Synthesis of Translatable mRNA for Phytochrome during Imbibition in Embryonic Axes of Pisum sativum L.

The activity of translatable mRNA for phytochrome was measuredin excised embryonic axes of Pisum sativum L. during imbibitionboth in the dark and under continuous irradiation with whitelight. When measured in cell-free protein synthesis systemsof both rabbit reticulocyte lysate and wheat germ extract, theactivity of translatable mRNA for phytochrome was not detectedin dry quiescent axes but increased rapidly after imbibitionin the dark. After 24 h imbibition, the level of translatablemRNA for phytochrome, in terms of the incorporation of [³⁵S]methioninein the wheat germ system, was ca. 0.0034% of total translatablemRNA. In the presence of 0.5 µg ml^–1 -amanitin,the appearance of translatable mRNA for phytochrome was inhibitedby 60%, while 2 µg ml^–1 -amanitin was almost completelyinhibitory. This indicates that the synthesis of translatablemRNA for phytochrome in embryonic axes begins upon imbibition. When the axes were imbibed under continuous white light, theactivity of phytochrome mRNA increased as rapidly during thefirst 3 h as in the dark. After this time, the activity wasmarkedly lower than in the dark. Nevertheless, during the 24h of imbibition, activity in the light was always found to bemore than half of that in the dark. These results indicate thatin germinating pea axes the level of translatable mRNA for phytochromeis partially repressed by light. (Received June 5, 1985; Accepted September 2, 1985)     

Effects of divalent cations and EDTA on spectral properties of phytochrome in particulate fractions from etiolated pea epicotyls

The photoreversible absorbance change of phytochrome in suspensionsof a 20,000xg particulate fraction (20kP) prepared from a 1,000xgsupernatant (1kS) of etiolated pea epicotyl extracts decreasedremarkably in the presence of 5 mM Cu²⁺, Zn²⁺ and Co²⁺, butremained unchanged in 5 mM Ca²⁺, Mg²⁺, Fe²⁺ or Mn²⁺. This spectraldistortion of phytochrome was more evident in soluble preparationsand in suspensions of pellets prepared from red light (R)-irradiatedtissues than it was in suspensions of pellets prepared in thedark from etiolated tissues that received no actinic irradiation. When Cu²⁺ was added to the red-light-absorbing form of phytochrome(Pr) in resuspended pellets prepared from R-irradiated tissues,the distortion of its difference spectrum took place after irradiationwith the first actinic R. In contrast, when Cu²⁺ was added tothe far-red-light-absorbing form of phytochrome (Pfr) in thesame resuspended pellet, no distortion was seen, unless thePfr in the pellet was first photoconverted to Pr and then photoconvertedback to Pfr. Spectral distortion of Pr remained small during dark incubationat 25°C when suspensions of 20kPs were prepared and incubatedwith a buffer containing EDTA, whether the 20kP was preparedfrom nonirradiated tissue or from R-irradiated tissues. But,when EDTA was added to a suspension of 20kP prepared from 1kS,after the 1kS was irradiated with R in the presence of 10 mMCaCl₂, the spectral distortion of Pr in 20kP occurred instantaneously. (Received April 14, 1980; )     

Changes in the Content of Phytochrome I and II Apoproteins in Embryonic Axes of Pea Seeds during Imbibition

The contents of phytochrome I and II in crude extracts fromembryonic axes of Pisum sativum cv. Alaska seeds were immunochemicallydetermined using purified pea phytochrome I and II as standards.We have produced and used three different types of mouse monoclonalanti-pea phytochrome antibodies (mAP) such as one reacting preferentiallywith phytochrome I, one with phytochrome II, and one with bothI and II. Phytochrome II was separated from I in the samplesusing immobilized column chromatography with mAPl. The amountsof two phytochrome species were quantitatively measured withwestern blotting and ELISA. Ca. 0.2 µg /axis of phytochromeI and ca. 0.05 µg /axis of phytochrome II were detectedby ELISA after imbibition for 12 h in the dark, though smallamounts of both were detected in dry axes. Ca. 0.05 µg/axis each of phytochrome I and II were detected by ELISA afterimbibition for 12 h in the light, and the results were confirmedby western blotting. This study showed that phytochrome II isnot green-tissue-specific, being also found in dark-imbibedembryonic axes, and that although light significantly lowersthe content of phytochrome I in the axis, it does not significantlyaffect that of phytochrome II. (Received June 10, 1987; Accepted August 27, 1987)     

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.     

