The Inhibition of Growth in Higher Plants by a Homologous Series of Guanidines and its Reversal by Spermine

The effects of guazatine, synthalins A and B and a homologousseries of aliphatic monoguanidines on the growth of cress, barleyand oat seedlings, and apple cell suspension cultures have beenstudied. In the homologous series of aliphatic monoguanidines[NH₂C(=NH)NH(CH₂)_x–1CH₃] greatest inhibition was foundwith x = 8-10 for cress, barley and oats and x= 10–14for apple cells. Spermine partially reversed the inhibitionin the light for cress and barley but in the dark no reversalwas found. Technical guazatine inhibited growth to a greaterextent than pure guazatine, and was comparable in toxicity tosynthalin B in cress, barley and oats. Reversal by spermineof inhibition due to guazatine and synthalin B was greater inthe light than in the dark in these plants. Calcium ions didnot reverse the toxicity of guazatine, synthalin B or dodine.Reversal of the inhibition by guazatine, synthalin B and dodineof the growth of the apple cells was considerably greater withspermine than with spermidine. Lepidium sativumcress, Hordeum vulgarebarley, Avena sativaoat, Malus sylvestrisapple, guanidines, guazatine, synthalins, dodine, spermine, spermidine     

Arginine Decarboxylase Is Localized in Chloroplasts

Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC.     

Regulation of arginine decarboxylase by spermine in osmotically-stressed oat leaves

Biosynthesis of polyamines in plants is controlled primarily by the enzymes ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (ADC: EC 4.1.1.19), which are responsible for the production of putrescine, and S -adenosyl-L-methionine (SAM) decarboxylase (EC 4.1.1.50) that is necessary for the formation of spermidine and spermine (Spm). Little is known about the metabolic or molecular mechanisms regulating the synthesis of these enzymes. We have studied the regulation of ADC synthesis by Spm in osmotically-stressed oat ( Avena sativa L. ev. Victory) leaves, using a polyclonal antibody to oat ADC and a cDNA clone encoding oat ADC. Treatment with Spm in combination with osmotic stress resulted in increased steady-state levels of ADC mRNA, yet the levels of ADC activity decreased. This absence of correlation is explained by the fact that Spm inhibits processing of the ADC proenzyme, which results in increased levels of this inactive ADC form and a consequent decrease in the ADC-processed form. Spermine treatment leads to delayed loss of chlorophyll in dark-incubated and osmotically-treated oat leaves. Thus, post-translational regulation of ADC synthesis by Spm may be important in explaining its anti-senescence properties.     

Polyamines inhibit lipid peroxidation in senescing oat leaves

Exogenous diaminopropane, spermidine, spermine and guazatine, an inhibitor of polyamine oxidase (EC 1.5.3.3) activity, were able to inhibit loss of chlorophyll and reduce the level of malondialdehyde in dark-incubated Avena sativa L. cv. Victory leaves, as well as in leaves subjected to osmotica used for protoplast isolation. Both lipoxygenase (EC 1.13.11.12) activity and enzyme-protein levels were reduced by incubation with spermine. The results provide support for the contention that the inhibition of lipid peroxidation by polyamines may be one of the mechanisms responsible for their anti-senescence effects.     

Effect of Stress Conditions Induced by Temperature, Water and Rain on Senescence Development

The effect of different stress conditions was studied in segmentsof oat leaves (Avena sativa L. cv. Suregrein). Temperature and water (water dificit and rain) stress conditionsaccelerated senescence development. Any stress condition leadingto an increase in membrane permeability accelerated photooxidativephenomena in light, and senescence development including chlorophyllbreakdown and hydroperoxides increase. On the contrary, in darkness,any stress condition prevented chlorophyll breakdown and senescencedevelopment except for proteolysis, despite an increase in permeability. Temperature stress did not increase proteolysis. Water stressinduced in a humid chamber increased proteolysis, but it didnot increase proteolysis when water stress was induced by floatingin osmotic solution. (Received November 17, 1986; Accepted August 19, 1987)     

