Onset Of Imbibition Research Articles

Temporal accumulation and ultrastructural localization of dehydrins in Zea mays

Immunolocalization using polyclonal antibodies raised against a conserved dehydrin amino acid sequence was used to establish the temporal and spatial patterns of dehydrin accumulation in embryo tissue of Zea mays L. (var. Ohio 43) kernels imbibed in the presence of abscisic acid. The temporal pattern of accumulation indicated an increase in dehydrins over time (particularly between 15 and 30 h) and with maximum levels detected 48 h after the onset of imbibition. Dehydrins were first evident, and also the most concentrated, in the cytosol throughout the accumulation period suggesting that the primary function of dehydrins involves the cytosol and the structures contained therein. Only after an accumulation of dehydrins in the cytosol was there an increase in the abundance of nuclear dehydrins. In addition, dehydrins were also observed in association with the proteinaceous matrix of protein bodies and membranes of protein and lipid bodies; these findings have not been reported previously. The observed localization at a number of sites indicates that the specific biochemical roles of dehydrins are likely to be diverse.

Changes in RNA and Protein Metabolism Associated with Alterations in the Germination Efficiency of Sunflower Seeds

Precise knowledge of seed quality after harvest and during storage is of particular importance for seed producers. We analysed different sunflower seed lots (Helianthus annuusL.) characterized by extremes of germination ability. We used RNA analysis to study possible changes in gene expression in seeds unable to germinate. Total RNA content was very small in dry seeds showing a low germination ability. Capacity for total RNA synthesis at the onset of imbibition was also reduced in these seeds. In addition, correlations were found between these parameters and germination ability at 19 °C. We demonstrated a high correlation between the amount of total RNA in the dry seed, the capacity of RNA synthesis at the onset of imbibition and the seed moisture content at the time of the harvest. The ability of dry seed mRNAs to be translatedin vitrowas also reduced and seven polypeptides, from stored mRNAs, were characteristic of the cotyledons from high germinability seeds. Germination can thus be affected at several levels including membrane, enzymatic and nucleic acid deteriorations.

The effect of electron acceptors on the phytochrome‐controlled germination of Paulownia tomentosa seeds

The effect of substances with different redox potentials in the phytochrome‐controlled germination of Paulmtnia tomentosa seeds was examined. Up to 25% of water‐imbibed seeds germinated upon irradiation with 5 min red light The seeds irradiated with 5 min red light and incubated in substances with redox potentials equal or higher than E′0= 360 mV [potassium nitrate, potassium hexacyanoferrate (III) or potassium hexachloroiridate (IV)] and sodium nitroprusside germinated up to 80%. The optimal concentration was between 1 and 10 mM. Other electron acceptors such as 2.6‐dichlorophenol‐indoplhenol, phenazine methosulfate and methylene blue failed to produce and effect. The germination‐promoting effect was obtained when the substances were supplied to the incubation medium from the onset of imbibition or at the onset of irradiation. Delay of application of these chemicals until after the red light treatment diminished their effectiveness and the effect was lost after 48 h. The escape from far‐red reversibility was evident in the presence of substances which stimulated germination. The results presented support the view that phytochrome‐broken dormancy of Paulownia tomentosa seeds is closely connected with redox changes.

Staging Soybean Seedling Growth from Germination to Emergence

Scientists engaged in seed research are often forced to refer to sequential developments of seedling growth in terms of days or hours after planting. Such terms are imprecise, because seedlings develop at different rates depending on seed condition, treatment, and environment. Fehr and coworkers in 1971 published an accurate and consistent description of soybean [Glycine max (L.) Merr.] development from emergence to harvest maturity. Our objective was to devise a system of staging soybean seedling development up to emergence. Laboratory germination was conducted on four soybean cultivars, representing three maturity groups, in a specially designed germination apparatus. Rate of growth and seedling development were recorded at 6‐h intervals between onset of imbibition and full seedling development. Six developmental stages were identified, based on distinct and consistent morphological changes. Adoption of this developmental staging system will allow more precise scientific communication regarding soybean development from the initiation of germination to emergence.

