Phytochrome A and phytochrome B1 control the acquisition of competence for shoot regeneration in tomato hypocotyl

 We analysed the light-dependent acquisition of competence for adventitious shoot formation in hypocotyls of phytochrome A (fri) and phytochrome B1 (tri) mutants of tomato and their wild type by pre-growing the seedlings under different light quality. The regenerative response in vitro of explants from etiolated seedlings was reduced in comparison to that displayed by light-grown ones. Our results indicate that the light-dependent acquisition of competence for shoot regeneration in the tomato hypocotyl is regulated by phytochrome and antagonistically by a blue-light receptor. By using phytochrome mutants and narrow wave band light we showed that it is mediated at least by two distinct phytochrome species: phytochrome B1 and phytochrome A. The action of phytochrome B1 during seedling growth was sufficient to induce the full capacity of the subsequent regenerative response in vitro in explants from all positions along the hypocotyls. In contrast far-red light acting through phytochrome A did not induce the full capability of shoot regeneration from middle and basal segments of the hypocotyl when phytochrome B1 was absent (tri mutant). A few middle and basal hypocotyl explants pre-grown in blue light regenerated shoots. Received: 12 April 1999 / Revision received: 5 July 1999 · Accepted: 6 August 1999     

The Role of Phytochrome in Anthocyanin Synthesis in Seedlings of Vigna radiata (L.) Wilczek

Previously, it has been demonstrated that the red light-inducedanthocyanin accumulation in mung bean seedlings is mediatedby phytochrome [Dumortier and Vendrig Z. Pflanzenphysiol. 87:313 (1978)]. In this paper the importance of phytochrome forthe accumulation of anthocyanins in seedlings of mung beanswas studied in non-irradiated seedlings and in seedlings irradiatedwith 5 min R. A short FR-irradiation given early after sowing reduced theamount of anthocyanins which were normally found in non-irradiatedseedlings. This indicates that P_FR may be important for at leastpart of the anthocyanin synthesis in the dark. As for the redlight-mediated anthocyanin accumulation, irradiation appearedto be most effective when given to seedlings at the age of 36–48hr. Although the seedlings were sensitive to red light irradiationbefore that time, they were not able to synthesize anthocyaninsuntil they had reached the age of 36 hr. Complete escape ofred/far-red reversibility occurred only when far-red was given12 hr after red, although partial escape could be observed witha shorter time-interval. Furthermore, the time-course of anthocyaninaccumulation after a two-fold R-irradiation was compared withthe effect of a single R-exposure. From the results could beconcluded that the pattern of anthocyanin accumulation is dependenton the time during which P_FR is present in the seedlings. Theseexperiments also indicate that P_FR not only plays a role inthe synthesis of anthocyanins but probably also in their degradation. The results of our study show that phytochrome is importantfor anthocyanin accumulation in non-irradiated mung bean seedlingsas well as in R-irradiated, and that it probably is also involvedin the degradation of the pigment. (Received January 18, 1982; Accepted April 30, 1982)     

Osmotic relations during elongation growth in hypocotyls of Helianthus annum L.

The relationship between growth, change in cell osmotic pressure and accumulation of osmotic solutes was investigated in hypocotyls of sunflower (Helianthus annum L.) seedlings. During growth in darkness the osmotic pressure decreased by 50% between days 2 and 6 after sowing. After irradiation of dark-grown seedlings with continuous white light (WL) an inhibition of hypocotyl growth was measured, but the osmotic pressure of the growing cells was not lower than in the dark-grown control. Growth in darkness and after WL irradiation was accompanied by an increase in the amount of osmotic substances (soluble sugars) which was proportional to the increase in length of the organ. During growth in continuous WL the cell osmotic pressure decreased by 45 % between days 2 and 6 after sowing. The transfer of WL-grown seedlings to darkness (“re-etiolation”) resulted in a rapid acceleration of hypocotyl growth, but the cell osmotic pressure was the same as that of the WL grown control. Growth in continuous WL was accompanied by a corresponding accumulation of osmotic substances (soluble sugars). The transition from WL to darkness resulted in an enhanced accumulation of osmotica and an increase in cell-wall extensibility. The results indicate that the relative maintenance of cell osmotic pressure during rapid hypocotyl growth in darkness is caused by an enhanced accumulation of soluble sugars into the growing cells of the organ.     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