Molecular forms of arginine decarboxylase in oat leaves

Arginine decarboxylase (ADC, EC 4.1.1.19) is the first enzyme in one of the two biosynthetic pathways of putrescine in plants and has been characterized from a number of species, beginning with the work of Smith (1979; Phytochemistry 18: 1447–1452) who suggested that oat ADC had native sizes of 118 and 195 kDa. There are several studies showing a lack of correlation between changes in enzyme activity and mRNA or protein levels. In oats ( Avena sativa L.) a posttranslational modification of ADC has been described. The protein is synthesized as a 66-kDa precursor that is proteolytically processed into two polypeptides of 42 and 24 kDa with an associated gain of enzyme activity. In the present work, we have studied the existence of different ADC molecular forms in oat leaves by determination of enzymatic activity and using polyclonal antibodies obtained against an amino acid sequence near the C terminus deduced from the nucleotide sequence. In Avena sativa L. cv. Victory, we demonstrate the existence of 5 different molecular forms of ADC, with approximate molecular mass of 195, 115, 66, 38 and 23 kDa, that react with our antibodies and have enzymatic activity. Our results agree with previous work, but this is the first report showing all molecular forms simultaneously and might be a preliminary contribution to solve the question about the distribution of multiple forms of ADC and their importance in the correlation between the enzymatic activity and mRNA levels.     

Thigmomorphogenesis: XXIII. Promotion of foliar senescence by mechanical perturbation of Avena sativa and four other species

Mechanical perturbation (MP, gentle tubbing) promoted the senescence of detached oat ( Avena sativa L. cv. Victory) leaf segments in the dark. The promotion of senescence increased with increase in the number of rubbings and could be seen after 24 h of dark incubation; the maximum effect was reached on day 3. The effect (% of control) of MP on the loss of protein was greater than the effect on chlorophyll (Chl) loss on day 1. However, on day 3 the effect of MP on the loss of Chl became greater than the effect on the loss of protein. Ethephon and 1-aminocyclopropane-1-carboxylic acid (ACC) marginally promoted the loss of Chl by both control and rubbed oat leaf segments, and the effect was additive with MP. Chloramphenicol (CAP), spermine, aminoethoxyvinylglycine (AVG) and Ca²⁺ marginally delayed the loss of Chl and protein in both control and rubbed segments. Kinetin greatly retarded the senescence of all segments. Even in the presence of these substances, the amounts of Chl and protein in the rubbed segments were always less than in their respective controls, thus retaining the effect of the MP. However, abscisic acid (ABA) and cycloheximide (CHI) caused the rubbed oat leaf segments to retain more Chl and protein than their respective control segments. The effect of CHI was actually enhanced by MP. Rubbing promoted the senescence of attached leaves of oats ( Avena sativa L. cv. Victory), maize ( Zea mays L. cv. Early Belle) and pumpkin ( Cucurbita pepo L. cv. Jack-o-lantern) cotyledons in the dark. Rubbing promoted the senescence of oat leaf segments even in light, although to a lesser extent compared to the effect in the dark. The senescence of leaves of pumpkin and cocklebur ( Xanthium strumarium Wallr. var. Pennsylvanicum ) in situ was also enhanced by MP.     

Effect of polyamines on stabilization of molecular complexes in thylakoid membranes of osmotically stressed oat leaves

