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The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm.

Preparations of potato leafroll virus in aphid diet solution were made from aphids ( Myzus persicae Sulz.) or from stems of Datura stramonium L. When the preparations were placed between membranes and offered to aphids at 8°, the aphids acquired the virus by feeding and subsequently transmitted it to test plants. The efficiency of transmission was slightly higher when the aphids received the virus by injection. Preparations from aphids which contained much residual host material usually gave fewer transmissions than those containing less host material. Preparations from D. stramonium were at least as infective as those from aphids.

Tobacco (Nicotiana tabacum L.) leaves were found to contain an extracellular proteinase that endoproteolytically cleaves tobacco pathogenesis-related (PR) proteins. This proteinase was partially purified from tobacco leaves and characterized as an aspartyl proteinase with a pH optimum around pH 3 and a molecular mass of 36,000 to 40,000 daltons. In vitro, the enzyme cleaved purified tobacco and tomato PR proteins into discrete fragments. The characteristics of this proteinase were similar to pepsin and identical to those displayed by a previously described tomato 37-kilodalton aspartyl proteinase active against tomato PR proteins (I Rodrigo, P Vera, V Conejero [1989] Eur J Biochem 184: 663-669), suggesting that these extracellular proteases could play a role in a conserved mechanism for PR protein turnover in plants.

Chapter 12; International audience; Iron is an essential micronutrient for plants and associated microorganisms. Iron nutrition of these organisms relies on the soil supply. However, bioavailability of iron in cultivated soils is low. Plants and microorganisms have thus evolved active strategies of iron uptake based on acidification, chelation, and/or reduction processes. Iron acquisition by these organisms leads to complex interactions ranging from mutualism to competition. In the rhizosphere, plants support abundant and active microbial communities through the release of rhizodeposits. Iron uptake by these microorganisms and by the host plant decrease even more the concentration of iron in solution. Therefore, there is an intense competition for iron among rhizosphere microorganisms, favoring those with the most efficient iron uptake strategy. This is the case for fluorescent Pseudomonas bacteria that synthesize siderophores, called pyoverdines or pseudobactines, which have a high affinity for iron and suppress fungal phytopathogens and deleterious microorganisms. Pyoverdines also elicit plant defense reactions and contribute to plant iron acquisition. Taken together, these mutual effects promote plant growth and health. However, competition for iron may also occur between plants and microbes during pathogenesis. Siderophores contribute to the iron uptake of the host plant and to the virulence of pathogens; conversely, host plants activate mechanisms aimed at depriving pathogens of nutritional iron. The iron-withholding mechanisms of the host plant rely on controlling its iron homeostasis. In this chapter, we describe the strategies of iron uptake of plants and microorganisms, the resulting complex interactions between them, and the challenges represented by their monitoring in agroecology.

An important question in plant defense signaling research is: how are plants capable of integrating signals induced by pathogenic micro-organisms and herbivorous insects into defenses that are specifically active against the attacker encountered? Plant defenses against pathogens and insects are differentially regulated by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling and genome-wide transcriptome changes in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium ( Pseudomonas syringae pv. tomato ), a pathogenic leaf fungus ( Alternaria brassicicola ), tissue-chewing caterpillars ( Pieris rapae ), cell-content-feeding thrips ( Frankliniella occidentalis ), or phloem-feeding aphids ( Myzus persicae ). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. The transcriptional alterations were predominantly attacker-specific, but the processes affected surprisingly similar. This indicates that the different attackers induce changes in similar plant processes through largely non-overlapping transcriptional alterations. Yet, infestation by M. persicae induced a transcriptional response that was opposite to those induced by the other attackers or exogenous application of MeJA. We concluded that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk and additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.Little is known about the spectrum of effectiveness of the different types of induced resistance that are expressed upon attack by pathogens or insects. Because the signaling pathways that control induced resistance against pathogens and insects partly overlap, we decided to investigate the effectiveness of microbially induced resistance against the tissue-chewing herbivorous insects Pieris rapae and Spodoptera exigua in Arabidopsis. Two types of microbially induced resistance were studied: systemic acquired resistance (SAR), which is induced upon predisposal infection by necrotizing pathogens, and rhizobacteria-mediated induced systemic resistance (ISR), which is triggered by selected strains of non-pathogenic, root-colonizingrhizobacteria. No effect of SAR or ISR was evident on herbivore-induced attractiveness of the parasitic wasp Cotesia rubecula , indicating that neither type of induced resistance influenced indirect defense against these insects. In feeding experiments on whole plants, induction of SAR and ISR significantly reduced growth and development of the generalist herbivore S. exigua , whereas the performance of the specialist P. rapae was unaffected. The JA- and ET-responsive genes PDF1.2 and HEL , which were activated upon feeding by either of the two herbivores, showed a strongly potentiated expression pattern in SAR- and ISR-expressing plants upon feeding by S. exigua , but not upon feeding by P. rapae .This differential priming for enhanced herbivore-induced gene expression was confirmed in microarray experiments using a dedicated cDNA microarray containing 111 insect-responsive Arabidopsis genes. These results suggest that the effectiveness of microbially induced SAR and ISR against S. exigua feeding is associated with priming for enhanced defense-related gene expression.In conclusion, this thesis highlights the complexity of the defense signaling interactions between plants, pathogens and insect herbivores.

Keywords: Earliness, Foliage maturity type, Late blight, Phytophthora infestans , Potato, QTL analysis, Resistance, Solanum tuberosumPotato ( Solanum tuberosum ) is grown throughout the world and is among the most important crops for global food supply. Late blight, caused by Phytophthora infestans , is the most important and destructive disease wherever potato is grown. Potato varieties with resistance to late blight can be used to contest the disease. The application of race-specific resistance has turned out not to be durable, because P. infestans is able to overcome this type of resistance. Race-non-specific resistance is expected to be more durable but, unfortunately, this type of resistance against P. infestans is consistently associated with late foliage maturity. The purpose of the research described in this thesis was to unravel the nature of the association between race-non-specific resistance to late blight and foliage maturity type in potato. The main goal was to determine whether the association between the two traits is genetic or physiological. As part of the physiological studies, single-node cuttings of potato were tested for their potential to serve as a model to predict foliage maturity type at any time during the cropping season. Single-node cuttings did not reflect the stage of tuber development or tuber induction of the plants from which they were taken, and they do not appear to reflect the physiological state of the whole plant adequately. Consequently, single-node cuttings cannot be used to predict foliage maturity type.A possible physiological association between late blight resistance and foliage maturity type was studied by relating the effects of plant age, leaf age, and leaf position on resistance. Leaf position had the largest and most significant effect: apical leaves were far more resistant to late blight than basal leaves. Plant age and leaf age had only minor effects. Thus, the resistance of a specific leaf remained about the same during its entire lifetime.Genetic studies were performed with seven different diploid potato progenies. Recombinant genotypes with early foliage maturity and resistance against P. infestans were not identified. Therefore, it cannot be concluded that the association between late blight resistance and foliage maturity type is due to closely linked genes. QTL analyses revealed loci for resistance to late blight on chromosomes 3, 5, and 10, and for foliage maturity type on chromosome 5. The locus for foliage maturity type could not be distinguished from the most important QTL for resistance: the allele of molecular marker GP21 that is associated with late blight resistance is also associated with late foliage maturity.

