Although the physiological and molecular bases of light-mediated tropisms in vegetative organs have been thoroughly investigated, the mechanisms that control inflorescence orientation in response to light gradients under natural conditions are not well understood. In this work, we have used a combination of laboratory and field experiments to investigate light-mediated re-orientation of Arabidopsis thaliana inflorescences. We show that inflorescence phototropism is promoted by photons in the UV and blue spectral range (≤500 nm) and depends on multiple photoreceptor families. Experiments under controlled conditions show that UVR8 is the main photoreceptor mediating the phototropic response to narrowband UV-B radiation, and phototropins and cryptochromes control the response to narrowband blue light. Interestingly, whereas phototropins mediate bending in response to low irradiances of blue, cryptochromes are the principal photoreceptors acting at high irradiances. Moreover, phototropins negatively regulate the action of cryptochromes at high irradiances of blue light. Experiments under natural field conditions demonstrate that cryptochromes are the principal photoreceptors acting in the promotion of the heliotropic response of inflorescences under full sunlight.

Trading direct for indirect defense? Phytochrome B inactivation in tomato attenuates direct anti-herbivore defenses whilst enhancing volatile-mediated attraction of predators.

Under conditions of competition for light, which lead to the inactivation of the photoreceptor phytochrome B (phyB), the growth of shade-intolerant plants is promoted and the accumulation of direct anti-herbivore defenses is down-regulated. Little is known about the effects of phyB on emissions of volatile organic compounds (VOCs), which play a major role as informational cues in indirect defense. We investigated the effects of phyB on direct and indirect defenses in tomato (Solanum lycopersicum) using two complementary approaches to inactivate phyB: illumination with a low red to far-red ratio, simulating competition, and mutation of the two PHYB genes present in the tomato genome. Inactivation of phyB resulted in low levels of constitutive defenses and down-regulation of direct defenses induced by methyl jasmonate (MeJA). Interestingly, phyB inactivation also had large effects on the blends of VOCs induced by MeJA. Moreover, in two-choice bioassays using MeJA-induced plants, the predatory mirid bug Macrolophus pygmaeus preferred VOCs from plants in which phyB was inactivated over VOCs from control plants. These results suggest that, in addition to repressing direct defense, phyB inactivation has consequences for VOC-mediated tritrophic interactions in canopies, presumably attracting predators to less defended plants, where they are likely to find more abundant prey.

Fruit-localized photoreceptors increase phenolic compounds in berry skins of field-grown Vitis vinifera L. cv. Malbec.

Sunlight exposure has multiple effect on fruits, as it affects the light climate perceived by fruit photoreceptors and fruit tissue temperature. In grapes (Vitis vinifera L.), light exposure can have a strong effect on fruit quality and commercial value; however, the mechanisms of light action are not well understood. The role of fruit-localized photoreceptors in the control of berry quality traits was evaluated under field conditions in a commercial vineyard in Mendoza (Argentina). Characterization of the diurnal dynamics of the fruit light environment in a vertical trellis system indicated that clusters were shaded by leaves during most of the photoperiod. Supplementation of the fruit light environment from 20 days before veraison until technological harvest showed that red (R, 660 nm) and blue (B, 470 nm) light strongly increased total phenolic compound levels at harvest in the berry skins without affecting sugar content, acidity or berry size. Far-red (FR, 730 nm) and green (G, 560 nm) light supplementation had relatively small effects. The stimulation of berry phytochromes and cryptochromes favored accumulation of flavonoid and non-flavonoid compounds, including anthocyanins, flavonols, flavanols, phenolic acids and stilbenes. These results demonstrate that the chemical composition of grape berries is modulated by the light quality received by the clusters under field conditions, and that fruit photoreceptors are not saturated even in areas of high insolation and under management systems that are considered to result in a relatively high exposure of fruits to solar radiation. Therefore, manipulation of the light environment or the light sensitivity of fruits could have significant effects on critical grape quality traits.

Phytochrome B increases drought tolerance by enhancing ABA sensitivity in Arabidopsis thaliana.

Phytochrome B (phyB) can adjust morphological and physiological responses according to changes in the red:far-red (R:FR) ratio. phyB-driven acclimation of plants to open environments (high R:FR ratio) increases carbon gain at the expense of increased water loss. This behaviour alleviates stressful conditions generated by an excess of light, but increases the chances of desiccation. Here we evaluated how phyB modulates this drought-tolerance response by comparing wild-type Arabidopsis thaliana adult plants to the null phyB in response to water shortage. phyB wilted before the wild type, and this was due to phyB maintaining open stomata under a reduction in soil water availability. Although phyB presented enhanced ABA levels under well-watered conditions, this mutant was less sensitive than the wild type in diminishing stomatal conductance in response to exogenous ABA application. Reduced sensitivity to ABA in phyB correlated with a lower expression of ABCG22, which encodes a putative ABA influx transporter, and PYL5, which encodes a soluble ABA receptor. Furthermore, the expression of RAB18 and RD29A, both typical ABA-induced genes, was lower in phyB than the wild type after ABA treatment. We propose that phyB contributes to the acclimation of plants to open environments by enhancing ABA sensitivity when soil water becomes limiting.