Impaired stem-growth responses to blue-light irradiance in light-grown transgenic tobacco seedlings overexpressing Avena phytochrome A

The effects of blue light (B) on stem extension-growth were compared in light-grown seedlings, of tobacco overexpressing Avena phytochrome A and its isogenic wild type (WT). Under natural radiation, lowering the levels of B reaching the whole shoot promoted stem extension growth in WT but not in transgenic seedlings. Under controlled conditions, the seedlings were exposed to white light (WL) or WL minus B, each one provided at two different irradiances. In WT seedlings stem extension growth was promoted by lowering B at both irradiance levels. In transgenic seedlings a reduction of B was promotive only at low irradiance levels. The seedlings were also grown under WL, WL minus B, WL minus red light (R) and far-red light (FR) or WL minus R, FR and B. In the WT, lowering B promoted stem extension growth irrespective of R+FR levels. In the transgenics, B was effective only at very low levels of R+FR (i.e. at low phytochrome cycling rates). Lowering the Pfr levels at the end of the day promoted extension growth in wild type and transgenic seedlings. Responses to B were not observed in transgenic seedlings having low Pfr levels at the end of the day. The results suggest that the overexpressed phytochrome A acts mainly via irradiance-dependent reactions. When these reactions are highly expressed, B responses are not observed.     

Metabolic Adaptations of Plant Respiration to Nutritional Phosphate Deprivation

Shoots of the lazy-2 (lz-2) gravitropic mutant of tomato (Lycopersicon esculentum Mill.) have a normal gravitropic response when grown in the dark, but grow downward in response to gravity when grown in the light. Experiments were undertaken to investigate the nature of the light induction of the downward growth of lz-2 shoots. Red light was effective at causing downward growth of hypocotyls of lz-2 seedlings, whereas treatment with blue light did not alter the dark-grown (wild-type) gravity response. Downward growth of lz-2 seedlings is greatest 16 h after a 1-h red light irradiation, after which the seedlings begin to revert to the dark-grown phenotype. lz-2 seedlings irradiated with a far-red light pulse immediately after a red light pulse exhibited no downward growth. However, continuous red or far-red light both resulted in downward growth of lz-2 seedlings. Thus, the light induction of downward growth of lz-2 appears to involve the photoreceptor phytochrome. Fluence-response experiments indicate that the induction of downward growth of lz-2 by red light is a low-fluence phytochrome response, with a possible high-irradiance response component.     

Enhancement of red-light-induced anthocyanin synthesis in sorghum first internodes by moderate low temperature given in the pre-irradiation culture period

Anthocyanin synthesis in the broom sorghum, Sorghum bicolor Moench cvs. Acme Broomcorn and Sekishokuzairai-Fukuyama, is mediated separately or synergistically by an ultraviolet light-B (UV-B) photoreceptor and phytochrome. When seedlings were exposed to moderate low temperatures ranging from 12 to 20° C before irradiation, only the phytochrome-mediated anthocyanin synthesis was markedly enhanced compared with the control, which was kept throughout at 24° C; synthesis mediated by the UV-B photoreceptor was unaffected. The effectiveness of an exposure to 20° C increased as the duration of exposure increased up to 24 h and as the time of exposure became closer to the time of irradiation. However, when seedlings were exposed to 20° C from after irradiation until harvest, anthocyanin syntheses induced by both UV-B and red light were equally suppressed, probably due to the general reduction of metabolism involved in anthocyanin synthesis that is a consequence of lower temperature. The results support the view that the signal transduction of the pyhtochrome system is different from that of the UV-B photoreceptor, and indicate that the phytochrome system may involve a step or steps which are amplified by a previous exposure to the moderate low temperature.Abbreviations FR far-red light - LT low temperature - MLT moderate low temperature - Pfr far-red-light-absorbing form of phytochrome - R red light - UV ultraviolet light - UV-B ultraviolet light-B We thank Drs. Y. Takeuchi (Shionogi Pharmaceutical Company, Aburahi, Shiga) and K. Hosaka (the Experimental Farm, Kobe University, Kasai) for seeds; Dr. M. Watanabe and Mr. M. Kubota (the National Institute for Basic Biology, Okazaki) for operation of the spectrograph. This work was supported by grants from the Yamada Science Foundation, Ministry of Education (No. 63480015 and 03454048), and the National Institute for Basic Biology (Large Spectrograph grant No. 91-523).     

