Inhibition of phytochrome synthesis by the transaminase inhibitor, 4-amino-5-fluoropentanoic Acid

Gardner and Gorton (1985 Plant Physiol 77: 540-543) demonstrated that the transaminase inhibitor gabaculine (5-amino-1,3-cyclohexadienyl-carboxylic acid) inhibits the initial synthesis and resynthesis of spectrophotometrically detectable phytochrome in vivo. Another mechanism-based transaminase inhibitor, 4-amino-5-fluoropentanoic acid (AFPA), is examined in this report for its effects on phytochrome synthesis in developing etiolated seedlings. Preemergence treatment with AFPA was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis. These results confirm those with gabaculine, indicating that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein. AFPA was a more effective inhibitor of both chlorophyll and phytochrome synthesis than was gabaculine, suggesting that AFPA may be the preferred tool with which to probe the physiological consequences of the inhibition of phytochrome biosynthesis.     

Inhibition of phytochrome synthesis by gabaculine

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a transaminase inhibitor, also inhibits chlorophyll formation in plants, and the effect of this compound can be counteracted by 5-aminolevulinic acid (ALA) (Flint, personal communication, 1984). Since it is probable that ALA also serves as a precursor to phytochrome, the effects of gabaculine on phytochrome synthesis in developing etiolated seedlings were examined using in vivo spectrophotometry. Preemergence treatment with gabaculine was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis, perhaps due to preexisting phytochrome in the seed. Foliar treatment of etiolated pea seedlings prior to light-induced destruction of phytochrome inhibited subsequent phytochrome resynthesis in the dark. These results suggest that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein.     

Phytochrome Chromophore Biosynthesis : Both 5-Aminolevulinic Acid and Biliverdin Overcome Inhibition by Gabaculine in Etiolated Avena sativa L. Seedlings

Etiolated Avena sativa L. seedlings grown in the presence of gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid) contained reduced levels of phytochrome as shown by spectrophotometric and immunochemical assays. Photochromic phytochrome levels in gabaculine-grown plants were estimated to be 20% of control plants, while immunoblot analysis showed that the phytochrome protein moiety was present at approximately 50% of control levels. Gabaculine-grown seedlings administered either 5-aminolevulinic acid or biliverdin exhibited a rapid increase of spectrophotometrically detectable phytochrome. Phytochrome concentrations estimated immunochemically did not similarly increase throughout treatment with either compound. Similar experiments with 5-amino[4-¹⁴C] levulinic acid showed radiolabeling of phytochrome with kinetics that paralleled the spectrally detected increase. These results are consistent with (a) the intermediacy of both 5-aminolevulinic acid and biliverdin in the biosynthetic pathway of the phytochrome chromophore and (b) the lack of coordinate regulation of chromophore and apoprotein synthesis in Avena seedlings.     

Loss of phytochrome photoreversibility in vitro: I. Extraction and partial purification of killer

Crude pea (Pisum sativum L. var. Alaska) phytochrome extracts contain a substance, “Killer,” which interacts with the far red-absorbing form of phytochrome causing a net loss of spectrophotometrically detectable phytochrome in vitro. Killer is absent from crude extracts of Avena phytochrome, is separable from pea phytochrome by gel filtration, and is alcohol-extractable from etiolated pea seedlings. Killer activity in alcohol extracts behaved, during partial purification, in a manner identical to that derived from pea phytochrome preparations. The mass extraction and partial purification of Killer are described.     

Response of tissue with different phytochrome contents to various initial photostationary States

Pretreatment of etiolated pea plants with red light and with red combined with far-red light produced morphologically similar plants having 4-fold differences in spectrophotometrically detectable phytochrome. Stem segments from the variously pretreated plants respond in the same way to different percentage conversions of phytochrome to P_FR. These results suggest that the P_FR./P_R ratio, rather than the concentration of P_FR, governs pea stem segment elongation. However, the ratio hypothesis does not explain contradictions between spectrophotometric and physiological assays previously obtained with this tissue, nor does it explain similar contradictions obtained in other systems. The only hypothesis consistent with the data to date is that of the existence of bulk and active phytochrome fractions, with the latter present in insufficient quantities to be spectrophotometrically detectable.     

