Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.)

Vicilin and legumin, the storage globulins of mature dry vetch (Vicia sativa L.) seeds, are found in protein bodies which are present not only in the cotyledons, but also in the radicle, axis and shoot (together, for reasons of simplicity, here called axis). When at 24 h after the start of imbibition (hai) the radicle breaks through the seed coat a major part of the globulins in the axis has already been degraded, whereas in the cotyledons globulin breakdown cannot yet be detected. Globulin mobilization starts with the degradation of vicilin. At 48 hai when globulin mobilization in the cotyledons just begins, the axis is already nearly depleted of globulins. Mobilization of storage globulin is probably brought about by a complex of different cysteine proteinases (CPRs). The papain-like CPR2 and CPR4, and the legumain-like VsPB2, together with their mRNAs, are already present in axes and cotyledons of dry seeds. This means that they must have been formed during seed maturation. Additional papain-like CPRs are formed later during germination and seedling growth. CPR4 and VsPB2 together with their corresponding mRNAs become undetectable as germination and seedling growth proceed. VsPB2 and VsPB2-mRNA are substituted by the homologous legumain-like proteinase B and its mRNA. The composition of stored and newly formed CPRs undergoes developmental changes which differ between axes and cotyledons. It is concluded that storage globulin mobilization in germinating vetch seeds is started by stored CPRs, whereas the mobilization of the bulk of globulin is predominantly mediated by CPRs which are formed de novo.     

Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination

Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination. Received: 14 February 2000 / Accepted: 16 August 2000     

The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns,intracellular localization and functions in globulin proteolysis

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V. sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (VPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

Distinct ethylene- and tissue-specific regulation of β-1,3-glucanases and chitinases during pea seed germination

The expression of β-1,3-glucanase (βGlu) and chitinase (Chn) was investigated in the testa, cotyledons, and embryonic axis of germinating Pisum sativum L. cv. `Espresso generoso' seeds. High concentrations of βGlu and Chn activity were found in the embryonic axis. Treatment with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that an early, 4-fold induction of βGlu activity in the embryonic axis during the first 20 h after the start of imbibition is ethylene-independent. This initial increase was followed by a later 4-fold ethylene-dependent induction in the embryonic axis starting at 50 h, which is after the onset of ethylene evolution and after completion of radicle emergence. The βGlu activity in cotyledons increased gradually throughout germination and was ethylene-independent. In contrast, the ethylene-independent Chn activity increased slightly after the onset of radical emergence in the embryonic axis and remained at a constant low level in cotyledons. Immunoinactivation assays and immunoblot analyses suggest that early βGlu activity in the embryonic axis is due to a 54-kDa antigen, whereas late induction is due to a 34.5-kDa antigen, which is likely to be the ethylene-inducible class I βGlu G2 described for immature pea pods. Increases in Chn in the embryonic axis were correlated with a 26-kDa antigen, whereas amounts of the additional 32- and 20-kDa antigens remained roughly constant. Thus, ethylene-dependent and ethylene-independent pathways regulate βGlu and Chn during pea seed germination. The pattern of regulation differs from that of leaves and immature pods, and from that described for germinating tobacco seeds. The functional significance of this regulation and its underlying mechanisms are discussed. Received: 12 January 1999 / Accepted: 22 March 1999     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

Pumpkin (Cucurbita sp.) seed globulin VII. Immunofluorescent study on protein bodies in ungerminated and germinating cotyledon cells

Protein bodies of pumpkin cotyledon cells were oval (about 10?7µm), and each was composed of a crystalloid, a globoidand proteinaceous matrix. They started to swell and fuse with1 day of imbibition. The proteinaceous matrix region expandedat the expense of crystalloids, and its electron density decreased.Finally, the protein bodies became central vacuoles includingmany small protein particles in about 8 days of germination. Fluorescent microscopy using antibodies raised against pumpkinseed globulin showed that fluorescence could not be observedin either protein bodies of ungerminated seeds or crystalloidsof germinating cotyledons, and only the proteinaceous matrixof germinating cotyledons became fluorescent. Probable causesof no fluorescence on crystalloids of seed globulin depositionwere considered. (Received November 9, 1979; )     

Purification and partial characteristic of a major gliadin-degrading cysteine endopeptidase from germinating triticale seeds

The endopeptidase of the highest electrophoretic mobility was the main endopeptidase hydrolyzing gliadin in the endosperm of germinated triticale (X Triticosecale Wittm.) grains after three days of imbibition. Activity of this endopeptidase, named EP8 starts to be detectable after two days of imbibition. The appearance of its activity in the endosperm on a second day of imbibition may suggest that EP8 is synthesized in aleurone during germination and/or secreted into the starchy endosperm as an inactive polypeptide during grains development and then activated. EP8 was isolated from the endosperm of germinating triticale seeds and purified 257-fold using ammonium sulphate, ion exchange chromatography on DEAE Sepharose CL-6B and gel filtration on Sephadex G-100. The enzyme was totally inhibited by E-64—class-specific cysteine proteinases inhibitor and activated by thiol compounds. Molecular weight estimated by SDS-PAGE was 39.5 kDa. The optimum pH for the hydrolysis of gliadin was 4.2 and for hemoglobin 5.2. High activity of EP8 against wheat gliadin in vitro suggests that this cysteine endopeptidase plays a major role in the mobilization of storage proteins in the endosperm of germinating triticale grains.     

