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Experiments are reported on the association number of magnesium bromide, diethylmagnesium, mixtures of these two compounds, the Grignard reagent prepared from magnesium and ethyl bromide and mixtures of this Grignard reagent with magnesium bromide, all dissolved in pure diethyl ether at concentrations from 10−3 to 10−2 molar and measured at the same temperature. No trace of the equilibrium: was observed. Only one magnesium atom is incorporated in the molecules of the Grignard reagent, even in the presence of a large amount of magnesium bromide.

If one molar equivalent of 2-methyl-, 2-ethyl- or 2-n-propyl-1,3-dioxolane is used for the preparation of Grignard-reagents in benzene, the five-membered hetero-ring is opened with formation of a magnesium halide 2-alkoxyethan-1-olate. The steric and electronic effects of the alkyl groups of the Grignard-reagent and those of the dioxolane molecule are discussed.

The reaction of methylmagnesium iodide with allyl phenyl ether was investigated. Contrary to expectation2 1-butene, the product of the ether cleavage reaction, was formed only to a small extent, the main gaseous product being methane. The mechanism proposed for the formation of this product is: Claisen rearrangement of allyl phenyl ether under the influence of magnesium iodide and reaction of methylmagnesium iodide with the rearrangement product, o-allylphenol with formation of methane. The formation of methane as well as of 1-butene indicates that two competitive reactions (rearrangement and ether cleavage reaction) take place.

The possibility of the existence of an equilibrium in diethyl ether between ethylmagnesium bromide - prepared from 14C-labelled ethyl bromide and magnesium - and diethylmagnesium was investigated. The result was inconclusive because complete exchange of ethyl groups was found which might be attributed either to the existence of an equilibrium or to a transmetallation reaction.

The influence of oxygen on the number of individual particles in 10−3 · 10−2 molar diethyl ether solutions of ethylmagnesium bromide, diethylmagnesium, magnesium bromide and a mixture of diethylmagnesium and magnesium bromide was investigated. The ethoxide formed from the ethylmagnesium compounds is present as a trimer.

Potato plants (Solanum tuberosum L., cv. Désirée) were transformed with the polyphosphate kinase gene from Escherichia coli fused to the leader sequence of the ferredoxin oxidoreductase gene (FNR) from Spinacea oleracea under the control of the leaf specific St-LS1 promoter to introduce a novel phosphate pool in the chloroplasts of green tissues. Transgenic plants (cpPPK) in tissue culture developed necrotic lesions in older leaves and showed earlier leaf senescence while greenhouse plants showed no noticeable phenotype. Leaves of cpPPK plants contained less starch but higher concentrations of soluble sugars. The presence of polyphosphate in cpPPK leaves was demonstrated by toluidine blue staining and unambiguously verified and quantified by in vitro 31P-NMR of extracts. Polyphosphate accumulated during leaf development from 0.06 in juvenile leaves to 0.83 mg P g-1 DW in old leaves and had an average chain length of 18 residues in mature leaves. In situ 31P-NMR on small leaf pieces perfused with well-oxygenated medium showed only 0.036 mg P g-1 DW polyphosphate that was, however, greatly increased upon treatment with 50 mM ammonium sulfate at pH 7.3. This phenomenon along with a yield of 0.47 mg P g-1 DW polyphosphate from an extract of the same leaf material suggests that 93% of the polyphosphate pool is immobile. This conclusion is substantiated by the observation that no differences in polyphosphate pool sizes could be discerned between darkened and illuminated leaves, leaves treated with methylviologen or anaerobis and control leaves, treatments causing a change in the pool of ATP available for polyPi synthesis. Results are discussed in the context of the chelating properties of polyphosphates for cations and its consequences for the partitioning of photoassimilate between starch and soluble sugars.

Sucrose synthase is usually localized by immunocytochemistry, but this method does not show the actual activity of the localized enzyme. A histochemical assay is presented here showing the activity of sucrose synthase by tetrazolium salt precipitation on sections of developing maize kernels. The advantages of the assay are a high sensitivity for low amounts of active sucrose synthase and the independence of specific antibodies. In this study the activity of endosperm sucrose synthase is shown to move gradually from the apical part of the endosperm to the basal endosperm during kernel development. This shift in sucrose synthase activity correlates well with the localization of starch synthesis during kernel development. The assay also shows the early loss of activity in the aleurone layer bordering the embryo, and a loss of activity in the apical aleurone during the final stage of kernel development while the enzyme was still found by immunocytochemistry. This is in contrast to a high sucrose synthase activity in the epithelium of the scutellum, where hardly any labelling was found with antibodies against maize sucrose synthase. Low sucrose synthase activities were found in the pericarp and pedicel parenchyma. Possible functions of the high and low activity patterns in the developing maize kernels and differences between the enzyme assay and immunocytochemistry are discussed.

Summary Tuberized one-leaf cuttings of potato ( Solanum tuberosum L.) were kept under conditions inducing tuber regrowth (35 °C, leaf removed) or conditions allowing normal tuber growth (20 °C, leaf removed). The level of starch decreased substantially under conditions that induced tuber regrowth with a concomitant rise in the levels of sucrose and glucose. As a result, water was taken up and the dry matter content of the tubers decreased. Immediately after leaf removal, the level of ABA in the tubers decreased significantly in both treatments. It remained low under conditions that induced regrowth, whereas in the control treatment the level of ABA increased again. The changes in carbohydrate levels and in ABA content between the two treatments both preceded the occurrence of visible second growth, but the carbohydrate differences occurred first.

The tubers on one potato stem vary greatly in size and their size distribution may be described by their number and their average size and its variation. These components are mutually and closely related. The variation is partly caused by stolon characteristics, including their date of initiation, position and size. Tuber size is therefore partly determined before tuberization. Tuber-specific factors, such as the position and activity of the tuber initial also play a role, but the date of initiation of an individual tuber is not crucial. Later on, during tuber bulking, the activity of enzymes involved in starch metabolism, the levels of hormones, the mineral composition and the turgor potential are associated with the tuber growth rate. The growth characteristics of individual tubers may also vary since they are exposed to different conditions because of differences in position or growing period.

