Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth

The tripeptide antioxidant glutathione (γ-l-glutamyl-l-cysteinyl-glycine; GSH) essentially contributes to thiol-disulphide conversions, which are involved in the control of seed development, germination, and seedling establishment. However, the relative contribution of GSH metabolism in different seed structures is not fully understood. We studied the GSH/glutathione disulphide (GSSG) redox couple and associated low-molecular-weight (LMW) thiols and disulphides related to GSH metabolism in bread wheat (Triticum aestivum L.) seeds, focussing on redox changes in the embryo and endosperm during germination. In dry seeds, GSH was the predominant LMW thiol and, 15?h after the onset of imbibition, embryos of non-germinated seeds contained 12 times more LMW thiols than the endosperm. In germinated seeds, the embryo contained 17 and 11 times more LMW thiols than the endosperm after 15 and 48?h, respectively. This resulted in the embryo having significantly more reducing half-cell reduction potentials of GSH/GSSG and cysteine (Cys)/cystine (CySS) redox couples (E_GSSG/2GSH and E_CySS/2Cys, respectively). Upon seed germination and early seedling growth, Cys and CySS concentrations significantly increased in both embryo and endosperm, progressively contributing to the cellular LMW thiol-disulphide redox environment (E_{thiol-disulphide}). The changes in E_CySS/2Cys could be related to the mobilisation of storage proteins in the endosperm during early seedling growth. We suggest that E_GSSG/2GSH and E_CySS/2Cys can be used as markers of the physiological and developmental stage of embryo and endosperm. We also present a model of interaction between LMW thiols and disulphides with hydrogen peroxide (H₂O₂) in redox regulation of bread wheat germination and early seedling growth.     

Abscisic acid controls embryo growth potential and endosperm cap weakening during coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> cv. Rubi) seed germination

The mechanism and regulation of coffee seed germination were studied in Coffea arabica L. cv. Rubi. The coffee embryo grew inside the endosperm prior to radicle protrusion and abscisic acid (ABA) inhibited the increase in its pressure potential. There were two steps of endosperm cap weakening. An increase in cellulase activity coincided with the first step and an increase in endo--mannanase (EBM) activity with the second step. ABA inhibited the second step of endosperm cap weakening, presumably by inhibiting the activities of at least two EBM isoforms and/or, indirectly, by inhibiting the pressure force of the radicle. The increase in the activities of EBM and cellulase coincided with the decrease in the force required to puncture the endosperm and with the appearance of porosity in the cell walls as observed by low-temperature scanning electronic microscopy. Tissue printing showed that EBM activity was spatially regulated in the endosperm. Activity was initiated in the endosperm cap whereas later during germination it could also be detected in the remainder of the endosperm. Tissue printing revealed that ABA inhibited most of the EBM activity in the endosperm cap, but not in the remainder of the endosperm. ABA did not inhibit cellulase activity. There was a transient rise in ABA content in the embryo during imbibition, which was likely to be responsible for slow germination, suggesting that endogenous ABA also may control embryo growth potential and the second step of endosperm cap weakening during coffee seed germination.     

Phytin is synthesized in the cotyledons of germinated castor-bean seeds in response to exogenously supplied phosphate

Following germination of the castor bean (Ricinus communis L.) seed, levels of phytin decline in both the endosperm and the embryo. However, as seedling growth continues, phytin increase in the latter to a level exceeding that present in the mature dry embryo, while phytin declines concomitantly in the endosperm. It is likely that phosphate mobilized from phytin in the endosperm acts as a substrate for phytin synthesis in the embryo. This is supported by the observation that isolated embryos supplied with phosphate accumulate phytin, particularly in the cotyledons. This increase is enhanced whenmyo-inositol is provided concurrently as a carbon source. Phytin synthesis in the cotyledons of the isolated embryos can occur without the attached axis. Whether initially exposed to exogenous phosphate or not, the isolated cotyledons remain competent in their ability to synthesize phytin for an extended post-germinative period, even though the major reserves are being mobilized at this time.     

