Absence of phyB inhibits hypocotyl elongation in dark-grown Ih cucumber seedlings: an active role for PrB

Cucumber (Cucumis sativus L.) seedlings carrying the long hypocotyl (Ih) mutation, which confers a lack of B-type phytochrome (phyB), were significantly shorter than their near-isogenic wild-type counterparts when grown in complete darkness. Relative growth rates determined for 5 mm hypocotyl regions were lower in Ih seedlings in all growing regions, and the zone of elongation was less extensive in Ih hypocotyls. Digital imaging microscopy revealed that the pattern of epidermal cell lengths along the stem axis differed between the Ih mutant and the iso-genic wild-type. These findings (and the fact that experiments were conducted under conditions where phytochrome photoconversion to the far-red-absorbing form does not occur) suggest that the red-absorbing form of phyB (PrB) is an active positive regulator of development in etiolated plants.     

Inhibition of hypocotyl elongation by ultraviolet-B radiation in de-etiolating tomato seedlings. II. Time-course, comparison with flavonoid responses and adaptive significance

UV-B radiation inhibits hypocotyl elongation in etiolated tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) seedlings acting through a photoreceptor system with peak apparent effectiveness around 300 nm. In order lo further characterize the response and gain insight into its potential ecological significance, the time-course of inhibition was measured and compared with the time-course of flavonoid accumulation in the same seedlings. When a background of strong (> 620 μmol m^?2 s^?1) white light (WL) was supplemented with low irradiance UV-B (～ 3 μmol m^?2 s^?1). substantial (～ 50%) inhibition of elongation occurred within 3 h of the light treatment. The magnitude of UV-B-induced elongation inhibition was similar in wild type (WT) and au-mutant seedlings, in spite of the large differences between genotypes in rate and temporal pattern of elongation. In comparison to the effect of UV-B on elongation, induction of flavonoid accumulation in WT and au seedlings undergoing de-etiolation was a much slower response. Several UV-absorbing compounds appeared to be specifically induced by light, and some of them accumulated faster under the WL + UV-B treatment than under WL alone. However, there was little or no delectable effect of WL on flavonoid levels until up to 3 h of treatment, and the specific UV-B effect was measurable only after 6 h of continuous treatment. Indeed. UV-B-screening properties of crude alcoholic extracts were not different between WL and WL + UV-B treatments until after 9 or 24 h. When the light treatments were applied to seedlings that were just breaking through the soil surface. UV-B was found to consistently retard seedling emergence. These results suggest that the rapid inhibition of elongation in de-etiolating seedlings is an evolved response lo UV-B, which may serve to minimize seedling exposure to sunlight until protective pigmentation responses (triggered by WL and UV-B) have taken place in the seedlings epidermis.     

A double enhancement of greening in the cotyledons of cucumber seedlings by excision and red light

Cotyledons excised without the hypocotyl hook from 6-day-old etiolated cucumber ( Cucumis sativus L. var. Elem) seedlings accumulated a significantly higher amount of chlorophyll than cotyledons excised with hooks or intact cotyledons. It was found that maximum ehancement of greening was achieved after 2 h of dark incubation following excision. Pretreatments with red light effected an additive rise in chlorophyll level in subsequent white light after a dark incubation, suggesting that the effects of excision and phytochrome on greening act independently. Etiolated seedlings were variously dissected before greening and it was found that enhancement occurred only when cotyledons were excised at the level of the hypocotyl hook or above it. Similar results were obtained when the dissected plants were pre-treated with red light.     

Phytochrome-mediated control of respiratory activities of mitochondria in cotyledons of dark-grown cucumber seedlings

Mitochondria isolated from cotyledons of dark-grown cucumber ( Cucumber sativus L., cv. Shimotsuki-Aonaga) seedlings after illumination with continuous far-red light showed an increased capacity for oxidation of malate or α-ketoglutarate, as compared with those from cotyledons of non-illuminated seedlings. This increase is supposed to be caused by phytochrome action (high irradiance response). Exogenous NAD⁺ had no effect on the rate of the oxidation of α-ketoglutarate or malate by mitochondria isolated from far-red light-treated cotyledons, but it enhanced the oxidation rate of mitochondria from control cotyledons to the level of mitochondria from light-treated ones. The NAD (NAD⁺+ NADH) content was higher in mitochondria isolated from continuously far-red light-treated cotyledons than in mitochondria from controls. The NAD content was also increased by the treatment with a red light pulse and this response was reversed by a subsequent far-red light pulse. It is proposed that phytochrome controls respiratory activities of cucumber mitochondria by changing the size of the NAD pool in the mitochondria.     