Tuber-size distribution is regulated by many diverse, interacting mechanisms and is therefore difficult to understand and manipulate. It is determined by plant density, number of stems per plant, number of tubers per stem, and yield. Seed size and plant number per unit area are easy to control, but stem number is affected by less controllable factors. Interactions between stems of different types are important for tuber-size distribution. The hormonal regulation of stolonization and tuberization is still unknown, but under the conditions of north-west Europe the process of tuber set (which is also poorly understood) makes a greater contribution to the final number of tubers than tuberization. The total yield is also relevant, because it affects both the average tuber size and its variation. Tubers on the same stem differ in timing, rate and duration of growth. The resulting hierarchy in sink strength is not consistent over time. Several mechanisms are suggested for this hierarchy.

Using the cDNA-AFLP method, we have isolated a transcript-derived fragment (TDF) which shows a differential expression pattern during tuber organogenesis of Solanum tuberosum L. The TDF was used to isolate a cDNA clone carrying a 1.5 kb insert and potentially coding for a 32.5 kDa peptide which, by homology, represents a potato homologue of an alpha-snap gene and has been designated Stsnap. Northern analysis showed that the Stsnap gene is expressed in actively dividing tissues throughout the potato plant. Analysis of genomic DNA from potato revealed that the Stsnap gene is likely to be a single-copy gene. The expression of antisense Stsnap cDNA under the control of the CaMV 35S promoter results in plants with an altered morphology such as curled leaves. Several of these transgenic lines also display cellular and developmental abnormalities with distinct changes in assimilate transport including accumulation of starch and soluble sugars in source leaves. We argue that these findings are consistent with the hypothetical function of the StSNAP gene product in vesicle targeting and fusion during plant development.

Tuber formation in the strictly short day-dependent potato species Solanum demissum Lindi. was studied. In order to accurately determine the developmental stage of individual stolon tips, a reliable parameter describing tuber formation is defined. Dry matter percentage of stolon tips was highly correlated with starch concentration and with swelling of the stoIon tips, independent of plant age and stolon diameter. Based on this parameter, changes in carbohydrate metabolism during the initial stages of tuber formation were analyzed. Glucose and fructose levels decreased significantly upon tuber formation. This decrease could be explained by a change in sucrose hydrolysis, dominated by acid invertases before tuber formation, and by sucrose synthase afterwards. A model of the temporal and spatial changes in sucrose unloading and hydrolysis in swelling stolon tips is discussed. The observed changes in carbohydrate metabolism are similar to changes observed in a simplified in vitro system, indicating that such model systems adequately reflect tuber development in intact plants.

Summary Swelling stolon tips (diameter >2mm) and small developing tubers of up to 7.5 mm diameter from glasshouse-grown potato plants (cv. Desiree) contained 5–7 fold more L-ascorbic acid (AsA) compared with non-swelling stolon tips (diameter

The effects of abscisic acid (ABA) on the uptake of sucrose into discs from tobacco (Nicotiana tabacum) source leaves were studied. ABA stimulated an increase in sucrose uptake up to three-fold, depending on the osmotic potential of the medium. Maximal stimulation was observed around 200 mOsm, and at 1 μm ABA. A tentative explanation for the discrepancy between these results and the previously described inhibition by ABA of sucrose uptake into leaf discs from other plant species is presented

All the concepts examined in the student research project were rated as useful by practitioners. Application of these concepts could improve interactions with your elderly patients, and ultimately, their health care.

The binding of NAA to particulate fractions isolated from tobacco pith callus was studied. The presence of a specific auxin receptor with high affinity was demonstrated. NAA-binding was maximal after 30 min of incubation at 36°C (pH 5). The affinity constant for NAA was approx. 3 × 106/mol and the concentration of binding-sites was approx. 75 pmol/g fresh weight (60 pmol/mg protein). The affinities for various auxin analogues were roughly correlated with their activities in auxin bio-assays. The same type of receptor was found in freshly excised tobacco pith, but at a lower concentration.

Peroxycarboxylic acids are increasingly used as bleaching agents for the laundering process. These peroxy acids can be added as such to the wash liquor or may be generated in situ by reaction of a precursor component with hydrogen peroxide. Bleaching effectiveness appears to be determined by a delicate balance of chemical reactions of peroxy acid with the staining dyes and the rate of peroxy acid decomposition under the prevailing conditions. To improve our understanding of the chemistry involved we have carried out a systematic study using a range of aromatic and aliphatic mono- and diperoxycarboxylic acids with various carbon chain lengths and degrees of branching. We investigated peroxy acid decomposition, bleaching of stained fabric and bleaching of a water-soluble dye, and showed that the alkyl chain length of the peroxy acids, the presence of detergent components and the pH of the medium are important factors. Combination of the kinetic data for decomposition and bleaching reactions allows the selection of structures and conditions for optimum performance.

In vitro binding of 1-naphthaleneacetic acid (NAA) to particulate fractions from tobacco leaf protoplasts was studied. In freshly isolated protoplasts no specific binding could be detected, whereas it was present in particulate fractions from tobacco leaves. It is concluded that the NAA-binding-sites are probably located at the external face of the plasma membrane; they are destroyed during protoplast isolation by proteolytic enzymes in the cellulase and macerozyme preparations. After culturing the protoplasts for 3-4 d, the first cell divisions were observed and at the same time specific NAA-binding became detectable. The affinity constant for NAA was approx. 2·10(6) mol(-1) and the number of binding sites increased during further culture.

Investigations of the genetics of self-compatibility and self-incompatibility in dihaploids and diploid derivatives from cv. Gineke revealed the presence of S1, S2 and S3 at the S-locus of Gineke and in addition an S1-allele on a translocation. By means of a complete tester set involving the S-alleles S1, S2 and S3 (all from Gineke) and S4 (from Black 4495) it was demonstrated that some Gineke dihaploids were compatible with all six testers. This indicated a fourth S-allele in Gineke, which differs from those in the tester series and was therefore assigned S5. Additional evidence was obtained from an analysis of F1's from crosses of two S5-bearing dihaploids and one of the testers. So the S-genotype of cv. Gineke was identified as S1S2S3S5/S1, the second S1 being the S-allele on a translocated fragment.

