Blue-Light-Induced Shrinking of Protoplasts from Maize Coleoptiles and Its Relationship to Coleoptile Growth

Protoplasts isolated from red-light-grown maize (Zea mays L.) coleoptiles shrank transiently upon brief exposure (e.g. 30 s) to blue light under background irradiation with red light. The maximal volume reduction (about 4% at a saturating fluence) occurred about 5 min after blue-light stimulation. The response was prevented by the anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Red light and far-red light did not induce any comparable response. Protoplasts of different sizes and those isolated from different coleoptile positions showed similar responses. After treatment with a saturating blue-light pulse, the protoplasts became responsive to a second pulse and gained full responsiveness within 5 min, suggesting that the photoreceptor system involves a dark-reversible component. The response to continuous blue light was also found to be transient. The protoplast volume was reduced during about 6 to 9 min of irradiation and returned within the next 30 min to the control level. The response to continuous blue light was saturated at 30 [mu]mol m-2 s-1. However, when the fluence rate was enhanced 10-fold after a period of irradiation at 30 [mu]mol m-2 s-1, the protoplasts showed another shrinking response. These and other kinetic results indicate that the photoreceptor system undergoes a photosensory adaptation. Growth in different zones of the coleoptile was inhibited by blue light transiently after pulse stimulation, as well as during continuous stimulation. It was concluded that the observed protoplast shrinking is related to the blue-light-induced inhibition of coleoptile growth.     

Photomorphogenic effects of UV-B radiation on hypocotyl elongation in wild type and stable-phytochrome-deficient mutant seedlings of cucumber

Hypocotyl elongation responses to ultraviolet-B (UV-B) radiation were investigated in glasshouse studies of de-etiolated seedlings of a long-hypocotyl mutant ( lh ) of cucumber ( Cucumis sativus L.) deficient in stable phytochrome, its near isogenic wild type (WT), and a commercial cucumber hybrid (cv. Burpless). A single 6- or 8-h exposure to UV-B applied against a background of white light inhibited hypocotyl elongation rate by ca 50% in lh and WT seedlings. This effect was not accompanied by a reduction in cotyledon area expansion or dry matter accumulation. Plants recovered rapidly from inhibition and it was possible to stimulate hypocotyl elongation in plants exposed to UV-B by application of gibberellic acid. In all genotypes inhibition of elongation was mainly a consequence of UV-B perceived by the cotyledons; covering the apex and hypocotyl with a filter that excluded UV-B failed to prevent inhibition. These results indicate that reduced elongation does not result from assimilate limitation or direct damage to the apical meristem or elongating cells, and strongly suggest that it is a true photomorphogenic response to UV-B. The fact that UV-B fluences used were very low in relation to total visible light, and the similarity in the responses of lh and wild-type plants, are consistent with the hypothesis that UV-B acts through a specific photoreceptor. It is argued that, given the weak correlation between UV-B and visible-light levels in most natural conditions, the UV-B receptor may play an important sensory function providing information to the plant that cannot be derived from light signals perceived by phytochrome or blue/UV-A sensors.     