In vivo Studies on the Occurrence of Stored mRNA in Embryonic Axes of Vigna unguiculata Seeds

-Amanitin and cordycepin at various concentrations were testedfor their inhibitory effect on the fresh weight increase ofVigna unguiculata embryonic axes after the onset of imbibitionand on the incorporation rate of ³H-labeled leucine into proteinin axes of the 36–38 h stage. -Amanitin at 0.5–5µ/Kg/ml clearly exerted an inhibitory effect on both thefresh weight increase and the protein synthesis. This drug at1 µg/ml, however, showed no significant effect on theprotein synthesis at an early stage of imbibition (4 h), whereascycloheximide was a very potent inhibitor. By experiments inwhich ‘dry’ axes were allowed to imbibe 3H-lebeledadenosine solution for 4 and 12 h in the presence of -amanitin,it was found that poly A⁺RNA was newly synthesized to some extentin axes as early as 4 h after the onset of imbibition and thatthe drug effectively inhibited the poly A⁺RNA synthesis. Theresults may indicate the occurrence of stored mRNA in embryonicaxes of V. unguiculata seeds. (Received June 11, 1983; Accepted August 16, 1983)     

ATP Accumulation in Peanut Seeds during Seed-Ripening and During the Dormancy-Breaking Process

Ripening and dormancy-breakage of peanut (Arachis hypogaea L.)seeds are accompanied by an increase, followed by a decrease,in the ability to accumulate ATP (from AMP + phospho-enolpyruvate(PEP)) in the embryonic axes as well as in the cotyledonarysegment powder. A similar increase and decrease profile in theconcentrations of the ATP precursors (AMP + ADP) in the embryonicaxes and in the cotyledonary segments is noted during dormancybreakage. The ability of the seeds to germinate during the ripeningprocess (after dormancy-breakage treatment) seems to correlatewith the decrease in the ability to accumulate ATP in the embryo,while dormancy breakage seems to be correlated with the samephenomenon in the cotyledon. Imbibition of seeds (as whole seeds or as embryonic axes andcotyledonary segments separately) causes a 10–40-foldincrease in the ability to accumulate ATP from AMP and PEP,and a decrease in the concentrations of the precursors of ATP.Ethylene has a stimulatory effect on ATP accumulation in embryonicaxes during imbibition. Key words: Arachis hypogaea, ATP, Dormancy, Peanut, Seed Ripening     

Photoregulation of Phytochrome Synthesis in Germinating Embryos of Avena sativa L.

Hilton, J. R. and Thomas, B. 1987. Photoregulation of phytochromesynthesis in germinating embryos of Avena sativa L.—J.exp. Bot. 38: 1704–1712. The effect of light on the accumulation of phytochrome in germinatingAvena embryos was determined. A quantitative ELISA using monoclonalantibody AFRC MAC 56 was used to measure specifically type 1(or dark) phytochrome. A pulse of red light given after 14 himbibition but prior to the onset of type 1 phytochrome synthesis,strongly inhibited subsequent type 1 phytochrome accumulation.This effect of red light at 14 h was reversible by far-red lightindicating the involvement of phytochrome. Red light also inhibitedphytochrome synthesis after 18 h and 24 h imbibition but after24 h, far-red light did not reverse the effect. The effect ofred light treatment at 18 h was reversed by giving a pulse offar-red light at any time up to 30 h. Seed germination was notinfluenced by light under the conditions of these experiments.It is proposed that type 2 (or light) phytochrome may be responsiblefor photoregulation of type 1 phytochrome synthesis in germinatingAvena embryos. Key words: Photoregulation, phytochrome, seed.     

Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction     

Synthesis of phytochrome apoprotein and chromophore are not coupled obligatorily

G Gardner and HL Gorton (1985 Plant Physiol 77: 540) demonstrated that gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid) inhibits the initial synthesis and resynthesis of spectrophotometrically detectable phytochrome in pea, maize, and oat. We show that the level of immuno-detectable phytochrome in pea is unaffected by the presence of gabaculine at a concentration that reduces spectrophotometrically detectable phytochrome up to 10-fold. This result indicates that gabaculine inhibits chromophore synthesis without affecting phytochrome apoprotein synthesis and that chromophore-less phytochrome is stable in the cell.     