Light requirement,phytochrome and photoperiodic induction of flowering of Pharbitis nil Chois

For dark-grown seedlings of Pharbitis nil capacity to flower in response to a single inductive dark period was established by 24 h white, far-red (FR) or ruby-red (BCJ) light and by a skeleton photoperiod of 10 min red (R)-24 h dark-10 min R. FR alone was ineffective without a brief terminal (R) irradiation, confirming that the form of phytochrome immediately prior to darkness is a crucial factor for flowering in Pharbitis. The magnitude of the flowering response was significantly greater after 24 h FR or white light (WL) (at 18° C and 27° C) than after two brief skeleton R irradiations, but the increased flowering response was not attributable to photosynthetic CO₂ uptake because this could not be detected in seedlings exposed to 24 h WL at 18° C. Photophosphorylation could have contributed to the increased flowering response as photosystem I fluorescence was detectable in plants exposed to FR, BCJ, or WL, but there were large differences between flowering response and photosystem I capacity as indicated by fluorescence. We conclude that phytochrome plays a major role in photoresponses regulating flowering. There was no simple correlation between developmental changes, such as cotyledon expansion and chlorophyll formation during the 24-h irradiation period, and the capacity to flower in response to a following inductive dark period. Changes in plastid ultrastructure were considerable in light from fluorescent lamps and there was complete breakdown of the prolamellar body with or without lamellar stacking at 27 or 18° C, respectively, but plastid reorganization was minimal in FR-irradiated seedlings.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing from of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

Photocontrol of anthocyanin formation in turnip seedlings

Summary As measured by in vivo spectrophotometry the phytochrome content in etiolated turnip seedlings was higher in cotyledons than in hypocotyls; in the latter, it is confined to the apical part. During early growth in darkness the amount increased in both tissues to a maximum, reached about 40 hours after sowing; the levels then gradually declined. Separation of seedlings into hypocotyl and cotyledons increased the rate of phytochrome loss in the former, but not in the latter.Following 5 minutes of red light P _frdecayed very rapidly in darkness; after 1.5 hours all of the phytochrome was present as P _r, which was presumably not converted initially. In continuous red light the total phytochrome was reduced to below the detection level within 3 hours. Seedling age markedly affected the loss of phytochrome following red light; more was destroyed in older than in younger hypocotyls and apparent new synthesis occurred only in young seedlings. The capacity to synthesise phytochrome differed in cotyledons and hypocotyl. In cotyledons, synthesis occurred following shots of red light varying from 10 seconds, to 6×I minute, but the amount of newly formed phytochrome was not related to the amount destroyed: after 5 hours of continuous red light no new synthesis occurred. In hypocotyls, the amount of phytochrome synthesised was related to the amount previously destroyed, and the phytochrome content after 24 hours of darkness was similar following all red light treatments of 1 minute or longer: new synthesis occurred following 5 hours of continuous red light.In far-red light phytochrome decayed very slowly, approaching the limit of detection after 48 hours. In cotyledons some loss was already observed after 5 hours of far-red and, in hypocotyls, after about 10 hours.These results are discussed in relation to the possible role of phytochrome as the pigment mediating anthocyanin synthesis in prolonged far-red light.     

Precursors and Enzymatic Development of Caucas Flavor Components

Although coloration in plants is ascribable to both the accumulation of anthocyanin pigments in vacuoles and to the acidification of vacuolar pH, the environmental factors causing the decrease in vacuolar pH are unknown. We found that blue-light irradiation of buckwheat seedlings using light-emitting diodes caused reddening on the surface of the hypocotyls. It has also been reported that light stimulation induces an accumulation of anthocyanin pigments. However, here we confirmed for the first time on the basis of real-time PCR analysis that light stimulation simultaneously triggers expression of the genes coding for subunit A of vacuolar H⁺-ATPase (V-ATPase) and vacuolar H⁺-pyrophosphatase (V-PPase).     