Peroxidases are probably the most extensively studied enzymes in higher plants. Various isoenzymes occur as soluble proteins in the apoplast and in the vacuole, or are bound to membranes and cell walls. Their occurrence is often organ-specific and developmentally controlled, and there is circumstantial evidence that they function in growth, differentiation and defence. In Chapter I biochemical, physiological and genetic aspects of higher-plant peroxidases are reviewed, particularly in relation to the peroxidase system in petunia ( Stimoryne Rafin., formerly Petunia Juss.). It is pointed out that only by studying genetically characterized systems a full understanding of the biochemical and physiological characteristics of the various peroxidase isoenzymes can be obtained.In petunia eight different peroxidase isoenzymes have been genetically characterized and the occurrence of two more is inferred (Chapters 1 and 2). Of these , the most prominent one in leaves and stems is peroxidase a (PRXa), which migrates upon gel electrophoresis as a fast-moving, anionic set of three (sometimes four) molecular forms (mozymes). PRXa also occurs in petals, but is absent from roots. Leaves also contain the intermediatemoving anionic isoenzyme PRXb and the fast-moving cationic peroxidase bands of PRXc. PRXa and PRXc were shown to be the major peroxidases present in extracellular extracts obtained by the vacuum infiltration method, whereas PRXb was the most active peroxidase in isolated mesophyll protoplasts. The three isoenzymes differed in their affinity for Concanavalin A (Con A)-Sepharose; whereas PRXb bound firmly, and was released only in the presence of mannose or glucose, PRXa and PRXc were only partly retained. Since all peroxidases are glycoproteins, these results suggest that the nature of the oligosaccharide chains could be important in determining the cellular location of the peroxidases (Chapter 3).The localization of PRXa in the apoplast was exploited for its purification by preferentially extracting the enzyme from leaves by the vacuum infiltration method (Chapter 4). The electrophoretic variant (allozyme) PRXa1, the product of the allele prxA1 , present in the petunia cv. Roter Vogel, was purified over 1300-fold by two sequential acetone precipitations, gel filtration and chromatofocusing. The purified enzyme had a RZ (Reinheits Zahl; A 404nm /A 280nm ) of 3.6, an apparent molecular weight of about 37 kD and an isoelectric point of 3.8. The three molecular forms had slightly different molecular weights and were separated by affinity chromatography on a Con A-Sepharose column. The absorption spectrum of PRXa showed maxima at 404 (Soret band), 496 and 636 nm. Further spectral analy sis revealed that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. The pH optimum for the reaction with hydrogen peroxide and guaiacol was 5.0 and the specific activity of the enzyme 61 mkat/g protein. The rate constants for its reaction with hydrogen peroxide and guaiacol were 2.6 x 10 6and 1.9 x 10 6M -1s -1, respectively. The reaction with guaiacol was inhibited by various aromatic compounds, notably by trihydroxylated and ortho - and para -dihydroxylated phenolic derivatives. Cinnamic-acid derivatives were more inhibitory than phenol or benzoic-acid derivatives. Coniferyl alcohol was completely inhibitory and was polymerized to presumed lignin-like material. Together with its localization in the apoplast, this substrate preference suggests that PRXa functions in the polymerization of lignin in the cell wall (Chapter 4).A specific antiserum raised in a rabbit against the purified PRXa was used to investigate as to how far peroxidases in other Solanaceous species are antigenically related to the petunia enzyme. Polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) followed by immunoblotting revealed the presence of cross-reacting proteins in all species tested. To determine whether these proteins represented peroxidases, thorn-apple ( Datura stramonium L.), tobacco ( Nicotiana tabacum L.), sweet pepper ( Capsicum annuum L.), potato ( Solanum tuberosum L.) and tomato ( Lycopersicon esculentum Mill.) were selected for further analysis. Immunoblots after native PAGE revealed that the antiserum specifically recognized the fastmoving anionic peroxidases that are localized in the apoplast. Despite their antigenic relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight, possibly as a