On the phytochrome phototransformation kinetics in mustard seedlings

Summary The in vivo phototransformation kinetics of mustard hook and cotyledon phytochrome exhibit a deviation from a single first order curve, quite similar to that for pumpkin hooks as reported in a previous paper (Boisard, Marmé and Schäfer, 1971). The P _frP_rkinetics can be characterized by the ratios _fr, ^I · P _fr ^I / _fr, ^II · P _fr, ^II and where P _fr ^I and P _fr ^II are two populations of phytochrome molecules which convert to P _rwith a first order half-life of and . These ratios depend on the length of time of etiolation. The ratio _fr, ^I · P _fr ^I / _fr, ^II · P _fr, ^II is independent of the amount of total P _frpresent at the beginning of the P _frP_rphototransformation after a non-saturating dose of red light. The half-lives of the two populations, however, depend on the concentration of total P _frinitially present. P _frP_rphototransformation kinetics with different light intensities show that reciprocity holds.     

Carotenoid synthesis in mustard seedlings as controlled by phytochrome and inhibitors

Summary Accumulation of carotenoids in the mustard seedling (Sinapis alba L.) is controlled by phytochrome (P_fr). Separation of the carotenoids shows that the control is quantitative rather than qualitative. Kinetic studies indicate that P_fr exerts a rapid and nearly reversible control over the rate of carotenoid accumulation. Whereas carotenoid accumulation between 36 and 60h after sowing is relatively insensitive towards Actinomycin D, the sensitivity towards cycloheximide and Puromycin is high. It is concluded that at least some of the enzymes required for carotenoid biosynthesis are made in the extraplastidal cytoplasm and it is suggested that P_fr acts at the level of carotenoid accumulation by providing a structural prerequisite for carotenoid accumulation in the plastid compartment. This latter suggestion is mainly based on the fact that carotenoid accumulation in light and dark is very sensitive towards chloramphenicol if the compound is applied at the time of sowing. If, however, the compound is applied 36 h after sowing, the effect of chloramphenicol is different in light and dark. In the dark there is no influence up to 200 g·ml^-1, whereas in the light there is a significant inhibition of carotenoid accumulation.Herrn Prof. Kurt Mothes mit guten Wünschen zum 70. Geburtstag.     

Kinetics of phytochrome decay in Amaranthus seedlings

Summary In Amaranthus seedlings the disappearance of the unstable P _fr form of phytochrome does not involve dark reversion to P _r . The rate constant for the decay of total phytochrome under continuous illumination is directly related to the proportion in the P _fr form. This relationship allows calculations to be made of the proportion of P _fr under continuous far-red illumination where the amount is too low to be measured directly.     

Appearance of nitrite-reductase mRNA in mustard seedling cotyledons is regulated by phytochrome

Nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) are the key enzymes of nitrate reduction. It is well established that the appearance of these enzymes is “induced” by nitrate, and it is generally believed that NR is cytosolic while NIR is plastidic. In mustard (Sinapis alba L.) cotyledons we observed two isoforms of NIR (NIR₁ and NIR₂) using a chromato-focusing technique. Only one of them (NIR₂) disappeared when the plastids were damaged by photooxidation in the presence of Norflurazon. It is concluded that NIR₂ is plastidic while NIR₁ is extraplastidic and not affected by photooxidation of the plastids. Both isoforms appear to have the same molecular weight (60 kilodaltons, kDa). Two distinct translation products which could be immunoprecipitated with NIR antiserum produced against total NIR from mustard were observed which differed slightly in molecular weight (60 versus 63 kDa). The 63-kDa polypeptide was considered to be the precursor of NIR₂. While synthesis of NIR protein depended largely on nitrate, the levels of in-vitro-translatable NIR mRNAs were found to be either independent of nitrate and light (NIR₁) or controlled by phytochrome only (NIR₂). It appears that phytochrome strongly stimulates the level of mRNA while significant enzyme synthesis (NIR₂) takes place only in the presence of relatively large amounts of nitrate. Since an increased enzyme level was strictly correlated with an increase of immunoresponsive NIR protein it is improbable that activation of a precursor plays a role. Rather, it is concluded that, in situ, nitrate controls translation.     

