Photoregulation of beta-Tubulin mRNA Abundance in Etiolated Oat and Barley Seedlings

The effect of light on the abundance of β-tubulin mRNA was measured in etiolated Avena sativa L. and Hordeum vulgare L. seedlings. Slot blot analysis employing an oat β-tubulin cDNA clone was used to measure β-tubulin mRNA levels. White light induced a 45% decrease in oat β-tubulin mRNA abundance by 2 hours after transfer. A saturating red light pulse induced 40 and 55% decreases in β-tubulin mRNA levels in oats and barley, respectively. Recovery of β-tubulin mRNA levels was observed after a red light pulse but not after transfer to continuous white light. The red light induced decrease in oat β-tubulin mRNA abundance was not reversible by a subsequent far-red light treatment. The mesocotyl portion of etiolated oat seedlings exhibited a more dramatic decrease in β-tubulin mRNA abundance in response to red light than did the coleoptile portion. The results indicate that the well-documented effects of red light on the growth of etiolated seedlings are accompanied by changes in the expression of the β-tubulin genes.     

Regulation of phytochrome mRNA abundance in green oat leaves

Abstract. Avena sativa L. (oat) seedings were grown 4 d in continuous white light followed by 3 d in darkness. Probes derived from an oat phytochrome cDNA clone (pAP 3.2) were used in slot blot analyses to measure the abundance of phytochrome mRNA in the distinct etiolated and green portions of the leaves produced by these seedlings. Both the green and etiolated portions accumulated phytochrome mRNA to a level of about 85% of the etiolated seedling level. Subsequent experiments with similar seedlings showed that both the green and etiolated portions were capable of inducing a dramatic decline in phytochrome mRNA abundance in response to a saturating red light pulse. Despite the ability of green portions of oat leaves to accumulate phytochrome mRNA and to down-regulate phytochrome mRNA abundance in response to light, no substantial variation in phytochrome mRNA abundance was observed in green oat seedlings maintained on a 12-h day/12-h night cycle. In the same oat seedlings, the abundance of chlorophyll a/b binding protein mRNA fluctuated dramatically during the day/night cycle.     

Isolation and characterization of rice phytochrome A mutants

To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception--far-red light-dependent high irradiance response and very low fluence response--and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings.     

Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Interactions between Light and the Circadian Clock in the Regulation of CAT2 Expression in Arabidopsis

In Arabidopsis seedlings germinated and grown in continuous light, CAT2 mRNA abundance peaks 1 d after imbibition, consistent with the role of catalase in detoxifying H2O2 generated during the [beta]-oxidation of fatty acids stored in the seed. A second peak of CAT2 mRNA abundance, of lower amplitude than the initial peak, appears 6 d after imbibition and may be associated with the development of photosynthetic competence and induction of photorespiration. This second peak in steady-state CAT2 mRNA abundance is regulated by light and is not seen in etiolated seedlings. CAT2 mRNA accumulation is induced by exposure to high-fluence blue or far-red light but not by red light. In addition, light induction is unaffected by several mutations that block blue light-mediated inhibition of hypocotyl elongation (blu1, blu2, blu3, hy4), suggesting phytochrome involvement. When etiolated seedlings are transferred to continuous white light, CAT2 mRNA rapidly (within 30 min) accumulates. It is interesting that in these seedlings CAT2 mRNA abundance undergoes pronounced oscillations with a circadian (24 h) periodicity, indicating control by the endogenous circadian clock. No such oscillations are detected in CAT2 mRNA abundance in etiolated seedlings prior to illumination. Control of CAT2 expression by the circadian clock is also seen in 5-week-old plants grown in a light-dark cycle and transferred either to continuous dark or to continuous light; in continuous light the circadian oscillations in CAT2 mRNA abundance persist for at least five circadian cycles, indicating the robustness of this circadian rhythm.     

Mass spectrometric characterization of oat phytochrome A: isoforms and posttranslational modifications.

At least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N-terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass-spectrometry (ESIMS). The total tryptic digest of iodoacetamide-modified phyA was fractionated by gel filtration chromatography followed by reversed-phase high-performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision-induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1,128 amino acid residues) was determined in the present study. Mass-spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595-10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark-grown and red-light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light-treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A-mediated light-signal transduction.     

Dynamic properties of endogenous phytochrome A in Arabidopsis seedlings.

