Lack of Functional Interrelationship between {beta}-amylase Photoregulation and Chloroplast Development in Mustard (Sinapsis alba L.) Cotyledons

In the cotyledons of mustard seedlings light mediates an increasein ß-amylase [EC 3.2.1.2 [EC] ] activity via agency of phytochrome.In order to understand the functional significance of abovephotoresponse, the relationship between light induced ß-amylaseincrease, chloroplast development and starch content of cotyledonwas investigated. The application of SAN 9789 a chlorosis inducinginhibitor to mustard seedlings, though destroyed chloroplast,had no effect on light mediated increase in ß-amylaseindicating the lack of functional interrelationship betweenchloroplast development and ß-amylase increase. Thesubcellular localization studies revealed that ß-amylaseis a cytosolic enzyme. Additionally, the increase in the levelof ß-amylase had no relationship with in vivo starchlevel, which was present only in trace amounts. The noncorrelationof the photoregulated ß-amylase increase with thestarch content and its extra-chloroplastic localization indicatesthat ß-amylase does not participate in the mobilizationof plastidic starch in mustard cotyledon. (Received December 28, 1988; Accepted September 8, 1989)     

Nutrients-mediated shift in temporal expression of phytochrome controlled β-amylase synthesis in mustard (Sinapis alba L.) cotyledons

Abstract. In cotyledons of mustard ( Sinapis alba L.) seedlings grown with distilled water (DW) phytochrome controlled increase in β-amylase (E.C. 3.2.1.2) level takes place at about 42 h after sowing (starting point), while the photoresponse escapes from photoreversibility at 30 h after sowing. The temporal onset of starting point is presumed to be determined by innate process of developmental homeostasis, which is not amenable to influence of environmental factors such as light and nutrients. However, the temporal appearance of onset of phytochrome controlled increase in β-amylase level (starting point) in seedlings grown with Hoagland's nutrient solution (HS) is delayed by 9 h as compared to DW-grown seedlings. Concomitantly, the temporal appearance of the loss of photoreversibility of phytochrome mediated increase in β-amylase level (coupling point) is also delayed by 9 h in HS-grown seedlings. HS does not influence the primary action of phytochrome, the lifetime of components involved in signal chain of above photoresponse and the turnover of β-amylase enzyme. These results indicate that HS-induced temporal shift in onset of starting point of above photoresponse is caused by interaction of nutrients with the process of developmental homeostasis.     

Accumulation of Phenylpropanoids in the Cotyledons of Morning Glory (Pharbitis nil) Seedlings during the Induction of Flowering by Low Temperature Treatment, and the Effect of Precedent Exposure to High-Intensity Light

Extracts from the cotyledons of seedlings of Pharbitis nil strain‘Violet’ cultured at low temperature, which inducestheir flowering even in continuous light, with or without precedentexposure to high-intensity light, which shortens the periodof low temperature required for flowering, were analyzed byHPLC for substances correlating with the flower-inducing process.The content of two phenylpropanoids were found to increase duringthe low-temperature, and were identified as 3-O-feruloylquinicacid and dehydrodiconiferyl alcohol-13-O-ß-D-glucoside.The increase was more rapid in the cotyledons exposed to high-intensitylight before the low-temperature. This suggests that the accumulationof these compounds is correlated to the promotive effect ofhigh-intensity light on the flower-induction by low temperature. (Received March 7, 1994; Accepted April 2, 1994)     

Development of {beta}-amylase activity and polymorphism in wheat seedling shoot tissues8

Increasing ß-amylase activity in wheat (Triticum aestlvum,var. Star) seedling shoot tissues was consistently accompaniedby the development of a characteristic polymorphism of the enzyme,as shown by electrophoresis employing amylopectin-containingpolyacrylamide gels. Very young shoot tissue contained one principalform of the enzyme (ß1), whereas two other major forms(ß2, ß3) appeared complementary to thisupon further growth. In vitro incubation experiments indicatedthat the polymorphism arose via a probably proteolytic conversionof ß1 into ß2 and ß3. The conversioninvolved neither an activation of ß-amylase nor asignificant modification of ß-amylase component plvalues. The electrophoretic ß-amylase patterns ofsubcellular leaf compartments suggested that ß1 issynthesized in the cytoplasm of leaf mesophyfi cells and thatthe other forms arise upon transfer of this ‘primary’form into the vacuole. The development of shoot ß-amylaseactivity did not require light, but appeared to be under thenegative control of the chloroplast and was stimulated by mineralnutrients. No clear relationship between ß-amylaseactivity and starch metabolism was evident, since the leaf activitywas largely absent from mesophyll protoplasts, could not beunequivocally demonstrated in the mesophyll chioroplasts, anddeveloped regardless of whether the tissues contained significantamounts of starch or not. Key words: Wheat, leaves, ß-amylase, polymorphism, compartmentation     