The kinetics of binding of 1-naphthylacetic acid to particulate fractions from tobacco-pith callus were studied. This binding site does not bind auxin at 0° C. Binding experiments performed at 25° C demonstrated an apparent K a of approx. 6.5·10(6) M(-1). A filtration method was developed in order to study non-equilibrium kinetics of this binding. Dissociation of the complex of auxin and binding site indicates the presence of at least two binding components with dissociation rate constants (k off) of 6.1·10(-3) min(-1) and 6.0·10(-2) min(-1). This binding behaviour was not independent, indicating that the binding of auxin to the particulate fractions was more complex than binding of one hormone molecule to one binding site. This complexity was further confirmed by experiments in which the initial velocity of complex formation was measured. A model was worked out into which our data fit without contradictions. It involves the binding of four hormone molecules to one receptor molecule.

Structure and composition of the walls of normal and apolar embryos ofFucus vesiculosus L. were studied. Fucoidin was found in an amorphous outer layer and in an inner fibrillar layer of the wall, mainly at the rhizoid pole. Also in apolar embryos this inner layer was present; it was markedly thickened at the presumptive site of rhizoid formation.

It was not until the late Middle Ages that the sea penetrated far into the interior of The Netherlands, thus flooding three quarters of a million hectares of land. Since then half a million hectares have been reclaimed from the sea. The Dutch Government chose to preserve the remaining quarter of a million hectares of shallow sea with mudflats of the Waddensea as a nature reserve. The management objectives are at one hand to preserve all characteristic habitats and species with a minimal interference by human activities in geomorphological and hydrological processes, and at the other hand to guarantee the safety against the sea of the inhabitants of the adjacent mainland and islands and to facilitate certain economic and recreational uses of the Waddensea without jeopardizing the natural qualities. These objectives are being elaborated in managementplans.

When young radish (Raphanus sativus L. cv, Novired RS) plants were exposed to low levels of ethylene, the fresh weight and dry weight of the tubers significantly increased. This was mainly because ethylene reduced the percentage of plants that hardly or not at all formed a tuber. Decaptated seedling cultured in vitro, were supplied with several plant growth regulators in order to determine a possible correlation between the induction of radial growth and the biosynthesis of ethylene in the hypocotyl. Indole-3-acetic acid, or in combination with 6-benzylaminopurine, induced growth of the hypocotyl and markedly enhanced ethylene biosynthesis. However, the application of a precursor and an inhibitor of ethylene biosynthesis revealed that there is no direct causal relationship between radial growth and ethylene evolution. It is suggested that ethylene favoured tuber growth in intact plants by changing the partitioning of assimilates in the plant, rather than by the induction of cambial activity.

Ethylene, applied as ethephon, inhibited the elongation of etiolated, axillary potato shoots cultured in vitro and it stimulated radial growth along the whole length of these shoots. The same phenomena were observed when ACC, the precursor of ethylene, was added to the medium, whereas silver ions reversed these effects. However, tuber formation in vitro was suppressed by ethephon. This indicates a dual role of ethylene in the induction of tuber formation in potatoes: it had a positive effect by blocking the elongation of stolons and it suppressed tuber initiation.

We studied the modulation of the number of membrane-bound naphthaleneacetic acid (NAA)-binding sites during the growth cycle of tobacco cells in batch culture. Both cell number and specific NAA-binding increased exponentially, but at different rates and for different periods. This caused a characteristic modulation of the number of binding sites per cell during the growth cycle: During the first day of the lag phase this number decreased; in the exponential phase it rose markedly, and in the stationary phase it was constant.

Durch Assoziationsmessungen wird festgestellt, das die aus 3.3′-Dibrom-di-n-propylather (3) erhaltliche Di-Grignard-Verbindung 1 in Tetrahydrofuran vollstandig zu monomerem 1-Oxa-5-magnesiacyclooctan (2) und Magnesiumbromid disproportioniert. Die Bevorzugung von 2 wird auf intramolekulare Koordination zwischen Magnesium und Sauerstoff zuruckgefuhrt. Bei hoherer Temperatur zersetzt sich 2 zu Cyclopropan und trimerem 1-Oxa-2-magnesiacyclopentan (6a). Bifunctional and Cyclic Organomagnesium Compounds, II. – 1-Oxa-5-magnesiacyclooctane From association measurements it is concluded that the di-Grignard compound 1 formed from 3,3′-dibromodi-n-propyl ether (3) in tetrahydrofuran completely disproportionates to monomeric 1-oxa-5-magnesiacyclooctane (2) and magnesium bromide. The preference for 2 is ascribed to intramolecular coordination between magnesium and oxygen. At higher temperatures 2 decomposes to cyclopropane and trimeric 1-oxa-2-magnesiacyclopentane (6a).

In co-oxidations of aldehydes and olefins with molecular oxygen in the liquid phase, epoxides are formed via two different routes: (1) by reaction of intermediate acylperoxy radicals with the olefin, and (2) by a non-radical reaction of intermediate peracid with the olefin. For the systems benzaldehyde cis-1, 2-D2-1-octene and benzaldehyde cis- and trans-2-octene the ratio of radical to non-radical epoxidation has been calculated with the aid of the kinetic relationships derived for this type of reaction and has been determined experimentally from the stereo selectivity of the expoxidation and from the production of carbon dioxide. The results show that the ratio of radical to non-radical epoxidation increases with increasing reactivity of the olefin as well with increasing olefin/aldehyde ratio.

Association numbers of magnesium compounds in tetrahydrofuran have been investigated. One Mg-atom is incorporated in the molecules of the Grignard reagent, of MgBr2, and of Et2Mg.

The potassium contents of bark strips of cassava (Manihot esculenta Crantz) and of phloem exudate of castor bean (Ricinus communis L.) were analyzed at different regions of the stem. In cassava, a peak in potassium content was observed near the first mature leaf, leveling off both above and below this point. In castor bean, only a downward decreasing gradient was observed. In both plants, the direction of the potassium gradient coincided with the presumed direction of assimilate flow.