Proteinase activities in resting and germinating seeds of Scots pine, Pinus sylvestris

Resting seeds of Scots pine contained a moderate amount of acid proteinase activity, about 90% of which was inhibited by pepstatin A and about 10% by p-hydroxymer-curibenzoate. In gel chromatography on Sephacryl S-200 the proteinase activity showed a complex elution pattern with poorly separated peaks at positions corresponding to mol. wts. 100,000 and 30,000 and several shoulders. The results suggested that pine proteinases I and II, which are the main proteinases in the endosperms of germinating seeds (Salmia 1981: Physiol. Plant. 51: 253–258), were not present in the resting seeds.—Seedling extracts showed a low level of acid proteinase activity, which separated into several peaks in chromatography on Sephacryl S-200. As none of the peaks had the catalytic properties of proteinase I or II, it seems that these endospermal enzymes are also lacking in the seedling tissues.—In the endosperms of germinating seeds the activity of the pepstatin-sensitive acid proteinase(s) remained at a constant level throughout the period of reserve protein mobilization (lasting up to the stage when the length of dark-grown seedlings was 60 mm). Proteinases I and II were absent from resting seeds, showed a small increase up to the 20-mm stage, and then increased rapidly up to the 60-mm stage.—Resting embryos contained relatively higher acid proteinase activity than resting endosperms, and again about 90% of it was inhibited by pepstatin A and about 10% by p-hy-droxymercuribenzoate. During germination the former activity decreased, the latter activity remained at approximately the same level, and the activity of the other acid proteinases increased continuously with the growth of the seedling.—It is concluded that the pepstatin-sensitive proteinase(s), which is not affected by endogenous proteinase inhibitors, plays a central role in the initiation of reserve protein mobilization in both the embryo and the endosperm. Proteinases I and II, on the other hand, seem to account for the greater part of reserve protein breakdown in the main protein storage tissue, the endosperm.     

The Relation of Storage Protein to Vigor and Its Mobilization Pattern in Germinating Peanut Seeds

The peanut (Arachis hypogaea L.) seeds harvested at the last stage of maturation were divided into five grades by size. The content of total protein, salt-soluble protein, arachin, conarachin I and 2s globulin in these seeds were measured. No obvious differences in germination percentage and the length of radicle and hypocotyl within 3d germination in dark were observed among the five grades of seeds. But there were significant differences in the seedling growth after two weeks of germination in light. There was a very close correlation between the storage protein in cotyledons and the seedling growth. When seeds germinated in light, the efficiency of mobilization of the salt-soluble protein in the cotyledons was higher than that in the cotyledons of the seeds germinating in dark. All of the salt-soluble protein in cotyledons was used up after 14d seedling growth in light. SDS-PAGE of salt-soluble protein showed that 23.5, 38.5 and 41 kD subunits of arachin were first mobilized during germination. The 18 kD subunits of arachin were not mobilized until the above-mentioned subunits were used up. The 60.5 kD subunit of conarachin I and 2s globulin were degradated within 2 to 3 days during germination.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases

Summary. Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied. mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development. Correspondence: C. S. Pereira, Institute for Molecular and Cell Biology, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.     

Weakening of muskmelon perisperm envelope tissue 4

Muskmelon (Cucumis melo L.) embryos are enclosed in an envelopeof tissue consisting of a layer of endosperm and a multi-cell-layeredperisperm that the radicle must penetrate for germination tooccur. The force and energy required to penetrate the perispermenvelope tissue were measured using an Instron universal testingmachine at a crosshead speed of 5 mm min^–1 after 0, 10,15, 22, 23, and 25 h of imbibition at 25C. The cellular structureof perisperm envelope tissue surrounding the radicle was observedafter 10, 15, 20, 25, and 48 h of imbibition using scanningelectron microscopy. The force required to puncture 5-mm-long,micropylar seed pieces declined steadily from 1.65 N in driedseeds to 0.65 N after 21 h of imbibition. The penetration energydeclined from 3.0 N mm in dry seeds to 1.1 N mm at 21 h afterthe start of imbibition when the first seeds germinated. Theforce and energy required to penetrate germinated seed pieceswere 0.55 N and 0.9 N mm, respectively, so the net punctureforce and energy needed to rupture the micropylar region ofthe perisperm envelope was roughly 0.10 N and 0.2 N mm at radicleemergence, respectively. Instron measurements of penetrationforce and energy decreased dramatically at crosshead speedsless than the 5 mm min^–1. Crosshead speeds greater than5 mm min^–1 may overestimate the pressure needed to ruptureperisperm and endosperm tissues. Intracellular cracks were firstobserved in SEM images 15 h after the start of imbibition, andafter 20 h cracking was apparent throughout the micropylar regionof the perisperm envelope. The perisperm envelope ruptured inone of two ways, coincident with radicle emergence. In approximately85% of muskmelon seeds, a large crack formed in the perispermenvelope adjacent to the radicle, while in roughly 15 % a circulararea of the perisperm envelope detached during radicle emergence.In dead seeds, the penetration force remained constant from10–24 h after the start of imbibition, and there wereno visible signs of tissue degradation. Cellular degradationand weakening of the perisperm envelope tissue precedes radicleemergence in muskmelon seeds. Key words: Seed, Instron, turgor, cell wall, electron microscopy, Cucumis melo     

