Identification and characterization of dehydrins in horse chestnut recalcitrant seeds

The fraction of heat-stable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stress-induced dehydrin-like polypeptides. In our experiments, in tissues of dormant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Low-molecular-weight heat-stable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not cross-react with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissue-specificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heat-stable proteins during the entire period of stratification and also radicle emersion. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cycloheximide, or α-amanitin, dehydrins did not disappeared from the fraction of heat-stable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could cross-react with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50-kD heat-stable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heat-sensitive proteins and was named as a dehydrin-like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heat-stable cytosolic proteins. The role and function of major heat-stable proteins in horse chestnut seeds are yet to be studied.     

Proteins of cotyledons of mature horse chestnut seeds

This is the first characterization of proteins from storage parenchyma of cotyledons of mature dormant recalcitrant horse chestnut (Aesculus hippocastanum L.) seeds and evaluation the cell protein-synthesizing capacity. It was established that the content of protein in cotyledons did not exceed 0.5% of tissue fresh weight. Soluble proteins (the proteins of the postmitochondrial supernatant or cytosol) comprised the bulk (up to 90%) of total proteins. Protein of subcellular structures (20000 g-pellet) comprised 5–7% of total protein. Cotyledon proteins were heterogenous in their charges and molecular weights of subunits. Cotyledon protein was easily extracted with a salt (1 M NaCl); they comprised 90% of water-soluble albumin-like proteins. The proportion of globulins was insignificant; it did not exceed 5%. Most water-soluble proteins (more than 80%) were tolerant to heat denaturing. Among these heat-stable proteins, two major groups of polypeptides dominated: an electrophoretically homogeneous component with a mol wt of 24–25 kD and a complex group from three to five polypeptides with mol wts in the range between 6 and 12 kD. Native heat-stable proteins had disulfide bonds. Four fractions of heat-stable proteins were obtained by ammonium sulfate fractionation; three of them were alike in their polypeptide composition and contained major components with mol wts of 24–25 and 5–12 kD. It was established that the active translational machinery functioned in the cells of storage parenchyma in cotyledons of mature dormant horse chestnut seeds. During each stage of stratification, cotyledon fragments incorporated ³⁵S-methionine into TCA-insoluble material more actively than axial organs. We discuss cotyledon protein composition, their function as a storage organ, and a possible role of heat-stable proteins.     

Growth Capacity of Embryo Axes Excised from Dormant and Germinating Horse Chestnut Seeds and Their Response to Exogenous Abscisic Acid

In embryo axes excised from mature horse chestnut (Aesculus hippocastanum L.) seeds, both freshly-fallen and subjected to cold stratification, the ability for growth was studied. While excised axes were kept on water at 28°C for 3 days, their fresh weight and length increased, the polypeptide composition of soluble proteins changed, the content of some heat-stable polypeptides decreased, and the capacity for protein synthesis in vivo retained. All these processes were similar to those in the axes of intact seeds during stratification until radicle protrusion. Growth of excised axes accelerated with the increasing duration of stratification. Cycloheximide (50 mg/l) and -amanitin (7 mg/l) inhibited axis growth, but an inhibitor of ABA synthesis fluridone (5 mg/l) and a natural cytokinin dihydrozeatin (10^–5 M) did not influence the growth rate. The growth capacity of axes excised from dormant and germinating horse chestnut seeds indicates the absence of dormancy in the axes of mature seeds. ABA (10^–5 M) suppressed completely the growth of axes detached from seeds experiencing cold stratification but still not germinating, although protein synthesis was not inhibited. The axes excised from the seeds after radicle emergence were insensitive to ABA and grew actively in its presence. ABA-induced growth inhibition might be related to the suppressed synthesis of minor polypeptides required for growth or to the activated synthesis of some growth-retarding proteins. The conclusion was drawn that the excised axes could be used as a model for studying the processes preceding visible germination of recalcitrant seeds.     