Monocotyledonous leaves subjected to osmotica used for protoplast isolation accumulate a massive amount of putrescine (Put), lose chlorophyll and senesce rapidly. Treatment with spermidine (Spd) or spermine (Spm) prevents the loss of chlorophyll, indicating preservation of the thylakoid membranes at the site of the chlorophyll-protein complexes. Using several recently produced antibody probes, the effects on the stabilization of thylakoid membranes of applying either difluoromethylarginine (DFMA), a specific inhibitor of putrescine synthesis via arginine decarboxylase, or the polyamines Spd, Spm, or diaminopropane (Dap) to osmotically shocked oat leaves (Avena sativa L.) have been investigated. High protein levels were maintained in thylakoid membranes of leaf tissue incubated in the dark in the presence of 0.6 M sorbitol when pretreated with DFMA. After 48 h incubation, the level of the thylakoid protein D1, at the core of photosystem II, was higher in the DFMA-pretreated leaves as was the stromal protein ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; as indicated by the level of large subunits). Applications of Spd, Spm or Dap were effective in retarding the loss of D1, D2 and cytochrome f from the thylakoid membranes as well as Rubisco large subunits and chlorophyll from the leaf tissue. The effects of polyamine applications may be mediated through Dap since most of the added Spd or Spm was converted to Dap within 6 h. The possible mechanisms of action of polyamine applications and DFMA-pretreatment on stabilizing the composition of the thylakoid membrane are also discussed.Abbreviations Cyt cytochrome - Dap diaminopropane - DFMA DL--difluoromethylarginine - LSU large subunit (of Rubisco) - Put putrescine - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Spd spermidine - Spm spermine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis This research was supported by the Agricultural and Food Research Council and by the British-Spanish joint research programme Acción Integrade HB-079 (R.T.B. and A.F.T.), British Council SPN/BAR/991 (R.T.B.) and Comision Interministerial de Cienica y Tecnologia 90-130 (A.F.T.). We thank Merrell Dow Research Center (Cincinnati, Ohio) for the gift of DFMA and Teresa Capell and Xavier Figueras (Univ. Barcelona) for help and suggestions.     

Changes in Chlorophyll Content and Organization During Senescence of the Primary Leaves of Phaseolus vulgaris L. in Relation to Photosynthetic Electron Transport

A large decrease was observed in the chlorophyll content ofthe primary leaves of Phaseolus vulgaris during senescence.Chloroplasts isolated from mature and senescent leaves gavevery similar light saturation curves for electron transportreactions involving either PS I or PS II, indicating that theaverage number of chlorophyll molecules associated with eachreaction centre did not change during senescence. It is concludedthat the reaction centres ceased to function at the same timeas, or perhaps before, their antenna chlorophylls were lostfrom the thylakoid membrane, and that the percentage decreasein the number of functional reaction centres per leaf was atleast as great as the percentage decrease in the leaf chlorophyllcontent. The chlorophyll-protein composition of thylakoid membrane preparationswas examined by electrophoresis of samples treated with sodiumdodecyl sulphate. In older leaves a smaller proportion of thechlorophyll applied to polyacrylamide gels was associated withthe P700- chlorophyll a-protein complex. There was also a declinein emission at 734 nm in the 77 °K fluorescence spectrumof intact leaf tissue during senescence. These results indicatethat older leaves contained a smaller proportion of chlorophyllsassociated with PS I, and this is consistent with the decreaseobserved in the leaf chlorophyll a/b ratio during senescence.The effect of these changes in chlorophyll content on the capacityof the chloroplast to carry out photosynthetic electron transportis discussed.     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)     

Control of Chlorophyll Degradation in Detached Leaves of Barley and Oat through Effect of Kinetin on Chlorophyllase Levels

Chlorophyllase seems to be responsible for the degradation of chlorophyll during senescence of detached leaves of barley (Hordeum vulgare) and oat (Avena sativa). Treatment at temperatures higher than 40°C — which protects against chlorophyll loss — lowers the level of chlorophyllase. Kinetin treatment lowers the level of chlorophyllase in barley leaves and prevents its rise in oat leaves after their detachment. Synthesis of proteins in both cytoplasm and chloroplast seems to be required in order to maintain a high level of Chlorophyllase in barley leaves after detachment.     

Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.     