Photoregulation of Phenylalanine Ammonia Lyase is not Correlated with Anthocyanin Induction in Photomorphogenetic Mutants of Tomato(Lycopersicon esculentum)

Photoregulation of phenylalanine ammonia lyase (PAL)(EC 4.3.1.5 [EC] )was analyzed in wild type (WT) and mutants: phytochrome dencient-awrea(au), high pigment exhibiting exaggerated phytochrome response(hp) and the double mutant (au.hp) of tomato (Lycopersicon esculentum(Mill.) cv. Ailsa Craig). Red light, acting via phytochrome,stimulates PAL activity in cotyledons and hypocotyls of tomatoseedlings. The time course of photoinduction of PAL in cotyledonsof the mutants (au and au.hp) and WT seedlings has a peak ofactivity at 4 h, after which the activity falls sharply, exceptin hp seedlings where activity is maintained at a high level.In hypocotyls, photoinduction of PAL also shows an initial rise,reaching a maximum at 3 h, followed by a sharp decline in themutants (au and au.hp) and WT seedlings. However in hp seedlingsphotoinduction of PAL is about 3 fold that in WT. The photoinductionof PAL appears to be dependent on de novo synthesis of proteinand nucleic acids. The use of a PAL specific inhibitor a-aminooxyß-phenylpropionic acid indicated that PAL is an essentialcomponent of the anthocyanin biosyn-thetic pathway in the tomatoseedlings. However, a comparison of anthocyanin biosynthesis[Adamse et al. (1989) Photochem. Photobiol. 50: 107] and PALphotoinduction data revealed that phytochrome mediated inductionof PAL and anthocyanin in the tomato seedlings are not correlated.While au and au.hp mutant seedlings show a similar increasein PAL level as in the WT, there is little formation of anthocyaninin these mutant seedlings. The results indicate that, in contrastto the photoregulation of anthocyanin synthesis which is dependenton the presence of the labile phytochrome (IP) pool in tomatoseedlings, the photoinduction of PAL is mediated via a smallpool of phytochrome in au mutant: stable phytochrome (sP) ora residual /P pool. (Received August 6, 1991; Accepted September 27, 1991)     

Effect of light treatment and endogenous growth hormones on {alpha}-and {beta}-amylase activities in cotyledons of Phaseolus vulgaris L.

The influence of light and endogenous plant-growth regulatorson the evolution of - and ß-amylases in cotyledonsof Phaseolus vulgaris L. was investigated. Both enzymes, whichare not present in ungerminated seeds, appear during germinationof intact seedlings or incubation of excised cotyledons. -Amylaseactivity decreases upon exposure to light. This inhibition iscorrelated with a drastic increase in chlorophyll content anda change in the endogenous gibberellin-inhibitor balance. ß-Amylaseactivity was not affected by light treatment but was by thepresence of endogenous cytokinins. (Received February 3, 1977; )     

The Effect of Light and of 2-(4-chlorophenylthio)-triethylamine hydrochloride on Chlorophyll and Carotenoid Contents of Mung Bean Seedlings

Carotenogenesis in mung been seedlings has been shown to beunder phytochrome control. Dark-grown seedlings contain moreindividual carotenoids than light-treated ones, but total carotenoidsare greatly increased by light. As expected, chlorophyll wassynthesized after a lag period on transferring etiolated seedlingsto the light, and there was an increase in total chlorophylland a parallel increase in total carotenoids. CPTA [2-(4-chlorophenylthio)-triethylamine hydrochloride]-treatedseedlings showed a decrease in both total carotenoids and totalchlorophylls. The usual effect of CPTA was also observed withmung bean seedlings, namely that lycopene, normally absent,appeared and increased to 17 per cent of total carotenoids afteran eight-day treatment. A cis-lycopene, possibly poly-cis-lycopene,was observed for the first time in CPTA-treated tissues. Onlyin the presence of CPTA but not on its removal was it possibleto show an inverse relationship between ß-caroteneand lycopene. This suggests that CPTA may not act as a cyclaseinhibitor in green tissues, cyclase being the enzyme/s involvedin the cyclization of carotenes. Phaseolus aureus Roxb., mung bean, carotenes, lycopene, xanthophylls, chlorophyll, 2-(4-chlorophenylthio)-triethylamine hydrochloride     