Embryology in Trithuria submersa (Hydatellaceae) and relationships between embryo, endosperm, and perisperm in early-diverging flowering plants

? Premise of the study: Despite their highly reduced morphology, Hydatellaceae bear the unmistakable embryological signature of Nymphaeales, including a starch-rich maternal perisperm and a minute biparental endosperm and embryo. The co-occurrence of perisperm and endosperm in Nymphaeales and other lineages of flowering plants, and their respective functions during the course of seed development and embryo germination, remain enigmatic. ? Methods: Development of the embryo, endosperm, and perisperm was examined histologically from fertilization through germination in flowers and fruits of Trithuria submersa. ? Key results: The embryo of T. submersa initiates two cotyledons prior to seed maturity/dormancy, and their tips remain in contact with the endosperm throughout germination. The endosperm persists as a single layer of cells and serves as the interface between the embryo and the perisperm. The perisperm contains carbohydrates and proteins, and functions as the main storage tissue. The endosperm accumulates proteins and aleurone grains and functions as a transfer cell layer. ? Conclusions: In Nymphaeales, the multiple roles of a more typical endosperm have been separated into two different tissues and genetic entities: a maternal perisperm (nutrient acquisition, storage, mobilization) and a minute biparental endosperm (nutrient transfer to the embryo). The presence of perisperms among several other ancient lineages of angiosperms suggests a modest degree of developmental and functional lability for the nutrient storage tissue (perisperm or endosperm) within seeds during the early evolution of flowering plants. Finally, we examine the evolutionary developmental hypothesis that, contrary to longstanding assumptions, an embryo-nourishing perisperm along with a minute endosperm may represent the plesiomorphic condition for flowering plants.     

Thioredoxin and germinating barley: targets and protein redox changes

The endosperm and embryo of barley ( Hordeum vulgare L.) grain were investigated to relate thioredoxin h and disulfide changes to germination and seedling development. The disulfide proteins of both tissues were found to undergo reduction following imbibition. Reduction reached a peak 1 day earlier in the embryo than in the endosperm, day 1 vs. day 2. The profile in both cases resembled those observed with wheat and rice, i.e., the reduction of the storage proteins increased initially and then declined during the period of seedling growth. The extent of the increase in reduction observed with barley endosperm was, however, less pronounced than with the other cereals. Also, unlike wheat and rice, the storage proteins of the endosperm were highly reduced in the dry seed and the sulfhydryl content of glutelins showed no appreciable change during this period. The relative abundance of thioredoxin h during germination and early seedling growth differed in the embryo and endosperm: a progressive decrease in the endosperm (as seen with wheat) vs. an increase in the embryo. Thioredoxin h was found in the major seed tissues in characteristic forms. Three forms were found in the scutellum and aleurone, whereas two, which may represent isoforms, were identified in the root and the shoot. Using a recently developed strategy based on two-dimensional gel electrophoresis, several proteins were identified as specific targets for thioredoxin in the embryo following oxidation with H(2)O(2), among them barley embryo globulin 1, peroxiredoxin and acidic ribosomal protein P(3). The results confirm earlier findings with the endosperm of other cereals and extend the importance of thioredoxin-linked redox change to the germinating embryo for functions that potentially include dormancy, protection against reactive oxygen species, translation and the mobilization of storage proteins.     

