The Effect of Priming Treatments on the Viability and Accumulation of Chromosomal Damage in Aged Pea Seeds

A range of post-storage priming treatments were evaluated todevelop a protocol for priming pea seeds. Post-storage primingtreatments at 16 °C with PEG-8000 (-0·5, -1·0and -1·2 MPa), ABA (10^-1 M) and distilled water for 3,5 and 7 d ameliorated some of the damage which resulted fromageing. Most of the benefits occurred during the first 3 d withPEG or ABA and during the first 5 d distilled water. Primingtreatments increased the final germination and decreased themean germination time (MGT) and the frequency of chromosomalaberrations, possibly due to the repair of some age-induceddamage. The results of the priming experiment suggest that thecritical moisture content that facilitates repair of chromosomaldamage in pea seeds is likely to be between 32 and 46%. ABAhas been identified as a possible chemical which arrests germinationand facilitates repair of age-induced genetic damage.Copyright1995, 1999 Academic Press Pisum sativum, Pea, PEG, Polyethylene glycol, ABA, Abscisic acid, MGT, Mean germination time, seed priming, chromosome repair     

Nuclear Proteins During the Onset of Cell Proliferation in Pea Root Meristems

The DNA: proteins ratio in nuclei of root meristems of pea (Pisumsativum L. cv. Lincoln) changed considerably during germinationas cells moved from a quiescent to an actively proliferatingstate with a higher protein content in nuclei of the latter.Electrophoretic patterns of nuclear proteins extracted at differenttimes of germination were used to examine qualitative changes.The various patterns presented a substantial similarity butthere were some proteins whose content increased or decreasedand others which disappeared or appeared during germination.The bulk of these variations occurred between 24 and 48 h ofgermination, suggesting that they might be correlated to thetransition from quiescence to the proliferating state. The patternof nuclear proteins obtained from adult differentiated roottissue was also examined. We tried to purify five differentnuclear protein components from intact nuclei by a multi-stepextraction procedure using a series of different buffers toascertain the nature of proteins presenting major interestingvariations. Most of these proteins purified with the nuclearsap or ribosomal components. Key words: Cell proliferation, electrophoresis, Pisum sativum L, root meristems     

Activity of DNA topoisomerase I and quiescence of embryo cells in seeds of Pisum sativum L.

DNA topoisomerase activity is present at a very early stageof germination in nuclear extract of pea root meristems. Theactivity of this enzyme changes before the onset of replicativeDNA synthesis, thus suggesting the existence of a correlationbetween DNA topoisomerase I and events taking place during therelease of cells from a quiescent state. An antibody preparedagainst a human topoisomerase I is able to immuno-precipitatepart of the DNA topoisomerase activity present in pea nuclearextracts, and recognizes a protein with a molecular weight of45 kDa. We suggest that the 45 kDa protein is a DNA topoisomeraseI; its presence during embryogenesis and its storage in dryseeds would explain the presence of DNA topoisomerase I activityduring early stages of germination. Key words: Pisum sativum L, DNA topoisomerase, nuclei, quiescence, proliferation     

Changes in the activity of poly(adenosine diphosphate-ribose) polymerase during germination of pea

Poly(adenosine diphosphate-ribose) polymerase activity has been detected in nuclei from pea seedlings. The reaction is a specific polymerization of the adenosine diphosphate-ribose moiety of NAD, giving an average chain length of 2.4 residues. The enzyme shows marked changes in activity during germination: it exhibits a clear peak between 24 and 36 hr of germination, after which the activity declines to the level observed in ungerminated seeds.     