The Relationship Between Cyanide Assimilation and Asparagine Biosynthesis in Lupins

ß-cyanoalanine synthese in etolated seedlings of Lupinusangustifolius is found mainly in the mitochondrial fractionof the cotyledons and stems. In seedlings developing at 25°Cin the dark it reaches a maximal concentration after 5 days,at the same time as asparagine accumulation is most rapid andother physiological changes occur. However, maximal abiliatyto assimilate HCN gas to asparagine develops after 3 to 4 days,before asparagine accumulation begins. The possibility thatthe ß-cyanoalanine pathway is a major route for asparaginebiosynthesis in lupin seedlings is concluded to be unlikely.     

Changes in {alpha} and {beta}-Amylase Activities during Germination of Seeds of Alfalfa (Medicago sativa L.)

We examined the methods available for the assay of -amylasein alfalfa (Medicago sativa) and found the Phadebas test mostsuitable. The Phadebas assay and activity staining on ampholinegels after isoelectrofocusing revealed that an amylase is presentin the dry seeds of alfalfa and that its activity decreasesrapidly after the second day of seed germination. An amylasewas purified by affinity chromatography and gel filtration.The kinds of sugar generated from soluble starch by the purifiedamylase resembled those generated by other -amylases from plants,in particular those from mung bean (Vigna radiata). These resultsindicate that the amylase in alfalfa seeds belongs to the familyof -amylases. The molecular weight and isoelectric point ofthe -amylase were determined to be 43 kDa and 4.92, respectively. The Pantrac assay and activity staining on immobiline gels afterisoelectrofocusing revealed that the activities of ß-amylasesincreased during the initial 4 to 5 days of germination. Furthermore,treatment of whole seedlings with cycloheximide or actinomycinD inhibited the increase in activity of ß-amylasesbut did not affect the reduction in activity of -amylase. During germination of alfalfa seeds, -amylase activity decreaseswhile, in contrast, ß-amylase activity increases (inthe cotyledons of germinating seeds), changes that are specificto the germinating seeds of alfalfa. (Received September 8, 1990; Accepted February 20, 1991)     

cytokinin-induced amylolytic activity in bean cotyledons: Identification of the regulated enzyme

Separation of an extract of cotyledons of Phaseolus vulgarison a column of Sephadex G-200 revealed three amylolytic fractions.The slower migrating fraction hydrolyzed/ß-Limit dextrinazure and was inhibited by EDTA. The activity of this fractionwas enhanced by the embryo axis and this effect could be fullyreplaced by kinetin or benzyladenine. These results suggestthat the bean embryo axis exerts a promotive influence on theactivity of a-amylase in the cotyledons and that this effectis mediated by cytokinins. The other two amylolytic fractions did not show a-amylase activity.No effect of the embryo axis or of cytokinins on their activitycould be noted. ¹Present address: The Thimann Laboratories, University of California,Santa Cruz, California, U.S.A. (Received June 27, 1979; )     

Effect of Hypocotyl Excision on Red Light-Mediated Betacyanin Formation in Amaranthus Seedlings

The effect of hypocotyl excision on red light-mediated betacyanininduction in cotyledons of Amaranthus caudatus L. was studiedusing etiolated, 3-day-old half-seedlings and isolated cotyledons.The removal of the hypocotyl promoted betacyanin formation undersafelight conditions in a manner competitive with brief (5 mins),but additive with prolonged (6 h) red illumination. If a papersupport was provided in order to improve the aeration, betacyaninformation in safelight conditions was further stimulated, reducingthe inductive effect of brief, but not changing the action ofprolonged red illumination. These results demonstrate that betacyaninphotoregulation is restricted to cotyledons of A. caudatus seedlings,with no evidence for transmission of light signals between differentorgans. Excision and aeration appear to promote selectivelya very low fluence response (VLFR) induced by safelight, suggestingdifferent mechanisms of phytochrome phototransduction underVLFR, low fluence response induced by brief saturating red light(LFR) and high irradiance reaction (HIR) occurring under prolongedred illumination. Amaranthus caudatus L, betacyanin photoregulation, red light, very low fluence phytochrome reaction     