The Role of Water Uptake in the Transition of Recalcitrant Seeds from Dormancy to Germination

In recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) maintaining a high water content during winter, dormancy is determined by the presence and influence of the seed coat, while the axial organs of the embryos excised from these seeds are not dormant. Such axial organs were capable for active water uptake and rapid fresh weight increase, so that their fresh weights exceeded those in intact seeds at the time of radicle protrusion. Fructose plays an essential role in the water uptake as a major osmotically active compound. ABA interferes with the water uptake by the axial organs and thus delays the commencement of their growth. The manifestation of seed response to ABA during the entire dormancy period indicates the presence of active ABA receptors and the pathways of its signal transduction. The content of endogenous ABA in the embryo axes doubled in the middle of dormancy period, which coincided with a partial suppression of water uptake by the axes. During seed dormancy release and imbibition before radicle protrusion, the level of endogenous ABA in axes declined gradually. Application of exogenous ABA can imitate dormancy by limiting water absorption by axial organs. Fusicoccin A (FC A) treatment neutralized completely this ABA effect. Endogenous FC-like ligands were detected in the seed axial organs during dormancy release and germination. Apparently, endogenous FC stimulates water uptake via the activation of plasmalemmal H⁺-ATPase, acidification of cell walls, their loosening, and turgor pressure reduction. FC can evidently counteract the ABA-induced suppression of water uptake by controlling the activity of H⁺-ATPase. It is likely that, in dormant intact recalcitrant seeds, axial organs, maintaining a high water content, are competent to elevate their water content and to start their preparation for germination under the influence of FC when coat-imposed dormancy becomes weaker.     

Analyses to determine the role of embryo immaturity in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: synthesis and accumulation of storage proteins and proteins implicated in desiccation tolerance

Development of yellow cedar seeds is completed by about 17-21 months after pollination. Following dispersal from the parent plant, the seeds exhibit a low capacity for germination and typically require an additional year to meet their moist chilling requirements and break dormancy. Biochemical analyses were undertaken in order to address whether seed dormancy is imposed and maintained because the embryo or megagametophyte is immature at the time of seed shedding and hence requires time to complete developmental events before dormancy can be terminated. Major protein reserves of the embryo and megagametophyte are the buffer-insoluble crystalloid (legumin) storage proteins and the water-soluble albumin proteins. SDS-PAGE, fluorography of in vivo synthesized proteins and Western blot analyses showed that the greatest increase in protein reserve synthesis and accumulation occurred between the first and second years of development; deposition of soluble and insoluble storage protein was largely completed in seeds of second-year cones by August, 2-3 months prior to seed dispersal. The period associated with greatest accumulation of storage proteins was accompanied by an increased accumulation of two ER-resident proteins associated with post-translational maturation of storage proteins (binding protein and protein disulphide isomerase). Accumulation of proteins implicated in the acquisition of desiccation tolerance (dehydrins and the tonoplast intrinsic protein, -TiP) occurred between the first and second years of development. Several heat-stable proteins and some of the proteins associated with late development continued to be synthesized after seed shedding and in 13 d moist-chilled mature seeds. However, this did not include the major dehydrin-like protein of yellow cedar seeds. Further, the continued synthesis of heat-stable proteins does not appear to be a factor preventing the germination of yellow cedar seeds following dispersal from the parent plant; rather, the mechanism of dormancy is primarily coat-imposed.     

Recalcitrant seeds of horse chestnut lack protein bodies

In recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.), the bulk of protein in axial organs and cotyledons is accounted for by water-soluble proteins (albumins). In the cells of embryo, proteins are predominantly located in the cytosol, whereas the fraction of cell structures precipitate in the range from 1000 to 20000 g, accounting for only an insignificant part of total protein. Among the proteins of this fraction, there were no major components that could play a role of storage proteins. The aim of this work was to study deposition of protein in the vacuoles of cells of recalcitrant seeds of horse chestnut. Light microscopy and specific staining of protein and phytin did not detect protein bodies in the vacuoles of axial organs and cotyledons. Electron microscopy revealed traces of phytin in the vacuoles, but there were no formed globoids or considerable amount of protein therein. It is possible that precisely the absence of typical storage proteins and genetically determined desiccation in the course of maturation of recalcitrant seeds of horse chestnut stipulated preservation of the vacuoles that in mature recalcitrant seeds were not transformed into protein bodies.     

Changes in ubiquitin and ubiquitin-protein conjugates during seed formation and germination