An Analysis of Seed Development in Pisum sativum: VI. ABSCISIC ACID ACCUMULATION

The abscisic acid (ABA) content of wrinkled (rr) pea seed tissueshas been quantified during development using multiple-ion-monitoringcombined gas chromatography-mass spectrometry and a deuteratedinternal standard. The level of ABA in the embryo generallyincreased with increasing cotyledon fresh weight while thatin the testa showed a distinct maximum at the time of maximumendosperm volume and the slowing in the growth of the testa.Pods contained relatively little ABA on a fresh weight basis.The total seed ABA content showed a biphasic distribution, thefirst maximum following the maximum growth rate of the testaand the second that of the embryo. The biphasic distributionof ABA in the pea seed was confirmed using a second pea genotype,near-isogenic to the first except for the r locus, and by theanalysis of individual seeds using a radioimmunoassay for ABA.The first maximum was composed mainly of a testa component andthe second mainly of an embryo component. When plants were grownin different environments, wrinkled seeds were found to containslightly more ABA than round (RR) but this was only significantlate in development. Immature seeds were capable of metabolizing17'-deoxy ABA to ABA, as determined by incorporation of either³H or ²H, and the metabolite was present mainly in the testa.The production of ABA in pea seeds is discussed in relationto the development of the different seed tissues. Key words: Abscisic acid, peas, seed development     

Ethylene Production in Pea and Cocklebur Seeds of Differing Vigour

Relationships between seed vigour and ethylene (C₂H₄) productionwere studied using C₂H₄-responsive fatty cocklebur seeds (Xanthiumpennsyhanicum Wallr.) and C₂H₄-insensitive starchy pea seeds(Pisum sativum L. cv. Alaska), which had been harvested in differentyears and subjected to different storage conditions. In bothspecies, the seeds with the highest vigour evolved the largestamounts of C₂H₄ during a period of water imbibition. The reductionof C₂H₄ production in cocklebur seeds occurred concomitantlywith the reduction in the growth potentials of both axial andcotyledonary tissues. Similarly, the activity of ACC-C₂H₄ conversionincreased with soaking, and was greater in seeds of high vigourcompared with those of low vigour. However, the change in ACCcontent in pea seeds differed from that in cocklebur seeds.That is, pea seeds with high vigour accumulated less ACC duringan imbibition period than those with low vigour. From theseresults it was suggested that the inferior C₂H₄ production bylow vigour pea seeds is mainly attributable to low ACC-C₂H₄conversion, whereas that by low vigour cocklebur seeds is dueto the shortage of ACC supply in addition to the reduced ACC-C₂H₄conversion. However, germination of deteriorated cocklebur seedswas not restored by exposure to ACC or C₂H₄, suggesting thatthe loss of seed vigour reduces the responsiveness of seedsto C₂H₄ as well as C₂H₄ production. Key words: Pea, cocklebur, seed vigour, ethylene production, 1-aminocyclopropane-1-carboxylic acid     

Turnover of Storage Protein in Seeds of Soya Bean and Pea

We were interested in determining whether the low protein contentof pea seeds (Pisum sativum L.) as compared to soya bean seeds(Glycine max L. Merrill) might be due to faster degradationof the pea storage proteins during development of the seed.Pea and soya bean cotyledons were subjected to a ‘pulse-chase’experiment using [³H]glycine in in-vitro cultures. In peas,legumin had a half-life of 146 days, while vicilin had a half-lifeof 39 days. There was no measureable degradation of soya beanstorage proteins. Even with the pea storage proteins, the half-liveswere so much longer than the maturation time of seeds that degradationof storage proteins could not account for the lower proteincontent of peas as compared to soya beans. The validity of theseresults was indicated by the finding that non-storage proteinshad much shorter half-lives and that omission of a carbon ora nitrogen source greatly accelerated degradation. Labelledglycine was found to be a good probe for protein turnover studiesbecause it was very rapidly metabolized. Glycine max L. Merrill, soya bean, Pisum sativum, L. pea, protein turnover, storage proteins, legumin, vicilin     

Nuclear Replication Activity During Seed Development, Dormancy Breakage and Germination in Three Tree Species: Norway Maple (Acer platanoidesL.), Sycamore (Acer pseudoplatanusL.) and Cherry (Prunus aviumL.)