The dynamic behavior of phytochrome A (phyA) in seedlings of the model plant Arabidopsis was examined by in vivo spectroscopy and by western and northern blotting. Rapid accumulation of phyA was observed, reaching a steady state after 3 d. Both red and far-red light initiated a rapid destruction of the far-red-light-absorbing form of phytochrome (Pfr); the apparent half-life was only 4-fold longer in far-red than in red light. Furthermore, the Pfr-induced destruction of the red-light-absorbing form of phytochrome (Pr) of phyA occurred in darkness with a rate identical to that of Pfr destruction. A 2-fold decrease in mRNA abundance was observed after irradiation, irrespective of the applied light quality. However, reaccumulation occurred rapidly after far-red but slowly after red irradiation, indicating different modes of regulation of phytochrome expression after light-dark transitions depending on the light quality of the preceding irradiation. The wavelength dependency of the destruction rates was distinct from that of mustard, a close relative of Arabidopsis, and was explained on the basis of Pfr-induced Pr destruction and a simple kinetic two-step model. No dark reversion was detectable in the destruction kinetics after a red pulse. From these data we conclude that Arabidopsis phyA differs significantly in several aspects from other dicot phytochromes.     

Light-Dependence of Phospholipase D Activity in Oat Seedlings

The effect of light on the activity of phospholipase D (PLD) in oat (Avena sativa L.) seedlings and the dependence of this enzyme activity on the regime of their illumination were studied. The PLD activity in etiolated seedlings was 1.5–2.0-fold higher than in green plants. The illumination of etiolated seedlings with white light resulted in a decrease in PLD activity to its level in the seedlings grown under light. In contrast, the transfer of green seedlings to darkness enhanced the activity of the enzyme up to its level in etiolated seedlings. The illumination of etiolated seedlings with red light inhibited the PLD as well. It was shown that this photoeffect decreased with seedling aging and correlated with a phytochrome content in plants. Far-red light reversed the effect of red light. The involvement of phytochrome in the control of the PLD activity is discussed.     

Red/far-red light modulates phospholipase D activity in oat seedlings: relation of enzyme photosensitivity to photosynthesis

Phospholipase D (PLD) activity was found to be higher in etiolated oat seedlings than in green seedlings. White and red (R) light exposure inhibited PLD activity in etiolated seedlings. Far-red light eliminated R-light-induced decrease in PLD activity, indicating phytochrome participation in observed photomodulation. Inhibitor of electron transport in chloroplast 3-(3,4-dichlorophenyl)-1,1-dimethylurea stimulated and glucose suppressed PLD activity in green and etiolated oat seedlings, respectively. These results suggest that PLD activity in oat seedling is regulated by light with involvement of phytochrome photoreceptor, and associated with photosynthesis process.     

The Effects of Red, Far-red, and Blue Light on the Geotropic Response of Coleoptiles of Zea mays

The effects of red, far-red, and blue light on the geotropicresponse of excised coleoptiles of Zea mays have been investigated.Seedlings were grown in darkness for 5 or 6 days, exposed tovarious light treatments, and then returned to darkness fordetermination of the geotropic response. The rate of response of the coleoptiles is decreased after theyhave been exposed to red light (620–700 mµ, 560ergs cm^–2sec^–1 for the 24 hrs, but not for the 4hrs, preceding stimulation by gravity. Furthermore, their rateof response is greatly reduced if they are exposed to red lightfor 10 min and then returned to darkness for 20 hrs before geotropicstimulation. At 25° C an interval of 6 to 8 hrs elapses between a 10-minexposure to red light and the first detectable decrease in thegeotropic response of the coleoptile. This interval can be lengthenedby exposing the seedlings to low temperatures (0° to 2°C) after the light treatment but cannot be greatly shortenedby increasing the duration of exposure to red light. Using a standard procedure of exposing 5-day-old etiolated seedlingsto light for various times, replacing them in darkness for 20hrs and then determining the response of the coleoptiles to4 hrs geotropic stimulation, it has been found that: (a) Exposureto red light for 15 sec significantly decreases the geotropiccurvature of the coleoptiles and that further reduction occurson increasing the length of the light treatment to 2 and 5 min.(b) Far-red light has no effect on the geotropic response ofthe coleoptiles but it can completely reverse the effect ofred light. After repeated alternate exposure to red and far-redlight the geotropic response of the coleoptile is determinedby the nature of the last exposure, (c) Complete reversal ofthe effect of red light by far-red radiation only occurs whenexposure to far-red follows immediately after exposure to red.The reversing effect of far-red radiation is reduced if a periodof darkness intervenes between the red and far-red light treatments,and is lost after a dark interval of approximately 2 hrs. The effect of red light on the rate of geotropic response ofthe coleoptiles is independent of their age and length at thetime of excision. Blue light acts in a similar way to red light, but the seedlingsare less sensitive to blue than to red light. Coleoptiles grown throughout in a mixture of continuous, weak,red, and far-red light have a lower rate of geotropic responsethan etiolated coleoptiles.     