Effects of Gabaculine on Phytochrome Synthesis during Imbibition in Embryonic Axes of Pisum sativum L.

Effects of gabaculine, a transaminase inhibitor, on phytochromesynthesis in the embryonic axes of Pisum sativum during imbibitionat 25°C on 0.2% agar medium were investigated. The contentof phytochrome in crude homogenates (2kS) prepared from embryonicaxes was determined both by a spectrophotometric assay, withhighly purified phytochrome solution as the internal standard,and by an enzyme-linked immunosorbent assay (ELISA) (a sandwichmethod) with polyclonal and monoclonal antibodies against peaphytochrome. The-content of optically detectable phytochromein 2kS prepared from 12 h-imbibed embryonic axes was reducedin the presence of gabaculine at concentrations of 0.002 minor higher. The maximum inhibitory effect occurred at ca. 0.1IBM. The effect of 0.5 mM gabaculine on optically detectablephytochrome became noticeable 3 h after the start of imbibition.In contrast, the time course of the increase of apophytochromeduring imbibition was unaffected in the presence of gabaculineat concentrations below 1 miu. We conclude that the appearanceof holophytochrome in imbibed embryonic axes results from denovo synthesis of both the protein moiety of phytochrome andits chromophore. (Received July 18, 1986; Accepted September 8, 1986)     

The Phytochrome-Deficient pcd1 Mutant of Pea Is Unable to Convert Heme to Biliverdin IX[alpha

We isolated a new pea mutant that was selected on the basis of pale color and elongated internodes in a screen under white light. The mutant was designated pcd1 for phytochrome chromophore deficient. Light-grown pcd1 plants have yellow-green foliage with a reduced chlorophyll (Chl) content and an abnormally high Chl a/Chl b ratio. Etiolated pcd1 seedlings are developmentally insensitive to far-red light, show a reduced response to red light, and have no spectrophotometrically detectable phytochrome. The phytochrome A apoprotein is present at the wild-type level in etiolated pcd1 seedlings but is not depleted by red light treatment. Crude phytochrome preparations from etiolated pcd1 tissue also lack spectral activity but can be assembled with phycocyanobilin, an analog of the endogenous phytochrome chromophore phytochromobilin, to yield a difference spectrum characteristic of an apophytochrome-phycocyanobilin adduct. These results indicate that the pcd1-conferred phenotype results from a deficiency in phytochrome chromophore synthesis. Furthermore, etioplast preparations from pcd1 seedlings can metabolize biliverdin (BV) IX[alpha] but not heme to phytochromobilin, indicating that pcd1 plants are severely impaired in their ability to convert heme to BV IX[alpha]. This provides clear evidence that the conversion of heme to BV IX[alpha] is an enzymatic process in higher plants and that it is required for synthesis of the phytochrome chromophore and hence for normal photomorphogenesis.     

Spore germination and phytochrome biosynthesis in the fern Lygodium japonicum as affected by gabaculine and cycloheximide

We investigated whether the gradual increase in phytochrome content in the fern Lygodium japonicum (Thunb.) Sw. during dark imbibition results from hydration or from biosynthesis of phytochrome. Addition of gabaculine or cycloheximide to the culture medium caused inhibitions of both red light-induced spore germination and of the appearance of phytochrome in the spores. Fifty percent inhibition of both red light-induced germination and of the appearance of phytochrome in the spores occurred at ca 10⁻7 M cycloheximide. Red light-induced germination and phytochrome appearance were markedly inhibited by 10⁻4 M and completely by 10⁻3 M gabaculine, but germination induced by gibberellic acid was unaffected. Phytochrome was not detected in spores after forced hydration. These results suggest that the increase in phytochrome during imbibition was mainly due to de novo synthesis of the phytochrome apoprotein and to synthesis of the chromophore and/or proteins required for phytochrome formation, rather than to hydration of preexisting phytochrome molecules.     

