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PubMed1982

Changes in the activity and properties of trehalase during early germination of yeast ascospores: correlation with trehalose breakdown as studied by in vivo 13C NMR.

The regulation of trehalose breakdown during dormancy and the induction of germination in yeast ascospores was studied both by in vivo high-resolution NMR spectroscopy and in vitro assays of trehalase activity. Natural-abundance (13)C NMR spectra taken during the induction of germination with glucose and phosphate showed a rapid breakdown of part of the trehalose content. The presence of both glucose and phosphate was important for maximal trehalose breakdown. The (13)C NMR spectra showed that the externally added glucose and the internal trehalose were metabolized mainly to glycerol and ethanol. Under these conditions of nitrogen deprivation, full germination is not possible and trehalose breakdown stopped after approximately 1 hr. At this moment resynthesis of trehalose occurred while glycerol and ethanol production from the exogenous glucose continued. In complex media where full spore germination can occur, trehalose breakdown was more pronounced. Measurements of trehalase activity in spore extracts made after addition of varying amounts of glucose and phosphate to the spores revealed a sudden 10-fold increase in the activity of trehalase, within the first minutes of spore germination. The activation was transient: after reaching a maximum between 5 and 10 min, the activity declined back to low values during the next hours. The increase in trehalase activity was not inhibited by cycloheximide or by anaerobic conditions. The decline in trehalase activity that occurred after the initial activation could be correlated with the extent of trehalose breakdown as measured by (13)C NMR. In addition to the increase in trehalase activity, differences in the control properties were found between the enzymes from dormant and germinating spores. Trehalase from dormant spores was strongly inhibited by ATP at a concentration of approximately 0.5 mM, which corresponds with the ATP concentration found in dormant spores. On the other hand, trehalase from germinating spores was not inhibited by ATP up to the much higher ATP concentrations that are found in germinating spores. It is suggested that the low activity and the stringent ATP feedback inhibition of trehalase from dormant spores are responsible for the very slow mobilization of the huge amount of trehalose in dormant spores. Therefore, dormancy seems to be caused primarily by extreme curtailment of the energy production within the spore at one selective and primary point. The switch towards high activity and low ATP inhibition upon induction of germination is suggested to be responsible for the breaking of dormancy and for the rapid breakdown of trehalose that occurs during the initial phase of germination.

Disaccharides↗
PubMed2000

Temperature-dependent germination traits in oilseed rape associated with 5'-anchored simple sequence repeat PCR polymorphisms.

An experiment was conducted to test the hypothesis that phenotypes differing in germination rate and the presence or absence of secondary dormancy at low temperature were not genetically different. Seed of oilseed rape was germinated at 4, 10 and 19 degrees C, where selections were made in the percentile ranges 1-10 (early), 45-55 (intermediate) and 91-100 (late). Secondary dormancy occurred only in the late selections at the two lower temperatures. Thermal weighting of curves of cumulative germination on time gave circumstantial evidence that early percentiles were similar at all three temperatures and that seeds with secondary dormancy came largely from later percentiles above the 50th. To test for genetic differentiation between phenotypes, 5'-anchored simple sequence repeat primers were used to generate DNA marker profiles of seedlings raised from seed from each category. Principal coordinate analysis, and more detailed comparisons using the most discriminating markers, confirmed that the early germinators at the three temperatures were not associated with different banding profiles, but seeds entering secondary dormancy, particularly at 10 degrees C, were genetically distinct from germinators at the same temperature. Secondary dormant seeds at low temperature appear to originate mainly from the late germinating seed at higher temperature. Effects of temperature history and the requirement for alternating temperatures to break secondary dormancy were quantified. The results confirm the existence of genetically discrete sub-populations differing in ecologically significant traits.

Brassica↗
PubMed1977

Effects of Quality, Intensity, and Duration of Light Breaks during a Long Night on Dormancy in Blue Spruce (Picea pungens Engelm.) Seedlings.