Flow cytometric analyses of nuclear DNA levels were carriedout during development, stratification and germination of dormantseeds from three tree species with contrasting characteristics.Norway maple (Acer platanoides) and sycamore (Acer pseudoplatanus)have orthodox (desiccation-tolerant) and recalcitrant (desiccation-sensitive)storage behaviours, respectively, and require only a periodof cold to break dormancy, whereas, orthodox cherry (Prunusavium) seeds require an initial warm period before cold stratificationto fully stimulate germination. Whole embryos and radicle tipsof both Norway maple and sycamore were found to have stablehigh levels of 4C DNA during the latter stages of developmentand both contained nuclei arrested at the 2C and 4C levels atmaturity. Mature cherry embryos had nuclei predominantly arrestedat the 2C level. This suggests that the acquisition of desiccationtolerance is not correlated with the arrest of the cell cycleat any particular nuclear DNA level. Neither DNA replicationin radicle cells nor germination occurred when seeds were maintainedmoist at a constant 20 °C. However, in the late stages ofcold treatment during stratification, nuclear DNA levels inradicle cells changed in advance of radicle emergence in theorthodox Norway maple and cherry, whereas in the recalcitrantsycamore, change was not recorded until after radicle emergence.These results show that DNA replication has potential use asan indicator of the progress of tree seeds through stratificationtreatments used to break some types of dormancy. The ways inwhich this indicator could be exploited for seed quality andperformance testing are discussed.Copyright 1998 Annals of BotanyCompany Norway maple,Acer platanoidesL., sycamore,Acer pseudoplatanusL., cherry,Prunus aviumL., DNA replication, flow cytometry, seed dormancy, stratification     

Sunflower (Helianthus annuus) Leaves Contain Compounds that Reduce Nuclear Propidium Iodide Fluorescence

Sunflower leaves have unidentified compounds that interferewith propidium iodide (PI) intercalation and/or fluorescence.Independently prepared pea leaf nuclei show greater PI fluorescencethan nuclei from pea leaves simultaneously processed (co-chopped)with sunflower leaves. Differences in fluorescence persist aftermixing the PI-stained pea and the co-chopped pea/sunflower samples,i.e. PI staining protects the nuclei from the effects of theinhibitor. The current results are significant to practicalflow cytometric determination of plant nuclear DNA content.They show: (1) simultaneous processing of nuclear samples fromthe target and the standard species is necessary to obtain reliableDNA estimates; (2) a test for the presence of inhibitors shouldbe conducted; and (3) when inhibitors are present caution shouldbe taken in interpreting differences in estimated DNA content.The previously reported environmentally-induced variation inDNA content in sunflower populations is most simply explainedby variation in the amount of environmentally-induced inhibitorthat interferes with intercalation and/or fluorescence of PI.Intraspecific variation of DNA content for Helianthus annuusneeds to be re-evaluated using best practice techniques comparingphysiologically uniform tissues that are free of inhibitors.The best estimate for 2C DNA content of H. annuus used in thisstudy is 7.3 pg. Copyright 2000 Annals of Botany Company Helianthus annuus, DNA content, flow cytometry, propidium iodide, endogenous inhibitors     

Amylase Activities in Attached and Excised Cotyledons and in Embryonic Axes of Pisum sativum L.

Amylase activity increased in attached cotyledons of peas, Pisumsativum L. var. Bördi, only during imbibition and remainedalmost constant up to 96 h after germination, but in excisedcotyledons the activity increased slightly at first then markedly.In contrast, the content of the reducing sugars was higher inattached cotyledons than in excised ones. A similar inverserelationship has been found between the concentration of reducingsugars in axes (both attached and excised) and amylase activity. The leakage from intact seeds contained more reducing sugarsthan the leakage from excised cotyledons, whereas the amountof proteins released from the cotyledons was four times greaterduring imbibition. This increase in amylase activity in excisedcotyledons is not thought to be the result of axis excision,but to be the result of the leakage of sugars from the cotyledonsduring incubation. These results suggest that the concentration of reducing sugarsmay be a factor that regulates amylase activity in vivo in boththe cotyledons and axis during the germination of pea seeds. (Received August 4, 1982; Accepted December 14, 1982)     

Interruption of Water Delivery at Physiological Maturity is Essential for Seed Development, Germination and Seedling Growth in Pea (Pisum sativum L.)

