Manipulation of the polyamine content and senescence of apical buds of G2 peas

The effect of various treatments on the apical senescence and polyamine content of apical buds of G2 peas was analysed. Defruiting prevented senescence and increased bud size and polyamine content. Exogenous applications of GA₂₀ enhanced bud size and spermidine concentration. Applied spermidine had a slight effect on spermidine level but did not delay senescence. ACC strongly induced adecrease in bud size and, at 10 mM, apical senescence. This was accompanied by a steady decline in the level of all polyamines though their concentration remained constant until 10 mM ACC, where a drop was noted. Spermidine in the presence of ACC modulated the effect of ACC on the bud size while returning the internal polyamine content to control levels. AVG, an inhibitor of ACC synthesis produced pronounced increases in putrescine though no apparent effect on apical bud growth. Polyamine synthesis inhibitors were without effect on growth or internal polyamine content. The internal polyamine content appeared to correlate with apical bud size and vigor but did not show any consistent relationship to apical bud senescence.     

Correlation of spermine levels with ovary senescence and with fruit set and development inPisum sativum L.

Separation and quantitation of polyamines from unpollinated pea (Pisum sativum L.) ovaries and young fruits induced by application of gibberellic acid to unpollinated ovaries showed, in both cases, a decrease in putrescine and spermidine levels between anthesis and 4 d later. By contrast, spermine levels increased prior to the onset of senescence of the unpollinated ovaries (3 d post anthesis) and decreased during fruit development. Low levels of putrescine, spermidine and spermine were also observed in young fruits obtained by self-pollination and by treatment of unpollinated ovaries with 2,4-dichlorophenoxyacetic acid. In-vitro culture of ovary explants in a medium containing spermine showed that a reduction of the growth of gibberellic acid-treated unpollinated ovaries was associated with a rise in the level of spermine in the fruits. The results obtained indicate that changes in spermine levels are involved in the control of ovary senescence and of fruit set and development.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophen-oxyacetic acid - GA₃ gibberellic acid - HPLC high-performance liquid chromatography     

Evidence for a graft-transmissible substance which delays apical senescence in Pisum sativum L.

Apical senescence in an early flowering line of pea, G2, is greatly delayed by short days. This behavior is controlled by two dominant genes. Apical senescence of ungrafted, insensitive (I) lines is unaffected by photoperiod. When I-type scions with one of the two required genes were grafted onto G2, apical senescence of the I-type was delayed in short days, but not in long days. Flowering of the I-type was unaffected. The apex of the G2 stock was unaffected as well. Apical senescence of an I-type line lacking both photoperiod genes was not delayed when grafted on G2 in short days. It is concluded that G2 plants grown in short days produce a graft-transmissible factor which delays apical senescence of photoperiodically insensitive lines.     

The endogenous gibberellins of vegetative and reproductive tissue of G2 peas

The gibberellins (GAs) of both vegetative (leaves and stems) and reproductive (pods and seeds) tissue of the G2 strain of peas Pisum sativum L. were characterized in purified extracts by a combination of sequential silicic-acid partition column chromatography, and gas chromatography-mass spectrometry. Gibberellins A₁₉, A₂₀, A₂₉ and an A₂₉ catabolite were identified in both types of tissue. Gibberellins A₉, A₁₇ and A₄₄ were also found in pods and seeds.Abbreviations FID Ilame ionization detector - GA(s) gibberellin(s) - GC gas chromatograph(y) - HPLC high performance liquid chromatograph(y) - LD long day - MS mass spectrum(a) or mass spectrometer(ry) - SD short day     

Proteolytic activity in relationship to senescence and cotyledonary development in Pisum sativum L.

Changes in the weight and in the chlorophyll, free amino-acid and protein content of developing and senescing, vegetative and reproductive organs of Pisum sativum L. (cv. Burpeeana) were measured, and the proteolytic activity in extracts from the senescing leaf and the subtended pod was followed in relation to these changes. Protein content decreased in the ageing leaf and pod while it increased in the developing cotyledon. The proteolytic activity of the leaf did not increase as the leaf protein content decreased. In contrast, proteolytic activity in the subtended pod increased while the protein level decreased. The proteolytic activity in the extracts from the ageing organs was greater than the rates of protein loss. The proteolytic activity of leaf and pod extracts was greater on protein prepared from the respective organ than on non-physiological substrates. Proteolysis was increased by 2-mercaptoethanol and ethylenediaminetetraacetate but was not influenced by addition of ATP to the reaction mixture. The pH optimum was at 5.0. Free amino acids did not accumulate in the senescing leaf or pod when protein was degraded in each organ. It is suggested that these amino acids were quickly metabolized in situ or translocated to sink areas in the plant, especially to the developing seeds.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Gibberellins in developing fruits of Pisum sativum cv. Alaska: Studies on their role in pod growth and seed development