Changes in both free ubiquitin and ubiquitin-protein conjugateswere followed in cotyledons of lupin (Lupinus albus L.) duringthe course of seed formation, from the flower to the dry seed,and during germination and seedling growth, from the dry seedto the senescing cotyledons. The observed levels of ubiquitinconjugates, detected by immunoblotting using antiubiquitin antibodiesand by autoradiography using ¹²⁵I-labelled ubiquitin, suggestan intense involvement of the ubiquitin-mediated proteolyticpathway during the highly regulated phases of seed formationand germination. High amounts of free ubiquitin are presentat all stages in all tissues examined. With the exception ofthe dry seed, the high molecular mass ubiquitin-protein conjugatesare also present at all stages. Higher amounts of these conjugateswere found during the initial stages of pod development andseed germination and during the most active phases of storageprotein deposition and degradation. Germination and seedlinggrowth in total darkness not only delays the degradation ofthe storage proteins, but also extends the period characterizedby the presence of a high amount of these conjugates. No suchconjugates were detected in the dry seeds, probably reflectingthe extremely low metabolic activity observed in these organs.A number of smaller molecular mass polypeptides were also detectedat different stages of seed development, germination and seedlinggrowth. Of particular interest is the abrupt accumulation ofan abundant 20 kDa polypeptide in the cotyledons during the4th day after imbibition, which is maintained in high amountsin these organs, rapidly declining after about 12–14 d.The pattern of accumulation of the 20 kDa polypeptide is controlledneither by light nor by the embryo axes, and large variationsin its concentration are observed during heat shock. Key words: Ubiquitin, ubiquitin-protein conjugates, seed storage proteins, protein synthesis, protein degradation     

Transition from hormonal to nonhormonal regulation as exemplified by seed dormancy release and germination triggering

It is generally believed that seed dormancy release is terminated by germination and that this process is controlled by phytohormones. Most attention was paid to gibberellins (GAs) because treatment with GAs is most frequently applied for seed dormancy breaking. The review characterizes the hormonal regulation of seed dormancy and its release, as exemplified by arabidopsis seeds possessing non-deep physiological dormancy. Dormancy release occurs under the influence of low temperature and/or illumination with red light. Two main trends are typical of this process: (1) a decrease in ABA content and blocking of signal transduction from ABA, and (2) GA synthesis and activation of GA signaling pathway. Dormancy release ends with the GA-induced syntheses of some proteins, enzymes in particular, required for the start of germination. Quiescent seeds are capable of realizing the germination program without hormonal induction, due to nothing but seed hydration. In imbibing seeds, the triggering role of water lies in the successive activation of basic metabolic systems after attaining the water content thresholds characteristic of these systems and in preparing cells of embryo axial organs for germination. Thus, seed dormancy release is controlled by phytohormones, whereas subsequent germination manifesting itself as the initiation of cell elongation in embryo axes is controlled by water inflow.     

Water relations in germinating seeds

Life strategy of plants depends on successful seed germination in the available environment, and sufficient soil water is the most important external factor. Taking into account a broad spectrum of roles played by water in seed viability and its maintenance during germination, the review embraces early germination events in seeds different in their water status. Two seed types are compared, namely orthodox and recalcitrant seeds, in terms of water content in the embryonic axes, vacuole biogenesis, and participation of water channels in membrane water transport. Mature orthodox seeds desiccate to low water content and remain viable during storage, whereas mature recalcitrant seeds are shed while well hydrated but die during desiccation and cannot be stored. In orthodox Vicia faba minor air-dry seeds remaining viable at 8–10% water content in embryonic axes, the vacuoles in hypocotyl are preserved as protein storage vacuoles, then restored to vacuoles in imbibing seeds in the course of protein mobilization. However, in newly produced meristematic root cells, the vacuoles are formed de novo from provacuoles. In recalcitrant Aesculus hippocastanum seeds, embryonic axes have a water content of 63–64% at shedding and they lack protein storage vacuoles but preserve vacuoles preformed in maturing seeds. Independent of the vacuolar biogenetic patterns, their further trend is similar; they expand and fuse, thus producing an osmotic compartment, which precedes and becomes an obligatory step for the initiation of cell elongation. Prior to this, water moves in imbibing seeds through the membranes by diffusion, although the aquaporins forming water channels are present. In both seed types, water channels are opened and actively participate in water transport only after growth initiation. Aquaporin gene expression and their composition change in broad bean embryonic axes after growth initiation. This is the way how a mass water flow into growing seedling cells is achieved, independent of differences in seed water content and vacuole biogenesis patterns.     