result of differences in glycosylation. Apparently, the Solanaceae contain orthologous genes encoding peroxidase isoenzymes homologous to petunia PRXa. The antiserum did not react with peroxidases from horseradish, turnip, African marigold, maize and oats (Chapter 5).Since PRXa is encoded by a single gene, the occurrence of three molecular forms must result from post-transcriptional or post-translational modifications. In Chapter 6 the nature of this heterogeneity was further analysed. Upon incubation withα-mannosidase the larger PRXa1.1 and PRXa1.2 were converted into immunoreactive products resembling the smaller PRXa1.3, suggesting that the three forms of PRXa1 differ in the number of terminal mannose residues. Someα-mannosidese activity was found to be present in the apoplast of leaves and may be responsible for the conversion of PRXa1.1 into PRXa1-2 and PRXa1.3 during leaf development. Lectin-affinity blotting suggested that PRXa contains small, complex-type carbohydrate chains; gas-chromatographic analysis indicated the presence of xylose, arabinose, fucose, mannose, glucose and galactose (Chapter 6).In Chapter 7 the distribution of the soluble peroxidase activity in tissues from petals, leaves and stems was investigated. In petals from fully expanded flowers, the upper epidermis contained nearly 20 % of the peroxidase activity, but no activity was present in protoplasts isolated from this tissue. The peroxidase activity in the upper epidermis was extra-cellular and consisted of PRXa. In protoplasts from the remaining tissue (mesophyll and lower epidermis) PM was the only peroxidase present; it was responsible for about 45 % of the total activity in petals. In leaves, the lower epidermis contained 14 - 40 % of the peroxidase activity, depending on their stage of development. Up to half of this activity was localized extracellularly. In stems, the epidermis contained nearly 80 0 of the peroxidase activity and 30 % of this activity was localized extracellularly. All extracellular extracts contained merely PRXa, suggesting that PRXa is localized mainly in the epidermis. By pressing transversely cut leaves and stems on membrane filters and using these dip-blots to localize the enzyme insitu , it was shown that both total peroxidase activity and immunoreactive PRXa are localized mainly in the epidermis, in leaves particularly at the site of the midrib and veins. The epidermal localization of PRXa is suggestive of a role in modulating growth rate or in defence, either by the deposition of lignin, suberin or cutin, or by increased cross-linking of various cell-wall polymers, or both (Chapter 7).In order to analyse the developmentally-controlled regulation of PRXa synthesis, it was attempted to obtain a cDNA probe for PRXa mRNA. Using a Poly(A) +-RNA fraction isolated from leaves, a possible precursor of PRXa was detected upon immunoprecipitation of invitro translation products, and an extended product of about 110 nucleotides was detected after reverse transcription primed with a mixed oligonucleotide probe based on a conserved amino-acid sequence in other plant peroxidases. The incorporation of 35S-methionine and 32P-CTP, respectively, was very low, suggesting that peroxidase mRNA was present at a low concentration only. A cDNA library in the bacteriophageλgt11 was constructed and screened with the antiserum or the oligonucleotide probe, but no positive clones were detected. Similarly, no peroxidase-specific clones were found upon screening a corrola- specific cDNA library with the antiserum (Chapter 8).In Chapter 9 possible functions of PRXa are discussed. Based on its extracellular localization, its organ and tissue specificity, and its catalytic properties, a function in the rigidification of cell walls in response to internal and external signals seems likely. Petunia PRXa strongly resembles a supposedly lignin-forming peroxidase in tobacco, and seems to be different from a suberin- forming peroxidase induced in potato tubers and tomato fruits upon wounding. Accordingly, in contrast to the total peroxidase activity, PM activity does not increase in floating leaf discs, indicating that it is not induced upon wounding. The identification of genetically defined peroxidase isoenzymes in members of the Solanaceae as being involved in specific physiological processes will provide insight into the function and the regulation of the peroxidase system in this plant family.