Phytochrome A increases tolerance to high evaporative demand.

Stresses resulting from high transpiration demand induce adjustments in plants that lead to reductions of water loss. These adjustments, including changes in water absorption, transport and/or loss by transpiration, are crucial to normal plant development. Tomato wild type (WT) and phytochrome A (phyA)-mutant plants, fri1-1, were exposed to conditions of either low or high transpiration demand and several morphological and physiological changes were measured during stress conditions. Mutant plants rapidly wilted compared to WT plants after exposure to high evaporative demand. Root size and hydraulic conductivity did not show significant differences between genotypes, suggesting that water absorption and transport through this organ could not explain the observed phenotype. Moreover, stomatal density was similar between genotypes, whereas transpiration and stomatal conductance were both lower in mutant than in WT plants. This was accompanied by a lower stem-specific hydraulic conductivity in mutant plants, which was associated to lower xylem vessel number and transversal area in fri1-1 plants, producing a reduction in water supply to the leaves, which rapidly wilted under high evaporative demand. PhyA signaling might facilitate the adjustment to environments differing widely in water evaporative demand in part through the modulation of xylem dimensions.

Phototropins but not cryptochromes mediate the blue light-specific promotion of stomatal conductance, while both enhance photosynthesis and transpiration under full sunlight.

Leaf epidermal peels of Arabidopsis (Arabidopsis thaliana) mutants lacking either phototropins 1 and 2 (phot1 and phot2) or cryptochromes 1 and 2 (cry1 and cry2) exposed to a background of red light show severely impaired stomatal opening responses to blue light. Since phot and cry are UV-A/blue light photoreceptors, they may be involved in the perception of the blue light-specific signal that induces the aperture of the stomatal pores. In leaf epidermal peels, the blue light-specific effect saturates at low irradiances; therefore, it is considered to operate mainly under the low irradiance of dawn, dusk, or deep canopies. Conversely, we show that both phot1 phot2 and cry1 cry2 have reduced stomatal conductance, transpiration, and photosynthesis, particularly under the high irradiance of full sunlight at midday. These mutants show compromised responses of stomatal conductance to irradiance. However, the effects of phot and cry on photosynthesis were largely nonstomatic. While the stomatal conductance phenotype of phot1 phot2 was blue light specific, cry1 cry2 showed reduced stomatal conductance not only in response to blue light, but also in response to red light. The levels of abscisic acid were elevated in cry1 cry2. We conclude that considering their effects at high irradiances cry and phot are critical for the control of transpiration and photosynthesis rates in the field. The effects of cry on stomatal conductance are largely indirect and involve the control of abscisic acid levels.

Melatonin levels, determined by LC-ESI-MS/MS, fluctuate during the day/night cycle in Vitis vinifera cv Malbec: evidence of its antioxidant role in fruits.

The identification of melatonin in plants has inspired new investigations to understand its biological function and which endogenous and external factors control its levels in these organisms. Owing to the therapeutical and nutraceutical properties of melatonin, it should be important to develop reliable analytical methods for its quantification in vegetal matrices containing this indoleamine, such as grape and wine. The main objectives of the present study were to test whether melatonin levels fluctuate during the day in berry skins of Vitis vinifera L. cv Malbec, thereby possibly relating its abundance to its putative antioxidant function, to determine whether daylight reaching clusters negatively controls melatonin levels, and to evaluate whether total polyphenols and anthocyanins also change through a 24-hr period. Grapes were harvested throughout the day/night to determine the moment when high levels of these components are present in grapes. The presence of melatonin in grapes was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. It is shown for the first time that melatonin levels fluctuate during the day/night cycle in plants grown under field conditions in a fruit organ of the species Vitis vinifera. We also determined that the diurnal decay of melatonin in berry skins is induced by sunlight, because covered bunches retained higher melatonin levels than exposed ones, thus explaining at least part of the basis of its daily fluctuation. Evidence of melatonin's antioxidant role in grapes is also suggested by monitoring malondialdehyde levels during the day.

Phytochrome B enhances photosynthesis at the expense of water-use efficiency in Arabidopsis.