Perception of solar UV radiation by plants: photoreceptors and mechanisms

About 95% of the ultraviolet (UV) photons reaching the Earth’s surface are UV-A (315–400 nm) photons. Plant responses to UV-A radiation have been less frequently studied than those to UV-B (280–315 nm) radiation. Most previous studies on UV-A radiation have used an unrealistic balance between UV-A, UV-B, and photosynthetically active radiation (PAR). Consequently, results from these studies are difficult to interpret from an ecological perspective, leaving an important gap in our understanding of the perception of solar UV radiation by plants. Previously, it was assumed UV-A/blue photoreceptors, cryptochromes and phototropins mediated photomorphogenic responses to UV-A radiation and “UV-B photoreceptor” UV RESISTANCE LOCUS 8 (UVR8) to UV-B radiation. However, our understanding of how UV-A radiation is perceived by plants has recently improved. Experiments using a realistic balance between UV-B, UV-A, and PAR have demonstrated that UVR8 can play a major role in the perception of both UV-B and short-wavelength UV-A (UV-A_sw, 315 to ∼350 nm) radiation. These experiments also showed that UVR8 and cryptochromes jointly regulate gene expression through interactions that alter the relative sensitivity to UV-B, UV-A, and blue wavelengths. Negative feedback loops on the action of these photoreceptors can arise from gene expression, signaling crosstalk, and absorption of UV photons by phenolic metabolites. These interactions explain why exposure to blue light modulates photomorphogenic responses to UV-B and UV-A_sw radiation. Future studies will need to distinguish between short and long wavelengths of UV-A radiation and to consider UVR8’s role as a UV-B/UV-A_sw photoreceptor in sunlight.

In sunlight, UVR8 mediates the perception of both UV-B and short-wavelength UV-A radiation with its sensitivity moderated by blue light perceived through cryptochromes.     

Ultraviolet A-specific induction of anthocyanin biosynthesis in the swollen hypocotyls of turnip (Brassica rapa)

Ultraviolet A (UV-A)-mediated regulation of anthocyanin biosynthesis was investigated in swollen hypocotyls of the red turnip 'Tsuda'. The shaded swollen hypocotyls which contained negligible anthocyanin were exposed to artificial light sources including low fluence UV-B, UV-A, blue, red, far-red, red plus UV-A, far-red plus UV-A, and blue plus red. Among these lights, only UV-A induced anthocyanin biosynthesis and co-irradiation of red or far-red with UV-A did not affect the extent of UV-A-induced anthocyanin accumulation. The expression of phenylalanine ammonia lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), and anthocyanidin synthase (ANS; EC 1.14.11.19) genes was increased with time during a 24 h exposure to UV-A. In contrast, irradiation with red, blue, UV-B, and a combination of blue with red failed to induce CHS expression. Microarray analysis showed that only a few genes, including CHS and F3H, were induced significantly by UV-A, while a separate set of many genes was induced by low fluence UV-B. The UV-A-specific induction of anthocyanin biosynthesis and the unique gene expression profile upon UV-A irradiation as compared with blue and UV-B demonstrated that the observed induction of anthocyanin biosynthesis in red turnips was mediated by a distinct UV-A-specific photoreceptor, but not by phytochromes, UV-A/blue photoreceptors, or UV-B photoreceptors.     

INTERACTION BETWEEN CRYPTOCHROME AND PHYTOCHROME IN HIGHER PLANT PHOTOMORPHOGENESIS

At least three photoreceptors are involved in the mediation of light action in higher plant photomorphogenesis: cryptochrome (UV-A/blue light photoreceptor), UV-B photoreceptor, and phytochrome. The degree of photoreceptor interaction in photomorphogenesis can apparently vary depending on the response, the species, and the stage of development of the biological system. In most cases of interaction studied so far, Pfr, the physiologically active form of phytochrome, is apparently required for the final expression of the response. In some systems, the cryptochrome and/or UV-B photoreceptor mediated action of UV/blue radiation is required to establish/enhance/maintain responsiveness toward Pfr. There is no evidence for photoreceptor interaction in some response-system combinations. It is not known for sure if this apparent lack of photoreceptor interaction represents a real situation or just a failure to detect it because of experimental limitations. Practically nothing is known about the mechanism of photoreceptor interaction at the molecular level.     

