Regulation of pea epicotyl elongation by blue light : fluence-response relationships and growth distribution

Irradiation with blue light causes a rapid decrease in stem elongation in Pisum sativum. Growing plants under continuous red light allowed us to study the fluence dependence and spatial distribution of blue-induced growth effects without interference from large changes in the ratio of the far-red absorbing form of phytochrome to total phytochrome. The magnitude of the inhibition generated by a 30-second pulse of blue light was linearly related to the log of the fluence applied over two orders of magnitude. Reciprocity held for irradiations with a pulse length shorter than the lag time for the response. The spatial distribution of inhibition was studied by marking the growing zone and photographing the stem at 10-minute intervals before, during, and after a 1-hour exposure to blue light. The region just below the hook does not undergo any perceptible change in growth rate while growth is nearly 100% inhibited in the base of the third internode.     

Signal Integration by ABA in the Blue Light-Induced Acidification of Leaf Pavement Cells in Pea (Pisum sativum L. var. Argenteum)

Leaf pavement cell expansion in light depends on apoplastic acidification by a plasma membrane proton-pumping ATPase, modifying cell wall extensibility and providing the driving force for uptake of osmotically active solutes generating turgor. This paper shows that the plant hormone ABA inhibits light-induced leaf disk growth as well as the blue light-induced pavement cell growth in pea (Pisum sativum L.). In the phytochrome chromophore-deficient mutant pcd2, the effect of ABA on the blue light-induced apoplastic acidification response, which exhibits a high fluence phase via phytochrome and a low fluence phase via an unknown blue light receptor, is still present, indicating an interaction of ABA with the blue light receptor pathway. Furthermore, it is shown that ABA inhibits the blue light-induced apoplastic acidification reversibly. These results indicate that the effect of ABA on apoplastic acidification can provide a mechanism for short term, reversible adjustment of leaf growth rate to environmental change.Key Words: ABA, apoplastic acidification, blue light, epidermal pavement cell growth, leaf growth, pea (Pisum sativum L.), signal integration     

Two distinct blue-light responses regulate epicotyl elongation in pea

Blue light induces a long-term suppression of epicotyl elongation in red-light-grown pea (Pisum sativum L.) seedlings. The fluence-response characteristics are bell-shaped, indicating the possibility of two different blue-light responses: a lower fluence response causing suppression and a higher fluence response alleviating the suppression. To determine if two responses are in effect, we have grown pea seedlings under dark conditions hoping to eliminate one or the other response. Under these growth conditions, only the lower fluence portion of the response (suppression of elongation) is apparent. The kinetics of suppression are similar to those observed for the lower fluence response of red-light-grown seedlings. The response to blue light in the dark-grown seedlings is not due to the excitation of phytochrome because a pulse of far-red light large enough to negate phytochrome-induced suppression has no effect on the blue-light-induced suppression. Furthermore, treatment of the dark-grown seedlings with red light immediately prior to treatment with high fluence blue light does not elicit the higher fluence response, indicating that the role of red light in the blue high fluence response is to allow the plant to achieve a specific developmental state in which it is competent to respond to the higher fluences of blue light.     

Photophysiology and phytochrome content of long-hypocotyl mutant and wild-type cucumber seedlings

Photomorphogenetic responses have been studied in a cucumber (Cucumis sativus L.) mutant (lh), which has long hypocotyls in white light (WL). While etiolated seedlings of this mutant have a similar phytochrome content and control of hypocotyl elongation as wild type, deetiolation is retarded and WL-grown seedlings show reduced phytochrome control. Spectrophotometric measurements exhibit that WL-grown tissues of the lh mutant (flower petals and Norflurazon-bleached leaves) contain 35 to 50% of the phytochrome level in the wild type. We propose that this is a consequence of a lack of light-stable phytochrome, in agreement with our hypothesis proposed on the basis of physiological experiments. The lh mutant lacks an end-of-day far-red light response of hypocotyl elongation. This enables the end-of-day far-red light response, clearly shown by the wild type, to be ascribed to the phytochrome, deficient in the lh mutant. Growth experiments in continuous blue light (BL) and continuous BL + red light (RL) show that when RL is added to BL, hypocotyl growth remains inhibited in the wild type, whereas the lh mutant exhibits significant growth promotion compared to BL alone. It is proposed that the hypocotyls fail to grow long in low fluence rate BL because photosynthesis is insufficient to sustain growth.     