Action of red and far red light on ribosome formation in cabbage seedlings

The action of light on ribosome formation was examined in the cabbage seedlings, a system extensively used in the studies of anthocyanin synthesis. Ribosomes were extracted 18 h after the beginning of the irradiation and separated by sucrose gradient centrifugation. In the cotyledons of dark-grown cabbage seedlings, a brief red light induces an increase both in total ribosomes and in the fraction present as polysomes; the effect of red light is reversed by far red light, indicating the involvement of phytochrome in polysome formation in cabbage seedlings. Continuous red and continuous far red light are about equally effective in bringing about an increase of total ribosomes and of the polysome fraction. Streptomycin, which inhibits chlorophyll synthesis and chloroplast development, and enhances anthocyanin synthesis in cabbage seedlings, causes a decrease of total ribosomes and of the fraction present as polysomes. In hypocotyls, the red-far red reversibility is evident only for the polysome content and streptomycin does not decrease the polysome/monosomo ratio as it does in cotyledons.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.     

Anthocyanin Accumulation Mediated by Blue Light and Cytokinin in Arabidopsis Seedlings

It has been reported that pigmentation In plants Is stimulated by light and cytoklnln （CTK）; however, the signaling pathways and the relationship between light and CTK Involved In the regulation of anthocyanln accumulation remain to be elucidated. We Investigated （i） the role of blue light （BL） and CTK In anthocyanln accumulation ; and （ii） the relationship between BL and CTK In wild type （WT） and by4 mutants of Arabidopsis thaiiana. Two-d-old seedlings grown on medium with or without klnetln （KT） or zeatln （ZT） In darkness were Irradiated using BL at different fluence rates for 3 d before the anthocyanln content was determined using a spectrophotometrlc method. Anthocyanln accumulation was strongly Induced by BL In WT seedlings but not In hy4 seedlings, which demonstrated that CRY1 Is the main photoreceptor for BL. Both KT and ZT enhanced the response of the WT seedlings to BL In a dose-dependent manner, whereas they were not sufficient to promote anthocyanln eccumulatlon In darkness. In addition, data from experiments using the hy4 mutant showed that the CTK effect of BL was also CRYl-dependent. The results from experiments with three different treatment programs showed that the relationship between BL and KT In anthocyanln accumulation of Arabidopsis seedlings seems neither muItlpllcatlve nor additive coactlon, but rather Interaction. BL Is necessary for anthocyanln accumulation, and KT might be Involved In the BL signaling pathway.     

Lag-phase and rate of synthesis in light-mediated anthocyanin synthesis

Mustard (Sinapis alba L.) seedlings were irradiated with continuous far-red light either with or without a pretreatment with 3 or 6 h of the same far-red light, separated by a 15 h dark period. The pretreatment increases the initial rate of anthocyanin accumulation — as caused by the 2nd light treatment — at least 6-fold but leads to an earlier cessation of anthocyanin accumulation. Moreover, the pretreatment seems to shorten the apparent lag-phase of anthocyanin accumulation considerably but it does not eliminate the lag. If the pretreatment with far-red light is terminated before the seedling reaches competence (with regard to phytochrome and anthocyanin synthesis) the pretreatment has no effect on the apparent lag-phase even though the future capacity of anthocyanin biogenesis is considerably stimulated by the pretreatment. The time course of induction of anthocyanin and that of phenylalanine ammonia-lyase (PAL) (Acton et al. 1980, Fig. 1) is in line with the concept that induction of PAL by light is a prerequisite for the onset of light-mediated anthocyanin synthesis.Abbreviation PAL phenylalanine ammonia-lyase     

Photocontrol of anthocyanin formation in turnip seedlings

Summary When irradiated for a period of 48 hours with light of restricted wave-lengths in the red and far-red regions, maximum anthocyanin content in hypocotyls was found at 730 nm; in cotyledons the yield at 716 was almost equal to that at 730 nm.In hypocotyls the effectiveness of 703, 716 and 760 nm light was much increased if plants were transferred to red light after 6 hours. The intermediate wave-length band centred at 703 nm was ineffective when given alone but led to a high yield if followed by red light. When given before far-red light (standard far-red field), 6 hours at 703 nm reduced anthocyanin yield in the same way as red light; when given after 6 hours of far-red, however, 703 nm light did not act like red light to sustain a high rate of anthocyanin synthesis.It is concluded that two reactions are involved in the photocontrol of anthocyanin formation in turnip seedlings.     