As pea plants (Pisum sativum L.) cv. Finale and Solara developin the field, pods located at the second flowering node, increasein fresh and dry weight until 75 d after planting (DAP); thereafter,dry weight and moisture content decrease rapidly. Seed developmentconsists of three phases, all limited by low moisture content(MQ. The first phase (PI) corresponds to the formation of theembryo and surrounding structure (MC is stable at 85%). Duringthe second phase (P2) the cotyledons are filled (MC decreasesfrom 84% to 55%). The third phase (P3) entails desiccation onthe mother plant. Fresh and dry weight increase until 75 DAP(55% MC or physiological maturity, PM) and rapidly decreaseduring the third phase until 14% MC. Leakage conductivity ofimmature seeds reflects sap arrival in the testa and accumulationin the apoplast it is important during PI and the onset of P2,but during P2 it decreases slowly until physiological maturityand rapidly thereafter. Immature seed germination is noted from62 DAP, increases until 67 DAP, then decreases with a maximumat physiological maturity; some days after physiological maturitygermination is complete (up to 100%). Seedling growth from immatureseeds during P2 is low, but slowly increases until physiologicalmaturity. When germination (up to 100%) is attained seedlinggrowth is normal. Hydration of the immature seed indicates that55% MC is a highly specific value at which a transition in thehydration rate is occurring. Moisture loss from pea seeds duringthe P2 filling stage appears to be necessary for seed adaptationto the abrupt desiccation which occurs at physiological maturity.Physiological maturity is attained when disruption of the vascularconnection between the pod and the mother-plant occurs. Key words: Physiological maturity, germination, growth, pea     

Polyadenylation of pea seed RNA at the early stages of germination.

1. The RNA polyadenylating activity was found in embryo axes of dry, as well as imbibed and germinated pea seeds, both in nucleus and cytoplasm. 2. The total enzymatic activity remains unchanged during germination, but the intracellular distribution is altered; the activity in nuclei is increased about four-fold at the expense of the postmitochondrial fraction. 3. Specificity towards RNA primers of the polyadenylating system from pea embryo axes is low. 4. Cordycepin inhibits RNA polyadenylation only when [14C]ATP is used as a nucleotide donor, and has no visible influence on the activity of the system utilizing [14C]oligo(A)-nucleotides. 5. It seems that RNA in the pea embryo axes is polyadenylated by a two-step mechanism: synthesis of oligo(A)-nucleotides, and their addition to RNA.     

Protein Synthesis During Rehydration, Germination and Seedling Growth of Naturally and Precociously Matured Soybean Seeds (Glycine max)

Soybean seeds [Glycine max (L.) Merr.] synthesize de novo andaccumulate several non-storage, soluble polypeptides duringnatural and precocious seed maturation. These polypeptides havepreviously been coined ‘maturation polypeptides’.The objective of this study was to determine the fate of maturationpolypeptides in naturally and precociously matured soybean seedsduring rehydration, germination, and seedling growth. Developingsoybean seeds harvested 35 d after flowering (mid-development)were precociously matured through controlled dehydration, whereasnaturally matured soybean seeds were harvested directly fromthe plant. Seeds were rehydrated with water for various timesbetween 5 and 120 h. Total soluble proteins and proteins radio-labelledin vivo were extracted from the cotyledons and embryonic axesof precociously and naturally matured and rehydrated seed tissuesand analyzed by one-dimensional PAGE and fluorography. The resultsindicated that three of the maturation polypeptides (21, 31and 128 kDa) that had accumulated in the maturing seeds (maturationpolypeptides) continued to be synthesized during early stagesof seed rehydration and germination (5–30 h after imbibition).However, the progression from seed germination into seedlinggrowth (between 30 and 72 h after imbibition) was marked bythe cessation of synthesis of the maturation polypeptides followedby the hydrolysis of storage polypeptides that had been synthesizedand accumulated during seed development. This implied a drasticredirection in seed metabolism for the precociously maturedseeds as these seeds, if not matured early, would have continuedto synthesize storage protein reserves. Glycine max (L.) Merr, soybean, cotyledons, maturation, germination/seedling growth     

Studies on the Germination of the Seeds of Striga hermonthica: III. On the Nature of Pretreatment and after-ripening