Changes in late embryogenesis abundant proteins and a small heat shock protein during storage of beech (Fagus sylvatica L.) seeds

Beech seed physiology, including the effect of stress proteins like late embryogenesis abundant (LEA) and small heat shock proteins (sHSP) on viability during storage, is not fully understood. Four lots of beech (Fagus sylvatica L.) seeds have been stored for 1, 4, 6 and 8 years at −10 °C and 8–9% moisture content (MC). Under these conditions, the germination capacity ranges from 81.5% to 100% in the youngest seeds. However, the seeds decrease in vigour with prolonged time of storage. Dehydrins and dehydrin-like proteins were identified both in cotyledons and embryonic axes of the dry stored seeds. In general, decreased contents of LEA proteins as well as reduced content of total soluble protein were detected during prolonged storage. The contents of soluble proteins in embryonic axes and nearly all detected dehydrins and dehydrin-like proteins were correlated with germination capacity. Moreover a sHSP with molecular mass of approximately 22 kDa was identified. The largest content of this protein was observed in the oldest seeds, especially in embryonic axes. The proteins identified may play a protective role during water deficit and storage.     

Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L

Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M_r. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds.     

Seed germination during floatation and seedling growth of Carapa guianensis, a tree from flood-prone forests of the Amazon

Carapa guianensis Aubl. (Meliaceae), a hard wood tree from the Brazilian Amazon, has large recalcitrant seeds that can germinate and establish in both flood-free (terra-firme) and flood-prone (várzea) forests. These seeds, although large, can float. This study was designed to experimentally examine seed longevity under floating conditions ex-situ and its effects on subsequent germination and seedling growth. Many seeds germinated while floating, and radicle protrusion occurred from 3 to 42 d after the start of the floating treatment (tap water, room temperature 20–30 °C). Shoots of newly germinated floating seedlings may elongate up to 37.0 cm in 20 d without loss of viability. Epicotyl and first leaf emergence were delayed by floating. Seeds that did not germinate while floating were then placed on vermiculite and watered daily, where many seeds resumed germination. Germination during and after floating was affected by the length of the floating treatment: 88% germinated after 1 mo, 82% germinated after 2 mo and 70% germinated after 2.5 mo. These results indicate that Carapa guianensis has physiological variation regarding dormancy in response to seed floatation. The fact that floatation induces dormancy in recalcitrant seeds of this economically important species can be relevant to initiatives of ex situ storage of seeds.     

Protein Changes during the Stratification of Malus domestica Borkh. Seed

Apple seeds (Malus domestica Borkh. cv Golden Delicious) were stratified at 5 and 15°C for various lengths, weighed, and soluble protein of axis and cotyledon tissue was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only seeds treated at 5°C germinated; seeds treated at 15°C did not germinate. Optimal germination required 63 days of stratification. Excised embryos required less stratification time for germination than intact seeds. When stratification was less than 35 days, the resulting seedlings from 5°C stratified embryos were dwarfed and epinastic. After 63 days of stratification, axes from 5 and 15°C treated intact seeds had increased in fresh weight by 72 and 28% (w/w), respectively. The dry weights of the axes did not change significantly and both fresh and dry weights of cotyledons remained unchanged during stratification. Total soluble protein in axes and cotyledons changed very little during stratification. However, axis polypeptide profiles changed. Most obvious was the occurrence of a new polypeptide and the increase of four other clearly identifiable polypeptides during 5°C treatment. The levels of the five most predominant axis proteins decreased at the same time. We observed no changes in the profiles of soluble cotyledon proteins. Control seeds kept at −10°C showed none of the reported changes.     

Recalcitrancy and a new kind of epicotyl dormancy in seeds of the understory tropical rainforest tree Humboldtia laurifolia (Fabaceae, Ceasalpinioideae)

We report a new kind of seed dormancy and identify the storage behavior category for an important understory rainforest tree that also is used as an ornamental. While studying seed dormancy of Fabaceae species in Sri Lanka, we observed a considerable delay in emergence of the plumule following radicle emergence in Humboldtia laurifolia. Because epicotyl dormancy has not been reported in Fabaceae, we undertook a detailed morphological study of seed germination in this species. Our aims were to document desiccation tolerance/intolerance and epicotyl dormancy in seeds of H. laurifolia. Drying and low temperature storage were used to evaluate storage behavior of the seeds and imbibition, germination, and seed coat anatomy to categorize seed dormancy in two seed collections. Plumule development before its emergence and effects of light and temperature on plumule emergence were monitored. All seeds that were dried to 15% moisture content or stored at -1°C lost viability. Plumules began to grow 20 ± 5 d from radicle emergence and emerged after 40 ± 3 d. Dark and high illuminance further delayed plumule emergence. Seeds are recalcitrant and have a hitherto unreported kind of epicotyl dormancy, for which we propose the formula .     