A structural study of germination in celery (Apium graveolens L.) seed with emphasis on endosperm breakdown

Germination of celery seed occurred after 6 d of imbibition in light. During this time the embryo enlarged at the expense of the adjacent endosperm cells and at the time of germination was 2–3 times as long as in the dry seed. Breakdown of the endosperm cells near the root cap preceeded radicle emergence. None of these changes occurred in darkness.Endosperm digestion began adjacent to the embryo and spread radially. In degrading cells, the aleurone grains often became larger and fewer in number. The cell walls were modified and appeared to undergo partial degradation. Ultimately the cells seemed to lose their contents. In cells adjacent to the root cap, similar changes occurred except there was a transient appearance of starch grains. Radial progression of endosperm breakdown also occurred in isolated endosperm treated with gibberellin A₄₊₇.The results indicate that (1) the stimulus for breakdown of celery endosperm emanates from the embryo in response to light; (2) the stimulus may be a gibberellin because changes in endosperm cells and the sequence of endosperm digestion during germination resemble the responses of isolated endosperm to gibberellin; and (3) the radial progression of endosperm breakdown during germination may be the result of a sequential response of cells to a uniformly applied stimulus rather than the result of gradual embryo expansion.     

Control of macaw palm seed germination by the gibberellin/abscisic acid balance

The hormonal mechanisms involved in palm seed germination are not fully understood. To better understand how germination is regulated in Arecaceae, we used macaw palm (Acrocomia aculeata (Jacq.) Lodd. Ex Mart.) seed as a model. Endogenous hormone concentrations, tocopherol and tocotrienol and lipid peroxidation during germination were studied separately in the embryo and endosperm. Evaluations were performed in dry (D), imbibed (I), germinated (G) and non‐germinated (NG) seeds treated (+GA₃) or not treated (control) with gibberellins (GA). With GA₃ treatment, seeds germinated faster and to a higher percentage than control seeds. The +GA₃ treatment increased total bioactive GA in the embryo during germination relative to the control. Abscisic acid (ABA) concentrations decreased gradually from D to G in both tissues. Embryos of G seeds had a lower ABA content than NG seeds in both treatments. The GA/ABA ratio in the embryo was significantly higher in G than NG seeds. The +GA₃ treatment did not significantly affect the GA/ABA ratio in either treatment. Cytokinin content increased from dry to germinated seeds. Jasmonic acid (JA) increased and 1‐aminocyclopropane‐1‐carboylic acid (ACC) decreased after imbibition. In addition, α‐tocopherol and α‐tocotrienol decreased, while lipid peroxidation increased in the embryo during germination. We conclude that germination in macaw palm seed involves reductions in ABA content and, consequently, increased GA/ABA in the embryo. Furthermore, the imbibition process generates oxidative stress (as observed by changes in vitamin E and MDA).     

Mechanism and control of Solanum lycocarpum seed germination

BACKGROUND AND AIMS: Solanaceae seed morphology and physiology have been widely studied but mainly in domesticated crops. The present study aimed to compare the seed morphology and the physiology of germination of Solanum lycocarpum, an important species native to the Brazilian Cerrado, with two species with endospermic seeds, tomato and coffee. METHODS: Morphological parameters of fruits and seeds were determined by microscopy. Germination was monitored for 40 d under different temperature regimes. Endosperm digestion and resistance, with endo-beta-mannanase activity and required force to puncture the endosperm cap as respective markers, were measured during germination in water and in abscisic acid. KEY RESULTS: Fruits of S. lycocarpum contain dormant seeds before natural dispersion. The best germination condition found was a 12-h alternating light/dark and high/low (20/30 degrees C) temperature cycle, which seemed to target properties of the endosperm cap. The endosperm cap contains 7-8 layers of elongated polygonal cells and is predestined to facilitate radicle protrusion. The force required to puncture the endosperm cap decreased in two stages during germination and showed a significant negative correlation with endo-beta-mannanase activity. As a result of the thick endosperm cap, the puncture force was significantly higher in S. lycocarpum than in tomato and coffee. Endo-beta-mannanase activity was detected in the endosperm cap prior to radicle protrusion. Abscisic acid inhibited germination, increase of embryo weight during imbibition, the second stage of weakening of the endosperm cap and of endo-beta-mannanase activity in the endosperm cap. CONCLUSIONS: The germination mechanism of S. lycocarpum bears resemblance to that of tomato and coffee seeds. However, quantitative differences were observed in embryo pressure potential, endo-beta-mannanase activity and endosperm cap resistance that were related to germination rates across the three species.     