Gibberellins A₁, A₈, A₂₀ and A₂₉ were identified by capillary gas chromatography-mass spectrometry in the pods and seeds from 5-d-old pollinated ovaries of pea (Pisum sativum cv. Alaska). These gibberellins were also identified in 4-d-old non-developing, parthenocarpic and pollinated ovaries. The level of gibberellin A₁ within these ovary types was correlated with pod size. Gibberellin A₁, applied to emasculated ovaries cultured in vitro, was three to five times more active than gibberellin A₂₀. Using pollinated ovary explants cultured in vitro, the effects of inhibitors of gibberellin biosynthesis on pod growth and seed development were examined. The inhibitors retarded pod growth during the first 7 d after anthesis, and this inhibition was reversed by simultaneous application of gibberellin A₃. In contrast, the inhibitors, when supplied to 4-d-old pollinated ovaries for 16 d, had little effect on seed fresh weight although they reduced the levels of endogenous gibberellins A₂₀ and A₂₉ in the enlarging seeds to almost zero. Paclobutrazol, which was one of the inhibitors used, is xylem-mobile and it efficiently reduced the level of seed gibberellins without being taken up into the seed. In intact fruits the pod may therefore be a source of precursors for gibberellin biosynthesis in the seed. Overall, the results indicate that gibberellin A₁, present in parthenocarpic and pollinated fruits early in development, regulates pod growth. In contrast the high levels of gibberellins A₂₀ and A₂₉, which accumulate during seed enlargement, appear to be unnecessary for normal seed development or for subsequent germination.Abbreviations GA_(a) gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFK perfluorokerosene - PVP polyvinylpyrrolidone     

The source of gibberellins in the parthenocarpic development of ovaries on topped pea plants

The role and source of gibberellins (GAs) involved in the development of parthenocarpic fruits of Pisum sativum L. has been investigated. Gibberellins applied to the leaf adjacent to an emasculated ovary induced parthenocarpic fruit development on intact plants. The application of gibberellic acid (GA₃) had to be done within 1 d of anthesis to be fully effective and the response was concentration-dependent. Gibberellin A₁ and GA₃ worked equally well and GA₂₀ was less efficient. [³H]Gibberellin A₁ applied to the leaf accumulated in the ovary and the accumulation was related to the growth response. These experiments show that GA applied to the leaf in high enough concentration is translocated to the ovary. Emasculated ovaries on decapitated pea plants develop without application of growth hormones. When [³H] GA₁ was applied to the leaf adjacent to the ovary a substantial amount of radioactivity accumulated in the growing shoot of intact plants. In decapitated plants, however, this radioactivity was mainly found in the ovary. There it caused growth proportional to the accumulation of CA₁. Application of LAB 150978, an inhibitor of GA biosynthesis, to decapitated plants inhibited parthenocarpic fruit development and this inhibition was counteracted by the application of GA₃ (either to the fruit, or the leaf adjacent to the ovary, or through the lower cut end of the stem). All evidence taken together supports the view that parthenocarpic pea fruit development on topped plants depends on the import of gibberellins or their precursors, probably from the vegetative aerial parts of the plant.Abbreviations FW flesh weight - GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A₂₀ (GA₂₀) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A₂₉ and GA₂₉-catabolite were the major metabolites of GA₂₀ in the genotype na le. However, low levels of GA₁, GA₈ and GA₈-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a leaky mutation. Gibberellin A₂₀ was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3-hydroxylated metabolite of GA₂₀, GA₈ (2-hydroxy GA₁), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA₈ levels. A similar relationship was found on comparing GA₂₀ metabolism in the three genotypes le ^d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3-hydroxylation of GA₂₀ to GA₁). These results indicate that GA₂₀ has negligible intrinsic activity and support the contention that GA₁ is the only GA active per se in promoting stem growth in pea.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Lipoxygenase heterogeneity in Pisum sativum

Antibodies raised against two pea (Pisum sativum L. cv. Birte) seed lipoxygenases have been used to analyze lipoxygenase heterogeneity in seeds and in other organs. At least seven different polypeptides were identified in vivo; five of these were identified as precursors synthesized in vitro. The developmental appearance of the seed polypeptides has been analyzed and early and late forms were identified. Limited N-terminal sequence data indicated further heterogeneity when compared with sequences predicted from cDNAs.Abbreviations cDNA complementary DNA - DAF days after flowering - HPLC high-performance liquid chromatography - Ig immunoglobulin - kb kilobase - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SSC 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0 This work was supported by the Agricultural and Food Research Council via a grant-in-aid to the John Innes Institute. We acknowledge financial support from the Commission of the European Communities Biotechnology Action Programme; grant No. 0063-UK.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Photosynthesis and photosynthetic electron transport