Phytochrome Levels in Light-Grown Avena Change in Response to End-of-Day Irradiations

The effect of 15-minute end-of-day irradiations on photoreversible phytochrome levels in light-grown oat (Avena sativa L., cv Garry) seedlings was investigated. Oat seedlings were grown in a cycle of 8 hours of natural daylight and 16 hours of complete darkness, from sowing until harvest at day 10. The level of extractable, photoreversible phytochrome per unit fresh weight was 60% higher after end-of-day far-red irradiation than after either end-of-day red irradiation or end-of-day far-red followed by end-of-day red. Seedlings irradiated with end-of-day far-red also exhibited a small but significant increase in shoot height and fresh weight per seedling. Extracts of seedlings given each of these end-of-day treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted, and immunostained with monoclonal antibodies specific to different phytochromes. Regardless of end-of-day light treatment, phytochrome that is abundant in etiolated tissue was below the limit of detection, indicating that one or more of the phytochromes predominating in green tissue changes in abundance.     

The roles of phytochromes in elongation and gravitropism of roots

Gravitropic orientation and the elongation of etiolated hypocotyls are both regulated by red light through the phytochrome family of photoreceptors. The importance of phytochromes A and B (phyA and phyB) in these red light responses has been established through studies using phy mutants. To identify the roles that phytochromes play in gravitropism and elongation of roots, we studied the effects of red light on root elongation and then compared the gravitropic curvature from roots of phytochrome mutants of Arabidopsis (phyA, phyB, phyD and phyAB) with wild type. We found that red light inhibits root elongation approximately 35% in etiolated seedlings and that this response is controlled by phytochromes. Roots from dark- and light-grown double mutants (phyAB) and light-grown phyB seedlings have reduced elongation rates compared with wild type. In addition, roots from these seedlings (dark/light-grown phyAB and light-grown phyB) have reduced rates of gravitropic curvature compared with wild type. These results demonstrate roles for phytochromes in regulating both the elongation and gravitropic curvature of roots.     

Effect of red light on the phototropic sensitivity of corn coleoptiles

The effect of red light in alteration of the phototropic sensitivity of corn coleoptiles (Zea mays L., cultivar Burpee Barbecue Hybrid) is investigated. Phototropic dosage-response curves for etiolated coleoptiles are compared with those for coleoptiles receiving 1 hour of continuous red light immediately prior to phototropic induction. In the former case, only curvature comparable to the first positive curvature of oat coleoptiles is obtained. There is no evidence for first negative curvature and only minimal second positive curvature. The reciprocity law proved valid for all curvatures obtained. With red light, the sensitivity of the first positive curvature was decreased over ten-fold and there was clear appearance of second positive curvature for which the reciprocity law was not valid. Once again there was no evidence for negative curvature. Time course studies indicated that within 1 hour of the beginning of red light treatment at 25°, reactions leading to the decrease in phototropic sensitivity of the first positive component had gone to completion whether the red light was continuous or consisted of a single 1 second exposure followed by a 1 hour dark period. An action spectrum for the red-induced change in phototropic sensitivity showed a marked peak near 660 mμ with a small broad shoulder between 610 and 630 mμ, characteristic of phytochrome-mediated responses. The effect of red light could be fully reversed by low dosages of far-red light, but longer doses of far red were less effective. Large dosages of far-red light alone induced the same alteration in phototropic sensitivity as did red light.     

Oscillations of acetylcholine in oat seedlings

Using gas chromatography it was shown that acetylcholine (ACh) was present in both etiolated and green oat (Avena sativa L. cv. Diadem) seedlings. In etiolated seedlings the ACh level was low, but increased rapidly during exposure to sunlight and red light (RL). The stimulative influence of RL was reversed by far-red light (FRL). The RL- and FRL- changes in ACh level were correlated to changes in acetylcholinesterase (AChE) localization. Using Karnovsky's method, it was found that in the etiolated coleoptiles the products of enzymatic reaction showing AChE activity accumulated selectively on the external side of plasma membrane. After exposure of seedlings to RL AChE activity disappeared. Subsequent FRL made it reappear on the external side of the plasma membrane. When the plants became green, oscillations of ACh were clearly observable. For plants grown under a LD 16:8 cycle the changes were circadian.     

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR far-red light; Pfr - Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - R red light     

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.