The effect of abscisic acid (ABA) on the uptake of sucrose by discs from castor bean (Ricinus communis L. cv. Zanzibarensis) cotyledons was investigated. Incubation on ABA solutions for one hour or longer significantly inhibited sucrose uptake. The effect was measurable at ABA concentrations as low as 0.1 μM. The inhibition was due to a lowering of the apparent Vmax of the sucrose-carrier system, the Km being unaffected. Uptake of sucrose was coupled to proton uptake but ABA had no detectable effect on the latter process. It is suggested that ABA exerts an effect on the phloem loading of sucrose by enhancing the efflux of sucrose. In leaf discs from three other plant species (Beta vulgaris L., Petunia hybrida L. and Phaseolus vulgaris L.) ABA exerted a similar effect. The results are discussed with respect to source-sink relationships.

Plants synthesize a myriad of metabolites, far more than those produced by most other organisms. Plant hormones are a unique set of compounds, with distinctive metabolism and properties that affect physiological processes during development and growth. Their underlying genetic variation will have attracted much research attention by virtue of junctions of NGS technologies and metabolomics. The aim of this thesis was to study natural variation of plant hormones in Arabidopsis. To obtain reliable quantitative data of levels of hormone using LC-ESI-MS, I first developed a set of methodological processes related to extraction and purification. A set of Arabidopsis ecotypes was chosen, and studied to earn various aspects of natural variation with traits of mature roots. Based on results of these works, my study has moved to classical linkage mapping analysis to find genetic loci responsible for levels of a few sets of hormone in roots and leaves of Arabidopsis. Chapter 2 describes the problem of matrix effects caused by impurities in semi-purified extracts, on the accuracy of data derived from LC-ESI-mass spectrometry. Matrix effects may result in both ion suppression and enhancement, and severely affects quantitative data of hormone levels. Without proper ways to minimize matrix effects, hormone data would be unreliable, and would not allow accurate QTL mapping. I validated a few key-points that are critical for determining the levels of a wide range of plant hormones in Arabidopsis extracts based on a one-step solid phase extraction (SPE) method. For the simplified purification of Arabidopsis extracts, a commercially available HLB sorbent was chosen to recover a wide range of chemically diverse series of hormone analytes. Dilution using a much smaller starting sample (e.g., 2.5 mg) reduced the matrix effects considerably but additional measures were required for most of the analytes. Flushing the HLB-SPE column with acidic methanol was more effective to reduce matrix effect than acetonitrile based eluent. At the end, I proposed a series of steps and procedures to optimize the protocol for hormone analysis in LC-ESI-MS. Chapter 3 describes natural variation of hormone levels found in 13 Arabidopsis ecotypes. Variations of hormone level among the accessions were remarkably small, viz., less than three-fold difference between extremes. For CKs, relatively larger variations were found for ribosides and glucosides, as compared to the free bases. Root phenotypic traits of these accessions were also measured, using a new parameter (mature root unit) for complex root systems, and correlation analyses were done between hormone data and mature root traits. For root phenotyping, length-related traits—lateral root length and total root length—showed larger variations than lateral root number-related ones. Antagonistic interactions between hormones (IAA and trans-zeatin) were detected for root weight. These findings provide enough basis to warrant a quantitative genetic analysis in plant hormone metabolism and crucial information for the choice of a proper segregating population. Chapter 4 reports diverse QTLs that are responsible for hormone levels of CKs, SA and JA in roots of Arabidopsis Ler×Cvi RIL population. QTL analysis of two sub-populations, viz., vegetative and flowering plants revealed that many of the QTLs were development-specific, suggesting that the transition to flowering has a profound effect on hormone metabolism. Using near-isogenic lines, several significant QTLs were confirmed; three co-localized QTL regions were responsible for determining several CK metabolites. Using a knock-out plant, a functional role of zeatin N-glucosyltransferase gene (UGT76C2) underlying a large-effect QTL for levels of tZ N-glucosides and tZRMP was evaluated in CK metabolism. Pleotropic effects of this gene were found for levels of CK in both roots and leaves, but significant changes of morphological traits were observed only in roots. This suggests that CK N- glucosides play an important role in root development. I also advocated the possibility of genetic regulation of concentration ratio between tZ7G and tZ9G based on a newly observed QTL of the trait. As a further step of QTL analysis after Chapter 4, the study was extended to leaves. Chapter 5 describes the analysis of 5 groups of hormones in rosette leaves of the same RIL population. QTL analysis showed a multitude of significant loci for levels of IAA, ABAs and CKs. Also for leaves, development-specific QTLs were detected in two sub-populations, vegetative and flowering lines. QTLs for ratios between hormone metabolites belonging to the same group but also to different groups were found and some of them partly co-localized with those of single compounds, implying that QTLs for single hormones may also affect the balance between hormones. The detection of QTLs for ratios between structurally unrelated hormones (e.g. auxin : ABA) and further fine mapping may help unravelling genetic elements underlying hormone interactions in the regulation of plant development and stress responses. Finally, in Chapter 6, several issues arising from the separate experiments are taken into consideration. The main significance of this PhD thesis is the experimental confirmation that finding QTLs for hormone metabolism is feasible and worth being extended to other populations of Arabidopsis and to crop plants. I anticipate that in the near future metabolomics study towards natural variation of plant hormones will be part of interesting theme in quantitative genetics. It will provide us to gain a better understanding of the complexity of molecular mechanism underlying hormone metabolism in plants.