In open places, plants are exposed to higher fluence rates of photosynthetically active radiation and to higher red to far-red ratios than under the shade of neighbor plants. High fluence rates are known to increase stomata density. Here we show that high, compared to low, red to far-red ratios also increase stomata density in Arabidopsis (Arabidopsis thaliana). High red to far-red ratios increase the proportion of phytochrome B (phyB) in its active form and the phyB mutant exhibited a constitutively low stomata density. phyB increased the stomata index (the ratio between stomata and epidermal cells number) and the level of anphistomy (by increasing stomata density more intensively in the adaxial than in the abaxial face). phyB promoted the expression of FAMA and TOO MANY MOUTHS genes involved in the regulation of stomata development in young leaves. Increased stomata density resulted in increased transpiration per unit leaf area. However, phyB promoted photosynthesis rates only at high fluence rates of photosynthetically active radiation. In accordance to these observations, phyB reduced long-term water-use efficiency estimated by the analysis of isotopic discrimination against (13)CO(2). We propose a model where active phyB promotes stomata differentiation in open places, allowing plants to take advantage of the higher irradiances at the expense of a reduction of water-use efficiency, which is compensated by a reduced leaf area.

PHYTOCHROME KINASE SUBSTRATE4 modulates phytochrome-mediated control of hypocotyl growth orientation.

Gravity and light are major factors shaping plant growth. Light perceived by phytochromes leads to seedling deetiolation, which includes the deviation from vertical hypocotyl growth and promotes hypocotyl phototropism. These light responses enhance survival of young seedlings during their emergence from the soil. The PHYTOCHROME KINASE SUBSTRATE (PKS) family is composed of four members in Arabidopsis (Arabidopsis thaliana): PKS1 to PKS4. Here we show that PKS4 is a negative regulator of both phytochrome A- and B-mediated inhibition of hypocotyl growth and promotion of cotyledon unfolding. Most prominently, pks4 mutants show abnormal phytochrome-modulated hypocotyl growth orientation. In dark-grown seedlings hypocotyls change from the original orientation defined by seed position to the upright orientation defined by gravity and light reduces the magnitude of this shift. In older seedlings with the hypocotyls already oriented by gravity, light promotes the deviation from vertical orientation. Based on the characterization of pks4 mutants we propose that PKS4 inhibits changes in growth orientation under red or far-red light. Our data suggest that in these light conditions PKS4 acts as an inhibitor of asymmetric growth. This hypothesis is supported by the phenotype of PKS4 overexpressers. Together with previous findings, these results indicate that the PKS family plays important functions during light-regulated tropic growth responses.

PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism.

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.

Secret message at the plant surface.

In general, stomata open during the day and close at night. This behavior has a crucial importance because it maximizes the update of CO(2) for photosynthesis and minimizes the water loss. Blue light is one of the environmental factors that regulates this process. Certainly, when either entire plants or epidermal strips adapted to the dark are exposed to blue light, the stomata open widely their pores. But, what does happen if we illuminate individual stomata instead of peels or entire plants? In the inaugural issue of PLoS ONE, we have answered this question by irradiating individual stomata with a laser attached to a confocal microscope. Our study not only demonstrates that the stomata function independently from the behavior of their neighbors, and illuminates the implication of the blue light receptors PHOTOTROPIN1 and PHOTOTROPIN2 in such response. It also gives clues about the physiological relevancy of this behavior.

Use of confocal laser as light source reveals stomata-autonomous function.

In most terrestrial plants, stomata open during the day to maximize the update of CO(2) for photosynthesis, but they close at night to minimize water loss. Blue light, among several environmental factors, controls this process. Stomata response to diverse stimuli seems to be dictated by the behaviour of neighbour stomata creating leaf areas of coordinated response. Here individual stomata of Arabidopsis leaves were illuminated with a short blue-light pulse by focusing a confocal argon laser. Beautifully, the illuminated stomata open their pores, whereas their dark-adapted neighbours unexpectedly experience no change. This induction of individual stomata opening by low fluence rates of blue light was disrupted in the phototropin1 phototropin2 (phot1 phot2) double mutant, which exhibits insensitivity of stomatal movements in blue-illuminated epidermal strips. The irradiation of all epidermal cells making direct contact with a given stoma in both wild type and phot1 phot2 plants does not trigger its movement. These results unravel the stoma autonomous function in the blue light response and illuminate the implication of PHOT1 and/or PHOT2 in such response. The micro spatial heterogeneity that solar blue light suffers in partially shaded leaves under natural conditions highlights the physiological significance of the autonomous stomatal behaviour.

Promotion of photomorphogenesis by COP1.