An attempt has been made to elucidate some of the changes whichtake place in seeds of Striga hermonthica (immature, ripe andoverripe), (a) during moisture-treatment at three temperatures(15°C., 22°C., and 32°C.), and (b) during after-ripeningin dry storage. Observations on the drift of respiration of immature seed throughoutpretreatment at 15°C. show that an inverse relationshipexists between germination and respirational activity. Thissuggests that germination depends on the accumulation of somemetabolite during pretreatment. It would seem that this metabolitemay also be used as a substrate for respiration during pretreatment. After-ripening of the immature seeds has been shown to changethe nature of the seeds in some way so that their peak respirationon exposure to moisture is at first progressively reduced. Thisreduction has been ascribed to the presence of some barriercapable of restricting gaseous diffusion. The data presented, togetherwith those of the previous paperof this series, suggest that the specific metabolite requiredfor germination might be proteinaceous. Certain other data appearto rule out the suggestion that germination capacity is conditionedby the sugar content of the seeds. Reasons are given which supportthe view that one of the main effects of the stimulating solutionon the seed is to increase the permeability of the seeds togaseous diffusion.     

Long-lived Messenger RNA and its Relationship to Protein Synthesis During Germination of Pea (Pisum sativum L.) Seeds

Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Results from in vitro and in vivo protein synthesis experimentsand from studies of polysome formation suggest that much ofthe long-lived mRNA present in the embryo axis does not directprotein synthesis. The increase in the rate of protein synthesisduring germination is thus dependent on recruitment of newlysynthesized mRNA molecules. Pea, Pisum sativum L., germination, mRNA, protein synthesis     

Advantageous and Detrimental Effects of Osmotic Pre-Sowing Treatment on the Germination Performance of Agrostemma githago Seeds

Dormant and after-ripened seeds of Agrostemma githago (corn-cockle)were pretreated in polyethylene glycol 400 (PEG) solutions attemperatures which would have allowed germination if the seedshad been imbibed in water, viz. 4?C or 20?C for after-ripenedseeds, and 4?C for dormant seeds. Pretreated seeds germinatedfaster than untreated seeds. The maximum decrease of the T₅₀(time to 50% germination) was 66%. Furthermore, pretreated seedswere capable of germination at supra-optimal temperatures whichotherwise had inhibited germination completely (20?C for dormantseeds and 30?C for after-ripened seeds). The percentage germinationat a supra-optimal temperature was considerably higher whenthe seeds had been primed at a temperature at which they developedmore extension power. The advantageous effects of the osmotic pretreatment were lessthan might be expected when the osmoticum had inhibited onlycell elongation. This was largely, if not fully, due to a generaldetrimental effect of osmotic stress and not to a selectiveinhibition of the processes which occur during the pregerminativephase in preparation for growth. Thus, during priming seedscomplete all or almost all processes which occur in water-imbibedseeds prior to radicle emergence. Key words: Agroatemma githago, dormancy, germination, germination performance, osmotic stress, priming     

Protein Synthesis in the Axes of Polyethylene Glycol-Treated Pea Seed and during Subsequent Germination

Germination of Alaska pea seeds is inhibited by –0.3 MPapolyethylene glycol but upon subsequent transfer to water, germinationis completed rapidly and radicle emergence occurs more quicklythan in water-imbibed seeds. Protein synthesis is reduced inthe axes of seeds imbibed on PEG but increases upon their returnto water, though not to the level exhibited by axes germinatedon water. Mobilization of proteins in the axes is retarded bytheir failure to complete germination on PEG, although somedoes occur. The quantitative reduction in protein synthesisresulting from incubation in osmoticum is not accompanied bymarked qualitative changes. The block to germination is notobviously associated with a restriction in synthesis of anyparticular protein or set of proteins; conversely, no ‘water-stress’proteins are synthesized in the presence of PEG. The synthesisof growth-specific proteins is prevented by PEG, but these increaseupon relief from the osmoconditioning treatments. These observationsdispute earlier claims for accelerated protein synthesis resultingfrom PEG treatments. Key words: Osmotic priming, Pisum sativum, germination, protein synthesis