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Potato tubers are vegetative structures aimed at survival of the period when conditions are not optimal for the growth of the potato plant. Understanding the process of tuber formation in potatoes ( Solanum tuberosum ) is important, both from a scientific view, and for practical applications. During the initial stages of tuber formation an enhanced synthesis of starch and storage proteins is observed at the site of the stolon that develops into a tuber. Besides an enhanced synthesis of storage products, longitudinal cell division and cell enlargement is observed resulting in a radial expansion of the stolon. Because cell division and the synthesis of storage products are two important events during tuber development we analyzed the expression of two sets of genes: two cell cycle genes (cycB1;1 and CDC2a) and two storage related genes (AGPaseS andlPat21). To analyze cell cycle and storage related gene expression we fused the promoter sequences of these genes to the firefly luciferase reporter gene from North American firefly ( Photinus pyralis ). Luciferase (LUC) activity can be observed non- destructively in individual tubers and correlates with the luciferase mRNA level in both in vitro grown as well as in soil grown tubers, indicating that in planta LUC activity reflects the ongoing expression level of the reporter gene. More important, LUC activity correlates with the mRNA level of the endogenous genes.In chapter 2 we describe the expression of cell cycle and storage related gene expression in in vitro grown explants in relation to the onset of swelling marking the morphological onset of tuber formation. The average activity quantified in multiple tubers indicated that the storage related genes were upregulated well before visible swelling, viz., four days before swelling for AGPaseS, and three days before swelling forlPat21, while the average activities of both cell cycle genes showed an upregulation after the onset of swelling. However with the ability to observe changes in gene expression in individual tubers a variable upregulation for both storage related genes could be shown in relation to the moment of visible swelling, varying between four days before till ten days after the onset of swelling. The results indicate that tuber development does not occur via a fixed sequential order of events, but consists of independent individual programs that occur in the same region of the stolon resulting in the formation of a potato tuber.When after tuber initiation, environmental conditions become adverse for tuber induction, second growth may occur. Adverse tuber-inducing conditions may result in the outgrowth of the apical bud (second growth) and a reduced growth of the tuber (primary growth). We mimicked second growth inducing conditions by transferring developing in vitro tubers to medium with gibberellin (chapter 3) or to medium with a low level of ethanol (chapter 4). GA-induced second growth resulted in the outgrowth of the apical bud followed by the formation of a secondary stolon. Transfer of tubers to medium supplemented with ethanol resulted in the formation of a secondary tuber when combined with 8% sucrose, whereas ethanol combined with 1% sucrose resulted in the formation of a secondary stolon. Preceding bud growth cycB1;1, AGPaseS andlPat21 expression declined in the complete tuber both in young (1-5 days after visible swelling) and in mature tubers (25-30 days after visible swelling) after transfer to medium with GA 4+7 or medium with ethanol, whereas CDC2a activity only declined when tubers were transferred to medium with ethanol. Second growth inducing conditions did not affect the activity of the CaMV35S promoter, indicating that the decline in the cell cycle and storage related promoter activity in the tubers during second growth is not related to a general effect on gene expression. Although a decline in cell cycle and storage related gene expression is observed before second growth, there is no causal relation between this decline and the outgrowth of the bud, as concluded from analysis of tubers transferred to medium with GA or ethanol combined with ABA which prevents outgrowth of the bud. Under these conditions cell cycle and storage related gene expression also declined. Transferring tubers to medium with different alcohols and combining ethanol with an inhibitor of alcohol dehydrogenase (4-methyl pyrazole) showed that ADH activity is a key factor during ethanol-induced second growth and the changes observed in gene expression in bud and tuber region.Preceding second growth, a higher cycB1;1 and AGPaseS expression is observed in the bud region as compared to the activity in the tuber region. CycB1;1 expression in the bud region indicates a specific induction of the cell cycle genes during second growth. However, a relatively higher cycB1;1 expression in the bud is not sufficient for bud growth both during GA or ethanol-induced second growth. These results indicate that bud growth is not controlled by cycB1;1. AGPaseS expression in the bud region during second growth possibly indicates the enhanced import of sugars into the bud region during second growth. Preventing bud outgrowth by combining GA with ABA counteracts the difference in AGPaseS expression between bud and tuber in mature tubers, indicating that tuber and bud region respond differentially upon GA and ABA. During ethanol-induced second growth, relatively higher AGPaseS expression was observed in the bud region in young tubers when transferred to medium with ethanol and 8% sucrose whereas tubers transferred to medium with ethanol and 1% sucrose showed a decline in AGPaseS expression both in bud and tuber region during second growth. These results indicate that the type of second growth and the enhanced AGPaseS expression in the bud region during ethanol-induced second growth are related to the amount of sugars available for the bud. Mature tubers transferred to medium with ethanol showed higher AGPaseS expression in the bud region, irrespectively of the sucrose concentration in the medium. Combining ethanol with ABA could not prevent AGPaseS expression in the bud region during ethanol-induced second growth. These results indicate that ABA affects sugar import in the bud region during GA-induced second growth whereas ABA does not affect sugar import during ethanol-induced second growth.In chapter 5 we described the relation between the expression of the cell cycle and storage related genes in the tubers and the differences in tuber growth rate of tubers growing on complete plants, to determine if the expression of these genes can be used as a measure for differences in sink strength and sink activity between tubers. Tuber growth rate was determined during the complete period of tuber development as an indication for sink strength and the correlation was determined with the level of gene expression quantified per tuber and per unit tuber area. By using hydroponics pot assemblies we were able to observe tuber growth and to quantify gene expression non-destructively during the complete period of tuber development. Three different stages of tuber growth were distinguished viz., initiation, linear tuber growth and maturation. During initiation, a low correlation was observed between tuber growth rate and the expression level of the cell cycle and storage related genes. During the period of linear tuber growth a good correlation was observed forlPat21 while during maturation the expression of cycB1;1, AGPaseS andlPat21 correlated well with the sink strength, as reflected by the growth rate. The results presented in chapter 5 indicate that patatin gene expression is a good predictive marker for differences in sink strength between tubers initiated on the same potato plant.It has become clear that non-invasive methods to study gene expression are useful tools for developmenta l studies as described in this thesis on potato tuber development. Changes in gene expression can be observed in individual tuber samples, showing the exact relation between changes in gene expression and developmental changes.