Blue spruce (Picea pungens Engelm.) seedlings grow continuously when exposed to photoperiods exceeding 16 hours and enter dormancy within 4 weeks under photoperiods of 12 hours or less. Dormancy was prevented under 12-hour photoperiods by 2-hour light breaks of red light (1.70 muw/cm(2) at 650 nm) or high intensity white light (2,164.29 muw/cm(2) at 400 to 800 nm) given in the middle of the 12-hour night, and by continuous low intensity white light (204.76 muw/cm(2) at 400 to 800 nm). Two-hour light breaks of far red light (1.80 muw/cm(2) at 730 nm), red light followed by far red light, or low intensity white light were not effective in delaying dormancy. The results imply that the phytochrome system mediates the photoperiodic control of dormancy in blue spruce seedlings. The similarity of results obtained using the low intensity, long duration as against the high intensity, short duration light treatments suggests that the law of reciprocity applies in this response.

Journal Article↗
PubMed2006

History of science--spores.

Bacterial endospores were first studied 130 years ago by Cohn in 1876 and independently by Koch in the same year. Although spore dormancy and resistance have been much studied since then, questions still remain concerning the basic mechanisms and the kinetics of heat inactivation in particular. Likewise, the extreme dormancy and longevity of spores was recognized early on and later greatly extended but still evade complete understanding. Evidence has accumulated for the involvement of specific spore components such as calcium, dipicolinic acid, small acid soluble proteins in the core and peptidoglycan in the cortex. Involvement of physical factors too, such as the relative dehydration of the core, maybe in a high-viscosity state or even in a glassy state, has added to appreciation of the multicomponent nature of dormancy and resistance. Spore-former morphology formed the basis for early classification systems of sporeformers from about 1880 and consolidated in the mid-1900s, well prior to the use of modern genetic procedures. With respect to sporulation, groundbreaking sequence studies in the 1950s provided the basis for later elucidation of the genetic control widely relevant to many cell differentiation mechanisms. With respect to the breaking of dormancy (activation and germination), the elucidation of mechanisms began in the 1940s following the observations of Hills at Porton who identified specific amino acid and riboside 'germinants', and laid the basis for the later genetic analyses, the identification of germinant receptor genes and the elucidation of key germination reactions. The nonexponential nature of germination kinetics has thwarted the development of practical Tyndallization-like processing. So inactivation by heat remains the premier method of spore control, the basis of a huge worldwide industry, and still relying on the basic kinetics of inactivation of Clostridium botulinum spores, and the reasoning regarding safety first evolved by Bigelow et al. in 1920 and Esty and Meyer in 1922. 'Newer' processes such as treatment with ionizing radiation (first proposed in 1905) and high hydrostatic pressure (first proposed in 1899) may be introduced if consumer resistance and some remaining technical barriers could be overcome.

Cell Death↗
PubMed1974

The Metabolism of Hormones during Seed Germination and Dormancy: IV. The Metabolism of (S)-2-C-Abscisic Acid in Ash Seed.

Embryos from dormant and stratified Fraxinus americana seed were incubated with (S)-2-(14)C-abscisic acid (ABA) under a variety of conditions. Both dormant and stratified embryos rapidly metabolize abscisic acid to phaseic acid, dihydrophaseic acid, and an unidentified polar metabolite apparently derived from dihydrophaseic acid. Although the stratified embryos may have an increased capacity to metabolize abscisic acid, our calculations suggest that such an increased capacity would probably not be physiologically significant.Dormant intact seeds also metabolize (S)-2-(14)C-abscisic acid during stratification at 5 C or incubation at 25 C. The metabolites appear to be similar to those observed in excised embryos although by 12 days of stratification a fourth metabolite is observed. More than 90% of the (14)C-abscisic acid was metabolized after 26 days of stratification at 5 C or after 12 days of incubation at 25 C. Stratification at 5 C leads to the breaking of dormancy while incubation at 25 C does not.