Effects of Blue Light Pretreatments on Internode Extension Growth in Mustard Seedlings after the Transition to Darkness: Analysis of the Interaction with Phytochrome

The effects of blue light (B) pretreatments on internode extensiongrowth and their possible interaction with phytochrome mediatedresponses were examined in Sinapis alba seedlings grown for11 d under 280 µmol m^–2 s^–1 of continuousblue-deficient light from low pressure sodium lamps (SOX). SupplementaryB (16 µmol m^–2 s^–1) caused no detectable inhibitionof the first internode growth rate under continuous SOX, butgrowth rate was inhibited after transfer to darkness. This effect,and the growth promotion caused by far-red bend-of-day' lightpulses were additive. The addition of B at 16 µmol m^–2s^–1 during 11 d, or only during the first 9 or 10 d orthe latest 0.75, 1 or 2 d of the SOX pretreatment caused approximatelythe same extent of inhibition after the transition to darkness.A single hour of supplementary B before darkness caused morethan 50% of the maximum inhibition. However, 24 h of lower fluencerates of B (4 or 7 µmol m^–2 s^–1) were ineffective.Covering the internode during the supplementary B period didnot prevent the response to B after the transition to darkness.Far-red light given simultaneously with B (instead of the SOXbackground) reduced the inhibitory effect of B. Above a given threshold fluence rate, B perceived mainly inthe leaves inhibits extension growth in subsequent darkness,provided that high phytochrome photo-equilibria are presentduring the irradiation with B. Once triggered, this effect doesnot interact significantly with the ‘end-of-day’phytochrome effect. Key words: Blue light, extension growth, phytochrome     

Starch Metabolism in Developing and Germinating Soya Bean Seeds is Independent of {beta}-Amylase Activity

Developing seeds of soya bean cultivars Chestnut and Altonahave only trace amounts of ß-amylase activity. Comparedto a standard variety, Wells, ß-amylase activitieswere 200–300 times lower in Chestnut and Altona. Nevertheless,Chestnut and Altona accumulate starch as a transient reservematerial which is utilized later in development. Seeds of Chestnutand Altona also produce starch early in germination which subsequentlydeclines after the 4th day of germination. Throughout germinationß-amylase levels in these cultivars are about 300-foldlower than that observed in Wells, which has a similar patternof starch metabolism. Widely varying levels of ß-amylasein both developing and germinating seeds appear to be unrelatedto starch metabolism which is very similar in all cultivarsstudied. Consequently, ß-amylase activity seems irrelevantto starch metabolism in the soya bean seed. starch, ß-amylase, Glycine max. (L.), Merr, soya bean     

Phytochrome Regulation of Expression of mRNA Encoding the Major Light-Harvesting Chlorophyll a/b-Binding Proteins of Photosystem II in the Haploid Phase of Adiantum capillus-veneris

Light regulates development in the fern Adiantum capillus-venerisduring both the haploid gametophyte and diploid sporophyte phases.In this study, we have investigated the role of the photoreceptorphytochrome in haploid spore germination and regulation of expressionof mRNA encoding the major light-harvesting polypeptides ofphotosystem II {Lhcb mRNAs). Both of these responses appearto be induced by phytochrome and inhibited by blue light, acting.throughseparate photoreceptor(s). Characteristics of phytochrome actionwere similar for both responses, showing no induction in thevery low fluence range and an ‘escape’ from far-redreversal for 50% of the response by 6 h. Overall, these resultsshow that phytochrome regulates both a morphological and a molecularprocess in haploid fern spores, and that these processes mayshare a common signal transduction pathway. (Received February 4, 1998; Accepted April 8, 1998)     

Dark transformations of phytochrome in cotyledons of Pharbitis nil

Pharbitis seedlings grown in total darkness or continuous far-redirradiation were exposed to 30 min of red irradiation followedby a dark period, and in vivo phytochrome in their cotyledonswas photometrically assayed at various times. Loss of photo-reversibilityof P_fr after the exposure to red light occurred without darkreversion to P_r in cotyledons of both seedlings. P_fr decay inthe former cotyledons was mostly prevented in the first 30 minunder red light illumination, while that in the latter occurredwithout such a lag phase. P_fr was no longer photometricallydetectable by the eighth hr after irradiation at both 18?C and25?C. No evidence has yet been obtained to show a correlation betweenphotometrically detectable phytochrome in vivo and the red far-redreversible responses of flowering. (Received August 6, 1974; )     