The loss of chlorophyll during the senescence of leaves is preceded by a decrease in protein content. Proteases responsible for the degradation of the proteins have been implicated in the regulation of the senescence process. The first leaf of the seedling of oats ( Avena sativa L.) demonstrates the typical pattern of leaf senescence in cereals and was chosen to study the properties and subcellular localization of proteases throughout leaf development.In Chapter 1, a general introduction is given that indicates the significance of the breakdown of proteins in leaves for seed yield and quality in cereals. Also in this chapter, protease classification is documented, and the possible role of changing protease activities in the regulation of protein degradation is discussed.Oat leaves contain two major proteases with pH optima at pH 4.5 ("acidic protease") and 7.5 ("neutral protease"). During natural development of greenhouse-grown plants both types of enzymes showed highest activities in young and fully-grown leaves, and decreased throughout the course of senescence. Also in detached leaves incubated in the dark and, therefore, subject to accelerated ageing, loss of protein was not accompanied by increases in protease activities. In the light, protease activities increased, but the rate of protein loss was greatly reduced. Thus, protein breakdown appears to be independent of the amount of protease present and additional synthesis of the major proteases is not required for protein loss during senescence (Chapter 1).This apparent paradox could be resolved if proteases and their substrates were spatially separated and brought into contact by a controlled decompartmentalization. Therefore, the distribution and subcellular localization of the major proteases were determined. Protoplasts were prepared as a first step in the isolation of vacuoles. However, the cell wall-degrading enzyme mixtures used for protoplast isolation were seriously contaminated by proteases interfering with the determination of the endogenous protease activity in the isolated protoplasts. These extra proteases could be inactivated by heating the cell well-degrading enzyme mixture at 50°C for 10 min at pH 6.5. This treatment did not impair the cell walldegrading activity. Protoplasts isolated with heated enzymes showed similar protease activities as washed protoplasts, isolated with untreated enzymes. This proved that contaminating proteases were effectively removed during protoplast washing, and that the protease activity measured in isolated protoplasts was derived from the protoplasts themselves. (Chapter 3).Vacuoles were isolated by osmotically lysing the protoplasts in the presence of K 2 HPO 4 . The maximum concentration of phosphate by which lysis occurred decreased progressively with increasing leaf age. By using the appropriate phosphate concentrations, it became possible to isolate clean vacuoles from leaves up to an advanced stage of senescence, when the leaves had lost more than 50% of their protein. From leaves older than 17 days, only vacuoplasts (vacuoles with adhering cytoplasm, within a resealed plasma membrane) could be obtained. The integrity of both the plasmalemma and the tonoplast decreased in these older leaves and this phenomenon might be linked with increased decompartmentalization at a late stage of senescence (Chapter 4).When the distribution of the proteases was determined in different subcellular fractions, on an average 16% of the acidic protease activity was washed out of the intercellular space of the leaves. The major part of the acidic activity was located within the vacuole. The neutral protease was absent from both these compartments and must, therefore, be cytoplasmic. During the course of leaf development, all of the acidic protease activity present in protoplasts was recovered in the vacuoles, as long as clean vacuoles could be isolated (i.e. up to 17 days). It seems most likely that protein degradation is controlled by import of protein substrates into the vacuole (Chapter 5).The acidic and neutral proteases were partly purified by gel filtration and anion-exchange chromatography (Chapter 6). The enzymes hardly separated, indicating that they have similar molecular weights and charges. The neutral activity was stabilized by merceptoethanol and inhibited by inhibitors of metallopeptidases, whereas the acidic one was not. Both activities were inhibited to varying extents by sulphydryl- and serinetype inhibitors. Both enzymes were endopeptideses. The instability of, in particular, the neutral protease seriously hampers its further purification and characterization.Furthermore, by activity staining after electrophoretic separation and using aminoacyl-2-naphthyl am ides as substrates, five aminopeptidases and one trypsin-like endopeptidase were identified (Chapter 7). The main aminopeptidase was largely unspecific. Two aminopeptidases showed preference for arginine and lysine. An iminopeptidase acted on proline. The fifth aminopeptidase was most active with methionine. All enzymes were active at pH 4.5, 6.0 and 7.5, but showed highest activities at low rather then at neutral pH. The trypsine-like endopeptidase was active at pH 4.5 and 7.5, was inhibited by o -phenanthroline and was not identical with either the acidic or the neutral protease described previously. Its activity decreased during leaf development. During storage of protein extracts in the cold this arginine-specific endopeptidase associated with ribulosebisphophate carboxylase, concomitant with a loss of this protein band. As to how far this enzyme has a function in protein breakdown invivo has to be further elucidated.The results of this study show that the levels of protease activities are not correlated with the rate of over all protein degradation. The acidic protease has no entry to the substrates to be degraded. In contrast, the neutral protease appears to be located together with protein substrates and organelles in the cytoplasm, but seems to selectively degrade only a few proteins. Whereas the mechanism of normal protein turnover remains to be elucidated, the slow, steady decline of protein during natural development may be controlled by, on the one hand, compartmentalization of the acidic protease in the vacuole and, on the other hand, by maintaining a pH removed from the optimum for exopeptidese action in the cytoplasm.