Characterization of the Destruction of Phytochrome in the Red-absorbing Form

Both the red-absorbing (Pr) and far red-absorbing (Pfr) forms of phytochrome undergo destruction, defined as the loss of photoreversibly detectable chromoprotein following actinic irradiation of dark-grown tissue, in 4-day-old etiolated oat seedlings. Pr and Pfr destruction follow the same time course, exhibit the same time delay after actinic irradiation when the plants are grown in sealed containers, result in a loss of antigenically detectable phytochrome, as determined by radial immunodiffusion assay, equal to the loss of spectrophotometrically detectable phytochrome, and have the same sensitivity to 2-mercaptoethanol and azide. We suggest that Pr destruction is a consequence of the same mechanism that is responsible for Pfr destruction.     

4-amino-5-hexynoic Acid-a potent inhibitor of tetrapyrrole biosynthesis in plants

4-Amino-5-hexynoic acid, a suicide inactivator of the mammalian pyridoxal phosphate-dependent 4-aminobutyric acid:2-oxoglutaric acid aminotransferase, inhibits phytochrome and chlorophyll synthesis in developing oat (Avena sativa L.), corn (Zea mays L.), pea (Pisum sativum L.), and cucumber (Cucumis sativus L.) seedlings. In Avena and Cucumis seedlings, respectively, inhibition of phytochrome and chlorophyll accumulation by 4-amino-5-hexynoic acid can be significantly reversed by application of 5-aminolevulinic acid. These results indicate that 4-amino-5-hexynoic acid inhibits the synthesis of 5-aminolevulinic acid in plants.     

Phytochrome photoreversibility: Empirical test of the hypothesis that it varies as a consequence of pigment compartmentation

Phytochrome of oat (Avena sativa L., cv. Garry) coleoptile cells in the red-light-absorbing form, Pr, is diffusely distributed while after conversion to the far-red-light-absorbing form, Pfr, it is observed only in very small areas within the cell. Comparison of phytochrome photoversibility measurements to the distribution of the pigment within the cell indicates that the spectral assay is not influenced by the observed compartmentalization of the chromoprotein. However, the observed compartmentalization of phytochrome is correlated with a loss in spectrophotometrically detectable Pr.Abbreviations Pr red-absorbing form of phytochrome - Pfr farred-absorbing form of phytochrome - R red light - FR far-red light C.I.W.-D.P.B. Publication No. 622     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

Effect of red light on geotropism in pea epicotyls

Dose response curves were determined for phytochrome phototransformation and for a phytochrome-controlled decrease in geotropic curvature in epicotyls of dark-grown Pisum sativum L. cv. Alaska. Ten times as much light was required to produce a spectrophotometrically detectable transformation of phytochrome as was required to produce a significant change in the geotropic response. The red light energy required for a 50% phytochrome transformation caused a 90% change in the physiological response.     

Rice Phytochrome Is Biologically Active in Transgenic Tobacco

To investigate the mechanisms of phytochrome action in vivo, we have overexpressed rice phytochrome in transgenic tobacco plants. A full-length rice phytochrome cDNA was fused to the cauliflower mosaic virus 35S promoter and transferred to tobacco. The progeny of some of the transgenic plants contain large amounts of rice phytochrome mRNA in green leaves. Extracts prepared from overexpressing plants contain twofold to fivefold more spectrophotometrically detectable phytochrome than extracts from control plants. Species-specific, anti-phytochrome monoclonal antibodies were used in immunoblots to discriminate between rice and tobacco phytochrome apoproteins in fractions eluted from a DEAE-Sepharose column. Red minus far-red difference spectra of the partially purified rice phytochrome from the transgenic plants indicate that the rice phytochrome assembles with chromophore and is photoreversible. Analysis of the circadian pattern of Cab mRNA levels in transgenic plants versus controls demonstrates that the overproduction of rice phytochrome extends the duration of the free-running rhythm of Cab gene expression. The rice phytochrome is, therefore, biologically active in the transgenic tobacco plant, which establishes a system for in vivo functional analysis of phytochrome.     