Mobilisation of the major stored reserves in the embryo of fenugreek (Trigonella foenum-graecum L., Leguminosae), and correlated enzyme activities

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10–14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.     

Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998     

Cell Cycle Activation During Imbibition and Visible Germination in Embryos and Megagametophytes of Jack Pine (Pinus banksiana Lamb.)

Flow cytometric determination of cell cycle activation duringimbibition and visible germination in five families of jackpine (Pinus banksiana Lamb.) embryos and megagametophytes revealedthat in seeds that had undergone no imbibition the majorityof cells were in the 2C state. As the imbibition period increased,less of the nuclei were blocked in the G0/G1 state and morebecome active in the cell cycle. The augmentation in the nucleiactive in the 2C–4C cycle as well as those with DNA levelshigher than the 4C state occured gradually and preceeded radicleemergence. In megagametophyte tissue examined at various stagesof imbibition, cell cycle activity became apparent rapidly followingimbibition. In nuclei of green and white embryos examined separatelythe ²frequency distributions were significantly different forall three families after 144h. As imbibition period increased,fewer nuclei from the green embryos were blocked in the 2C state,and more became active in the 2C–4C cell cycle. This wasnot the case for white embryos where no significant linear relationwas noted. Cell cycle activity in the hypocotyl+cotyledons regionand the emerging radicle were examined separately. Functionalrelations found in the hypocotyl+cotyledons region were notevident in the radicle. As visible germination proceeded, cellcycle activity in the hypocotyl + cotyledons region for thisperiod of germination showed a reversal of the activity notedduring imbibition: fewer nuclei were active and once again ahigher proportion were found in the 2C state. cell cycle; C levels; DNA content; flow cytometry; germination; imbibition; jack pine; megagametophyte; Pinus banksiana Lamb     

Proteinase A-like enzyme from germinated kidney bean seeds. Its action on phaseolin and vicilin

A cysteine proteinase that possibly participates in the degradation of phaseolin, the main storage protein of kidney bean ( Phaseolus vulgaris L. cv. Moldavian) was isolated from germinating kidney bean seeds and partially characterized. According to its properties it may be classified as a member of a group of homologous cysteine proteinases A, also present in germinating seeds of a number of other plants. The proteinase of this group hydrolyze storage proteins to short peptides. Similarly, the kidney bean proteinase hydrolyzes vicilin, the reserve protein of vetch ( Vicia sativa ). However, its action on phaseolin is limited to the cleavage of subunits into two approximately equal parts and to the splitting off a small number of short peptides. An explanation of phaseolin resistance to the action of this proteinase is proposed on the basis of the differences of its structure from that of other homologous 7S proteins.     

Inter-nucleosomal DNA fragmentation and loss of RNA integrity during seed ageing

The germination of viable seeds is the basis for new plant growth and development. Seeds lose viability during storage, but the biochemical mechanisms of seed death are not fully understood. This study aimed to investigate degradation patterns of nucleic acids during seed ageing and subsequent water uptake. Seeds of Pisum sativum L. were artificially aged at 50°C and 12% seed water content (WC). Nucleic acids degradation was studied during ageing and during imbibition of four seed lots with differential viability from highly viable to dead. As seeds lost viability during ageing, DNA was gradually degraded into internucleosomal fragments, resulting in ‘DNA laddering’, in conjunction with disintegration of 18S and 28S rRNA bands. During imbibition, non-aged controls had high levels of DNA and RNA integrity through to radicle protrusion. In an aged seed lot with 85% total germination (TG) DNA fragmentation decreased upon imbibition probably due to nucleosome degradation, while rRNA integrity did not improve. In an aged seed lot with 44% TG, neither DNA nor rRNA integrity improved upon imbibition. Dead seeds showed DNA degradation as laddering throughout imbibition along with extensive degradation of rRNA. We present a model in which interlinked programmed and non-programmed events contribute to seed ageing, and suggest that protection of nucleic acids during ageing is key to seed longevity.