The development of the first leaf of oats ( Avena sativa L.) comprises four phases. About 4 day after sowing the leaf emerges and starts to expand. Expansion is complete and maturity is reached at 7 to 9 days. Thereafter, senescence ensues, as expressed by a gradual loss of chlorophyll over the period from 12 to 37 days. Finally the leaf withers, and dies by about 37 days.RNA and protein contents start to decrease before the onset of chlorophyll loss, and during the phase of senescence all three parameters decline in a coordinated manner. Since proteins are essential to the functioning of the leaf, their loss is considered to be a determining factor in the rate at which senescence proceeds. However, inhibitors of protein synthesis have been shown to block loss of protein and chlorophyll, indicating that also synthesis of proteins is required for senescence to progress. These proteins are not major proteases, nor are they likely to be membrane-associated, because membrane systems remain intact until a late stage of senescence.The present study was undertaken to search for proteins that are specific to each of the developmental stages in the life of the leaf, and to determine specific properties of proteins that function particularly during the stage of senescence. Changes in the pattern of soluble proteins during leaf development were followed by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (Chapter 2). Phenol-soluble protein samples were separated in up to 500 spots by isoelectric focusing between pH 5 and 8 in the first dimension and 12.6% SDS slab gel electrophoresis in the second dimension. The gels were stained with silver. A number of 122 protein spots were further characterized by their presence in isolated chloroplasts. The large and the small subunits of ribulosebisphosphate carboxylase/oxigenase (Rubisco) were identified by immunoblotting.Major changes occurred during the period of leaf expansion: 39 mostly low-molecular-weight polypeptides disappeared and 34 mostly high-molecular-weight spots became apparent. Of these, 16 and 13, respectively, were associated with chloroplasts. During the subsequent loss of protein and chlorophyll, indicative of senescence, the number of spots decreased gradually without new polypeptides becoming apparent, 75% of the total number being still detectable in yellow, 37 days-old leaves. About half of the disappearing spots were lost before the leaf had expanded to its final length. No polypeptides were detected that were present exclusively in the later stages of leaf development: maturity, senescence, and final collapse.It was considered that such proteins might be present in amounts below the limit of detection by general protein staining, either because they are synthesized in very small amounts, or because they are subject to rapid turnover. To be able to follow the synthesis and degradation of individual proteins, while avoiding effects of leaf detachment or wounding, a method was developed to label the proteins at different stages of development of intact leaves attached to the plants (Chapter 3). The following methods were tested: growth of seedlings on 35S-sulfate-containing Knop medium, labelling with 35S-methionine by vacuum infiltration of the leaf, injection into the leaf base or into the seed near the embryo, or wiping the surface of the leaf with ethanol and subsequent incubation in the labelled solution. The first four methods were unsuitable because of insufficient uptake, preferential transport to other plant parts, too much dilution by endogenous aminoacids or radiation damage. A specific activity of 10 5dpm / 20 μg of leaf protein, minimally necessary to obtain a distinct fluorogram of the gels, was reached only upon the treatment with ethanol. Under these conditions no more than 170 kBq of 35S-methionine was required in the uptake solution.When the 4.5 cm distal parts of the first leaf were thus labelled for 12 h at different stages of development, similar amounts of label were retained in the segments, but incorporation into protein decreased from 28% in 7-days- to 3% in 27-days-old plants. Even at a late stage of senescence a great many proteins were still being synthesized. Synthesis of Rubisco and the other chloroplastassociated proteins declined more rapidly than general protein synthesis. Although Rubisco ceased to be synthesized by 13 days, it was still clearly present in 27-days-old leaves indicating that, once synthesized, the protein is very stable. During senescence two sets of relatively high -molecularweight proteins became more pronounced. In addition, three proteins with a molecular weight around 67 kDa and isoelectric points between 6.5 and 6.8 became the most prominently synthesized proteins in older leaves. Since these senescence-associated proteins were hardly visible on silver-stained