Seed viability and seed dormancy of non-native Phragmites australis in suburbanized and forested watersheds of the Chesapeake Bay, USA

The non-native, invasive haplotype of Phragmites australis is rapidly invading tidal and non-tidal wetlands across North America. Phragmites has the potential to spread by seeds and rhizomes. Seed viability and dormancy differences were quantified among 18 patches of non-native Phragmites in subestuarine wetlands in developed (i.e., suburbanized) vs. forested watersheds of the Chesapeake Bay. We used tetrazolium and germination assays to assess seed viability and compared germination percentages and rate of germination among fresh seeds, cold–moist treated seeds, and warm–dry treated seeds to evaluate seed dormancy. Seed viability was <1% in most patches but a few patches produced abundant viable seeds (5–21%). Seed viability, however, did not differ significantly between wetlands in forested vs. developed watersheds. Contrary to studies of Phragmites seed dormancy in European populations, some Phragmites seeds were dormant at maturity; cold–moist treated seeds germinated faster and to higher percentages than fresh seeds or warm–dry treated seeds.     

Seed Dormancy in Acer: The Role of Testa-imposed and Embryo Dormancy in Acer velutinum

The embryo dormancy shown in freshly harvested samples of Acervelutinum seeds is weakly established and very short-lived.Loss of this embryo dormancy occurred during post-harvest fruitstorage at either 5 or 17 C. In contrast, the dormancy of intactfruits and seeds was overcome only during storage at the lowertemperature. Removal of the cotyledons from embryos of freshlyharvested fruits allowed more rapid germination of the embryonicaxes, indicating that the cotyledons exert an inhibitory effect,although the axes still retained a measure of innate dormancy.The inhibitory effect of the cotyledons became less marked withincreasing duration of fruit storage, this loss of inhibitoryeffect occurring at both storage temperatures. Applied ABA stronglysuppressed germinative capacity in intact embryos and isolatedembryonic axes from freshly harvested fruits, but when ABA wasapplied to embryos of fruits that had been stored for variousperiods at 5 or 17 C, the inhibitory effect was first weakenedand then lost with increased storage. Although dormancy in the seeds of A. velutinum may be describedas intermediate between testa-imposed dormancy and true dormancy,it is perhaps more properly included in the former category. Acer velutinum Boiss. var. vanvolxemii, abscisic acid, embryo dormancy, germination, seed storage, testa-imposed dormancy, tissue sensitivity     

Dynamics of carbohydrates in the embryo axes of horse chestnut seeds during their transition from dormancy to germination

It was shown that the content of carbohydrates and their composition in embryo axes of horse chestnut seeds changed as seeds acquired a capability of dormancy release and germination. Sucrose prevailed among carbohydrates, comprising to 150–160 mg/g dry wt. During the first half of the seed imbibition time, oligosaccharides, namely raffinose and stachyose, degraded, whereas the contents of glucose and fructose were very low. The second half of the imbibition period (until radicle protrusion) was characterized by a cessation of oligosaccharide breakdown and accumulation of monosaccharides. Carbohydrate balance showed that the contribution of oligosaccharide breakdown to sucrose and monosaccharide accumulation was rather small, and monosaccharides accumulated mostly at the expense of sucrose gradually coming from cotyledons during imbibition. The trend of carbohydrate metabolism in imbibing axial organs was similar during the entire period of a seed dormancy release in the course of stratification. A readiness for the commencement of these processes during the entire dormancy period implies that carbohydrate conversions in embryo axes are not a trigger for a dormancy release. Monosaccharide accumulation in embryo axes before radicle protrusion produces an increase in the osmotic pressure, as compared to that provided by sucrose, by approximately 20%. Recalcitrance of the horse chestnut seeds is discussed in relation to the role of carbohydrates and other endogenous osmotica in the establishment of osmotic properties.     

Biochemical genetics of some seed proteins of Pinus radiata

In a high-salt soluble fraction of the total protein from single seeds of Pinus radiata, up to 45 polypeptides were resolved on SDS-polyacrylamide gels. At least one-fifth of these polypeptides showed variation between seeds. In the 27,000–29,000 dalton region, two polypeptides were inherited as codominant alleles at a single locus and were shown to assort independently of another seed protein locus and three allozyme loci. A survey of 120 individuals from the five known native populations of P. radiata in California detected only the 27K and 29K alleles at the locus. In all populations, the 29K allele predominated, and the two island populations were monomorphic for the 29K allele. The 27 and 29 kdalton polypeptides were shown to have very similar amino acid sequences, and the allelic difference at this locus is most probably in the gene sequence for the polypeptide.