Repository hosted by TU Delft Library

The endogenous ABA contents of dormant and nondormant barley grains were determined following application of different compounds to break dormancy. The chemicals used for breaking of dormancy in intact dormant grains were weak and strong acids, alcohols,. hydrogen peroxide, cyanide, nitrate, salicylic acid, gibberellic acid and fusicoccin. The dormancy-breaking compounds could be classified into two major groups: compounds that caused a decrease in endogenous ABA (class I) and compounds which did not affect endogenous ABA (class II). Class I compounds included gibberellic acid, ethanol, hydrogen peroxide, nitrate, salicylic acid; class II compounds were fusicoccin, acid (H2SO4), sodium azide, n-caproic acid. in addition, these dormancy-breaking compounds were able to stimulate the germination rate when applied to embryos isolated from dormant grains. The concentrations necessary for stimulation of germination of isolated embryos were much lower than the concentrations for breaking the dormancy of intact grains. After embryos were isolated from dormant grains and incubated in water, ABA was determined in both embryos and in the incubation media. The class I compounds stated above also reduced ABA content in the incubation medium of isolated embryos, while class II compounds had no effect on ABA content of the medium. External application of ABA could overcome the effect of dormancy-breaking compounds of class I but not of class II. The results suggest that in the presence of the agents belonging to class II, ABA responsiveness of isolated embryos from dormant grains is decreased, compared to nontreated embryos.

Uniform and fast germinating seeds are of prime importance for agriculture. To improve the germination properties of seeds, different treatments called priming are used. These methods generally involve the controlled uptake of water by the seeds. To be able to understand the processes involved in different priming methods the aim of the research was to study the relationship between the methods of water supply, water uptake rate by seeds, seeds moisture content and germination of China aster 'Jolanta' and tomato 'Janosik' seeds. Seeds of these species were primed by: (i) soaking in excessive amount of distilled water for 0-1440 min in the ratio of seeds to water (v:v) of 1:3; (ii) soaking in limited amount of distilled water 10-1280 ml kg-1of seeds; (iii) matriconditioning in the ratios of 1:0.4:0.2-2.0 (w:w:w) for seeds, Calflo and water, respectively. The imbibed seeds were incubated for 1 day at 20°C and seed moisture content was measured after incubation. Additionally, in seeds moistened up to 37% (China aster) and 35% (tomato), dynamics of water uptake were examined. Seeds, after imbibition and incubation, were dried to the initial moisture content (m.c.). Percentage of the germinated seeds, maximum germination (Gmax), time for the first seed to germinate (T 1), time to reach 50% germination (T50), time between 10 and 90% of Gmax (T90-T10), at 5, 20 or 35°C for the China aster seeds and at 15, 20 or 35°C for tomato were evaluated. The results showed that water supply methods significantly affected seed imbibition rates and dynamics of seed germination, although these depended on plant species. It was found that water uptake was fastest in seeds soaked in excessive water amount and also that China aster took water faster than tomato. Imbibition of seeds during matriconditioning in Calfo and in limited water amounts was slower. Imbibition of seeds up to 37% m.c. for China aster and 35% m.c. for tomato, irrespective of the conditioning method, was the most favorable for germination. The earliest germination was observed in seeds soaked in excessive amount of water and incubated for 1 day. Germination of seeds matriconditioned, and those conditioned in limited water amount for 1 day was slower. We conclude that although the optimal final moisture content is independent of the different water supply methods used, the water supply method itself strongly affect the germination properties (T1, T50 and T90-T10 dynamics of germination). © 2005 Elsevier B.V. All rights reserved.