Mechanism and control of Genipa americana seed germination

Genipa americana (Rubiaceae) is important for restoration of riparian forest in the Brazilian Cerrado. The objective was to characterize the mechanism and control of germination of G. americana to support uniform seedling production. Morphology and morphometrics of seeds, embryo and endosperm were assessed by light and scanning electron microscopy during germination. Imbibition and germination curves were generated and over the same time interval endosperm digestion and resistance were measured by puncture force analysis and activity assay of endo-β-mannanase (EBM) in water and in abscisic acid (ABA). The gene encoding for EBM was partially cloned and its expression monitored by quantitative real-time-polymerase chain reaction. Embryos displayed growth prior to radicle protrusion. A two-phase increase in EBM activity coincided with the two stages of weakening of the micropylar endosperm. The second stage also coincided with growth of the embryo prior to radicle protrusion. Enzyme activity was initiated in the micropylar endosperm but spread to the lateral endosperm. ABA completely inhibited germination by inhibiting embryo growth, the second stage of weakening and expression of the EBM gene, but EBM activity was not significantly inhibited. This suggests that a specific isoform of the enzyme is involved in endosperm weakening. EBM may cause a general 'softening' of micropylar endosperm cell walls, allowing the embryo to puncture the endosperm as the driving force of the decrease in puncture force.     

Investigating seed dormancy in cotton (Gossypium hirsutum L.): understanding the physiological changes in embryo during after‐ripening and germination

• The dormancy of seeds of upland cotton can be broken during dry after‐ripening, but the mechanism of its dormancy release remains unclear.
• Freshly harvested cotton seeds were subjected to after‐ripening for 180 days. Cotton seeds from different days of after‐ripening (DAR) were sampled for dynamic physiological determination and germination tests. The intact seeds and isolated embryos were germinated to assess effects of the seed coat on embryo germination. Content of H₂O₂ and phytohormones and activities of antioxidant enzymes and glucose‐6‐phosphate dehydrogenase were measured during after‐ripening and germination.
• Germination of intact seeds increased from 7% upon harvest to 96% at 30 DAR, while embryo germination improved from an initial rate of 82% to 100% after 14 DAR. Based on T₅₀ (time when 50% of seeds germinate) and germination index, the intact seed and isolated embryo needed 30 and 21 DAR, respectively, to acquire relatively stable germination. The content of H₂O₂ increased during after‐ripening and continued to increase within the first few hours of imbibition, along with a decrease in abscisic acid (ABA) content. A noticeable increase was observed in gibberellic acid content during germination when ABA content decreased to a lower level. Coat removal treatment accelerated embryo absorption of water, which further improved the accumulation of H₂O₂ and changed peroxidase content during germination.
• For cotton seed, the alleviation of coat‐imposed dormancy required 30 days of after‐ripening, accompanied by rapid dormancy release (within 21 DAR) in naked embryos. H₂O₂ acted as a core link between the response to environmental changes and induction of other physiological changes for breaking seed dormancy.

     

The breakdown of lipid reserves in the endosperm of germinating castor beans.

1. Lipid extracts were obtained from castor-bean endosperm tissue at various times during germination and, after purification, the total lipid content was determined. Quantitative measurements of the triglyceride and phospholipid content together with the fatty acid composition were made. 2. The total lipid content of the endosperm rapidly decreased during germination; after 10 days less than 20% of the original weight of lipid remained. In contrast, the phospholipid content (initially less than 0.5% of the total lipid) increased slightly during this time. The fatty acid composition and the relative proportions of the triglyceride species of the total lipid extract remained constant during 10 days of germination. 3. Gibberellic acid (0.3 mM) markedly stimulated the rate of lipid breakdown but did not alter either the fatty acid composition or the relative proportion of triglyceride species. 4. The embryo had little effect on lipid metabolism in the endosperm tissue; only after 6 days of germination were differences observed in the rate of fat utilization in the presence and absence of the embryo.     