UV-B response of cucumber seedlings grown under metal halide and high pressure sodium/deluxe lamps

UV-B-sensitive (Poinsett) and -insensitive (Ashley) cultivars of cucumber ( Cucumis sativus L.) were grown in growth chambers at 600 μmol m⁻²s⁻¹ of photosynthetically active radiation provided by metal halide (MH) or high pressure sodium/deluxe (HPS/DX) lamps. Plants were irradiated 15 days from seeding for 6 h per day under 18. 2 kJ m⁻² day⁻¹ of biologically effective UV-B (UV-B_BE) radiation. One of the most pronounced effects of UV-B was a 27 to 78% increase in phenylalanine ammonialyase (PAL) activity. UV-B also increased total polyamines. Catalase and superoxide dismutase varied greatly in their response to UV-B. There were no interactive effects on PAL or catalase activity, or total polyamines. There was a UV × PAR source interaction for superoxide dismutase activity. UV-B increased chlorosis and decreased height, dry weight and leaf area. Stem elongation, biomass production, leaf enlargement and chlorosis were greater under HPS/DX lamps than under MH lamps. Chlorosis was greater in Poinsett than in Ashley and in lower leaves than in upper ones. Aside from chlorosis, there were no interactive effects of UV-B, PAR source or cultivar on any of the growth parameters measured, suggesting that the growth response of cucumber seedlings to UV-B is unaffected by PAR source or cultivar. Similarly, except for SOD activity, the biochemical response to UV-B was also not influenced by PAR source or cultivar.     

Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axrl-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.     

Effect of UV-B (290–320 nm) irradiation on growth and metabolism of cucumber cotyledons

Cucumber ( Cucumis sativus L. cv. Natsusairaku 3) seedlings were grown in a growth cabinet under UV-B (290–320 nm) irradiation (equivalent to the UV-B radiation normally incident at Tokyo, 36°N latitude, during clear sky conditions in mid-april on a weighted daily fluence basis) and a UV-B-free control condition. UV-B irradiation inhibited the growth of the cotyledons, i.e. the increase in area, and increase in fresh and dry weights of the cotyledons. The greatest inhibition rate was observed in the increase in area, causing a significant increase in specific leaf weight (the ratio of weight to area). UV-B irradiation had no significant effect on DNA and RNA contents in the cotyledons, but decreased protein content slightly. In contrast, the irradiation reduced the amounts of organic acids and soluble sugars, indicating that primary carbon metabolism was very sensitive to UV-B radiation. UV-B irradiation lowered the photosynthetic activity in the cotyledons without any effect on chlorophyll content and respiratory activity. These results indicate that UV-B radiation at the ambient level may act as a physiological stress in some UV-sensitive plants.     

Photocontrol of the hypocotyl hook opening of Sinapis alba seedlings. Involvement of phytochrome and a high irradiance response

Baumgartner, N. and Fondeville, J. C. 1989. Photocontrol of the hypocotyl hook opening of Sinapis alba seedlings. Involvement of phytochrome and a high irradiance response.
A statistical evaluation of the hypocotyl hook opening (hook opening index) was used for measurement of the hook angle in lots of etiolated Sinapis alba L. cv. Albatros seedlings. Studies of the kinetics for hook opening were carried out in continuous fluorescent white, blue and red light (6, 15 and 40 μmol m^-2s^-1) with 2-day-old dark-grown seedlings. At the beginning of the irradiation period the photoresponse in red light was the opposite to that in blue (low photon fluences). Blue rapidly induced the hook opening (in less than 20 min), while red produced hook tightening (photon fluences up to 70 mmol m^-2), which precedes the normal progressive hook opening. For low fluences, the data were consistent with the involvement of phytochrome and a specific blue light photoreceptor. A phytochrome effect was observed in the hook opening, dependent upon a high irradiance response (HIR). This HIR (like that for the inhibition of the hypocotyl elongation) was characterized by a wavelength response curve with maxima in the blue and far-red regions of the spectrum.     