Potato tuber dormancy is part of the vegetative life cycle of potato. It refers to a period, in which no growth is occurring, although the tuber is stored under conditions that are favourable for growth. Factors in the tuber are responsible for this growth arrest; soon after dormancy is broken, sprouting occurs. Dormancy is already initiated during tuber formation.Potato tubers are often placed in short- to long-term storage for a stable year round supply. The occurrence of sprouting can be detrimental for further (industrial) processing of the tubers. On the other hand, sometimes sprouting of the tubers is already necessary soon after harvest, e.g. when tubers need to be planted for the next growing season. Therefore, it is of major interest to investigate the physiological regulation of potato tuber dormancy, dormancy breaking and sprouting. In this research, microtubers obtained with an in vitro system were used; it was concluded (chapter 6) that microtubers were a good model for soil-grown tubers.Some physiological processes during tuber dormancy initiation (tuber formation) and dormancy breakage (just before visible sprouting), seemed to be reversed, e.g. increasing levels of GA were necessary to induce sprouting during dormancy initiation, while at the end of dormancy lower doses were sufficient. Also the endogenous levels of ABA, known as a dormancy hormone, are reported to increase during the initiation of dormancy and to decrease again at the end of dormancy, thus initiating sprouting. Starch, which is the major carbohydrate reserve in potato, is synthesized during tuber formation and degraded again during sprouting to supply carbohydrates for growth of the new plant. The hypothesis that the developmental processes of initiation leading to dormancy (tuber formation) and dormancy breakage / sprouting were more or less the opposite of each other in terms of levels of endogenous hormones, carbohydrates and intermediates and with respect to the involved enzymatic conversions, is reviewed in chapter 2. Especially the endogenous hormone levels during tuber formation and dormancy breakage / sprouting are extensively discussed (summarised in figure 2, chapter 2) and it was concluded that these hormone levels were partly the reverse in the two different developmental stages.Carbohydrates and enzymes involved in sugar and starch metabolism are also discussed in chapter 2, although litterature about data during dormancy breaking was relatively scarce. It was concluded that indeed carbohydrate metabolism related processes might be reversed when dormancy initiation and dormancy breaking are compared.The effect of GA application on dormancy breaking is undisputed; however, the observed changes in carbohydrate metabolism after GA-application could be the result of the developmental process of dormancy breaking, or a direct result of the hormone and not related to sprout induction. Therefore, another way of inducing sprouting, by the use of ethanol, is investigated and discussed in chapter 3. In this chapter, the role of ADH in ethanol-induced dormancy breaking was investigated. It was concluded that ADH seemed to be involved in dormancy breaking, but a role for the (acidic) products could not be shown. Therefore, a role for ADH in the NAD +to NADH conversion was suggested. Applying ethanol with 1% sucrose in the medium resulted in a sprout while ethanol with 8% sucrose in the medium resulted in a secondary tuber. We hypothesized, based on a theory developed for tuber formation, that sucrose could affect endogenous GA levels in a negative way. This would mean that high sucrose would result in low endogenous GA, which in its turn would result in a secondary tuber and vice versa. Determinations of endogenous GA levels did not confirm this hypothesis, low sucrose levels even lead to a decrease of GA 1 levels. Apparently, the role of sucrose and GA in the regulation of sprouting differed from their role in the regulation of tuber formation. Also ethanol, by no means, increased the level of GA 1 .As already mentioned, data on sugar and starch metabolism during dormancy and sprouting were rare. In chapter 4, starch cycling during tuber formation, dormancy and sprouting was investigated by determining enzyme activities involved in starch synthesis and degradation. AGPase activity appeared to increase before visible sprouting, but this was only shown in histochemical staining and not in activity measurements. Also AGPase S- luc tranformants showed an elevated expression in sprouting tubers. Amylase activity was also high during tuber formation and decreased during dormancy. It was only after sprouting that the activity of β-amylase increased. STP was high during tuber formation, decreased during dormancy and remained constant during sprouting. STP was suggested to be involvedin starch degradation and starch cycling was suggested to be active during all developmental stages of the tuber.The fact that a switch in sucrose breakdown from an invertase to a susy-regulated one occurs during tuber formation made us investigate sucrose breakdown during dormancy and sprouting. Also enzymes involved in the conversions of the sucrose degradation products were investigated, as well as the levels of sugars and intermediates. The results are discussed in chapter 5. Both invertase and susy were very low in the tuber both during dormancy and sprouting. As only susy showed some activity in histochemical staining, sucrose degradation in the tuber was suggested to be supported by susy. At the end of dormancy, glucose-1-phosphate, derived from starch breakdown by STP, is supposed to be converted by UGPase, SPS and SPP into sucrose, which is being exported from the sink tuber to the growing sprout. Invertase showed a huge activity in the sprout and hexokinase showed almost no activity. As a result, high levels of glucose were found in the sprout. The results of chapter 4 on starch cycling are implemented in the results on sucrose metabolism in chapter 5. A schematic model shows the roles of starch and sucrose cycling in both the tuber and the sprout, during dormancy breakage.In the general discussion (chapter 6), starch and sucrose metabolism during tuber formation and sprouting are compared and it could be concluded that both developmental processes use the same pathways (sucrose and starch cycling), with net fluxes in opposite directions, in the tuber. The conclusion of chapter 2, could be extended with the above conclusion. Carbohydrate metabolism in stolon and sprout are largely similar, also with respect to the flux direction.Furthermore, carbohydrate metabolism during induced sprouting (by GA or ethanol) is compared to the one occurring during spontaneous sprouting (chapter 4 and 5). It could be concluded that both methods for induced sprouting showed too many differences compared to spontaneous sprouting to use them as a model for the processes occurring during spontaneous sprouting (chapter 6).