Journal Article↗
PubMed2001

Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster.

Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5'-->3' direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 degrees C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 degrees C) and PPN (35-40 degrees C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of beta-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification.

Aminohydrolases↗
PubMed1994

Promoter tagging with a promoterless ipt gene leads to cytokinin-induced phenotypic variability in transgenic tobacco plants:implications of gene dosage effects.

Tobacco plants have been transformed with a T-DNA construct harboring a promoterless cytokinin-synthesizing ipt gene close to the right T-DNA border. Eighteen out of 85 transgenic clones displayed phenotypic alternations typical for an enhanced cytokinin production. Northern blot analysis confirmed the transcriptional activation of the introduced gene by tagged plant promoters. The concentration of cytokinins, expressed as zeatinriboside equivalents, was increased up to sevenfold in transgenic tissues. These increases in cytokinin levels resulted in major developmental changes. Transgenic clones exhibited to different levels traits of a general cytokinin-syndrome, i.e. reduced root growth, reduced apical dominance, reduced leaf surface, reduced growth of the stem and retarded leaf senescence or displayed localized and developmentally specific cytokinin-induced alterations in otherwise normally developing plants. These traits were in particular a simultaneous break of dormancy in all axillary buds before or at the onset of flowering or the reorientation of the developmental pathway of secondary meristems or terminally differentiated cells. This indicates that endogenously produced cytokinins not only influence different growth parameters but have the potential to alter differentiation pattern. The results show that stably inherited developmental alterations due to a general or localized cytokinin overproduction can be obtained by the promoter-tagging approach. The investigation of gene dosage effects in homozygote plants readdresses the question of threshold levels for cytokinin effects on the developmental program of plants.

Adenosine↗
PubMed1996

Isolation and characterization of the cDNA for an A-like cyclin in Adiantum capillus-veneris L.

We have isolated and characterized the cDNA for a homolog of a cyclin from the fern Adiantum capillus-veneris L. Three fragments of cDNAs for cyclin homologs were amplified by the PCR from the cDNA of germinating spores with degenerate primers that encoded the highly conserved region in the so-called cyclin box. We isolated a full-length cDNA for cyclin from a lambda ZAPII-cDNA library that has been derived from spores using an amplified fragment as a probe. The cDNA insert (Cyc-Ac1) in one positive clone was 2.0 kb in length, having an open reading frame of 1,599 bp that encoded 532 amino acids. The putative CycAc1 protein had a cyclin box and a destruction motif, and it was homologous to A-type cyclins. Northern blot analysis was performed to study the expression of CycAc1 during the first cell cycle in the haploid generation of the fern. CycAc1 mRNA was not detectable in dormant spores, during the first G1 phase, and at the onset of the S phase in imbibed spores after breaking of dormancy. This mRNA became detectable after the termination of the S phase, and it accumulated during the second G1 phase. Although CycAc1 was classified as an A-like cyclin, the failure to detect CycAc1 mRNA at the onset of the S phase suggests that CycAc1 might not play a role in the replication of DNA during the S phase.

Amino Acid Sequence↗
PubMed2004

Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds.

Exposure of imbibed seeds to low temperature (typically 4 degrees C) is widely used to break seed dormancy and to improve the frequency of germination. However, the mechanism by which temperature accelerates germination is largely unknown. Using DNA microarray and gas chromatography-mass spectrometry analyses, we found that a subset of gibberellin (GA) biosynthesis genes were upregulated in response to low temperature, resulting in an increase in the level of bioactive GAs and transcript abundance of GA-inducible genes in imbibed Arabidopsis thaliana seeds. Using a loss-of-function mutant, the cold-inducible GA biosynthesis gene, AtGA3ox1, was shown to play an essential role in mediating the effect of low temperature. Besides temperature, AtGA3ox1 also is positively regulated by active phytochrome and negatively regulated by GA activity. We show that both red light and GA deficiency act in addition to low temperature to elevate the level of AtGA3ox1 transcript, indicating that multiple signals are integrated by the AtGA3ox1 gene to control seed germination. When induced by low temperature, AtGA3ox1 mRNA was detectable by in situ RNA hybridization in an additional set of cell types relative to that in red light-induced seeds. Our results illustrate that the GA biosynthesis and response pathways are activated during seed imbibition at low temperature and suggest that the cellular distribution of bioactive GAs may be altered under different light and temperature conditions.