The photosynthetic capacity of detached leaves of a non-yellowing mutant of Festuca pratensis Huds. declined during senescence at a similar rate to that in a normal cultivar. Respiratory oxygen uptake in the dark continued at similar rates in both genotypes during several days of senescence. In chloroplasts isolated from leaves at intervals after excision, the rate of photosystem I (PS I)-mediated methyl viologen reduction using reduced N,N,N,N-tetramethyl-p-phenylene diamine as electron donor also declined in both genotypes, possibly due to loss of integrity of the photosynthetic apparatus in the cytochrome f-plastocyanin region. There was a similar fall in PS II electron transport using water as electron donor and measured at the rate of reduction of 2,6-dichlorophenolindophenol. Partial restoration of this activity by the addition of diphenyl carbazide was evidence for lability of the oxygen-evolving complex during senescence. An accentuated difference between mutant and normal material in this case indicated that the mutant retains a greater number of functional PS II centres. Changes in the light-saturation characteristics of the two photosystems have been discussed in relation to the organization of the photosynthetic membranes during senescence.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DMSO dimethyl sulphoxide - DPC diphenyl carbazide - MV methyl viologen - PS I, PS II photosystem I, II - TMPD N,N,N,N-tetramethyl-p-phenylene diamine     

Nature of the effect of ther locus on the lipid content of embryos of peas (Pisum sativum L.)

The aim of this work was to discover why pea (Pisum sativum L.) embryos recessive at the r locus (rr) have a higher lipid content than embryos dominant at this locus (RR). The r locus is a gene encoding starch-branching enzyme, rr embryos have a much lower activity of this enzyme than RR embryos, and hence a reduced rate of starch synthesis. The higher lipid content of rr embryos must be a consequence of this. We suggest that neither differences in the availability of substrate for lipid synthesis as a consequence of different rates of starch synthesis, nor differences in the capacity of the pathway for malonyl-CoA synthesis, account for the different lipid contents of RR and rr embryos. Lipid contents of the two sorts of embryo first diverge at a much later stage in development than divergence in starch content. Amounts of pyruvate and acetate, and activities of enzymes that convert triose phosphate to malonyl CoA are the same in the two sorts of embryo. Most of the lipid in developing embryos is polar, structural lipid, and polar lipid accounts for a large proportion of the difference in lipid content between the two sorts of embryo. This difference in structural-lipid content reflects considerable structural differences between the two sorts of embryo and is presumably the consequence of differences in rates of lipid turnover.Abbreviations DW dry weight - FW fresh weight - FAME fatty-acid methyl esters This work was supported by a grant-in-aid from the Agricultural and Food Research Council to the John Innes Institute. We are very grateful to Alan Jones for his valuable advice on lipid analysis and to Dr. Kay Denyer (Advanced Technologies, Cambridge, UK) for valuable discussions. We thank Dr. Cliff Hedley for the gift of the seed of the peas used in this work.     

Synthesis and properties of glutathione reductase in stressed peas

We have subjected peas (Pisum sativum L.) to four different oxidative stresses: cold conditions (4 °C) in conjunction with light, treatment with paraquat, fumigation with ozone, and illumination of etiolated seedlings (greening). In crude extracts of leaves from stressed plants, an increase (up to twofold) in activity of glutathione reductase (GR) was observed which was consistent with previous reports from several laboratories. In all cases, except for ozone fumigation, the increase in activity was not due to an elevation in the steady-state levels of GR protein. None of the applied stresses had any effect on steady-state levels of GR mRNA. In contrast to the small increase in GR activity, the K_m of GR for glutathione disulphide showed a marked decrease when determined for extracts of stressed leaves, compared with that from unstressed plants. This indicates that GR from stressed plants has an increased affinity for glutathione disulphide. The profile of GR activity bands fractionated on non-denaturing acrylamide gels varied for extracts from differently stressed leaves and when compared with GR from unstressed plants. The changes in GR-band profiles and the alteration in the kinetic properties are best explained as changes in the isoform population of pea GR in response to stress.Abbreviations GR glutathione reductase - GSSG glutathione disulphide - Rubisco Ribulose-1,5-bisphosphate carboxylase-oxygenase - RNase A/T1 ribonucleases A and T1We are grateful to Prof. Alan Wellburn and Dr. Phil Beckett (Division of Biological Sciences, University of Lancaster, UK) for providing ozone-fumigated material and Dr. Jeremy Harbinson for providing material grown at 4° C. This work was supported by a grant-in-aid to the John Innes Institute from the Agricultural and Food Research Council. E.A.E. and C.E. gratefully acknowledge the support of a John Innes Foundation studentship and a European Molecular Biology Organisation Fellowship respectively.     