Phytochrome Photoconversion in Vivo: Comparison between Measured and Predicted Rates

The measured rates of phytochrome photoconversion in vivo, in etiolated cabbage (Brassica oleracea L.) seedlings and cucumber (Cucumis sativus L.) cotyledons, under blue, red, and far red irradiation, are significantly different from those predicted on the basis of the spectral photon flux distributions of the light sources and optical parameters of purified phytochrome. The geometrical relationships between the light source and the irradiated sample affect the rate of phytochrome photoconversion, which is significantly faster in cabbage seedling laying flat on white, wet filter paper than in seedlings in a vertical position. Light reflected from the white filter paper on the bottom of the dish contributes significantly to phytochrome photoconversion. Substituting the white filter paper with a less reflective black one results in a significant decrease of the rate of phytochrome photoconversion in cucumber cotyledons.     

Developmental and photobiological factors affecting photoperiodic induction in Arabidopsis thaliana Heynh. Landsberg erecta

We have tested whether the promotion of flowering by long days(LD) in Arabidopsis thaliana is a consequence of photoperiodicinduction. To achieve this, the flowering responses of Arabidopsisthaliana (L.) Heynh. Landsberg erecta (Ler) and the long-hypocotylmutants hy2, hy3 and hy4 were determined with respect to age,daylength and light quality. Ler was capable of distinguishingbetween short days (SD) and long days (LD) from about 4 d aftersowing at 20 C, the time at which cotyledons were expandingand greening. At this stage, the critical daylength was between8 h and 10 h. At 7 d, seedlings required five LD for inductionand, as the seedlings aged, they became more sensitive so thatby day 20, one LD was fully inductive. The response to SD innewly germinated seedlings was to delay flowering without alteringleaf number, but after about 10 d, delay of flowering by SDwas accompanied by extra leaves. In light quality experiments,blue light (B) was inductive for 5-d-old plants and in all subsequenttreatments, far-red (FR) caused induction in treatments at 12d and 18 d and low pressure sodium, equivalent to red, was notinductive at 5 d and 12 d, but partially inductive at day 18.Hence, both a specific blue-light photoreceptor and phytochromeA in High Irradiance Response mode promote floral induction.In daylength transfer experiments all three hy mutants respondedto LD by earlier flowering. Both hy2 and hy3 produced substantiallyfewer leaves than Ler in SD and hy3 flowered slightly earlierthan Ler. The hy4 mutants flowered later than Ler in SD andhad a higher leaf number. A scheme is proposed in which photoperiodicinduction depends on the ability of the plant to sense photoperiod,the stage of development and the photobiological input. We alsopropose that phytochrome A and the blue photoreceptor promoteflowering whereas phytochrome B promotes vegetative development. Key words: Arabidopsis thaliana, blue-absorbing photoreceptor, flowering, photoperiodic induction, phytochrome     

Synthesis and Turnover of {alpha}-Amylase in Cotyledons of Germinating Vigna mungo Seeds: Effects of Exogenously Applied End-Products and Plant Hormones

Pulse-chase experiments indicated that the higher levels ofa-amylase in detached and incubated cotyledons of Vigna mungothan those in cotyledons attached to the embryonic axis weredue to both faster synthesis and slower degradation of the enzymein the detached cotyledons than in the attached cotyledons.Levels of a-amylase in the cotyledons were examined in termsof possible effects of end-products and the effects of exogenouslyapplied plant hormones and growth regulators. Levels of a-amylaseactivity and content were reduced by high concentrations ofglucose and sucrose, and it is suggested that this effect wascaused mostly by osmotic stress and partly by end-product repression.The level of a-amylase was nearly twice that in controls after1 to 10µM GA₃ had been applied to the cotyledons. In addition,0.1 mM kinetin, 0.1 mM 2,4-D and 0.1 to 0.S mM naphthaleneaceticacid also increased the level by 34% to 66% as compared to thecontrol. ABA and uniconazole both prevented the synthesis ofa-amylase. (Received July 4, 1994; Accepted November 14, 1994)