Control by phytochrome of urate oxidase and allantoinase activities during peroxisome development in the cotyledons of mustard (Sinapis alba L.) seedlings

The peroxisomal enzyme, urate oxidase (EC 1.7.3.3), and the next enzyme of the urate pathway, allantoinase (EC 3.5.2.5), demonstrate a lightmediated rise of activity in the cotyledons of mustard (Sinapis alba L.). The capacity of the peroxisomes for urate breakdown, marked by the time course of urate oxidase, develops distinctly later than the two other peroxisome functions (fatty acid breakdown, glyoxysomal function; glycolate breakdown, leaf peroxisomal function). The light effect on urate oxidase and allantoinase is mediated through the phytochrome system in all three seedling organs (cotyledons, hypocotyl, radicle), as revealed by induction/reversion experiments with red/far-red light pulses and continuous irradiation with far-red light (high irradiance reaction of phytochrome). Both enzyme activities can be induced by phytochrome in the seedling cotyledons only during a sensitive period of about 48 h prior to the actual light-mediated rise of activity, making it necessary to assume the existence of a long-lived intermediate (transmitter) in the signal response chain connecting enzyme formation to the phytochrome system. Detailed kinetic investigation, designed to test whether urate oxidase and allantoinase are controlled by phytochrome via the same signal response chain (coordinate induction), revealed large differences between the two enzymes: (i) a different onset of the loss of reversibility of a red light induction by a far-red light pulse (=onset of transmitter formation=coupling point; 48 h/24 h after sowing for urate oxidase/allantoinase); (ii) a different onset of the response (=onset of competence for transmitter= starting point; 72 h/48 h); (iii) full loss of reversibility (=completion of transmitter formation) is reached at different times (independence point, 90 h/52 h). These differences show that phytochrome controls urate oxidase and allantoinase via separate signal response chains. While urate oxidase can be localized in the peroxisomal fraction isolated from crude organelle extracts of the cotyledons by density gradient centrifugation, most of the allantoinase activity found in the peroxisomal fraction did not appear to be an integral part of the peroxisome but originated presumably from adhering membrane fragments.Abbreviations AL allantoinase, EC 3.5.2.5 - CAT catalase, EC 1.11.1.6 - GO glycolate oxidase, EC 1.1.3.1 - ICL isocitrate lyase, EC 4.1.3.1 - UO urate oxidase, EC 1.7.3.3. P_r - P_fr red and far-red absorbing forms of phytochrome On the occasion of his 80th birthday we dedicate this paper to Prof. Dr. phil., Dr. mult. h.c. Kurt Mothes, pioneer in research on metabolism of urates     

Evidence for bound phytochrome in oat seedlings

Phytochrome is consistently observed in pellets centrifuged from homogenates of etiolated, 5-day-old oat seedlings. The majority of pigment associated with the pellet cannot be removed by buffer washes, nor can appreciable quantities of additional phytochrome be adsorbed onto the sedimented material. Over 70% of phytochrome in the pellet is released by 1% Triton X-100.     

Control by light of hypocotyl growth in de-etiolated mustard seedlings

After sowing, mustard (Sinapis alba L.) seedlings were grown for 48 h in white light (25°C). These fully de-etiolated, green seedlings were used as experimental material between 48 and 72 (84) h after sowing. The question researched was to what extent control by light of hypocotyl elongation is due to phytochrome in these seedlings. It was found that the light effect on hypocotyl growth is very probably exerted through phytochrome only. In particular, we found no indication for the involvement of a specific blue light photoreceptor pigment.Abbreviations HIR high irradiance reaction - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - Pot total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_tot] - red red light - fr far-red light - wl white light - bl blue light - di dichromatic irradiation - l hypocotyl length     

Overexpression of rice phytochrome A in tobacco seedlings modifies responsiveness but not competence to phytochrome

To analyse the control of rice phytochrome A (phyA) overexpression (wild type or variously mutated) on gene regulation, transgenic tobacco lines overexpressing various rice phyA constructs were crossed with transgenic tobacco lines containing mustard Lhcb1 or Chs1 promoters fused to the uidA reporter gene (-glucuronidase). It was demonstrated that the temporal pattern of competence to respond to phytochrome was not altered by rice phyA overexpression. Also, overexpression of rice phyA did not change the spatial pattern of gene expression. The responsiveness to red and far-red light, on the other hand, depended on the type of overexpressed rice phyA in a structure-function relation: the serine-to-alanine mutant mediated an enhanced response both under continuous red and far-red light, whereas the N-terminal deletion mutant showed a dominant negative effect under continuous far-red light and even after red light pulses. However, the effectiveness of rice phyA overexpression depended on the promoter construct and the developmental stage of the seedlings. The Lhcb1 promoter also conferred -glucuronidase activity in etiolated seedlings. This dark expression could be decreased by a long-wavelength farred light pulse given early in development (24 h after sowing), indicating that this phenomenon is under the control of stable types of phytochrome.Abbreviations Chs1 chalcone synthase - GUS -glucuronidase - Lhcb1 type 1 light-harvesting chlorophyll a/b-binding protein - NTD N-terminal deletion mutant of rice phyA - phyA phytochrome A - phyB phytochrome B - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - RW rice wild-type phyA - S/A serine-to-alanine mutant of rice phyA - XAN wild-type tobacco cv. Xanthi We thank N.-H. Chua (Rockefeller Univ., New York, USA) and J. Stockhaus (Heinrich-Heine-Universität, Düsseldorf, Germany) for providing seeds from tobacco lines overexpressing the diverse rice phyA proteins. The work was supported by a grant from the Human Frontier Science Program and a grant from Deutsche Forschungsgemeinschaft (SFB 388). K.E. is a recipient of a Landesgraduierten-förderung fellowship     