Evidence for two photoreceptors controlling growth in de-etiolated seedlings

The effect of blue light on hypocotyl extension in de-etiolated seedlings of lettuce, cucumber and tomato was investigated under conditions which precluded the involvement of phytochrome. Small but highly inhibitory amounts of blue light were added to a high intensity background illumination from low pressure sodium lamps. A log-linear response for inhibition of hypocotyl extension against the blue light fluence rate was obtained for lettuce and cucumber, and inhibition in tomato was also related to the blue light fluence rate. The added blue light did not alter phytochrome photostationary state and its effect was independent of the total fluence rate. Growth inhibition by Pfr could be demonstrated in tomato and cucumber but not in lettuce. The results indicate that two photoreceptors may normally be involved in the control of seedling growth but their relative importance varies greatly between species.Abbreviations HIR high irradiance reaction - Pfr far red absorbing form of phytochrome - Pr red absorbing form of phytochrome     

Blue- and red-light action in photoorientation of chloroplasts in Adiantum protonemata

An action spectrum for the low-fluencerate response of chloroplast movement in protonemata of the fern Adiantum capillus-veneris L. was determined using polarized light vibrating perpendicularly to the protonema axis. The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm, the blue peaks being higher than the red one. The red-light action was suppressed by nonpolarized far-red light given simultaneously or alternately, whereas the bluelight action was not. Chloroplast movement was also induced by a local irradiation with a narrow beam of monochromatic light. A beam of blue light at low energy fluence rates (7.3·10^-3-1.0 W m^-2) caused movement of the chloroplasts to the beam area (positive response), while one at high fluence rates (10 W m^-2 and higher) caused movement to outside of the beam area (negative response). A red beam caused a positive response at fluence rates up to 100 W m^-2, but a negative response at very high fluence rates (230 and 470 W m^-2). When a far-red beam was combined with total background irradiation with red light at fluence rates causing a low-fluence-rate response in whole cells, chloroplasts moved out of the beam area. When blue light was used as background irradiation, however, a narrow far-red beam had no effect on chloroplast distribution. These results indicate that the light-oriented movement of Adiantum chloroplasts is caused by red and blue light, mediated by phytochrome and another, unidentified photoreceptor(s), respectively. This movement depends on a local gradient of the far-red-absorbing form of phytochrome or of a photoexcited blue-light photoreceptor, and it includes positive and negative responses for both red and blue light.Abbreviations BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - UV ultraviolet     

Stimulation of growth and ion uptake in bean leaves by red and blue light

Red and blue light both stimulate growth and ion accumulation in bean (Phaseolus vulgaris L.) leaves, and previous studies showed that the growth response is mediated by phytochrome and a blue-light receptor. Results of this study confirm that there is an additional photosynthetic contribution from the growing cells that supports ion uptake and growth. Disc expansion in the light was enhanced by exogenous K⁺ and Rb⁺, but was not specific for anions. Light increased K⁺ accumulation and the rate of ⁸⁶Rb⁺ uptake by discs, over darkness, with no effect of light quality. The photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, inhibited light-driven ⁸⁶Rb⁺ uptake by 75%. Light quality caused differences in short-term kinetics of growth and acidification of the leaf surface. At comparable fluence rates (50 μmol m⁻² s⁻¹), continuous exposure to blue light increased the growth rate 3-fold after a 2-min lag, whereas red light caused a smaller growth response after a lag of 12 min. In contrast, the acidification of the leaf surface normally associated with growth was stimulated 3-fold by red light but only slightly (1.3-fold) by blue light. This result shows that, in addition to acidification caused by red light, a second mechanism specifically stimulated by blue light is normally functioning in light-driven leaf growth.     

Photomorphogenic mutants of Arabidopsis thaliana reveal activities of multiple photosensory systems during light-stimulated apical-hook opening

Fluence rate-response curves were generated for red-, far-red-, and blue-light-stimulated apical-hook opening in seedlings of several photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. Compared to wild-type plants, hook opening was reduced in the phytochrome-deficient hy1, hy2, and hy6 mutants in red and far-red light at all fluence rates tested, and in low-fluence blue light, but was normal under high-irradiance blue light. In contrast, the blue-light-response mutants (blu1, blu2, and blu3) lacked the high-irradiance-dependent hook-opening response in blue light while hook opening was normal in low-fluence blue light and in red and farred light at all fluence rates tested. Hook opening in the phytochrome-B-deficient hy3 mutant was similar to wild type in all light conditions tested. The effects of the different mutations on light-induced hook opening indicate that a phytochrome(s) other than phytochrome B mediates hook opening stimulated by red, far-red and lowfluence blue light, while a blue-light-absorbing photoreceptor mediates the blue-light-sensitive high-irradiance response. Although the phytochrome and blue-light photosensory systems appear to work independently for the most part, some of their signal-transduction components may interact since the hy4, and hy5 mutants showed reduced hook-opening responses under conditions dependent on the phytochrome and blue-light-photosensory systems.We thank Jeff Young and Brian Parks for their many helpful suggestions during the progress of this research. This work was supported by National Science Foundation Grant No. DCB-9106697.     