In Vivo Photomodification of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Holoenzyme by Ultraviolet-B Radiation (Formation of a 66-Kilodalton Variant of the Large Subunit)

Increased levels of solar ultraviolet (290-320 nm) (UV-B) radiation could have profound effects on plant proteins because the aromatic amino acids in proteins absorb strongly in this spectral region. We have investigated the effects of UV-B radiation on plant proteins and have observed a novel 66-kD protein. This product was formed in vivo when Brassica napus L. plants grown for 21 d in 65 [mu]mol m-2 s-1 photosynthetically active radiation were subsequently exposed to 65 [mu]mol m-2 s-1 photosynthetically active radiation plus UV-B radiation (1.5 [mu]mol m-2 s-1). The protein appeared after 4 h of UV-B irradiation and accumulated during the next 16 h in UV-B. The 66-kD protein cross-reacted with an antiserum against the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme. Analysis of soluble leaf proteins revealed that the 66-kD product had a number of isoforms corresponding closely to those of the large subunit of Rubisco (LSU). Partial proteolytic digests of the LSU and the 66-kD protein resulted in an equivalent pattern of protein fragments, leading to the conclusion that the 66-kD protein was a photomodified form of the LSU. A similar high molecular mass variant of Rubisco was observed in soluble protein extracts from leaves of tomato (Lycopersicon esculentum), tobacco (Nicotiana tabacum), and pea (Pisum sativum L.) plants treated in vivo with UV-B, suggesting that it might be a common product, at least among C3 plants. It is interesting that the 66-kD product appears to be generated after incorporation of the LSU into holoenzyme complexes. This conclusion was drawn from two lines of evidence. First, the LSU variant co-purified with holoenzyme complexes isolated by nondenaturing polyacrylamide gel electrophoresis. Second, a UV-B-specific 66-kD protein did not accumulate in a tobacco mutant that synthesizes the Rubisco subunits but does not assemble them into normal holoenzyme complexes.     

Light-dependent anthocyanin synthesis: A model system for the study of plant photomorphogenesis

The biosynthesis of anthocyanins in plant tissues either requires light or is enhanced by it. Light-dependent anthocyanin synthesis has been extensively used as a model system for studies of the mechanism of photoregulation of plant development. Two components can be distinguished in the action of light on anthocyanin production. The first component is the red-far red reversible, phytochrome-mediated response induced by short irradiations; the amount of anthocyanin formed in response to a single, short irradiation is small. The second component is the response to prolonged exposures; the formation of large amounts of anthocyanin requires prolonged exposures to high fluence rates of visible and near-visible radiation (290 to 750 nm) and shows the typical properties of the “High Irradiance Reaction” (HIR) of plant photomorphogenesis. Phytochrome is involved in the photoregulation of the HIR response and is the only photoreceptor mediating the action of prolonged red and far red irradiations. The response to prolonged ultraviolet and blue radiation is probably mediated, at least in some systems, by two photoreceptors: phytochrome and cryptochrome, the latter being a specific ultraviolet-blue-light photoreceptor. The nature of the interaction between phytochrome and cryptochrome in the regulation of plant photomorphogenic responses is still unclear.     

UV-B-induced photomorphogenesis in Arabidopsis thaliana

Relatively little is known about the types of photomorphogenic responses and signal transduction pathways that plants employ in response to ultraviolet-B (UV-B, 290–320 nm) radiation. In wild-type Arabidopsis seedlings, hypocotyl growth inhibition and cotyledon expansion were both reproducibly promoted by continuous UV-B. The fluence rate response of hypocotyl elongation was examined and showed a biphasic response. Whereas photomorphogenic responses were observed at low doses, higher fluences resulted in damage symptoms. In support of our theory that photomorphogenesis, but not damage, occurs at low doses of UV-B, photomorphogenic responses of UV-B sensitive mutants were indistinguishable from wild-type plants at the low dose. This allowed us to examine UV-B-induced photomorphogenesis in photoreceptor deficient plants and constitutive photomorphogenic mutants. The cry1 cryptochrome structural gene mutant, and phytochrome deficient hy1, phyA and phyB mutant seedlings resembled wild-type seedlings, while phyA/phyB double mutants were less sensitive to the photomorphogenic effects of UV-B. These results suggest that either phyA or phyB is required for UV-B-induced photomorphogenesis. The constitutive photomorphogenic mutants cop1 and det1 did not show significant inhibition of hypocotyl growth in response to UV-B, while det2 was strongly affected by UV-B irradiation. This suggests that COP1 and DET1 work downstream of the UV-B signaling pathway.     