Developmental control of small heat shock protein expression during pea seed maturation

SummaryPlants synthesize four classes of small heat shock proteins (sHSPs); two classes are targeted to the plastid and endoplasmic reticulum, respectively, and two are found in the cytoplasm. In this paper, we describe a new role for the two classes of cytoplasmic HSPs in maturing embryos of developing seeds. The expression of each class of sHSPs was examined in pea seeds grown under non‐stress conditions using Western and Northern analysis. Class I and class II cytoplasmic sHSPs are coordinately expressed in the embryo and accumulate to levels seen in moderately heat‐stressed leaves. Their induction in cotyledons coincides with the mid‐maturation phase of seed development, and induction in axes roughly coincides with abscission of the seed from the ovary wall. Both classes of sHSPs persisted in cotyledons for 4 days after the onset of imbibition, but disappeared from axes shortly after germination. Neither class of cytoplasmic sHSP is expressed in non‐embryonic organs associated with the seed. The timing and organ specificity of sHSP expression is paralleled by the expression of the corresponding mRNAs. Neither the plastid nor the endoplasmic reticulum sHSPs were consistently expressed during seed development, but both could be induced by heat‐stressing the developing seed. Developmental regulation of the cytoplasmic sHSPs is evidence that these proteins function not only in responding to heat‐stress but also during seed development and/or germination.

Temporal and light regulation of the expression of three phytochromes in germinating seeds and young seedlings of Avena sativa L.

An oat (Avena sativa L.) plant contains at least three phytochromes, which have monomeric masses of 125, 124, and 123 kilodaltons (kDa) (Wang et al., 1991, Planta 184, 96-104). The 124-kDa phytochrome is most abundant in dark-grown seedlings, while the other two phytochromes predominate in light-grown seedlings. Using three monoclonal antibodies, each specific to one of the three phytochromes, we have monitored by immunoblot assay the expression of these three phytochromes in the 5 d following onset of imbibition of seeds. On a per-organism basis, each of these three phytochromes increased in abundance for the first 3 d in the light, or for the first 4 d in darkness, after which they each began to decrease in quantity. When 3-d-old dark-grown seedlings were transferred to the light, the abundance of each of these three phytochromes decreased both in absolute amount and relative to the phytochrome levels in control seedlings kept in darkness. In contrast, when 3-d-old light-grown seedlings were transferred to darkness, the abundance of the 124-kDa and 125-kDa phytochromes increased while that of 123-kDa phytochrome remained unchanged. In each case, the level of phytochrome was greater than that of control seedlings maintained in the light. Thus, in addition to temporal regulation, all three phytochromes exhibit photoregulated expression at the protein level, although the magnitude of this photoregulation varies substantially.

Ethanolic Fermentation in Hypoxic Cotton Seed

Cotton (Gossypium hirsutum L.) roots die within a few hours in the total absence of O2, but can continue growing for several hours at a reduced rate very low at O2 concentrations, even <10 mmol O2 mol−1. The physiology of this adaptive response has not been well characterized. The objective of this study was to determine if ethanolic fermentation occurs in cotton seeds under hypoxic conditions that inhibit but do not prevent radicle growth. Germinating seeds, after 23 h imbibition at 32 °C, were incubated for ≤4.5 h at 28 °C in sealed flasks. Hypoxic conditions (6‐9 mmol O2 mol−1) were imposed by gassing flasks with either N2 or CO2. Fermentation was measured as the production of ethanol and acetaldehyde, which were quantified by gas‐liquid chromatography. Both ethanol and acetaldehyde were readily produced during hypoxia at an average rate of 439 and 9.69 nmol h−1 seed−1, respectively, at 28 °C. Acetaldehyde‐and ethanol‐forming capacity increased substantially during the first 16 h following onset of imbibition. Seeds imbibed under hypoxia for only 3 h produced little ethanol or acetaldehyde. Small amounts of acetaldehyde and ethanol were produced by hydrated seeds imbibed in air. Ethanol produced during fermentation was rapidly assimilated if O2 stress was relieved. During a 2‐h hypoxic stress that promoted fermentation, radicle growth was halted, but resumed at a reduced rate. Inhibition was short‐lived and radicle growth recovered fully in air. Results indicate that ethanolic fermentation is a major metabolic response to hypoxia in germinating cotton seeds.