Effect of Light on Root Hair Formation in Arabidopsis thaliana Phytochrome-Deficient Mutants

Arabidopsis thaliana lacking phytochrome A, phytochrome B or both (double mutant) were analyzed by comparing their photoresponse with that of the wild type. Results indicate that root hair formation in Arabidopsis was strongly stimulated by light irradiation. Both phytochrome A and phytochrome B are responsible for photoinduction by continuous red light irradiation, while only phytochrome A mediates the response under continuous far-red light. The fluence response relationships to a red light pulse in the wild type displayed a biphasic trend similar to that previously observed in lettuce seedlings, with the first phase showing a sharp maximum at 78.3 Jm⁻², and the second one operating over a wider fluence range (3,100–9,400 Jm⁻²) two orders of magnitude higher than the first one. Analysis of the fluence response curves for red light induction in the phytochrome mutants revealed that phytochrome A is responsible for the first phase in the wild type, while the second is the result of the combined action of both phytochrome A and phytochrome B. Received 13 August 1999/ Accepted in revised form 22 December 1999     

Phytochrome-mediated phototropism in de-etiolated seedlings : occurrence and ecological significance

Phototropic responses to broadband far red (FR) radiation were investigated in fully de-etiolated seedlings of a long-hypocotyl mutant (lh) of cucumber (Cucumis sativus L.), which is deficient in phytochrome-B, and its near isogenic wild type (WT). Continuous unilateral FR light provided against a background of white light induced negative curvatures (i.e. bending away from the FR light source) in hypocotyls of WT seedlings. This response was fluence-rate dependent and was absent in the lh mutant, even at very high fluence rates of FR. The phototropic effect of FR light on WT seedlings was triggered in the hypocotyls and occurred over a range of fluence rates in which FR was very effective in promoting hypocotyl elongation. FR light had no effect on elongation of lh-mutant hypocotyls. Seedlings grown in the field showed negative phototropic responses to the proximity of neighboring plants that absorbed blue (B) and red light and back-reflected FR radiation. The bending response was significantly larger in WT than in lh seedlings. Responses of WT and lh seedlings to lateral B light were very similar; however, elimination of the lateral B light gradients created by the proximity of plant neighbors abolished the negative curvature only in the case of lh seedlings. More than 40% of the total hypocotyl curvature induced in WT seedlings by the presence of neighboring plants was present after equilibrating the fluence rates of B light received by opposite sides of the hypocotyl. These results suggest that: (a) phytochrome functions as a phototropic sensor in de-etiolated plants, and (b) in patchy canopy environments, young seedlings actively project new leaves into light gaps via stem bending responses elicited by the B-absorbing photoreceptor(s) and phytochrome.     

Light-Regulated Gravitropism in Seedling Roots of Maize

Red light-induced changes in the gravitropism of roots of Zea mays variety Merit is a very low fluence response with a threshold of 10⁻⁹ moles per square meter and is not reversible by far red light. Blue light also affects root gravitropism but the sensitivity of roots to blue is 50 to 100 times less than to an equal fluence of red. In Z. mays Merit we conclude that phytochrome is the sole pigment associated with light-induced changes in root gravitropism.     

Genetic analysis of the roles of phytochromes A and B1 in the reversed gravitropic response of the lz-2 tomato mutant

The lz-2 mutation in tomato ( Lycopersicon esculentum ) causes conditional reversal of shoot gravitropism by light. This response is mediated by phytochrome. To further elicit the mechanism by which phytochrome regulates the lz-2 phenotype, phytochrome-deficient lz-2 plants were generated. Introduction of au alleles, which severely block chromophore biosynthesis, eliminated the reversal of hypocotyl gravitropism in continuous red and far-red light. The fri ¹ and tri ¹ alleles were introduced to specifically deplete phytochromes A and B1, respectively. In dark-grown seedlings, phytochrome A was necessary for response to high-irradiance far-red light, a complete response to low fluence red light, and also mediated the effects of blue light in a far-red reversible manner. Loss of phytochrome B1 alone did not significantly affect the behaviour of lz-2 plants under any light treatment tested. However, dark-grown lz-2 plants lacking both phytochrome A and B1 exhibited reduced responses to continuous red and were less responsive to low fluence red light and high fluence blue light than plants that were deficient for phytochrome A alone. In high light, full spectrum greenhouse conditions, lz-2 plants grew downward regardless of the phytochrome deficiency. These results indicate that phytochromes A and B1 play significant roles in mediating the lz-2 phenotype and that at least one additional phytochrome is involved in reversing shoot gravitropism in this mutant.     