The distribution of hydroxyproline-rich glycoprotein mRNAs in shoots of cucumber and pea

The distribution of hydroxyproline-rich glycoprotein (HRGP) mRNAs in the shoots of dark-grown and irradiated cucumber ( Cucumis sativus L. cv. Burpee pickler) and pea ( Pisum sativum L. cv. Alaska) was studied. A cloned genomic DNA fragment encoding carrot ( Daucus carota ) root extensin (pDC5A1) was used to measure HRGP mRNAs from cucumber and pea along the length of dark-grown and irradiated shoots. There was a marked difference in the levels of HRGP mRNAs isolated from apical and basal regions of cucumber. Whereas apical, elongating regions had low levels of HRGP mRNAs, basal regions of the shoot had high levels. Levels of HRGP mRNAs were also compared in shoots of dark-grown and irradiated cucumber. Although light inhibits hypocotyl growth, it had no effect on levels of HRGP mRNAs. There was no gradient in the distribution of HRGP mRNAs along the epicotyl of dark-grown pea. As was the case with cucumber, light did not affect the accumulation of HRGP mRNAs in pea shoots. We conclude that light does not affect elongation by regulating the accumulation of HRGP mRNAs. The gradient of accumulation of HRGP mRNAs along the hypocotyl of cucumber probably reflects differences in cellular differentiation along the shoot.     

Nitrate reductase in cotyledons of cucumber seedlings as affected by nitrate, phytochrome and calcium

Regulation by the active form of phytochrome (P_FR) and the effect of Ca²⁺ was examined with nitrate reductase (NR) in etiolated cucumber ( Cucumis sativus cv. Beilpuig). Nitrate reductase activity (NRA) was studied in excised cotyledons of cucumber seedlings grown in distilled water and in darkness for seven days at 24 ± 0.5°C. All experiments were performed in the dark and a dim green safelight was used during analyses. In etiolated cucumber cotyledons NRA was induced by nitrate and a brief irradiation (15 min) with red light (R) resulted in 62% increase in NRA. This effect was nullified when R was followed immediately by a brief (5 min) far-red light (FR). NRA also showed a semidian (12 h) rhythmicity. Both P_FR, and nitrate effects were age dependent. Calcium seemed to be involved since the phytochrome effect was only observed when calcium was supplied in the external solution. The effect of R on NRA depended on the period of calcium nitrate incubation. An external supply of calcium ionophore mimicked the effect of R and, if supplied to R-irradiated cotyledons, produced a higher NR level than that caused by R alone. This suggested that intracellular free calcium was involved.     

A morphological and cytological study of Petunia hybrida exposed to UV-B radiation

The aim of this study was to investigate whether the cytoskeleton, and in particular the microtubular system, is affected by enhanced levels of ultraviolet-B (280–320 nm, 9 kJ m⁻² day⁻¹ biologically effective UV-B radiation) radiation in epidermal cells of Petunia x hybrida Vilm, isolated from leaves of plants grown under UV-B radiation and visible light. In addition, morphological changes during development were monitored. In a previous study microtubules were depolymerized and delays in the different stages of the cell cycle were found when protoplasts of Petunia were irradiated with UV-B radiation (Staxén et al. 1993. Protoplasma 173: 70–76). Thus it was of interest to ascertain whether the cytoskeleton would be similarly affected in an intact system. Assuming an effect of UV-B radiation on the microtubular system, we wished to determine whether this could be correlated to concomitant changes in leaf morphology. Plants of Petunia hybrida were grown in greenhouse conditions in the presence or absence of UV-B radiation. During the course of the experiment, samples were taken from young, expanding leaves and from older, fully expanded leaves and prepared for localization and analysis of microtubules from the adaxial epidermal cells. Morphology rather than the cytoskeleton was affected by UV radiation, despite the fact that the epidermal cytoskeleton would most likely be affected, since it is located in the cells which form the first intercepting layer for incident radiation.
Morphological changes under UV-B radiation, as compared to those under control conditions, were reflected in earlier flowering and an increase in leaf number. Cell division was thus stimulated as was also evidenced from the increased leaf area. Our results indicate that the number of stomata differentiated on a leaf area basis was not altered although the number of stomata per epidermal cell was reduced.     