Control by the embryo axis of the breakdown of storage proteins in cotyledons of germinating seeds of citrus limon

AbstractAzocaseolytic (endopeptidase) and carboxypeptidase activities in cotyledons of germinating seeds of Citrus limon (L) Burm f (cv Verna) showed a peak on the fourth day after the onset of imbibition and then decreased. A second peak of activity in both enzymes appeared 16 days after imbibition. The activities of leucine‐aminopeptidase and alanine‐aminopeptidase increased until 12 days after imbibition and then declined.When cotyledons were detached from seedlings 8 days after imbibition and then incubated in water, protein breakdown was accelerated and α‐amino N accumulation increased. In detached and incubated cotyledons, the azocaseolytic activity decreased whereas the carboxypeptidase activity continued to increase more markedly than when the axis remained attached. The effect of the axis on azocaseolytic activity was replaceable by kinetin. On the other hand, kinetin, GA3 and IAA showed no significant effect on the carboxypeptidase and aminopeptidase activities in detached cotyledons.

Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25 degrees C growth chamber cultivated, or from plants given 38 degrees C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25 degrees C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42 degrees C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25 degrees C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25 degrees C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25 degrees C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times.

Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination

By differential hybridization screening, we previously selected a class of cDNA clones from a lambda gt10 cDNA library that was constructed from the total poly(A)+ RNA of mature cowpea cotyledons (Plant Cell Physiol 31: 39-44, 1990). pSAS10, a clone of this class, hybridized with a cDNA probe complementary to poly(A)+ RNA from cotyledons collected 1 day after the onset of imbibition (DAI), but not with the cDNA probe from cotyledons at developmental stage II (13 to 15 days after flowering, DAF). pSAS10 mRNA was detectable only in cotyledons at developmental stage III (17 to 19 DAF) or later, and its level began to decline when seeds germinated. We have suggested that pSAS10 mRNA is likely to belong to the class of 'stored' mRNA or the mRNA that is formed at the late stage of seed maturation, is conserved in quiescent seeds and becomes functional at the early stage of germination. We determined the nucleotide sequence of pSAS10 cDNA consisting of 459 bp and an approximately 36 bp poly(A) tract, and deduced the amino acid sequence of its product, a 10-kDa cysteine-rich polypeptide. Synthesis of pSAS10 mRNA was induced just before germination began, not only in mature seeds but also in immature seeds even at stages I (9 to 11 DAF) and II (13 to 15 DAF) if they were placed under conditions suitable for germination.

Light and nitrate interaction in phytochrome-controlled germination ofPaulownia tomentosa seeds

Pulsed light and nitrate exhibit an interactive effect on the germination ofPaulownia tomentosa Steud. seeds that require long periods of light irradiation. Two pulses of red light (R), separated by an adequately long dark interval, substitute for continuous prolonged irradiation. A far-red (FR) pulse given at the beginning of the dark interval inhibits germination, while it has no effect if given at the end. The requirement for certain ratios of the far-red-absorbing form of phytochrome/total phytochrome (Pfr/Ptot) differs when a FR+R-pulse is given as the first or second of two pulses (FR+R or R) separated by a dark interval. An equal decrease of the Pfr/Ptot ratio leads to a more pronounced decrease in germination when the pulse of the same FR+R ratio is given as the second pulse at the end of the dark interval. The length of dark interval between light pulses needed for maximal germination, differed in (i) seeds with a natural requirement for long periods of light irradiation from that in (ii) seeds with their long light requirement imposed by two weeks of imbibition in darkness or by (iii) imbibition in 40% heavy water. However, a single R pulse was sufficient to induce a high percentage of germination if the seeds were supplied with KNO3 (10 mM) from the onset of imbibition up to the onset of light. This effect decreased with a delayed time of application, and was prevented if FR preceded the KNO3 application.