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L.

An interaction is demonstrated between the effects of phytochrome and cryptochrome (the specific blue-light photoreceptor) in the inhibition of hypocotyl elongation of light-grown cucumber (Cucumis sativus L.) cv. Ridge Greenline seedlings. At certain fluence rates of blue light the total inhibition response is greater than the sum of the separate responses to each photoreceptor. The threshold for response to blue light is reduced at least 30-fold by additional red-light irradiation. The synergistic effect is demonstrated for two different fluence rates of red light. Synergism is mediated by phytochrome in both the cotyledons and the hypocotyl.Abbreviations and symbols BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - R red light - photostationary state of phytochrome - _c calculated      

Effects of Blue Light Pretreatments on Internode Extension Growth in Mustard Seedlings after the Transition to Darkness: Analysis of the Interaction with Phytochrome

The effects of blue light (B) pretreatments on internode extensiongrowth and their possible interaction with phytochrome mediatedresponses were examined in Sinapis alba seedlings grown for11 d under 280 µmol m^–2 s^–1 of continuousblue-deficient light from low pressure sodium lamps (SOX). SupplementaryB (16 µmol m^–2 s^–1) caused no detectable inhibitionof the first internode growth rate under continuous SOX, butgrowth rate was inhibited after transfer to darkness. This effect,and the growth promotion caused by far-red bend-of-day' lightpulses were additive. The addition of B at 16 µmol m^–2s^–1 during 11 d, or only during the first 9 or 10 d orthe latest 0.75, 1 or 2 d of the SOX pretreatment caused approximatelythe same extent of inhibition after the transition to darkness.A single hour of supplementary B before darkness caused morethan 50% of the maximum inhibition. However, 24 h of lower fluencerates of B (4 or 7 µmol m^–2 s^–1) were ineffective.Covering the internode during the supplementary B period didnot prevent the response to B after the transition to darkness.Far-red light given simultaneously with B (instead of the SOXbackground) reduced the inhibitory effect of B. Above a given threshold fluence rate, B perceived mainly inthe leaves inhibits extension growth in subsequent darkness,provided that high phytochrome photo-equilibria are presentduring the irradiation with B. Once triggered, this effect doesnot interact significantly with the ‘end-of-day’phytochrome effect. Key words: Blue light, extension growth, phytochrome     

Four distinct photoreceptors contribute to light-induced side branch formation in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Side branch formation in the moss, Physcomitrella patens, has been shown to be light dependent with cryptochrome 1a and 1b (Ppcry1a and Ppcry1b), being the blue light receptors for this response (Imaizumi et al. in Plant Cell 14:373, 2002). In this study, detailed photobiological analyses were performed, which revealed that this response involves multiple photoreceptors including cryptochromes. For light induction of branches, blue light of a fluence rate higher than 6 μmol m⁻² s⁻¹ for period longer than 3 h is required. The number of branches increased with the increase in fluence rate and in the irradiation period. The number of branches also increased when red light was applied together with the blue light, although red light alone had a very few effect. By partially irradiating a cell, both receptive sites for blue and red light were found to be located around the nucleus. Further, both red and blue light determine the positions of branches being dependent upon the vibration plane of polarized light. Red light control of branch position was nullified by simultaneous far-red light irradiation. A blue light effect on branch position was not found in lines with disrupted phototropin genes. Thus, dichroic phytochrome and phototropin, possibly on the plasma membrane, regulate branch position. These results indicate that at least four distinct photoreceptor systems, namely, cryptochromes and red light receptor around or in the nucleus, dichroic phytochrome and phototropin around the cell periphery, are involved in the light induction of side branches in the moss Physcomitrella patens.     