Effects of ethylene and some other environmental factors on different stages of germination in red root pigweed (Amaranthus retroflexus L.) seeds*

Abstract. Ethylene was found to promote two distinct processes during germination of redroot pigweed (Amarantus retroflexus L.) seeds: embryo expansion that splits the seed coat (incomplete germination), and radicle penetration through the more elastic endosperm (complete germination). The two events can be separated in time by subjecting seeds to low water potential or low CO₂ levels, which arrest germination of some seeds at the incomplete stage. Ethylene applications to incompletely germinated seeds promote complete germination, with a response threshold near 0.02 cm³ m^?3 and saturation near 0.5 cm³ m^?3. Higher ethylene concentrations (0.5 to 50 cm³ m^?3) given during the first day of seed imbibition also increase the percentage of seeds which initiate embryo expansion and split the seed coat. Light and elevated CO₂ also promote radicle penetration of the endosperm in seeds incubated under water stress. The results support the view that the germination pause at the incomplete stage is an adaptation to environmental stresses that can be overcome with exogenous ethylene or certain other stimuli.     

Differential gene expression during seed germination in barley (Hordeum vulgare L.)

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process. Electronic Publication     

Studies on the Physiology of Rice: XV. CHANGES IN THE NITROGEN METABOLISM OF SEEDS DURING GERMINATION AND THEIR RELATION TO THE APPLICATION OF GROWTH REGULATORS

Seeds of rice variety ‘Chinsurah Boro’ were treatedwith two concentrations of indoleacetic acid (IAA) and maleichydrazide (MH), 0.1 mg. per litre and 0.01 mg. per litre, duringthe first 7 days of germination, and the resulting changes innitrogen metabolism of both endosperm and embryo were determined.The seedling growth at different times during the treatmentwas recorded. Uptake of water by the embryo increased up to 72 hours, butthereafter the percentage water content fell, owing to large-scaletranslocation of dry matter to it. The endosperm showed increasingwater content for 5 days. There are two marked stages of nitrogen metabolism of the seedduring germination. During the first 72 hours hydrolysis ofprotein in the endosperm and translocation of soluble nitrogento the embryo are the predominant features. Thereafter the embryoactively synthesizes protein from the products of translocation. IAA and MH affected the seedling growth similarly. Initiallythere was a small retardation of leaf growth, but at later periodsrecovery took place. Root growth was more adversely affectedthan shoot growth, the effect also persisting longer. Thesechanges in growth were reflected in the nitrogen metabolismof the seedlings. For the first few days IAA and MH retardedthe supply of soluble nitrogen from the endosperm to the embryo,consequently there was less soluble nitrogen in the embryo thoughprotein synthesis there was affected only slightly. The differencein soluble nitrogen between the treated and untreated embryospersisted throughout the seven days. An attempt is made to explainthis on the basis that primarily IAA and MH retard the enzymaticactivity responsible for the hydrolysis of protein in the endosperm.     

Changes in Cytokinin Activity during Seed Germination in Rice (Oryza sativa L.)

Cytokinin activity was determined in dry mature rice seeds,in endosperm and embryo tissues 24, 48 and 72 over imbibitionand in radicles 96 h after germination. Cytokinins with chromatographicproperties similar to zeatin, zeatin riboside, zeatin glucosideand zeatin riboside glucoside were datected in embryo and endosperm,but only the latter two were detected in mature seeds. Cytokininactivity was low during early toges of germination. Qualitativeand quantitative changes in cytokinins were observed in bothembryo and endosperm. The presence of higher cytokinin activityin the endosperm than in the embryo during the first 24 h aftergermination suggests that the endosperm may supply cytokininsuntil the embryo is able to synthaize its own cytokinins. Thepossible significance of high cytokinin glucoside activity inthe embryo early during germination and high cytokinin activityin the radicle during the later stages is discussed. Oryza sativa L., rice, cytokinin, germination, seed     