Spontaneous Chemiluminescence of Soybean Embryonic Axes during Imbibition

Isolated soybean (Glycine max L. var Hood) embryonic axes have a spontaneous chemiluminescence (about 150 counts per minute per embryo) that increases showing two phases, upon water imbibition. The first photoemission burst was measured between 0 and 7 hours of imbibition with a maximum of about 350 counts per minute per embryo after 2 hours. The second photoemission phase, between 7 and 30 hours, increased from about 220 to 520 counts per minute per embryo. Both chemiluminescence phases were inhibited by infused butylated hydroxyanisole while only the second phase was inhibited by infused salicylhydroxamic acid. On the basis of the sensitivity of the lipoxygenase reaction to both inhibitors (about 90%), the first burst is tentatively assigned to oxy-radicals mobilized upon water uptake by the embryonic axes, and the second phase is tentatively identified as due to lipoxygenase activity. The in vivo lipoxygenase activity of the embryonic axes was estimated by both the fraction of total oxygen uptake that was inhibited by butylated hydroxyanisole and by the fraction of photoemission that was inhibited by butylated hydroxyanisole and by salicylhydroxamic acid. Both approaches indicated marked increases (5-fold and 12-fold, respectively) of lipoxygenase activity between 2 and 30 hours of imbibition. The measured chemiluminescence per O₂ uptake ratio (the experimental quantum yield) for the lipoxygenase reaction (3.3 × 10⁻¹⁴ counts per O₂ molecule) was used to estimate the O₂ uptake due to lipoxygenase activity from the photoemission of the embryonic axes after 30 hours of imbibition. The value (0.54 microliters per minute per axis) was close to the butylated hydroxyanisole-sensitive O₂ uptake (1.2 microliters O₂ per minute per axis) of the same embryonic axes. Chemiluminescence may afford a noninvasive assay for lipoxygenase activity in intact plant tissues.     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Changes in Phytochrome Content during Imbibition in Spores of the Fern Lygodium japonicum

The phytochrome content was determined in intact fern sporesof Lygodium japonicum (Thunb.) Sw. by difference spectrophotometry.The spectral characteristics thus estimated in spores whichhad been imbibed for 9 days in darkness were: far-red maximumat 730?2.5 nm, red maximum at 662?1.5 nm and isosbestic pointat 684.5?1.4 nm. A detectable amount of phytochrome first appearedafter 3 days of dark imbibition, and the level then increasedduring the rest of the imbibition period. On the 7th day, thephytochrome content leveled off. During the dark imbibitionperiod, the phytochrome was revealed to be in the P_R form. (Received February 22, 1982; Accepted July 9, 1982)     

A Comparison of Seed and Seedling Phytochrome in Avena sativa L. using Monoclonal Antibodies

Hilton, J.R. and Thomas, B. 1985. A comparison of seed and seedlingphytochrome in Avena saliva L.using monoclonal antibodies.—J.exp. Bot. 36: 1937–1946.The kinetics of phytochrome accumulationduring imbibition and germination of seeds of Avena saliva L.cv. Saladin has been determined in vivo by spectrophotometryand in extracts by using Enzyme–Linked Immunosorbent Assay(EL1SA). In vivo measurements show two phases of phytochromeincrease. The first occurs during the initial 2 h of imbibitionand is associated with the hydration of the seed proteins; thesecond larger increase begins after 16 h, due probably to denovo synthesis. An ELISA using monoclonal antibodies purifiedfrom dark grown Avena seedlings detected only the second increasein phytochrome content. Mixing experiments indicate that theinability to detect phytochrome by ELISA during the first 16h is not due to the presence of inhibitors in the extracts.It is concluded that pre–existent seed phytochrome isantigenically dissimilar to seedling phytochrome. These twopools of phytochrome are stable and unstable respectively withregard to Pfr destruction. Key words: Immunology, phytochrome, seed     

Isolation of cDNA for Pea Phytochrome Using an Expression Vector

Partially purified phytochrome mRNA was obtained from etiolatedpea epicotyls by polyribosome immunoprecipitation or by sizefractionation of total poly(A)⁺RNA, and used for the synthesisof double-stranded complementary DNA (cDNA). cDNA librarieswere constructed using an Escherichia coli expression vector,pUC9, and screened for phytochrome cDNA by colony immunologicalassay. Nine colonies were found to produce a 27 kDa polypeptidethat was reactive to both polyclonal and monoclonal antipeaphytochrome antibodies. The plasmids from these colonies containedcDNA inserts of 1.2 or 2.0 kbp. Hybridization-arrest translationassay verified that the cDNA clones contained a sequence codingfor phytochrome polypeptide. RNA blot hybridization analysisindicated that the cDNA hybridized to a 4.1 kb poly(A)⁺RNA indark-grown pea. (Received March 22, 1986; Accepted June 13, 1986)     