Far-red light-insensitive, phytochrome A-deficient mutants of tomato

We have selected two recessive mutants of tomato with slightly longer hypocotyls than the wild type, one under low fluence rate (3 mol/m²/s) red light (R) and the other under low fluence rate blue light. These two mutants were shown to be allelic and further analysis revealed that hypocotyl growth was totally insensitive to far-red light (FR). We propose the gene symbol fri (far-red light insensitive) for this locus and have mapped it on chromosome 10. Immunochemically detectable phytochrome A polypeptide is essentially absent in the fri mutants as is the bulk spectrophotometrically detectable labile phytochrome pool in etiolated seedlings. A phytochrome B-like polypeptide is present in normal amounts and a small stable phytochrome pool can be readily detected by spectrophotometry in the fri mutants. Inhibition of hypocotyl growth by a R pulse given every 4 h is quantitatively similar in the fri mutants and wild type and the effect is to a large extent reversible if R pulses are followed immediately by a FR pulse. After 7 days in darkness, both fri mutants and the wild type become green on transfer to white light, but after 7 days in FR, the wild-type seedlings that have expanded their cotyledons lose their capacity to green in white light, while the fri mutants de-etiolate. Adult plants of the fri mutants show retarded growth and are prone to wilting, but exhibit a normal elongation response to FR given at the end of the daily photoperiod. The inhibition of seed germination by continuous FR exhibited by the wild type is normal in the fri mutants. It is proposed that these fri mutants are putative phytochrome A mutants which have normal pools of other phytochromes.     

Far-red light-induced changes in intracellular potentials of spinach mesophyll cells: interaction with red light

In green plants, the large bioelectric changes that photosynthetically active light stimulates make it difficult to observe electrical potential changes related to phytochrome photoconversion. As a first step towards distinguishing between photosynthetic and phytochrome effects, we showed that red light enhances far-red stimulated intracellular potential changes in spinach (Spinacia oleracea) leaf mesophyll cells.

For a dark-adapted leaf, the response to far-red light increased during the first 10 to 30 exposures of 2.5 minutes, after which it was constant. The intracellular potential depolarized by an average of 0.3 millivolts during each 2.5-minute far-red light period, and returned to the resting value during each subsequent dark period. Continuous supplementary red light (at 1-5% of the fluence rate of the far-red light that stimulated the depolarizations) increased the response to far-red 2- to 3-fold. Supplementary red light did not amplify the response to alternating 702 nanometers light and dark periods. The Emerson enhancement effect thus does not seem to explain amplification of the response to 730 nanometers light by supplementary red light. This does not prove that photosynthetic pigments are not involved in some other way.

     

Dual effect of phytochrome A on hypocotyl growth under continuous red light

The role of phytochrome A in the control of hypocotyl growth under continuous red light (Rc) was investigated using phyA and phyB mutants of Arabidopsis thaliana, which lack phytochrome A (phyA) or phytochrome B (phyB), respectively, and transgenic seedlings of Nicotiana tabacum overexpressing Avena phyA, compared to the corresponding wild type (WT). In WT seedlings of A. thaliana, hypocotyl growth inhibition showed a biphasic response to the fluence rate of Rc, with a brake at 10^?2μmol m^?2 s^?1. At equal total fluence rate, hourly pulses of red light caused slightly more inhibition than Rc. The response to very low fluences of continuous or pulsed red light was absent in the phyA and phyA phyB mutants and present in the phyB mutant. The second part of the response was steeper in the phyA mutant than in the WT but was absent in the phyB mutant. In WT tobacco the response to Rc was biphasic. Overexpression of Avena phyA enhanced the response only at very low fluence rates of Rc (< 10^?2μmol m^?2 s^?1). In both species, the effect of hourly pulses of far-red light was similar to the maximum inhibition observed in the first phase of the response to Rc. Using reciprocity failure (i.e. higher inhibition under continuous than pulsed light) as the operational criterion, a ‘true’ high-irradiance reaction occurred under continuous far-red light but not under Rc or red plus far-red light mixtures. Native and overexpressed phyA are proposed to mediate very low fluence responses under Rc. In WT A. thaliana, this effect is counteracted by a negative action of phyA on phyB-mediated low-fluence responses.     

Interactions of phytochromes A,B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.Abbreviations HIR: High irradiance response - LFR: Low fluence response - Pfr: Far-red absorbing form of phytochrome - phyA: Phytochrome A - phyB1: Phytochrome B1 - phyB2: Phytochrome B2 - phyA(B1, B2): Phytochrome mutant deficient in phyA (B1, B2) - phyAphyB1(B1B2,AB2): Double phytochrome mutant deficient in phyA and phyB1(B1, B2) - phyAphyB1phyB2: Triple mutant deficient in phyA, phyB1 and phyB2 - VLFR: Very low fluence response - WT: Wild-type tomato Communicated by R. Reski