Senescence of Rice Leaves XXX Levels of Endogenous Polyamines and Dark-Induced Senescence of Rice Leaves

The role of endogenous polyamines in the control of dark-inducedsenescence of detached rice leaves was investigated by quantitatinglevels of various polyamines by HPLC. Putrescine, spermidineand spermine were all present throughout senescence. Neithercadaverine nor 1,3-diaminopropane was detected. During dark-inducedsenescence, there was a marked decrease in levels of putrescineand an increase in those of spermidine and spermine. The rateof production of ethylene increased markedly upon excision ofleaves. -Difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO) caused a reduction in levels of putrescine, yet had noeffect on levels of spermidine and spermine. Neither DFMA norDFMO had any effect on senescence or on the production of ethylene.Treatment with dicyclohexylamine (DCH) and methylglyoxal bis-(guanylhydrazone)(MGBG) reduced levels of spermine and increased those of putrescinein detached leaves. After treatment with DCH or MGBG, both senescenceand the production of ethylene were significantly promoted.The current results suggest that endogenous polyamines may notplay a significant role in the control of dark-induced senescenceof rice leaves. This conclusion is supported by the furtherobservations that (a) benzyladenine, which is known to retardsenescence, decreased levels of putrescine but had no effecton those of spermidine and spermine; and (b) ABA, which promotedsenescence, increased levels of putrescine and had no effecton those of spermidine and spermine. (Received March 30, 1991; Accepted June 27, 1991)     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Identification of Hinesol as a Chlorophyll-Preserving Substance

A chlorophyll-preserving substance was isolated from rhizomesof Atractylodes lancea DC. and identified as (—)-hinesolon the basis of spectroscopic data. Hinesol at more than 0.22DIH was effective for preserving chlorophyll of oat (Avena sativaL. cv Victory) leaves in the dark with 40 to 50% of the initialchlorophyll content being retained at 2.2 mM. However, hinesolstimulated chlorophyll loss with light exposure and at 4.5 mMcaused complete bleaching when measured 4 days after treatment. ¹Dedicated to the memory of the late Professor Joji Ashida. (Received December 9, 1982; Accepted April 22, 1983)     

Senescence-promoting effect of arabidopside A

Arabidopside A isolated from Arabidopsis thaliana is a rare oxylipin, containing 12-oxophytodienoic acid (OPDA) and dinor-oxophytodienoic acid (dn-OPDA) which are known as precursors of jasmonic acid (JA) and methyl jasmonate (MeJA). The senescence-promoting effect of arabidopside A was examined by an oat (Avena sativa) leaf assay under dark or continuous light condition. Arabidopside A promoted senescence of oat leaves, and the promoting activity was more effective than for JA and OPDA, and as strong as for MeJA, which was well known to be a senescence promoter. These results suggest that arabidopside A plays important roles in leaf senescence.     

Stimulation of chlororespiration by heat and high light intensity in oat plants

High irradiance and moderate heat inhibit the activity of the photosynthetic apparatus of oat (Avena sativa L.) leaves. The incubation of oat leaves under high light intensity in conjunction with high temperatures strongly decreased the maximal quantum yield of photosystem (PS) II, indicating the close synergistic effect of both stress factors on PS II inhibition and the subsequent irreversible damage to the photosynthetic apparatus. The PS I A/B protein levels remained similar to control values in leaves incubated under high light intensity or moderate heat, and decreased only when both stress factors were simultaneously applied. Immunoblot analysis of thylakoid membranes using specific antibodies raised against the NDH-K subunit of the thylakoidal NADH dehydrogenase complex (NADH DH) and against plastid terminal oxidase (PTOX) revealed an increase in the amount of both proteins in response to high light intensity and/or heat treatments. In addition, these stress treatments were seen to stimulate the activity of electron donation by NADPH and ferredoxin to plastoquinone, the PTOX activity in plastoquinone oxidation and the NADH DH activity in thylakoid membranes. Incubation with n-propyl gallate (an inhibitor of PTOX) inhibited the increase of NDH-K and PTOX levels under high light intensity and heat, and slightly stimulated the activity of electron donation by NADPH and ferredoxin to plastoquinone. Antimycin A (an inhibitor of cyclic electron flow) increased the NADH DH activity and preserved the levels of NDH-K and PTOX in thylakoid membranes from leaves incubated under high light intensity and heat. The up-regulation of the PTOX and the thylakoidal NADH DH complex under these stress conditions supports a role for chlororespiration in the protection against high irradiance and moderate heat.     