Heat shock proteins in Sorghum bicolor and Pennisetum americanum. II. Stored RNA in sorghum seed and its relationship to heat shock protein synthesis during germination

Abstract. During the germination of sorghum, heat shock proteins (HSPs) are synthesized in response to high‐temperature stress at the onset of imbibition. HSP72 and HSP83 are apparently synthesized without a heat shock induction in the first 2 h of imbibition. The presence of RNA encoding HSPs in the quiescent embryo, and in the 2 h imbibed embryo, was confirmed by in vitro translation of RNA isolated from dry mature sorghum seeds. By 24 h of imbibition at 35°C, no translatable RNA was evident for these HSPs unless a heat shock treatment was also given. Treatment of imbibing embryos with cordycepin inhibited RNA synthesis. Protein synthesis was not affected so greatly and HSP synthesis was still inducible although at reduced levels. The involvement of RNA stored in the seed and of de novo transcription in protein synthesis during imbibition is discussed.

Influence of ageing on photo- and thermo-control of seed germination in grand rapids lettuce (Lactuca sativa L.).

The reversible photoreaction of R (Red light) and FR (Far-red light) controlling the seed germination in Grand Rapids lettuce (Lactuca sativa L.) varies markedly not only with germination temperature, but also with the physiological state of seed caused by ageing. The photoreversibility in Grand Rapids lettuce seeds is more evident in the dormant (fresh) and deteriorating (60 and 66 months after harvest) seeds. The reversibility in the non-dormant seeds, however, can be induced again by a prolonged expooure to FR light or high (40°C) temperature pretreatment after the onset of imbibition. The velocity and the viability in germination of the seeds exposed to the prolonged FR light or heat-sensitized at 40°C for 48 hr maintained for a longer time than in the seeds having no exposure to FR light or heat-sensitization. The photoreversible induction by the prolonged exposure to FR light is due to the secondary photo-dormant stage of the seeds. The photo-reversible induction by a heat-sensitization after the onset of imbibition, on the other hand, is due to the two different physiological states of the seed; a, the secondary thermo-dormant stage caused at 40°C for 48 hr, and b, the deteriorating stage caused by the thermal injury at 40°C for 144 hr. Germination induction effects in the heat-sensitized seeds with high temperature pretreatment after the onset of imbibition are greater in GA3 than in R light.

Gibberellins, Amylase, and the Onset of Heterosis in Maize Seedlings

Rood, S. B. and Larsen, K. M. 1988. Gibberellins, amylase, and the onset of heterosis in maize seedlings.—J. exp. Bot. 39: 223-233. The possible involvement of gibberellins and amylase in heterosis of maize seedlings was investigated in two parental inbreds, CM7 and CM49, and their single cross F, hybrid, CM7 x CM49. Germination of all three genotypes was complete within 36 h after the onset of imbibition. By 48 h, heterosis (hybrid vigour) for increased shoot and root length was consistently observed. The endogenous concentration of gibberellin (GA;) was measured in 48 h seedlings by gas chromatography-mass spectrometry with selected ion monitoring (GC-SIM) using pHJ-GAj as an internal standard. The GA3 concentration was highest in the hybrid (59 ng g~' dry wt.), intermediate in CM49 (9-Ongg-1), and lowest in CM7 (<50ngg_1). Amylase activities in all three genotypes were very low at 24 h, but increased during the next 24 h, after which time amylase activity in the hybrid was significantly higher than that of either parental inbred. Inhibitors of gibberellin (GA) biosynthesis, AMO-1618 or CCC, inhibited germination, shoot and root growth, and amylase activity in all three genotypes. Conversely, exogenous gibberellic acid (GA3) increased amylase activity, particularly in the inbred CM7. Amylase isozymes were separated through polyacrylamide gel electrophoresis and generally similar profiles of starch degrading enzymes were observed in the three genotypes. Since GA is known to control a-amylase biosynthesis in some cereals, these results are consistent with the hypothesis that GAs are involved in the regulation of heterosis in maize. A higher endogenous GAt concentration in the hybrid could result in increased amylase activity in the hybrid seedlings and consequently, more rapid starch hydrolysis which fuels heterosis for early growth.