Elimination of the Far-Red-Induced Lag Phase of Chlorophyll a Accumulation by a Brief Light in Pharbitis nil Cotyledons

In dark-grown Pharbitis nil seedlings, far-red light (FR) irradiationof 48 h or less promotes Chl a accumulation in the first 2-hof a subsequent white light (WL) period, without a lag phaseof Chl a accumulation. However, continuous FR irradiation of72 h or more, causes the so-called FR-induced lag phase. A 5-minWL given 4 h before the onset of the continuous WL promotesChl a accumulation irrespective of the length of the precedingFR irradiation period, if a 4-h dark period is inserted betweenthe 5-min WL and continuous WL. This suggests that the effectof the brief WL is independent of and additive to the effectof the preceding FR irradiation, although the effect of theFR irradiation changes from promotive to inhibitory with anincrease in the irradiation period. Red light (R) is more active than blue light (B) in this brieflight effect. The R effect is reversed by subsequent exposureto FR when the period of the preceding FR irradiation is 24h, but not when it is 72 h. The relative effectiveness of Bto R increases after prolonged FR irradiation. (Received August 6, 1986; Accepted March 12, 1987)     

Functional analysis of yeast-derived phytochrome A and B phycocyanobilin adducts

Investigations of phytochrome mutants of Arabidopsis suggested that the expression of chalcone synthase ( chs ) and anthocyanin accumulation is predominantly controlled by phytochrome A. To test the functionality of phytochrome A and B at the molecular level recombinant, yeast-derived phytochrome-phycocyanobilin adducts (phyA*, phyB*) and oat phytochrome A (phyA) were microinjected into etiolated aurea tomato seedlings. Subsequent to microinjection anthocyanin and chlorophyll accumulation was monitored as well as β-glucuronidase (GUS) expression mediated by light-regulated promoters ( chs , chlorophyll a/b binding protein ( lhcb1 ) and ferredoxin NADP+ oxidoreductase ( fnr )). Microinjection of phyA* under white light conditions caused anthocyanin and chlorophyll accumulation and mediated chs —GUS, lhcb1 —GUS and fnr —GUS expression. Microinjection of phyB* under identical conditions induced chlorophyll accumulation and mediated lhcb1 —GUS and fnr —GUS expression but neither anthocyanin accumulation nor chs —GUS expression were observed. The characterization of Arabidopsis phytochrome mutants and the microinjection experiments suggested that phyB cannot induce the accumulation of juvenile anthocyanin. Microinjections under far-red light conditions demonstrated that phyA can act independently of other photoreceptors. By contrast, phyB* injections under red light conditions indicated that phyB* needs interactions with other photoreceptors to mediate a rapid and efficient de-etiolation signal.     

蓝光调节高粱突变体har1幼苗的去黄化反应

以航空诱变高粱突变体har1为材料,对其幼苗去黄化过程进行研究。萌发的种子在远红光下预培养6小时后,置于12小时蓝光/2小时黑暗条件下培养。测量幼苗的各器官伸长,结果表明,与野生型R111相比,harl的胚芽鞘、中胚轴、第一叶鞘以及第二叶鞘的伸长均受到蓝光的明显抑制,而蓝光对叶片生长影响不明显。3天龄har1黄化苗在连续蓝光下中胚轴花色素苷的积累明显增高,红光和远红光无此效应。此外,蓝光促进har1叶片叶绿体发育,且在蓝光照射24小时后叶片中叶绿素含量升高。Westernblot检测结果显示,7天龄R111和har1幼苗隐花色素SbCRY1b蛋白水平呈现蓝光下低、黑暗中高的变化趋势,har1的SbCRY1b蛋白水平在黑暗中高于R111。研究结果表明,高粱har1在去黄化过程中具有蓝光超敏感表型,SbCRY1b的作用值得进一步深入研究。