The potato tuber is a vegetative storage organ, which is initiated at the tip of an underground diagravitropic growing stem, called a stolon. The process of tuber formation comprises the induction, initiation and growth of a stolon, cessation of the longitudinal growth of the stolon, followed by the induction, initiation and growth of the tuber. Tuber initiation is characterized by swelling of the stolon tip. The formation of tubers is accompanied by a large accumulation of starch and of a specific set of storage proteins. How these major changes in carbohydrate metabolism interact and are coordinated with the developmental process of tuber formation is an important question from a fundamental point of view, not only for potato tubers, but also for the formation of starch-accumulating plant tissues in general. When a potato plant has started to form tubers, in principle all stolons present on the plant can be induced to initiate this developmental programme. However, this does not occur synchronously, creating problems for research as it is not possible to select externally those stolons, which are going to tuberize. Another problem is that the different developmental stages on one plant can influence each other. Therefore, for our research of tuberisation-related changes in carbohydrate metabolism, we have used a highly synchronous in vitro tuberisation system, providing plant material in well-defined developmental stages, which are not influenced by each other.Sucrose is the starting point of carbohydrate metabolism in growing stolons and tubers. It is synthesised in the leaves (source organs) and transported towards the underground parts via the phloem. When it arrives in a growing stolon it is most likely unloaded in an apoplastic way. In the apical region of the stolon a high invertase activity is present, which can be attributed to the cell wall-bound form (CWI). This apoplastic invertase isoform hydrolyses sucrose, resulting in the presence of glucose and fructose in the apoplast. The hydrolysis of sucrose steepens the sucrose gradient between phloem and the recipient cells, which is thought to play an important role in driving the unloading and short-distance transport of sucrose in apoplastically-supplied tissues. After the hexoses have entered the cells, they can be fed into carbohydrate metabolism by the activity of specific phosphorylating enzymes. During stolon growth, one of these enzymes, hexokinase (HK), shows a high correlation with CWI at the level of overall activity, suggesting a coordinated coarse control between these two enzymes.Furthermore, localisation of HK by specific enzyme activity staining showed, like CWI, a high activity in the apical region of the stolon. In this particular region of the stolon cell divisions occur. Therefore, the predominance of these two enzymes in this region in comparison with the rest of the stolon suggests a functional link with cell division.Cellular growth is sustained by a high flux of carbon through the respiratory(-related) pathways producing energy in the form of ATP, NAD(P)H, and carbon intermediates for important anabolic routes, such as the synthesis of DNA, RNA, proteins, lipids and cell wall-components. Measurements of activity of the main enzymes involved in glycolysis and the oxidative pentose phosphate pathway (OPPP) indicated that the extra demand for energy and carbon intermediates by dividing cells is accompanied with an elevated activity of these enzymes. Furthermore, from the patterns of activity of these enzymes it could be deduced that they are probably subject to a coordinated coarse control. Such a coordinated control mechanism may regulate the potential of respiratory(-related) pathways in response to the demand for their products. The high activities of CWI and HK in the mitotic-active region could ensure that sufficient carbon is directed into the respiratory(-related) pathways by enhancing the flow of carbon towards the dividing cells and by rapid conversion of sucrose into hexose-phosphates, the substrates for glycolysis and OPPP. In addition, these two enzymes may also be involved in the process of glucose-sensing, which is thought to mediate the transcriptional (and/or translational) regulation of genes involved in cell division. Interestingly, during stolon growth the enzyme alkaline pyrophosphatase (APPase) shows a similar pattern of overall activity as the main enzymes involved in glycolysis and OPPP.Moreover, in situ hybridisation with a RNA probe derived from a potato APPase revealed a clear presence of the corresponding transcript in all stolon tissues. This particular isoform has been proposed to be localised in the cytosol, which is in agreement with its temporal and spatial pattern of expression and with the pattern of overall APPase activity during stolon-to-tuber transition. In the dividing cells the biosynthesis of cell components produces a large amount of PPi, which is the substrate of this enzyme. Rapid conversion of PPi by a cytosolic APPase or another enzyme (e.g. tonoplast bound pyrophosphatase) is required to drive the biosynthetic reactions in the synthetic direction. The Pi released by this activity may serve as a metabolic pool of Pi for the conversion of ADP into ATP. In this way the observed apparent coordination between level of APPase activity and of respiratory(-related) pathways could be explained.When the cells have left the cell cycle and are no longer involved in cell division, their main cellular activity comprises enlargement, which continues until the cells are fully elongated and mature. In the region just below the apical part of the stolon, where cell divisions no longer occur, less invertase and HK staining is present. The reduction in the levels of activity of CWI and HK in this region, can also be deduced from their patterns of overall activity during stolon growth. The activity of a soluble vacuolar invertase, however, does not decrease but remains rather constant throughout the whole growing stolon. In these elongating parts of the stolon the combined activity of the soluble invertases will establish the sink strength, i.e. the capacity to draw sucrose from the phloem. Besides this function in maintaining a sucrose gradient between phloem and elongating cells and allowing sucrose to enter carbohydrate metabolism, the vacuolar invertase may also be involved in the process of cell elongation. Cell elongation is mechanistically driven by enlargement of the vacuole, which depends on the osmotic pressure of the vacuole. The hydrolysis of one osmotically-active molecule into two by the activity of this vacuolar isoform will enhance the osmotic pressure and the water influx into the vacuole. The elongation zone of the stolon probably has a lower requirement for energy and carbon intermediates in order to sustain the cellular growth than the mitotically active apical region, which is reflected by the decline in the overall activity of enzymes involved in glycolysis and OPPP during the period of consecutive stolon growth in our in vitro system.Tuber initiation is accompanied by a clear change in the patterns of several enzymes. In situ hybridisation revealed that the transcripts corresponding to the acid invertases progressively disappear from the parenchymatic tissues of the tuber. As a result, the overall activities of these two isoforms declined to very low levels, which was also indicated by the in situ activity staining. In contrast, the enzyme sucrose synthase (Susy) increases rapidly to high levels in the parenchymatic tissues of the initiated tuber as indicated by the in situ analysis at mRNA level and activity level and by overall activity measurements. This developmental change in the sucrose-degrading pathway is clearly associated with tuberisation and the onset of massive starch accumulation as it was absent in the two non-tuberising