Changes of polyamines and ethylene in cucumber seedlings in response to chilling stress

Cucumber ( Cucumis sativus L. cv. Victory) seedlings were exposed to chilling at 5°C and endogenous levels of polyamines and 1-aminocyclopropane-1-carboxylic acid (ACC) were measured after chilling and after warming at 20°C. The level of spermidine was higher in the chilled seedlings than in the non-chilled seedlings. Treatment with a plant bioregulator, (2RS,3RS)-1-(4-cholorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol (paclobutrazol), reduced the chilling injury and the levels of spermidine in the chilled seedlings. The levels of ACC and production of ethylene showed sharp increases after warming following exposure to chilling. These increases were suppressed by the application of aminooxyacetic acid (AOA). However, AOA treatment did not reduce chilling injury or affect the levels of polyamines in the tissue. These data indicate that the increase in ACC and ethylene is a response of the tissue to the chilling exposure and is not a cause of the injury. The data also suggest that the syntheses of polyamines and ethylene are not competitive with each other even under chilling stress conditions.     

Identification in lettuce seedlings of a catecholamine active in synergistically enhancing the gibberellin effect on lettuce hypocotyl elongation

The occurrence of catecholamines in lettuce seedlings was examined by bioassay and gas chromatography-mass spectrometry (GC-MS), since synthetic catecholamines can synergistically enhance the stimulating effect of gibberellic acid (GA₃) on hypocotyl elongation of decotylized lettuce seedlings. The catecholamine fraction on TLC obtained from lettuce seedlings synergistically enhanced the GA₃ effect on hypocotyl elongation. The analysis of the catecholamine fraction from lettuce seedlings by GC-MS demonstrated the occurrence of dopamine.     

Short-term UV-B radiation and ozone exposure effects on aromatic secondary metabolite accumulation and shoot growth of flavonoid-deficient Arabidopsis mutants

The presence of UV-absorptive substances in the epidermal cells of leaves is thought to protect mesophyll tissues from the harmful effects of UV-B radiation. We examined the influence of short-term UV-B exposures on UV-absorptive (330 nm) sinapates and flavonols, and on shoot growth of the Arabidopsis wild type ecotype Landsberg erecta and two mutants. 114 deficient in chalcone synthase, and 115 , deficient in chalcone/flavonone isomerase. Sequential ozone exposures were used to determine the effects of oxidative stress The levels of sinapates and flavonols on a leaf fresh weight basis increased substantially in the wild type and sinapates increased in the 114 mutant in vegetative vegetative/reproductive transitional and reproductive stage plants in response to short-term (48h) UV-B radiation. When UV-B was discontinued the levels generally decreased lo pre-exposure levels after 48 h in vegetative/reproductive but not in reproductive plants. Exposure to ozone before or alter UV-B treatment did not consistently affect the levels of these UV-absorptive compounds. Dry matter accumulation was less affected by UV-B at the vegetative and reproductive stages than at the vegetative/reproductive stage. At the vegetative/reproductive stage, shoot growth of all 3 genotypes was retarded by UV-B. Growth was not retarded by short-term ozone exposure alone but when exposure to ozone followed UV-B exposure, growth was reduced in all genotypes. Leaf cupping appeared on 115 plants exposed to UV-B.     

Growth analysis of UV-B-irradiated cucumber seedlings as influenced by photosynthetic photon flux source and cultivar

A growth analysis was made of ultraviolet-B (UV-B)-sensitive (Poinsett) and insensitive (Ashley) cultivars of Cucuumis satives L. grown in growth chambers at 600 μmol m⁻² s⁻¹ of photosynthetic photon flux (PPF) provided by red- and far-red-deficient metal halide (MH) or blue- and UV-A-deficient high pressure sodium/deluxe f HPS/DX) lamps. Plants were irradiated 6 h daiiy with 0.2 f-UV-B) or 18.2 C+UV-B) kJ m⁻² day⁻¹ of biologically effective UV-B for 8 or 15 days from time of seeding. In general, plants given supplemental UV-B for 15 days showed lower leaf area ratio (LARs, and higher specific leaf mass (SLM) mean relative growth rate (MRGR) and net assimilation rate (NAR) than that of control plants, but they showed no difference in leaf mass ratio (LMR), Plants grown under HPS/DX lamps vs MH lamps showed higher SLM and NAR. lower LAR and LMR. hut no difference in MRGR. LMR was the only growth parameter affected by cultivar: at 15 days, it was slightly greater in Poinsett than in Ashley. There were no interactive effects of UV-B. PPF source or cultivar on any of the growth parameters determined, indicating that the choice of either HPS/DX or MH lamps should not affect growth response to UV-B radiation. This was true even though leaves of UV-B-irradiated plants grown under HPS/DX lamps have been shown to have greater chlorosis than those grown under MH lamps.     