Control by phytochrome of C-sucrose incorporation into buds of etiolated pea seedlings

When etiolated pea epicotyls are excised immediately above the cotyledons and dipped basally into ¹⁴C-sucrose, their terminal buds respond to red light by increased growth (IG) and enhanced incorporation of sucrose (EIS). Both phenomena are phytochrome controlled, showing typical kinetics, reversal by far-red light, escape from photochemical control and limitation to leaf tissue. EIS is of greater magnitude, occurs more rapidly and is saturated by lower energies of red light than IG, suggesting its possible importance as a controlling reaction in phytochrome-mediated growth. Both IG and EIS are best shown in the presence of a long epicotyl derived from a 5 to 6-day-old seedling in the presence of about 0.1 m unlabeled sucrose in the medium.     

A detailed analysis of phytochrome decay and dark reversion in mustard cotyledons

Summary During the first 10 min after a saturating dose of red light, 72 h dark-grown mustard cotyledons show no phytochrome decay. Within the same time interval there exists a transient form of P _fr (=P _fr ^T ) which is no longer photoconvertible at 0°C, but is at 25°C. This P _fr ^T converts in the dark to P _fr and P _r . These dark reversions take about 10 min. After a lag phase of 10 min the P _fr decay can be described by a single, first order kinetic curve. The time courses of these reactions are functions of the time of etiolation.Research supported by DAAD and by Deutsche Forschungsgemeinschaft (SFB 46).     

G-proteins in etiolated Avena seedlings. Possible phytochrome regulation

The molecular mechanism of light signal transduction in plants mediated by the photosensor phytochrome is not well understood. The possibility that phytochrome initiates the signal transduction chain by modulating a G-protein-like receptor is examined in the present work. Etiolated Avena seedlings contain G-proteins as examined in terms of the binding of GTP as well as by cross-reaction with mammalian G-protein antibodies. The binding of GTP was regulated in vivo by red/far-red light. The possible involvement of G-proteins in the phytochrome-mediated signal transduction in etiolated Avena seedlings has been implicated from the study of the light regulated expression of the Cab and phy genes.     

Aspects of phytochrome decay in etiolated seedlings under continuous Illumination

Summary The rate of total phytochrome decay in the dicotyledons Amaranthus caudatus, Mirabilis jalapa and Pisum sativum under continuous illumination with red, incandescent, and blue light depends on the P_FR/P_total maintained by each source. Amaranthus is an exception to this in that there is a deviation from firstorder decay kinetics under continuous illumination with incancdescent light. This deviation is probably not related to the chlorophyll present in the Amaranthus sample since chlorophyll-rich Pisum buds have the same phytochrome decay rate as epicotyl tissue under continuous incandescent light. Reports of a prolonged lag phase before the onset of first-order decay kinetics of phytochrome in Pisum have not been confirmed and the small lag phase observed in the present work can be accounted for by the time required to attain the P_FR/P_total ratio characteristic of blue light in a carotenoid rich tissue. In the monocotyledon, Avena sativa, and perhaps monocotyledons in general, decay rate is maximal at a low P_FR concentration and the decay curve is the same under continuous red, incandescent and blue light. This dicotyledon/monocotyledon difference with respect to saturation of phytochrome decay does not correlate with the other dicotyledon/monocotyledon difference, the presence or absence of dark reverions of P_FR to P_R, since the dicotyledons Amaranthus and Mirabilis that lack reversion still show no saturation of decay. Possible growth control by the P_FR/P_total ratio is discussed in relation to environmental changes in light quality.Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission.     

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.