controls. Sucrose synthase catalyses in vivo a reversible, low-affinity reaction, which is subject to a high degree of metabolic regulation. Sucrose degradation by invertases is irreversible, not subject to metabolic regulation, and it occurs already at a low sucrose level. These differences between the two different sucrolytic pathways make the Susy-catalysed pathway probably more suitable than invertases for the complex regulation of sucrose degradation in coordination with tuber growth, symplastic sucrose unloading, and starch synthesis, as is explained in chapters 2 and 5. Furthermore, there are indications that a certain sucrose influx and/or sucrose levels are required for the upregulation of the expression of genes involved in sucrose-starch conversion. The effectiveness of this apparent sucrose-modulated mechanism depends largely on the type of sucrolytic route predominantly present in the cell. The down-regulation of CWI and the switch of an invertase-catalysed sucrose-degradation route into a Susy-mediated sucrolytic system allow import of carbon in the form of sucrose and allow high sucrose levels in the cytosol. Moreover, hexoses released by the activity of invertases appear to have an inducing effect on respiratory(-related) metabolism, which is thought to occur at the expense of the carbon flux toward starch synthesis.Concomitant with the developmental switch in the sucrose-degrading pathway an increase in the overall activity of fructokinase can be observed. In situ analysis at transcript level and activity level indicated that the spatial and temporal pattern of this enzyme show a close resemblance to that of Susy. The apparently closely coordinated coarse control indicates a functional linkage between these two enzymes. As already mentioned Susy catalyses a reversible reaction, which is severely inhibited by accumulation of fructose. Fructokinase may downregulate the fructose content, thereby allowing a large net flux of sucrose degradation through the Susy-catalysed reaction. The other product of the degrading reaction of Susy is UDPGlucose (UDPGlc). UDPGlc can be converted by the enzyme UGPase, which does not show a development-related increase in our in vitro system, but is present at a high activity throughout the whole period of culture. The coarse control of this enzyme, just like that of the enzyme PFP, seems subject to modulation by high sugar levels. Both enzymes catalyse a reversible reaction, use PPi as an energy-donor and are able to convert the substrate of the sucrose synthesising enzyme SPS. In combination with Susy (and SPS), the reversible character of these two enzymes allows a process of sucrose/hexose-P and hexose-P/triose-P cycling, which offers the cell a mechanism to adjust rapidly and automatically the net rate of sucrose degradation in response to the supply of sucrose and demand for carbon in the respiratory and biosynthetic pathways in the tuber parenchyma cells.Measurements of the overall level of different hexose-P's during stolon-to-tuber transition indicated that tuber initiation and successive growth is accompanied with a decline in the hexose-P pool, which is in contrast with an expected increase due to the tuberisation-initiated rise in sucrose supply and degradation rate. A plausible explanation for this unexpected observation is that the level of the hexose-P pool has to remain low during tuber growth, because an increase in this pool might lead to an enhanced rate of sucrose resynthesis by Susy and SPS. Such an increase in unidirectional sucrose synthesis will lead to a decrease in the net rate of sucrose degradation, thereby lowering the net flux of carbon towards starch synthesis. Therefore, a low hexose-P pool might be an important prerequisite for a high, but adjustable net rate of sucrose degradation in Susy-mediated sucrolytic systems. A possible role for the enzyme PFP in the maintenance of a low hexose-P content is discussed in chapter 5.During successive tuber growth the overall activities of enzymes involved in respiratory(-related) metabolism declined, indicating that tuber growth and starch accumulation does not require an increase in the potential of respiratory(-related) metabolism. Concomitant with this decline the overall activity of APPase decreased also. In situ hybridisation with the presumed corresponding RNA probe revealed a substantial decline in mRNA content in the whole growing tuber. The Susy-mediated pathway of sucrose degradation depends on the availability of PPi, which is required by the UDPGlc-converting reaction of UGPase. The decline in activity of the assumed cytosolic APPase may prevent that the sucrose-degrading reaction of Susy becomes restricted by the accumulation of UDPGlc.Three other enzymes show a clear and large increase in activity after the onset of tuberisation, which is not observed in the non-tuberising controls. Two of these enzymes are known to be involved in starch synthesis. The enzyme ADPglucose pyrophosphorylase (AGPase) synthesises the precursor for starch synthesis and is thought to contribute to a large extent to the total flux control of carbon towards starch synthesis. The other enzyme is starch phosphorylase (STP), which catalyses a reversible reaction and may, therefore, be involved in the synthesis of starch or in its degradation. The patterns of both enzyme-activities correlate highly with the accumulation of starch in our in-vitro tuberisation system, which is consistent with their known role in the (cycling) process of starch synthesis. Starch accumulation seems to be a process of simultaneous synthesis and degradation. Two possible routes of degradation may exist. The first one can be catalysed by STP, the second one involves the activity of amylases. The (final) endproduct of amylases, glucose, can only be redirected into starch metabolism by the activity of a hexokinase. This enzyme shows a considerable increase in its level of activity after tuberisation onset. Activity staining revealed that this enzyme is present in the main starch-accumulating tissues of the tuber. The product of its reaction, Glc6P, has to be converted into Glc1P, which serves as the substrate for AGPase and STP.The enzyme responsible for this reaction, phosphoglucomutase (PGM) also shows a considerable increase in overall activity after the initiation of tuberisation, although not that large as AGPase, STP and HK. Furthermore, the tuberisation-related increase in Glc1P content in comparison with the decline in the content of the other hexose-P's, suggests that the reversible reaction catalysed by the plastidial isoform of PGM lies in the direction of Glc1P synthesis.From this study on changes of carbohydrate metabolism during stolon-to-tuber transition of potato, it has become clear that the sucrolytic switch itself forms a central and important step in the differentiation process of starch-storing tubers. The direct implications and consequences of this metabolic switch for the regulation of carbon fluxes towards starch synthesis and other carbon-demanding process are discussed in chapter 2, 3, 4 and 5. In addition in chapter 5 an effort has been made to place this tuberisation-related switch in a broader context of metabolic induction of growth-related and storage-associated differentiation processes, including the role and importance of glucose and sucrose as different signal-inducing molecules, modulating the expression of genes involved in cell division and respiratory(-related) metabolism (glucose-responsive) versus differentiation into storage cells and sucrose-starch converting potential (sucrose-responsive). In chapter 5 is also discussed how these mechanisms might affect sink strength and thus the capacity to enhance the flow of photosynthate towards the cells of the two different types of growing sink organs and its importance for growth and/or storage.