Ultraviolet B radiation enhances a phytochrome-B-mediated photomorphogenic response in Arabidopsis

Ultraviolet B radiation (UV-B, 290-315 nm) can cause damage and induce photomorphogenic responses in plants. The mechanisms that mediate the photomorphogenic effects of UV-B are unclear. In etiolated Arabidopsis seedlings, a daily exposure to 2.5 h of UV-B enhanced the cotyledon opening response induced by a subsequent red light (R) pulse. An R pulse alone, 2.5 h of UV-B terminated with a far-red pulse, or 2.5 h of continuous R caused very little cotyledon opening. The enhancing effect of UV-B increased with fluence rate up to approximately 7.58 micromol m(-2) s(-1); at higher fluence rates the response to UV-B was greatly reduced. The phyA, phyA cry1, and cry1 cry2 mutants behaved like the wild type when exposed to UV-B followed by an R pulse. In contrast, phyB, phyB cry1, and phyB phyA mutants failed to open the cotyledons. Thus, phytochrome B was required for the cotyledon opening response to UV-B --> R treatments, whereas phytochrome A and cryptochromes 1 and 2 were not necessary under the conditions of our experiments. The enhancing effect of low doses of UV-B on cotyledon opening in uvr1 uvr2 and uvr1 uvr3 mutants, deficient in DNA repair, was similar to that found in the wild type, suggesting that this effect of UV-B was not elicited by signals derived from UV-B-induced DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts). We conclude that low doses of UV-B, perceived by a receptor system different from phytochromes, cryptochromes, or DNA, enhance a de-etiolation response that is induced by active phytochrome B.     

Photobiological properties of the inhibition of etiolated Arabidopsis seedling growth by ultraviolet-B irradiation

Alteration of 'normal' levels of ultraviolet-B light (UV-B, 280–320 nm) can affect plant chemical composition as well as growth; however, little is known about how plants perceive UV-B light. We have carried out fluence response curves, and demonstrated that the growth inhibition of etiolated Arabidopsis thaliana seedlings by low fluence UV light is specific to UV-B and not UV-A (320–390 nm). The response shows reciprocity between duration and intensity, at least over a limited range, and thus depends only on photon fluence and not on photon flux. The action spectrum for this response indicates a peak of maximum effectiveness at 290 nm, and response spectra at different fluences indicate that the most effective wavelength at 30 000  µ mol m^–2 is 290 nm, whereas 300 nm light was the most effective at 100 000  µ mol m^–2. This response occurs in mutant seedlings deficient in cryptochrome, phytochrome or phototropin, suggesting that none of the known photoreceptors is the major UV-B photoreceptor. Some null mutants in DNA repair enzymes show hypersensitivity to UV-B, suggesting that even at low fluence rates, direct damage to DNA may be one component of the response to UV-B.     

Influence of high light and UV-B radiation on photosynthesis and D1 turnover in atrazine-tolerant and -sensitive cultivars of Brassica napus

An atrazine-tolerant mutant and an atrazine-sensitive cultivar of Brassica napus L. were grown under visible radiation (400 mumol m-2 s-1, photosynthetically active radiation, PAR) and then subjected to treatment conditions. These included short-term high PAR (1600 mumol m-2 s-1) which was given for 4 h either alone or in combination with an enhanced level of UV-BBE radiation (4.6 kJ m-2 h-1 biologically effective UV-B, 280-320 nm). Recovery from the radiation treatment was studied for 4 h under the light conditions for growth. Since it is known that the atrazine-tolerant mutant is susceptible to photoinhibition, one of the aims of the present study was to determine the effects of a supplemental, enhanced level of UV-B radiation with regard to the mutant. The results indicate an additive effect of UV-B radiation on Fv/Fm, photochemical yield and photosynthetic oxygen evolution during both exposure and recovery, and also a higher susceptibility of the mutant to photoinhibitory PAR conditions alone and in combination with UV-B, which may have implications in a changing environment. Both cultivars also showed a higher D1 turnover during the radiation stress than during recovery, as shown by immunoblotting and 35S-methionine incorporation measurements.     