Changes in Respiratory Metabolism during Aging in Seeds and Isolated Axes of Soybean

Cyanide-sensitive O₂ uptake was observed in the isolated embryonicaxis of soybean [Glycine max L. merr. cv. ‘Essex’]within 5 min after wetting and the O₂ uptake rate during thefirst h of imbibition was directly proportional to axis moisturecontent. Within 10 min after wetting, O₂ uptake was lower inaged low vigor (LV) axes than in high vigor (HV) axes imbibedeither in water (LV axes sustain imbibition injury) or in 30%w/v polyethylene glycol (LV axes do not sustain imbibition injury).Mitochondria isolated from LV axes after 4- and 24-h imbibitionwere characterized by lower O₂ uptake, ADP:O, and respiratorycontrol ratios relative to HV mitochondria. A plot of O₂ uptakevs temperature between 10 and 25?C revealed a break in the slopebetween 13 and 16?C for HV, but not LV, axes. Since LV axessustained water uptake injury at all temperatures tested, whereasHV axes sustained chilling injury only below 16?C, the dataindicate two temperature-O₂ uptake relationships: one for injuredtissues and one for non-injured tissues. We conclude that deteriorationper se involves impairment of respiratory metabolism in thesoybean embryonic axis. Chilling (10?C; HV and LV axes) andimbibitional (25?C; LV axes) injuries during the initial minof inbibition further disrupt respiratory metabolism and interferewith subsequent mitochondrial development and axis growth. ¹Scientific Articles No. A-3548, Contribution No. 6623 of theMaryland Agricultural Experiment Station. (Received May 25, 1983; Accepted September 28, 1983)     

Interactions of seed water content with phytochromeinitiated germination of Rumex crispus (L.) seeds

Light-initiated germination levels of Rumex crispus L. seedswere reduced equally by imbibition in mannitol or polyethyleneglycol 6000 (PEG 6000) solutions of the same w, indicating thatthe effects of each were through w. Reduction of the water contentof the seeds with these osmotica decreased the effectivenessof the far-red absorbing form of phytochrome (P_tr) in causinggermination. However, reduced water content had no effect onthe slopes or saturation points of fluence/response curves whichindicates that has no effect on the number of P_tr receptor sites.The time during which a portion of the seeds were still photoreversibleby far-red light was increased by imbibition in PEG 6000, indicatinga direct effect of w on a reaction involving phytochrome. Noqualitative effect of PEG 6000 on the Onset of secondary dormancywas seen; however, its effect on the relative rate of appearanceof secondary dormancy was equivocal. ¹ Mississippi Agricultural and Forestry Experiment Station cooperating. (Received February 23, 1978; )     

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.     

Desiccation of Phaseolus vulgaris Seeds During and Following Germination, and its Effect Upon the Translatable mRNA Population of the Seed Axes

Misra, S. and Bewley, J. D. 1986. Desiccation of Phaseolus vulgansseeds during and following germination, and its effect uponthe translatable mRNA population of the seed axes.—J.exp. BoL 37: 364–374. After imbibition and germination, seeds of P. vulgaris passfrom a stage where they are insensitive to desiccation to astage where they are sensitive. Desiccation of seeds duringthe sensitive stage results in an almost total impairment ofprotein synthesis upon subsequent rehydration. Seeds desiccatedduring the desiccation-tolerant stage, however, resume proteinsynthesis at almost control levels. The protein patterns obtained following in Vitro translationof bulk RNA from fresh imbibed, desiccated, and desiccated-rehydratedseed axes were qualitatively similar at 5 HAI (the desiccation-tolerant stage). The drying treatment resulted in increasedintensity of extant proteins at 5 and 12 HAI. At 12 HAI (thetransition stage between the desiccation-tolerant and desiccation-intolerantphases) desiccation and subsequent rehydration triggered synthesisof a unique set of proteins-the rehydration proteins. At 20HAI (the desiccation-intolerant stage) desiccation resultedin an overall decline in the intensity of proteins synthesizedin vitro. Also the rehydration proteins were not synthesizedin response to a drying and rehydration treatment at this time. Key words: Seed germination, desiccation, mRNA, in vitro translation, Phaseolus vulgaris     

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.