Enhancement of the Tolerance to Oxidative Stress in Cucumber (Cucumis sativus L.) Seedlings by UV-B Irradiation: Possible Involvement of Phenolic Compounds and Antioxidative Enzymes

Cucumis sativus L.) seedlings were irradiated or not irradiated with UV-B for several days in environment-controlled growth chambers. The first leaves irradiated with UV-B were retarded in growth but simultaneously acquired a remarkably high tolerance to oxidative stress, as induced by paraquat treatment, compared with the non-irradiated leaves. This enhanced tolerance was observed within 1d after the start of UV-B irradiation and was maintained during the 12 d period of UV-B treatment. The effects of UV-B on several antioxidative enzymes were examined, and activities of superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase, but not of glutathione reductase, were found to be enhanced. However, activation of these enzymes occurred only from 6 d after the start of irradiation. In contrast, accumulation of phenolic compounds was observed within 1d after the start of UV-B irradiation. HPLC analysis of phenolic compounds showed the distinct enhancement of a substance, which may have antioxidative properties in cucumber seedlings irradiated with UV-B. On the basis of these results, we conclude that not only antioxidative enzymes but also other factors in cucumber seedlings irradiated with UV-B, such as phenolic compounds, may participate in the enhanced tolerance to oxidative stress. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Photocontrol of stem elongation in plant neighbourhoods: effects of photon fluence rate under natural conditions of radiation

Abstract. We investigated the effects of photon fluence rate on internode elongation in fully de-etiolated plants growing under sunlight. Our goal was to find out whether perception by the stems of fluence rate changes related to canopy density may be involved in the modulation of internode growth in canopies formed by plants of similar stature (e.g. crop stands). Using Datura ferox L. and Sinapis alba L. seedlings growing under natural radiation, we found that internode elongation is promoted by localized shading. This effect was observed with internodes receiving light with a high (>0.9) or a low (0.3) red (R) to far-red (FR) photon ratio. Selective removal of the different wavebands from the light impinging on the internodes showed that part of the response to fluence rate is due to photons in the R + FR range. The blue (B) component, most likely acting through a specific photoreceptor, also inhibited elongation. However, changes in the fluence rate of B light did not have detectable effects on the response of the internodes to R:FR ratio. Fibre-optic studies and measurements with integrating-cylinder sensors in even-aged populations of seedlings showed that both the quality and quantity of radiation received by the stems are profoundly influenced by changes in canopy density. When density is very low (leaf area index = LAI ≥ 1) only the R:FR ratio is reduced, due to FR reflected from nearby leaves. In the LAI range of 1 to 2, though a large proportion of the leaf area is still receiving full sunlight, the photon fluence rate at the stem level drops dramatically. These results suggest that in even-aged populations of LAI > 1 elongation growth is promoted by the low R:FR ratio and the reduced fluence rate. Perception of these two factors at the stem level may elicit morphological adaptations in the canopy before the onset of severe competition among neighbours for the resource of light.     

Phytochrome A, phytochrome B and HY4 are involved in hypocotyl growth responses to natural radiation in Arabidopsis: weak de-etiolation of the phyA mutant under dense canopies

The roles of phytochrome A (phyA), phytochrome B (phyB) and a putative blue-light (BL) photoreceptor (HY4) in the control of hypocotyl growth by natural radiation were investigated using phyA, phyB and hy4 mutants of Arabidopsis thaliana. Full sunlight inhibited hypocotyl growth to a larger extent in wild-type (WT) than in phyA, phyB and, particularly, hy4 seedlings. In WT seedlings, hypocotyl growth was promoted by selectively lowering BL irradiance, lowering red-light (R) plus far-red-light (FR) irradiance or lowering the R/FR ratio (which was achieved either by increasing FR or by reducing R). The effects of lowering BL were reduced in hy4 and exaggerated in phyA seedlings. The effects of lowering R+FR were reduced in phyA and exaggerated in hy4 seedlings. Neither phyB nor hy4 mutants responded to low R/FR ratios. Neighbouring plants reflecting FR without shading caused subtle reductions of the R/FR ratio. This signal promoted hypocotyl growth in WT but not in phyA, phyB or hy4 seedlings. Intermediate canopy shade produced similar effects in all genotypes. Under deep shade, de-etiolation was severely impaired in phyA seedlings, which died prematurely. Thus, the FR ‘high-irradiance reaction’ mediated by phyA could be important for seedling survival under dense canopies.