Phytochrome Control of Its Own Synthesis in Pisum sativum

An analysis of phytochrome synthesis in Pisum seedlings by measuringthe activity of polysomal polyadenylated RNA (poly-A⁺-RNA) codingfor phytochrome apoprotein showed phytochrome control of itsown synthesis; brief red-light irradiation of pea seedlingsinhibited the activity of the RNA, and the red-light effectwas red/far-red reversible. ⁴ Permanent address: Biology Department, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received August 13, 1984; Accepted September 17, 1984)     

LOCALIZATION OF PHYTOCHROME DURING PEANUT (ARACHIS HYPOGAEA) GYNOPHORE AND OVULE DEVELOPMENT

Previous studies indirectly indicated that phytochrome plays a role in peanut (Arachis hypogaea L. cv. Virginia) gynophore elongation and in ovule and embryo development. Recent advances in the use of monoclonal antibody procedures used in this study have allowed precise localization of phytochrome in the developing peanut gynophore and ovular tissues. Peanut phytochrome from etiolated tissues was found to have a molecular weight of 124 kD as determined by immunoblotting procedures using a monoclonal antibody to pea (Pisum sativum L. cv. Alaska) phytochrome. Immunoblotting procedures revealed that no detectable phytochrome was present in the gynophore tissues or immature ovules during the elongation of the peanut gynophores. After the gynophores penetrated the soil for 8–12 d, phytochrome was detected in increasing amounts in the ovular tissues but not the gynophore tissues. Immunohistological analysis revealed that phytochrome was localized in the developing embryo and adjacent integument tissues. These findings contradict earlier reports that suggested phytochrome was initially present in the gynophore tissues after fertilization where it was believed to inhibit ovular development and stimulate gynophore elongation.     

Inhibitory effect of gabaculine on 5-aminolevulinate dehydratase activity in radish seedlings

We have compared the activity of 5-aminolevulinate dehydratase (5-ALAD) with the amount of protein detected by specific antibodies in rocket immunoelectrophoresis. Parallel kinetic evolutions of enzymic activity and amount of antigen were observed in radish (Raphanus sativus L.) cotyledons, both in complete darkness or under standard far red light involving phytochrome. However, the treatment of seedlings with gabaculine leads to an important decrease in enzymic activity, while the specific protein content is maintained. This inhibition is not overcome by the addition of glutamic acid, but by 5-aminolevulinic acid which points to a specific control of 5-ALAD activity by its substrate. As there is no discrepancy between the enzymic activity and the amount of antigen during the time course development of seedlings, this could confirm a coordinate cellular control between 5-aminolevulinic acid formation and 5-ALAD protein synthesis, both being amplified by the action of phytochrome.     

Comparative Phytochrome Immunochemistry as Assayed by Antisera against Both Monocotyledonous and Dicotyledonous Phytochrome