The effect of polyamines on ethylene synthesis during normal and pollination-induced senescence of Petunia hybrida L. flowers

Senescence of Petunia hybrida L. flowers is accompanied by a climacteric pattern in ethylene production and a rapid decline in the levels of putrescine and spermidine during the preclimacteric phase. The decrease in spermidine is caused by the decline in the availability of putrescine which is initially synthesized from L-arginine via agmatine and N-carbamoylputrescine. Inhibition of putrescine and polyamine synthesis resulted in a rapid drop in the levels of putrescine and spermidine without resulting in a concomitant increase in ethylene production. These results indicate that polyamine synthesis is not involved in the control of ethylene synthesis through its effect on the availability of S-adenosylmethionine, and is confirmed by the results obtained with pollinated flowers. Treatment with polyamines may stimulate or suppress ethylene production in the corolla, depending on the concentrations applied. In unpollinated flowers the onset of the climacteric rise in ethylene production was accelerated after treatment with polyamines. However, in pollinated flowers this process was delayed as a result of treatment with low concentrations of polyamines. The effects of exogenous polyamines on ethylene production in both pollinated and unpollinated flowers indicate that ethylene synthesis in these flowers is not regulated by a feedback control mechanism. Although polyamines do not play a key role in the control of ethylene production during the early stages of senescence through their effect on the availability of S-adenosylmethionine, it appears that they play an important role in some of the other processes involved in senescence.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MGBG methylglyoxal bis-(guanylhydrazone) - SAM S-adenosylmethionine     

Ribulose-1,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum L.

The polypeptide patterns obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis of undigested and autodigested extracts from pea (Pisum sativum L.) ovaries at the early stages of development or degeneration have been studied. Development of unpollinated ovaries was stimulated by application of different plant growth regulators (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and N⁶-benzyladenine) or by plant topping. Polypeptide bands of similar mobility to ribulose-1,5-bisphosphate carboxylase (RuBPCase) subunits (16 and 55 kDa) could be detected in all types of autodigested extracts from stimulated ovaries. However these bands were absent in electrophoretic patterns of autodigested extracts from unstimulated ovaries after 3 d post anthesis and in patterns of autodigested mixtures of these extracts with either those from stimulated ovaries or those from unstimulated ovaries before day 3. These observations indicate that a proteolytic activity which promotes the hydrolysis of RuBPCase appears in unstimulated ovaries about 3 d after anthesis. This event coincides with the loss of the capacity of unpollinated ovaries to develop in response to gibberellic acid and with the degeneration of the ovary wall.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis     

Changes in legumin messenger RNAs throughout seed development in Pisum sativum L.

The patterns of accumulation of three classes of legumin mRNA from Pisum sativum have been followed through seed development by cell-free translation and hybridization to complementary DNAs. Maximum amounts of mRNA were found at 19 days after flowering (DAF) for two classes and at 24 DAF for the third class. The proportions of the three classes varied through development: the RNA species which was 40% of the total legumin mRNA at 14 DAF represented 15–20% of the total at 25–27 DAF, whereas a second mRNA species represented approx. 30% and 70% at the same stages, respectively. Based on these results, some deductions about the possible contribution of individual genes within classes are made.Abbreviations cDNA complementary DNA - DAF days after flowering - IgG immunoglobulin G - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate)     

Enzymes of nitrogen mobilization in detached leaves of Lolium temulentum during senescence

During the senescence of Lolium temulentum leaf sections in the dark, asparagine and glutamine accumulated as the level of soluble protein declined. During the first 3–4 days after detachment, when the rate of protein loss was maximal, a four-fold increase in acid protease activity (EC 3.4.4.?) occurred. Subsequently this activity was replaced by proteases with a higher pH optimum. There was also a pronounced and continued activation of glutamate dehydrogenase (EC 1.4.1.2) during senescence. Glutamate pyruvate transaminase (EC 2.6.1.2), benzoylarginine-p-nitroanilide hydrolase (EC 3.4.?.?) and leucyl-p-nitroanilide hydrolase (EC 3.4.1.1) declined from high initial activities after 3–4 days. Glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1) was fairly stable although a marked increase occurred in the activity of one of two major GOT isoenzymes over the first two days. Glutamine synthetase (EC 6.3.1.2) was highly active in non-senescent leaves but fell sharply during the first three days of senescence. Little asparagine synthetase (EC 6.3.1.1) was detected. The role of these enzymes in the nitrogen metabolism of senescent detached leaves is discussed.