Leaf Development in Lolium temulentum: Photosynthesis and Photosynthetic Proteins in Leaves Senescing under Different Irradiances

Changes in carbon fixation rate and the levels of photosyntheticproteins were measured in fourth leaves of Lolium temulentumgrown until full expansion at 360 µmol quanta m^–2s^–1 and subsequently at the same irradiance or shadedto 90 µmol m^–2 s^–1. Ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco), light-harvesting chlorophylla/b protein of photosystem II (LHCII), 65 kDa protein of photosystemI (PSI), cytochrome f (Cytf) and coupling factor 1 (CF₁) declinedsteadily in amount throughout senescence in unshaded leaves.In shaded leaves, however, the decrease in LHCII and the 65kDa protein was delayed until later in senescence whereas theamount of Cyt f protein decreased rapidly following transferto shade and was lower than that of unshaded leaves at the earlyand middle stages of senescence. Decreases in the Rubisco andCF₁ of shaded leaves occurred at slightly reduced rates comparedwith unshaded leaves. These results indicate that chloroplastproteins in fully-expanded leaves are controlled individually,in a direction appropriate to acclimate photosynthesis to agiven irradiance during senescence. (Received August 20, 1992; Accepted January 5, 1993)     

燕麦叶片衰老与活性氧代谢的关系

燕麦连体叶片与高体叶片衰老中,过氧化氢酶和超氧物歧化酶(SOD)活性下降,脂类过氧化产物丙二醛(MDA)迅速积累,组织自动氧化速率显著加快。植物激素BA,GA_3,2,4—D及光、亚胺环己酮(CH),EDTA处理均不同程度地延缓离体叶片的衰老过程,同时抑制过氧化氢酶和SOD活性下降,阻止MDA的积累和组织自动氧化速率的提高.推测叶片衰老中活性氧起着重要的作用。     

Nitrate Effects on Growth of the First Four Main Stem Leaves of a Range of Temperate Cereals and Pasture Grasses

The effects of different applied NO₃^– concentrations onextension growth and final length and area of leaves 1–4of five cereals and six pasture grasses of temperate originwere examined. Increased applied NO₃^– in the range 0.1–0.5.0mol m^–3 caused decreased duration of growth but increasedgrowth rate and final length of leaves 2–4 of the cerealsAvena saliva, Hordeum vulgare, Secale cereale, x Triticosecaleand Triticum aestivum. For all cereals, increased NO₃^–resulted in increased area of leaves 1–4. Pasture grasseswere supplied either 0.5 or 50 mol m^–3 NO₃^–. Increasedapplied NO₃^– (0.5–5.0 mol m^–3) resulted indecreased duration of growth and increased growth rate and finalarea of leaves 1–4 of Bromus wiltdenowii, leaves 2–4ofFestuca arundinaceae and leaves 3 and 4 of Lolium muitiflorum.In addition, length of leaves 3 and 4 of B. witidenowii increasedwith increased NO₃^–. Increased NO₃^– resulted inincreased area of leaves 2–4 of Dactylis gtomerata andLolium perenne and leaves 3 and 4 of Phalaris aquaiica but hadno effect on extension growth of all three species. Avena sativa L, oat, Hordeum vulgare L, barley, Secale cereale L, rye, x Triticosecale Wittm, triticale, Triticum aestivum L, wheat, Bromus willdenowii Kunth, prairie grass, Dactylis gtomerata L, cocksfoot, Festuca arundinaceae Shreb, tall fescue, Lolium multijlorum Lam, Italian ryegrass, Lolium perenne L, perennial ryegrass, Phalaris aquatica L, nitrate, leaf extension, leaf expansion