Preparation and characterization of antisera against lettuce (Lactuca sativa L., cv. Grand Rapids) and pea (Pisum sativum L., cv. Alaska) phytochrome is described. These antisera, together with previously obtained antisera against zucchini (Cucurbita pepo L., cv. Black Beauty) and oat (Avena sativa L., cv. Garry) phytochrome, were used to compare by Ouchterlony double immunodiffusion phytochrome isolated from etiolated lettuce, pea, bean (Phaseolus vulgaris L., cv. Taylor Horticultural Bush), zucchini, oat and rye (Secale cereale L., cv. Balbo) seedlings. Cross reactivity between monocotyledonous phytochrome and antidicotyledonous-phytochrome serum and between dicotyledonous phytochrome and antimonocotyledonous-phytochrome serum was always weak or not perceptible by this assay. Among the four dicotyledonous phytochromes examined, pea and bean were the most similar immunochemically as anticipated. Pea and lettuce phytochrome somewhat unexpectedly also exhibited similar immunochemical reactivity. Zucchini phytochrome by contrast was immunochemically distinct from pea, bean, and lettuce phytochrome, although it did react with all three antidicotyledonous-phytochrome sera. Initial attempts to identify immunoglobulins that would recognize phytochrome regardless of its source indicated that they may exist. Such immunoglobulins are of interest because they might react with one or more determinants that could be part of an active site of phytochrome. These immunoglobulins, once isolated, could thus serve as a potential probe for the active site of phytochrome.     

Immunopurification and initial characterization of dicotyledonous phytochrome

Antiserum was prepared against proteolytically undegraded phytochrome obtained from etiolated zucchini squash (Cucurbita pepo L., cv. Black Beauty). The antiserum was prepared by injecting into a rabbit immunoprecipitates between zucchini phytochrome and specific antiserum against undegraded oat (Avena sativa L., cv. Garry) phytochrome. Specific antiphytochrome immunoglobulins were purified from this crude serum by an affinity column consisting of conventionally purified undegraded pea phytochrome covalently linked to cyanogen bromide-activated agarose. These purified immunoglobulins were also linked to cyanogen bromide-activated agarose and were used to immunopurify zucchini, pea (Pisum sativum L., cv. Alaska), and lettuce (Lactuca sativa L., cv. Grand Rapids) phytochrome. All three dicotyledonous phytochromes exhibited a monomer size near 120,000 daltons by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. Absorbance spectra of immunopurified zucchini phytochrome indicated that the ratio of visible to ultraviolet absorbance for purified zucchini phytochrome is lower than that observed for oat phytochrome. The isoelectric point of zucchini phytochrome, which was observed to be heterogeneous by this criterion, was found to be in the range of 6.5 to 7.0, higher than that observed for oat phytochrome. The electrophoretic mobility of zucchini phytochrome was found to be similar to that observed for oat and pea phytochrome under conditions that were nondenaturing and did not involve any molecular sieving effect. The amino acid analysis of zucchini phytochrome is similar to that reported previously for oat and rye (Secale cereale L., cv. Balbo) phytochrome.     

Phytochrome Control of Longitudinal Growth and Phytochrome Synthesis in Maize Seedlings

The active, far-red light absorbing, form of phytochrome was found to inhibit growth and phytochrome levels in the mesocotyl and coleoptile of 4- to 5.5-day-old seedlings of Zea mays L. Short, low-irradiance red or far-red light treatments were used to produce different proportions of active phytochrome at the end of highdirradiance white-light periods, which left different levels of total phytochrome in the plants. After light treatments which left relatively high levels of spectrophotometrically assayable phytochrome in the seedlings, apparent phytochrome synthesis in the subsequent dark period was low regardless of the proportions of each form of the pigment present at the beginning of the dark period. In light treatments producing relatively low levels of assayable phytochrome, levels of apparent phytochrome synthesis in both red and far-red treatments and differences between apparent synthesis in red and far-red treatments were maximal. No simple correlation was found between growth and apparent phytochrome synthesis. However, growth and total phytochrome levels were positively correlated in both organs. Using a subtractive method of correlation, in which only phytochrome effects were plotted, strong linear relationships between phytochrome levels or longitudinal growth and P_fr levels were found in those light treatments leaving greater than 8% of dark control levels of phytochrome in the tissues. Using this technique non-linear, inverse relationships between P_fr and apparent phytochrome synthesis was found, indicating that modes of phytochrome control over phytochrome synthesis and growth differ. Our results are consistent with the view that in vivo assays of “bulk’ phytochrome reflect levels and states of the physiologically active phytochrome fraction under our experimental conditions in maize.