The role of abscisic acid (ABA) in seed germination of two cultivars of lettuce (Lactuca sativa L.; light-sensitive Ritsa and light-insensitive Strada) was investigated. The inhibition of Ritsa seed germination by exogenous ABA was higher than that of Strada seeds, the extent of inhibition of both cultivars being reduced by a short light break. At 25°C the sensitivity of both cultivars to exogenous ABA was higher than at 15°C. The endogenous level of ABA was similar in dry seeds of both cultivars, increasing temporarily in Ritsa seeds during the first 4 h of imbibition in darkness but not in Strada seeds, nor in Ritsa seeds exposed to a short light break. The transitory increase of ABA content in Ritsa seeds imbibed in darkness was accompanied by increased expression of the gene responsive to ABA (Rab) under these conditions. Zorial (Norflurazone), an ABA-biosynthesis inhibitor, decreased ABA content and allowed dark germination of the light-requiring Ritsa seeds. A short light break induced germination of Ritsa seeds when applied at 24 and 48 h after imbibition onset, i.e. after the transitory increase of ABA. GA3, on the other hand was effective when applied at the beginning of imbibition. It seems that light induces germination of the photoblastic Ritsa seeds by both inhibiting ABA synthesis and decreasing seed sensitivity to ABA and inhibitory processes induced by it.

In isolated embryos from dormant barley grains, synergistic effects of fusicoccin (FC) and gibberellic acid (GA3) were observed on the induction of α-amylase mRNA expression. However, no α-amylase mRNA expression could be induced by both agents in embryos from non-dormant grains. Both light- and electron-microscopy studies demonstrated that there were large numbers of starch granules present in mature embryos (mainly in scutellum) from dormant barley grains but none or almost none in embryos from non-dormant grains. Furthermore, the content of reducing sugars in embryos from dormant grains was about half of that from non-dormant grains. In contrast to GA3, FC was able to induce a strong acidification of extracellular pH (pH(e)). Clamping the pH(e) to prevent FC-induced acidification, by using 50 mM MES buffer (pH 5.6), caused an inhibition of GA3- or FC-induced α-amylase mRNA expression but did not affect the germination of embryos from dormant grains. In addition, in MES buffer, addition of FC or a combination of FC and GA3 increased the germination rate of embryos isolated from dormant grains, though large numbers of starch granules were still present in these embryos. Based on these observations, the presence of starch granules and a low reducing sugar level in embryos from dormant grains is not a factor for control of grain dormancy and germination.

Intact grains of barley (Hordeum distichum cv. Triumph) germinated rapidly in the dark or when exposed to brief daily light breaks in the temperature range 15-25°C, although germination proceeded less rapidly at low temperatures. Prolonged illumination (16 h/day) or continuous light inhibited germination of grains most effectively when conditions were less than optimal for germination, i.e. under low temperatures, restricted water, and low water potential induced by mannitol. Embryos isolated from grains responded in a similar way to light applied when the amount of water was restricted; water stress did not affect germination percentages in the dark or after one brief tight break (3 min) but appreciably reduced the rate of germination. While light was only slightly inhibitory to embryos under optimal germination conditions, its inhibitory effect was intensified when other factors tending to delay germination were present. Water restriction also caused ABA contents in embryos to rise. Under conditions of water stress (water restriction or low osmotic potential), CaCl2 enhanced the inhibitory effect of light; however, addition of LaCl3 reversed this effect. The nature of light-induced inhibition of germination is discussed.

Besides the pre-existing lipoxygenase (LOX-1) present in quiescent grains, a new lipoxygenase (LOX-2) is induced in embryos of germinating barley [Holtman, W. L., Van Duijn, G., Sedee, N. J. A. and Douma, A. C. (1996) Plant Physiol. 111, 569-576]. The fact that LOX-1 and LOX-2 form different products after incubation with linoleic acid, the (9S)- and (13S)-hydroperoxides, respectively [Van Aarle, P. G. M., De Barse, M. M. J., Veldink, G. A. and Vliegenthart, J. E G. (1991) FEES Lett. 280, 159-162; Doderer, A., Kokkelink, I., Van der Veen, S., Valk, B. E., Schram, A. W. and Douma, A. C. (1992) Biochim. Biophys. Acta 1120, 97-104], and differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aimed to obtain more information about these functions by studying the substrate and product specificities of both isoenzymes. Analyses of the products formed from linoleic acid confirmed that LOX-1 oxygenated at C9, and LOX-2 at C13. When testing more complex substrates, it was found that both LOX-1 and LOX-2 were capable of metabolizing esterified fatty acids. K(m) values from both isoenzymes for free fatty acids were much lower than for esterified fatty acids (7-35-fold for LOX-1 versus 2-8-fold for LOX-2). interestingly LOX-1 showed significantly higher K(m) values for esterified fatty acids than did LOX-2. This was reflected by analyses of the products formed from di- and tri-linoleoylglycerol; LOX-2 formed higher amounts of oxygenated polyunsaturated fatty acids within the esterified lipids than did LOX-1, with a corresponding larger extent of oxygenation. In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extracts from barley after different periods of germination for LOX-derived products. The results indicated that esterified fatty acids were preferentially metabolized by LOX-2 activity. Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (13S)-hydroxy derivatives were formed, indicating the in vivo action of LOX-2. These data show that LOX-2 is capable of oxygenating storage lipids and suggest that during the onset of germination LOX-2 may be involved in oxygenation of esterified polyunsaturated fatty acids in barley seeds. We suggest that the oxygenation of these lipids precedes the onset of their catabolism and that the degradation product, (9Z,11E,13S)-13-hydroxy-octadecadienoic acid, serves as an endogenous substrate for β-oxidation and therefore as a carbon source for the growing barley embryo. Chemicals/CAS: Isoenzymes; Lipids; Lipoxygenase, EC 1.13.11.12

Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments these spore lost already a small but significant amount of DPA. When these spores were inoculated in nutrient medium-tryptone soy broth (TSA)-the non-lethally heated spores started to germinate. Results of classical optical density measurements showed that both phase darkening and subsequent outgrowth could be affected by sub-lethal heat. A study of single cells in TSB showed that lag times originating from exponentially growing cells followed a normal distribution, whereas lag times originating from spores followed a Weibull distribution. Besides classical optical density measurements were made to study the effect of previous heating on the kinetics of the first stages of germination. The germination kinetics could be described by the model as was proposed by Geeraerd et al. [Geeraerd, A.H., Herremans, C.H. and Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology 59, 185-209]. Two of the 4 parameters of the sigmoid model of Geeraerd were dependent on heating time and heating temperature, whereas the two other parameters were considered as independent of the heating conditions. Based on these observations, a secondary model could be developed that describes the combined effect of heating temperature and heating time on the kinetics of germination. To have more detailed information of the kinetics of germination samples incubated in TSB were tested at regular time intervals by flow cytometry. To that end the cells were stained with syto 9 to distinguish between the various germination stages. There was a qualitative agreement between the results of flow cytometry and those of optical density measurements, but there was a difference in quantitative terms. The results have shown that germination rate of spores is dependent on previous heating conditions both in the first stage when phase darkening occurs and also during the later stages of outgrowth when the phase dark spore develops to the vegetative cell. © 2008 Elsevier B.V. All rights reserved. Chemicals / CAS: dipicolinic acid, 17606-33-6, 499-83-2; Picolinic Acids; dipicolinic acid, 499-83-2

In this study, the impact of a range of organic acids and structurally similar alcohols with three to six carbon backbones and increasing lipophilic character, were tested on the germination behavior of B. cereus ATCC 14579 spores. This approach allowed substantiating whether the effectivity of the various compounds was largely dictated by membrane interference or a classic weak acid acidification effect. The octanol-water partition coefficient (log P oct/water) ranges from 0.25/0.33 to 2.03/1.96 for propanol/undissociated propionic acid and hexanol/undissociated hexanoic acid, respectively. Performance of germination assays at neutral (pH7) and acidic conditions (pH5.5) allowed for a comparative analysis of the action of dissociated versus undissociated acids, and the presumed pH-independent effect of the corresponding alcohols. Germination assays, based on both continuously measured optical density and time-based plating experiments, and microscopic observations demonstrated the correlation between the lipophilic character of the selected compounds and their inhibiting effect on spore germination. Real-time fluorescence based assays showed that membrane integrity in dormant spores was maintained in the presence of the tested inhibitors. Lowering the critical concentration of inhibitors by a one-step washing procedure resulted in the onset of nutrient-induced germination, indicating the reversible nature of the inhibition process. Furthermore, blocking of nutrient-induced germination in the presence of inhibitory concentrations of selected lipophilic acids and corresponding alcohols was by-passed upon addition of Ca-dipicolinic acid, pointing to loss of signaling capacity in germinant receptor-mediated germination activity. These findings show that lipophilicity is an important determinant for the ability of the selected acids and corresponding alcohols to accumulate in the spore inner membrane and their ability to act as a germination-inhibitor. © 2012 Elsevier B.V.

Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This sometimes allows surviving spores to germinate and grow out to vegetative cells causing food spoilage and possible intoxication. Similar issues though more pending toward spore toxigenicity are observed for the anaerobic Clostridia. The paper indicates the nature of stress resistance and highlights contemporary molecular approaches to analyze the mechanistic basis of it in Bacilli. A molecular comparison between a laboratory strain and a food borne isolate, very similar at the genomic level to the laboratory strain but generating extremely heat resistant spores, is discussed. The approaches cover genome-wide genotyping, proteomics and genome-wide expression analyses studies. The analyses aim at gathering sufficient molecular information to be able to put together an initial framework for dynamic modelling of spore germination and outgrowth behaviour. Such emerging models should be developed both at the population and at the single spore level. Tools and challenges in achieving the latter are succinctly discussed. © 2010 Elsevier Ltd.

Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Two full-length lipoxygenase cDNA sequences (LoxB and LoxC) from barley (Hordeum distichum cv. L. Triumph) are described. The cDNAs share high homology with the barley LoxA cDNA. Southern blotting experiments indicate single copy numbers of the three lipoxygenase genes. RFLP mapping revealed the presence of single lipoxygenase loci. LoxA and LoxB map on chromosome 4 and LoxC on chromosome 7. Two isoenzymes, LOX1 and LOX2, have been purified previously from germinating barley and characterized. LOX1 is encoded by LoxA, while LOX2 is encoded by LoxC. The product related to the third cDNA (loxB) has not been identified so far, suggesting a low protein abundance for the corresponding isoform in barley. Transcripts corresponding with these LOX genes are predominantly observed in grain and in seedling, whereas transcripts corresponding to LoxB and LoxC are also observed in mature vegetative tissue. No lipoxygenase mRNA could be detected in aleurone layer of germinating grain. No significant differences in lipoxygenase mRNA levels were observed in developing grains grown under dormant or non-dormant conditions, suggesting that LOX is not directly involved in induction of grain dormancy. Chemicals/CAS: Antibodies, Monoclonal; Isoenzymes; Lipoxygenase, EC 1.13.11.12; Recombinant Proteins; RNA, Messenger

The barley lipoxygenase 1 (Lox1) gene encodes a protein expressed in embryos during grain development and germination and in leaves after methyl-jasmonate (MeJA) treatment. Transient gene expression assays in germinating barley embryos were used to identify cis-regulatory elements involved in the embryo-specific expression of the Lox1 gene. Analysis of transcriptional or translational fusions between Lox1 5' upstream sequences and the gusA reporter gene indicated that the 5'-untranslated leader sequence was involved in embryo-specific expression. Replacement of the leader sequence from the aleurone-specific Chi26 gene with the Lox1 leader sequence resulted in a chimeric gene expressed at high levels in embryo as well as in aleurone cells. Insertion of the Lox1 leader sequence between the 35S minimum promoter (A domain -90/+8) and the gusA reporter gene greatly enhanced promoter activity in a tissue-specific manner. Deletion/replacement analysis of the Lox1 leader sequence, combined with transient expression in germinating embryos and in vitro transcription/translation assays, suggests that the Lox1 leader sequence contains cis-elements regulating qualitative (tissue-specific) and quantitative gene expression.