Arabidopsis↗
PubMed1982

Turnover of 32P-phosphate labelled poly(A) RNA during early development of Artemia.

The break of dormancy and the early development of Artemia are known to occur in the absence of any DNA and RNA synthesis. The presence and function of preformed messengers in the developing embryos were studied using 32PO4 to track the RNA species that turnover. The rapid labelling of the poly(A) tails of the particulate RNA by 32PO4 is found to be the predominant metabolic event accompanying initiation of development. Although these RNA populations represent a meagre percentage of the total poly(A) RNA of the cells, they nevertheless constitute more than 60% of the labelled poly(A) populations at early stages of development. Moreover the rise in the poly(A) RNA levels of the embryos observed during the first four hours of development could be attributed to the increase in the particulate poly(A) RNA. Prelabelled RNA of this fraction remained rather firmly associated with this fraction in chase experiments, indicating that once processed these RNA species function in association with membranes. The observed shift in the size of these RNAs from low to high molecular weight species further implies that they are being activated to take part in the early developmental programme.

Animals↗
PubMed1980

Effect of high pressure on the heat activation in vivo of trehalase in the spores of Phycomyces blakesleeanus.

The effect of pressure on the heat activation in vivo of trehalase in the spores of Phycomyces blakesleeanus has been investigated in order to obtain information about the molecular mechanism of the activation. For a protein conformational change directly induced in the enzyme by the heat treatment an upward shift with about 2-6 K/1000 atm (1.013 X 10(5) kPa) is to be expected in the moderate high-pressure region. On the other hand, for a phospholipid phase transition causing the activation, a continuous upward shift with about 20 K/1000 atm is to be expected. For trehalase activation we find a continuous upward shift of the activation temperature with about 5-9 K/1000 atm. The denaturation of trehalase, which occurs at slightly higher temperatures, is influenced by pressure completely as expected for a protein conformational change. The application of high pressure during spore heat activation makes it possibe to break the dormancy of the spores without concomitant activation of trehalase.

Alcohols↗
PubMed1988

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores.

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)

Centrifugation, Density Gradient↗
PubMed2006

Expression of genes coding for GerA and GerK spore germination receptors is dependent on the protein phosphatase PrpE.

The ability of Bacillus subtilis to form spores is a strategy for survival under unfavorable environmental conditions. It is equally crucial to break spore dormancy and return to vegetative growth at the appropriate time. Here we present data showing that the PrpE phosphatase is involved in the control of expression of genes coding for GerA receptors, which are necessary for L-alanine-induced spore germination. Moreover, PrpE is also involved in aspartic acid, glucose, fructose, and potassium (AGFK)-induced spore germination by controlling expression of genes coding for GerK receptors. In the absence of PrpE, the production of spores was essentially normal. However, L-alanine-induced spore germination and, to a lesser extent, the AGFK-induced pathway were abolished. In contrast, the germination pathway dependent on Ca2+-dipicolinate or dodecylamine remained intact. A protein phosphatase PrpE-green fluorescent protein fusion was localized to the prespore and to the dormant spore, consistent with a role in controlling expression of genes coding for GerA receptors. We propose that PrpE is an important element in a signal transduction pathway in Bacillus subtilis that controls the expression of genes coding for germination receptors.

Bacillus subtilis↗
PubMed1983

Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination.

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.

Antipain↗
PubMed2025

Speed breeding: protocols, application and achievements.