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) represses photomorphogenesis in darkness by targeting nuclear-localized transcription factors to proteasome-mediated degradation. Upon light exposure, COP1 migrates to the cytosol allowing photomorphogenesis to proceed but the residual nuclear pool down-regulates light signaling mediated by phytochrome A. Here we show that weak alleles of cop1 exhibit reverse photomorphogenic responses i.e. reduced rather than enhanced cotyledon unfolding under red light compared to darkness. Conversely, COP1 overexpressors which de-etiolate poorly under blue or far-red light, showed enhanced photomorphogenesis under red light. The positive relationship between COP1 and photomorphogenic response required phytochrome B. Thus, genetic manipulation of COP1 levels differentially affects phytochrome A- compared to phytochrome B-mediated responses. We hypothesize that COP1 could be involved in degradation of negative regulators of photomorphogenesis or in transcriptional activation, as observed for some E3 ligases in mammalian development.

A growth regulatory loop that provides homeostasis to phytochrome a signaling.

Phytochrome kinase substrate1 (PKS1) is a cytoplasmic protein that interacts physically with, and is phosphorylated by, the plant photoreceptor phytochrome. Here, we show that light transiently increases PKS1 mRNA levels and concentrates its expression to the elongation zone of the hypocotyl and root. This response is mediated by phytochrome A (phyA) acting in the very low fluence response (VLFR) mode. In the hypocotyl, PKS1 RNA and protein accumulation are maintained only under prolonged incubation in far-red light, the wavelength that most effectively activates phyA. Null mutants of PKS1 and its closest homolog, PKS2, show enhanced phyA-mediated VLFR. Notably, a pks1 pks2 double mutant has no phenotype, whereas overexpression of either PKS1 or PKS2 results in the same phenotype as the pks1 or pks2 single null mutant. We propose that PKS1 and PKS2 are involved in a growth regulatory loop that provides homeostasis to phyA signaling in the VLFR. In accordance with this idea, PKS1 effects are larger in the pks2 background (and vice versa). Moreover, the two proteins can interact with each other, and PKS2 negatively regulates PKS1 protein levels specifically under VLFR conditions.

Increased phytochrome B alleviates density effects on tuber yield of field potato crops.

The possibility that reduced photomorphogenic responses could increase field crop yield has been suggested often, but experimental support is still lacking. Here, we report that ectopic expression of the Arabidopsis PHYB (phytochrome B) gene, a photoreceptor involved in detecting red to far-red light ratio associated with plant density, can increase tuber yield in field-grown transgenic potato (Solanum tuberosum) crops. Surprisingly, this effect was larger at very high densities, despite the intense reduction in the red to far-red light ratios and the concomitant narrowed differences in active phytochrome B levels between wild type and transgenics at these densities. Increased PHYB expression not only altered the ability of plants to respond to light signals, but they also modified the light environment itself. This combination resulted in larger effects of enhanced PHYB expression on tuber number and crop photosynthesis at high planting densities. The PHYB transgenics showed higher maximum photosynthesis in leaves of all strata of the canopy, and this effect was largely due to increased leaf stomatal conductance. We propose that enhanced PHYB expression could be used in breeding programs to shift optimum planting densities to higher levels.

Light, phytochrome signalling and photomorphogenesis in Arabidopsis.

The phytochromes is a family of plant photoreceptors that control growth and development in response to environmental cues. Red and far-red light are the most efficient wavebands to induce conformational changes of phytochromes and consequently modify their kinetics, nuclear/cytoplasmic partitioning, ability to phosphorylate substrates, and physical interaction with proteins that bind DNA. Many players in phytochrome signalling have been identified and a complex, highly regulated network is envisaged. Here we describe the connection between different features of the phytochrome signalling network and the versatile relationship between light signals and physiological outputs shown by phytochromes.

Brassinosteroid mutants uncover fine tuning of phytochrome signaling.

Phytochromes (phy) A and B provide higher plants the ability to perceive divergent light signals. phyB mediates red/far-red light reversible, low fluence responses (LFR). phyA mediates both very-low-fluence responses (VLFR), which saturate with single or infrequent light pulses of very low fluence, and high irradiance responses (HIR), which require sustained activation with far-red light. We investigated whether VLFR, LFR, and HIR are genetically coregulated. The Arabidopsis enhanced very-low-fluence response1 mutant, obtained in a novel screening under hourly far-red light pulses, showed enhanced VLFR of hypocotyl growth inhibition, cotyledon unfolding, blocking of greening, and anthocyanin synthesis. However, eve1 showed reduced LFR and HIR. eve1 was found allelic to the brassinosteroid biosynthesis mutant dim/dwarf1. The analysis of both the brassinosteroid mutant det2 in the Columbia background (where VLFR are repressed) and the phyA eve1 double mutant indicates that the negative effect of brassinosteroid mutations on LFR requires phyA signaling in the VLFR mode but not the expression of the VLFR. Under sunlight, hypocotyl growth of eve1 showed little difference with the wild type but failed to respond to canopy shadelight. We propose that the opposite regulation of VLFR versus LFR and HIR could be part of a context-dependent mechanism of adjustment of sensitivity to light signals.