Expression and post-translational modification of barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, were investigated using isoform-specific antibodies. Although all three isoforms were shown to be present in the cytosolic, the nuclear and the microsomal cell fractions, differences in post-translational modification were identified for the different cell fractions. Germination-related modifications of 14-3-3 proteins were observed in the cytosol and the microsomal fraction, but not in the nucleus. In vitro proteolytic cleavage of 14-3-3 proteins using trypsin suggests that for 14-3-3A this change was caused by proteolytic cleavage of the unconserved C-terminal region. Copyright (C) 2000 Federation of European Biochemical Societies. Chemicals/CAS: 14-3-3 Proteins; Antibodies; Plant Proteins; Protein Isoforms; Proteins; Trypsin, EC 3.4.21.4; Tyrosine 3-Monooxygenase, EC 1.14.16.2

Recently, we have demonstrated by two different methods that lipoxgenases (LOXs) and 14-3-3 proteins form interactions in barley embryos [Holtman, Roberts, Oppedijk, Testerink, van Zeijl and Wang (2000) FEBS Lett. 474, 48-52]. It was shown by both co-immunoprecipitations and surface-plasmon resonance experiments that 13-LOX, but not 9-LOX, forms interactions with 14-3-3 proteins. In the present report we show that the presence of 13-LOX and 14-3-3 proteins was established in high-molecular-mass complexes. Amounts of 13-LOX and 14-3-3 proteins in high-molecular-mass fractions increased during germination, but were reduced after dephosphorylation of protein extracts or competition with the 14-3-3-binding peptide P-Raf-259, indicating that 13-LOX and 14-3-3 proteins interact in a phosphorylation-dependent manner. Chemicals/CAS: 13-lipoxygenase, EC 1.13.11.-; 14-3-3 Proteins; Lipoxygenase, EC 1.13.11.12; Tyrosine 3-Monooxygenase, EC 1.14.16.2

We have identified the major endo-β-l,4-xylanase (XYN-1) in the aleurone of germinating barley grain, and show that it is expressed as a precursor of Mr 61 500 with both N- and C-terminal propeptides. XYN-1 is synthesized as an inactive enzyme in the cytoplasm, and only becomes active at a late stage of germination when the aleurone ceases to secrete hydrolases. A series of processing steps, mediated in part by aleurone cysteine endoproteases, yields a mature active enzyme of Mr 34 000. Processing and extracellular release of the mature enzyme coincide with the programmed cell death (PCD)-regulated disintegration of aleurone cells. We discuss the significance of delayed aleurone cell-wall degradation by endoxylanases in relation to the secretory capacity of the aleurone, and propose a novel role for aleurone PCD in facilitating the export of hydrolases. Chemicals/CAS: alpha-Amylase, EC 3.2.1.1; Cysteine Endopeptidases, EC 3.4.22.-; Endo-1,4-beta Xylanases, EC 3.2.1.8; Green Fluorescent Proteins, 147336-22-9; Hydrolases, EC 3.-; Indicators and Reagents; Luminescent Proteins; Plant Proteins; RNA Precursors; Starch, 9005-25-8; Xylosidases, EC 3.2.1.-

Associations between lipoxygenases (Lox) and 14-3-3 proteins were demonstrated by two different methods. First, immunoprecipitation experiments, using isoenzyme-specific monoclonal Lox antibodies, showed that 14-3-3 proteins co-precipitate with 13-Lox, but not with the 9-Lox from barley. Second, interactions between 13-Lox and 14-3-3 were established by surface plasmon resonance studies, showing that 13-Lox binds with 14-3-3 proteins in a concentration-dependent manner. The interactions between 14-3-3 proteins and 13-Lox may reveal their role during plant development. Copyright (C) 2000 Federation of European Biochemical Societies. Molecular Sequence Numbers: GENBANK: L35931, L37358, X62388, X93170, Y14200; Chemicals/CAS: 13-lipoxygenase, EC 1.13.11.-; 14-3-3 Proteins; Antibodies, Monoclonal; Isoenzymes; Lipoxygenase, EC 1.13.11.12; Proteins; Recombinant Proteins; Tyrosine 3-Monooxygenase, EC 1.14.16.2

Many organisms have the ability to form spores, a remarkable phase in their life cycles. Compared with vegetative cells, spores have several advantages (e.g. resistance to toxic compounds, temperature, desiccation and radiation) making them well suited to various applications. The applications of spores that first spring to mind are bio-warfare and the related, but more positive, field of biological control. Although they are often considered metabolically inert, spores can also be used as biocatalysts. Other uses for spores are found in the fields of probiotics, tumour detection and treatment, biosensing and in the 'war against drugs'.

Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP® analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP® performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated. © 2007 Elsevier B.V. All rights reserved.