DNA Ligase in Pea (Pisum sativumL.) Seedlings: Changes in Activity during Germination and Effects of Deoxyribonucleotides

Daniel, P. P. and Bryant, J. A. 1988. DNA ligase in pea (Pisum sativum L.) seedlings: changes in activity during germination and effects of deoxyribonucleotides.—J. exp. Bot. 39: 481–486. Measurable amounts of ‘soluble’ and chromatin-bound DNA bgase are present in the embryo axis at least as early as 8 h after the onset of imbibition. Between 8 h and 48 h the activity of the chromatin-bound form increases nearly 10-fold, whilst the soluble activity increases 6-fold. There is then a slight decline in the chromatin-bound activity between 48 h and 72 h, whilst the soluble activity continues to increase. Both forms of ligase arc markedly inhibited by dTTP (but not by other deoxyribonucleoside triphosphates); the soluble form is significantly more sensitive to dTTP than the chromatin-bound form.

On the Role of Stored mRNA in Protein Synthesis in Embryonic Axes of Germinating Vigna unguiculata Seeds

Polyadenylated (poly A(+)) RNAs were prepared from both dry and incubated embryonic axes of Vigna unguiculata seeds and were translated by a wheat germ translation system. Analysis with gel electrophoresis and fluorography showed that translation products of poly A(+) RNA from dry embryonic axes were nearly the same as those from 2-hour incubated axes but somewhat different from those of 4- to 24-hour incubated axes, and that translation products remained almost unchanged between the 4- and 24-hour stages of postimbibition. The results indicate the possibility that the stored mRNA (poly A(+) RNA from dry embryonic axes) directs the protein synthesis required for early stages of germination. This is supported by comparison of the in vitro translation products of poly A(+) RNAs with those of polysomal RNAs. Experiments with alpha-amanitin, a specific inhibitor of RNA polymerase II (J. Jendrisak 1980 J Biol Chem 255: 8529-8533), suggested that the synthesis of some of the stored mRNA species is resumed as early as 4 hours after the onset of imbibition.

Changes in nuclear protein contents of root cells during germination.

Total protein content and area of nuclei were determined for cells of the primary root of Vicia faba during the first 144 hours following the onset of imbibition. We wished to determine what change, if any, occurs in protein contents of nuclei as meristematic cells move from a dormant into a proliferative condition. Nuclear proteins were stained with dinitrofluorobenzene and protein content, in arbitrary units, was determined by microspectrophotometry. In the first 50 hours mean protein content of nuclei increase; this, as we have shown, is the period in which cells and nuclei increase in size as they complete their first cell cycle. During subsequent cell cycles, cell area decreases markedly, nuclear area decreases to a lesser extent; accompanying these decreases, the mean and the range of protein contents of nuclei decreased significantly. Thus, as the proliferating population of cells of a root meristem becomes steady-state, there is an absolute decrease in the concentration of total proteins within the nucleus. This was revealed particularly clearly by comparing unclei of identical areas from roots of different ages. Increases in nuclear size were induced by treatment with 5-aminouracil for upto 24 hours: this did not result in a significant increase in protein content of nuclei. These results agree with those from untreated roots that area and protein content of nuclei can, to a considerable extent, vary independently of one another.