Physiological Studies with Heavy Water. II. Germination and Growth Inhibition of Barley by D2O

Seed germination and barley seedling growth in various D₂O concentrations have been studied. It was observed that the emergence of root and shoot was delayed, there being greater delay in shoot than in root emergence. A complete block was observed in germination in pure D₂O and the germination rate was slowed down significantly in lower concentrations. An initial germination delay by different D₂O concentrations seemed to cause a subsequent retardation in the growth measured as shoot and root length. A comparison of root and shoot length with their respective dry weights suggested that the growth by cell division/elongation might have been affected more than the transport of food materials from the endosperm to the embryo. Analysis of the total sugars of the endosperm and the embryo at 8 hour intervals showed that while the hydrolysis of starch to sugars was progressively decreased by increasing D₂O concentrations, the transport rate from endosperm to embryo showed a sharp inhibition in 50% D₂O and above. This indicated that the inhibition in the transport of materials, besides less hydrolysis of reserve food materials, may also be a causal factor of germination and growth inhibition in D₂O.     

Application of embryo rescue after interspecific crosses in the genus Rhododendron

Interspecific hybridization between evergreen pot azalea (Tsutsusi) cultivars and genotypes of other Rhododendron subgenera or sections (Rhododendron, Hymenanthes, Pentanthera, Vireya) is significantly hampered by many prezygotic and postzygotic barriers. The objective of our work was to overcome spontaneous abortion and lack of endosperm formation and to increase germination rates by establishing an embryo rescue protocol. The optimal germination medium for immature Rhododendron seeds was a basal medium supplemented with 145 μM GA₃. This medium induced germination of fertilized ovules after several sexual combinations of subgenera. Its use was clearly more efficient than in vivo sowing. The direction of the cross significantly influenced the occurrence of abortion, germination and albinism. The obtained seedlings were multiplied on Woody Plant Medium + 4.5 μM 2iP, and rooted afterwards. Finally, about 9% of the germinated ovules resulted in vigorously growing seedlings that were successfully acclimatized.     

水分胁迫条件下高粱种子的吸水机制及其可溶性糖代谢的变化

以高梁杂交种晋杂８６－１为材料研究了种子吸水萌动过程中胚与胚乳两部分的水分状况及可溶性糖变化的过程。结果表明：在吸水萌动过程中种胚和胚乳在水分及糖代谢方面具有不同的特点。     

Mobilisation of reserves and the earliest synthesis of sterols and latex triterpenes during germination and early seedling growth of Euphorbia lathyris

Seedlings of Euphorbia lathyris L. were grown in the dark at 25°C. Levels of amino acids, sugars and soluble phosphate in the endosperm increased upon germination after 4 days of imbibition, while the amounts of mineral reserves Mg²⁺ and K⁺ started decreasing. Ca²⁺ was not translocated from the endosperm to the seedling. Maximum values for amino acids were found on day 7, and the highest amounts of sugars were present on day 10. The endosperm was completely depleted by day 12.
Before germination (days 1–3) a low level of sterol synthesis in the embryo was detected with labeled sucrose and serine and to a lesser extent with labeled pyruvate. The label of [2-¹⁴C]-mevalonate proceeded exclusively to squalene. Laticifers started the synthesis of their triterpenes upon germination (day 4), using sucrose as a main substrate. A concurrent increase of sterol synthesis outside the laticifers was traced with labeled serine. Radioactive triterpenes, 4 α-methylsterols and sterols were detected in growing seedlings after [¹⁴C]-mevalonate uptake, but most of its label accumulated in squalene. The use of labeled mevalonate in sterol synthesis in growing seedlings is discussed.