One of the limiting factors in breeding and genetic research is the time required to develop pure lines. This is due, on the one hand, to the prolonged vegetative period of a single generation and, on the other hand, to the specifics of inbreeding, which typically requires 4-6 consecutive generations of self-pollination in plant material. Researchers have always sought approaches that enable the rapid development of homozygous plant lines. Consequently, methods such as greenhouse cultivation during the autumn-winter period, single-seed descent, shuttle breeding, embryo culture, and doubled haploid technology have been introduced into practice. All these methods have both advantages and limitations. One of the latest approaches facilitating a significant reduction in the vegetative period of plants is speed breeding (SB). This method is based on the application of factors that shorten the time from sowing to flowering, as well as techniques that accelerate the generative phase of development and overcome postharvest dormancy. This review provides a comprehensive list and characterization of all factors that influence the efficiency of speed breeding to varying degrees. Among the factors discussed that reduce the sowing-to-flowering period are photoperiod, light sources, spectral composition and light intensity, temperature, carbon dioxide levels, vernalization, mineral nutrition, substrate volume, mechanical shoot removal, and the use of plant growth regulators. To shorten the generative phase, the review summarizes the application of embryo culture and forced desiccation of immature seeds, along with methods to overcome postharvest dormancy. Additionally, applications of genetic approaches and genetic engineering for shortening generation time in speed breeding are described. The review also consolidates detailed protocols for approximately thirty crops. The high efficiency of speed breeding in reducing both the vegetative period per generation and the time required to develop pure lines has led to its increasing adoption in various research fields. This review highlights the application of speed breeding for hybridization and pure line development, introgression of target alleles, and genomic selection. A list of phenotypic traits exhibiting high correlation between controlled-environment and field conditions is provided.

accelerated flowering↗
PubMedDate not supplied

[Effect of growth regulators on proton transport through cytoplasmic membrane of potato tuber cells].

Effects of the growth regulators epibrassinolide-694 (EB), gibberellic acid (GA), and abscisic acid (ABA) on the ATP-dependent translocation of H+ through the membranes of plasma membrane vesicles of potato (Solanum tuberosum L.) tuber cells were studied. The ATP-dependent accumulation of H+ in the plasma membrane vesicles from dormant tubers was inhibited by EB and ABA and stimulated by GA. After the break of dormancy, the stimulatory effect of GA increased, the inhibitory effect of ABA decreased, and EB stimulated the accumulation of H+ in the vesicles. The data suggest that the plasma membrane H+ ATPase is a target of phytohormones that regulate the dormancy of potato tubers.

Abscisic Acid↗
PubMed1993

Molecular cloning and physiological analysis of an invertase isoenzyme in Helianthus tissues.

A soluble acid invertase activity isolated from Helianthus tuberosus (Jerusalem artichoke) shoots and analyzed by immunochromatography using polyclonal yeast antibodies, represents around 5% of the total invertase activity. This invertase isoenzyme was also isolated from dormant tuber parenchyma. In these partially dormant tissues, the specific activity of this isoenzyme is low suggesting a partial inactivation of the invertase molecules. Polyacrylamide gel electrophoresis of immunopurified fractions yields similar levels of the 58 kDa polypeptide both in shoots and dormant tubers, but with much lower activity of the enzyme in the tubers. A cDNA library was constructed in pUEX 1 from poly (A)+ RNA extracted from Jerusalem artichoke tubers. This library was screened for invertase using (i) a Bacillus subtilis invertase DNA probe and (ii) anti-yeast invertase antibodies. A recombinant clone of approximately 1.8 kb size was selected by these two methods. Using Northern blots, a temporal sequence in the expression of invertase gene was observed during the breaking of dormancy with the main level after 8 weeks of cold treatment at 4 degrees C. A 2.5 kb transcript was detected, translation of which would yield a 97 kDa polypeptide representing the precursor of Jerusalem artichoke invertase.

Bacillus subtilis↗