Does the Embryonic Axis Affect Respiration in Pea Cotyledons?

The conditions of imbibition crucially affected respiration of the excised pea cotyledons, Pisum sativum L. var. Bördi, during subsequent incubation. When excised cotyledons were immersed in water during imbibition, their respiration rate was lower than that of attached cotyledons up to 96 h of incubation. Hydration of excised cotyledons was faster than that of attached cotyledons, particularly at early stages of imbibition. The dry weight of excised cotyledons, however, decreased more rapidly than that of attached cotyledons. When excised cotyledons were kept on moist filter paper from the onset of imbibition, their respiration rate was the same as that of attached cotyledons during later incubation. In addition, the hydration rate of both excised and attached cotyledons was the same under such conditions, whereas the dry weight of the attached cotyledons decreased more rapidly than those of excised cotyledons. It was concluded that the respiration rate of the excised cotyledons during incubation was only dependent on the conditions used during imbibition. Hence, the embryonic axis had not effect on the development and maintenance of the respiratory activity in the cotyledons of germinating peas.

Lipid Mobilization in Dormant and Nondormant Caryopses of Yellow Foxtail (Setaria lutescens)

The major reserve lipid of yellow foxtail [Setaria lutescens(Weigel) Hubb.] caryopses is triacylglyceride stored in lipid bodies. Lipid bodies are heavily concentrated in the cells of the embryo axis, scutellum, and aleurone layer and are roughly spherical (average diam 0.5μm). Percentage lipid (hot isopropanol extract) on a fresh-weight basis of mature, unimbibed seed and caryopses collected locally was about 6.4 and 11%, respectively. About 95% of the total (0.23 mg/caryopsis) was saponifiable and distributed among fatty acid classes as follows: 16:0, 5.8%; 18:0, 1.5%; 18:1, 16.2%; 18:2, 73.7%; and 18:3, 2.8%. In nondormant caryopses, saponifiable lipid content declined (18:1 and 18:2 only) about 23% during the first 6 days after imbibition in the dark at 25 C. This modest decline was preceded by the appearance of an alkaline lipase at about 24 h after the onset of imbibition. Free fatty acids accumulated during these early stages of germination and accounted for about 22% of the total saponifiable lipid remaining (0.17 mg/caryopsis) 6 days after the onset of imbibition. Lipid content and lipase activity did not change in dormant caryopses treated similarly. The delayed initiation of lipid mobilization in germinating caryopses suggests that triacylglyceride degradation is not a pivotal metabolic pathway controlling dormancy.

Physiological significance of indoleacetic acid and factors determining its level in cotyledons of Lupinus albus during germination and growth

The results demonstrate the profile of the endogenous indole‐3‐acetic acid (IAA) in the cotyledons of Lupinus albus L. (L. termis Forssk.) during germination and seedling growth. The auxin level increases markedly after seed hydration, especially during the time of radicle emergence 24 h after the onset of imbibition. This rise is accompanied by a minimal IAA‐oxidase activity, formation of indoleacetylaspartic acid (IAAsp) and an increase in the endogenous tryptophan and tryptophan‐carboxyl‐14C degradation, though the latter cannot account for the high IAA level detected during early stages of germination. It is believed that cotyledons are a source of IAA to the developing embryonic axis. – The auxin level drops in the cotyledons during seedling growth, 2–18 days after sowing. This is true also for IAAsp and tryptophan‐degrading activity of enzyme extracts. Conversely, endogenous tryptophan is increasingly liberated up to day 14, and IAA‐oxidase activity climbs to a peak detected on day 12, prior to the appearance of senescence in the cotyledons. – The physiological significance of IAA and the factors regulating its level in the cotyledons during germination and growth are discussed.