UV-B辐射对8个大豆品种种子萌发率和 幼苗生长的影响

在生长房5种(暗处、可见光、低、中、高强度紫外线-B)处理下,研究了8个大豆品种的种子萌发率和萌发后幼苗的生长状况。结果表明,暗处种子萌发率高于自然光和UV-B辐射的种子。UV-B辐射增强对大豆种子的萌发率没有显著影响,仅使部分品种的最大萌发率降低和导致部分品种达到最大萌发率的时间延长。幼苗的生长对增强的UV-B辐射非常敏感。使大部分品种的胚根变短增粗,这可能是植物激素作用的结果。大豆的叶绿素a、叶绿素b和总叶绿素含量明显受到UV-B辐射的抑制。UV-B作用能促进类黄酮在幼苗中的积累,紫外吸收色素的增设有利于提高对UV-B的抵抗力。UV-B辐射的这种效应及大豆品种间的差异在自然情况下会产生深远的生物学和生态学意义     

Desensitization and recovery of phototropic responsiveness in Arabidopsis thaliana.

Phototropism is induced by blue light, which also induces desensitization, a partial or total loss of phototropic responsiveness. The fluence and fluence-rate dependence of desensitization and recovery from desensitization have been measured for etiolated and red light (669-nm) preirradiated Arabidopsis thaliana seedlings. The extent of desensitization increased as the fluence of the desensitizing 450-nm light was increased from 0.3 to 60 micromoles m-2 s-1. At equal fluences, blue light caused more desensitization when given at a fluence rate of 1.0 micromole m-2 s-1 than at 0.3 micromole m-2 s-1. In addition, seedlings irradiated with blue light at the higher fluence rate required a longer recovery time than seedlings irradiated at the lower fluence rate. A red light preirradiation, probably mediated via phytochrome, decreased the time required for recovery from desensitization. The minimum time for detectable recovery was about 65 s, and the maximum time observed was about 10 min. It is proposed that the descending arm of the fluence-response relationship for first positive phototropism is a consequence of desensitization, and that the time threshold for second positive phototropism establishes a period during which recovery from desensitization occurs.     

High pigment1 mutation negatively regulates phototropic signal transduction in tomato seedlings

Phototropins and phytochromes are the major photosensory receptors in plants and they regulate distinct photomorphogenic responses. The molecular mechanisms underlying functional interactions of phototropins and phytochromes remain largely unclear. We show that the tomato (Lycopersicon esculentum) phytochrome A deficient mutant fri lacks phototropic curvature to low fluence blue light, indicating requirement for phytochrome A for expression of phototropic response. The hp1 mutant that exhibits hypersensitive responses to blue light and red light reverses the impairment of second-positive phototropic response in tomato in phytochrome A-deficient background. Physiological analyses indicate that HP1 functions as a negative regulator of phototropic signal transduction pathway, which is removed via action of phytochrome A. The loss of HP1 gene product in frihp1 double mutant allows the unhindered operation of phototropic signal transduction chain, obviating the need for the phytochrome action. Our results also indicate that the role of phytochrome in regulating phototropism is restricted to low fluence blue light only, and at high fluence blue light, the phytochrome A-deficient fri mutant shows the normal phototropic response.     

Sugar and Organic Acid Accumulation in Guard Cells of Vicia faba in Response to Red and Blue Light

Changes in neutral sugar and organic acid content of guard cells were quantitated by high-performance liquid chromatography during stomatal opening in different light qualities. Sonicated Vicia faba epidermal peels were irradiated with 10 [mu]mol m-2 s-1 of blue light, a fluence rate insufficient for the activation of guard cell photosynthesis, or 125 [mu]mol m-2 s-1 of red light, in the presence of 1 mM KCl, 0.1 mM CaCl2. The low-fluence-rate blue light stimulated an average net stomatal opening of 4.7 [mu]m in 2 h, whereas the saturating fluence rate of red light stimulated an average net opening of 3.8 [mu]m in 2 h. Under blue light, the malate content of guard cells increased to 173% of the initial level during the first 30 min of opening and declined as opening continued. Sucrose levels continuously rose throughout the blue light-stimulated opening, reaching 215% of the initial level after 2 h. The starch hydrolysis products maltose and maltotriose remained elevated at all times. Under red light, guard cells showed very little increase in organic acid or maltose levels, whereas sucrose levels increased to 208% of the initial level after 2 h. Total measured organic metabolite concentrations were correlated with stomatal apertures in all cases except where substantial malate increases occurred. These results support the hypothesis that light quality modulates alternative mechanisms of osmotic accumulation in guard cells, including potassium uptake, photosynthetic sugar production, and starch breakdown.     

Photoregulation of growth and branching of plum shoots: Physiological action of two photosystems

Summary Plum shoot proliferation was investigated in terms of two distinct processes: axillary bud differentiation and axillary shoot development. Results showed that light quality influenced bud differentiation and interacted with apical dominance in determining shoot outgrowth, resulting in a differentiated structure of shoot clusters and type of branching. Results suggested that blue light, acting through its photoreceptor, increased the number of axillary buds differentiated from apical meristem, but did not remove the apical dominance. Red light removed apical dominance, while reducing the formation of axillary buds; both events appeared to be dependent on the putative amount of phytochrome active form, and independent of light photon fluence rate. On the contrary, blue light action appeared to be dependent on photon fluence rate. In addition, apparent blue-red interactions related to photomorphogenic events fit an antagonistic model for branching regulated by light via cryptochrome and phytochrome photoreceptors. Our results show that the dynamics of shoot cluster development is the product of two events: the formation of new axillary buds and their release from apical dominance.     

Inhibition of hypocotyl elongation by ultraviolet-B radiation in de-etiolating tomato seedlings. I. The photoreceptor

Broad-band UV-B radiation inhibited hypocotyl elongation in etiolated tomato ( Lycopersicon esculentum Mill. cv. Alisa Craig) seedlings. This inhibition could be elicited by < 3 μmol m⁻² s⁻¹ of UV-B radiation provided against a background of white light (> 620 μmol m⁻² s⁻¹ between 320 and 800 nm), and was similar in wild-type and phytochrome-1-deficient aurea mutant seedlings. These observations suggest that the effect of UV-B radiation is not mediated by phytochrome. An activity spectrum obtained by delivering 1 μmol m⁻² s⁻¹ of monochromatic UV radiation against a while light background (63 μmol m⁻² s⁻¹ showed maximum effectiveness around 300 nm, which suggests that DNA or aromatic residues in proteins are not the chromophores mediating UV-B induced inhibition of elongation. Chemicals that affect the normal (photo)chemistry of flavins and possibly pterins (KI, NaN, and phenylacetic acid) largely abolished the inhibitor) effect of broad-hand UV-B radiation when applied to the root zone before irradiation. KI was effective at concentrations < 10⁻⁴ M , which have been shown in vitro to be effective in quenching the triplet excited stales of flavins but not fluorescence from pterine or singlet states of flavins. Elimination of blue light or reduction of UV-A, two sources of flavin excitation, promoted hypocotyl elongation, but did not affect the inhibition of elongation evened by UV-B. Kl applied after UV-B irradiation had no effect on the inhibition response. Taken together these findings suggest that the chromophore of the photoreceptor system invoked in UV-B perception by tomato seedlings during de-etiolation may be a flavin.     

Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells.

UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species.