patterns, they seem to be subject to rapid turnover. A functioning of these proteins in senescence could explain the requirement of protein synthesis for senescence to proceed (Chapter 4).By labelling distal leaf parts of 7- and 15-days-old plants for 24 h and following the loss of label from individual protein spots, the rates of degradation of the synthesized proteins were determined over a 14-day period. Of the about 300 spots that could be distinguished, a large number turned over quickly (disappearing spots), while a smaller number was degraded very slowly (persisting spots). Thus, protein half-lifes varied from a few days to over at least a week. This pattern of in vivo breakdown was compared to that in vitro in soluble-protein extracts at pH 5.5 and 7.6, the optimal pH values of the acidic and neutral proteases, respectively. Although 75% of the proteins were degraded similarly in vivo and in vitro , substantial changes in the relative rates of degradation of the other proteins occurred under the different conditions. Notably, Rubisco was degraded very slowly in vivo , but rather quickly and in a different way in vitro at pH 5.5. As it has been found that there is no relationship between the level of protease activity and the rate of protein degradation in vivo, these results support our previous conclusion (Van der Valk and Van Loon 1988) that the proteases and their protein substrates are spatially separated in vivo . Whereas rapidly turning-over cytoplasmic proteins might be imported into the vacuole for degradation, long-lived chloroplastic proteins, such as Rubisco, are likely to be degraded by proteases within the organelle.To investigate in how far the changes observed in attached leaves also occur upon detachment, distal leaf segments were incubated vertically with their bases in water. Senescence of detached leaves in light did not differ significantly from senescence in attached leaves. In darkness, protein was lost at a higher rate, but several proteins showed relative increases. Notably, proteins previously characterized as high-molecular-weight and senescence-associated proteins increased in amount. Additional changes associated with incubation in light or darkness were not related to senescence. The cytokinin benzyladenine delayed and abscisic acid accelerated the changes compared to water. Besides changes previously identified in leaves senescing on the plant, detached leaves showed alterations that reflect their condition of incubation rather than their developmental stage. For this reason, detached leaves are less suitable for the study of senescence than leaves attached to the plant (Chapter 6).The comparison of many protein patterns was facilitated by the development of a computer program, GELSCAN, for the qualitative matching of any pair of gels. The program is written in PASCAL and makes use of a 640 kB personal computer. This program is a valuable tool for an objective analysis of 2D-PAGE patterns (Chapter 7).Finally, the regulation of protein synthesis and degradation during development of the first leaf of oats is discussed (Chapter 8). No specific properties of the proteins themselves, as identified by 2D-PAGE, act as determinants of their rate of degradation. The high-molecular-weight proteins, and particularly the senescence-associated proteins, are synthesized in increasing amounts during senescence and may play a role In mechanisms governing the senescence progress. De ontwikkeling van het eerste blad van haver is te verdelen in 4 fasen. 4 dagen na het zaaien komt het blad te voorschijn en begint te strekken, totdat het tussen 7 en 9 dagen volgroeid is. Daarna begint het blad te verouderen, hetgeen zich uit in een geleidelijk verlies van chlorofyl over een periode van 12 tot 37 dagen. Tenslotte verwelkt het blad, droogt uit en sterft af rond dag 37. In het onderzoek is gezocht naar eiwitten die specifiek zijn voor elk van de ontwikkelingsstadia tijdens het bladleven. Daarnaast is getracht specifieke eigenschappen te definieren van eiwitten, in het bijzonder voor het functioneren tijdens het verouderingsstadium. Veranderingen in het patroon van de oplosbare eiwitten gedurende de bladontwikkeling werden gevolgd m.b.v. 2-dimensionele polyacrylamide-gel-elektroforese. Om de synthese en afbraak van individuele eiwitten te volgen, maar tegelijk de effecten van afsnijding of verwonding te vermijden, werd een methode ontwikkeld om de eiwitten in intacte bladeren aan gehele bladeren in verschillende ontwikkelingsfasen te merken met een radioactieve precursor

Symptoms of viral infections in plants often resemble disturbances in growth and development. Therefore, symptoms appear to result from an interference of the virus with the regulation of growth and development of the host plant. Particularly the non-histone chromatin- associated proteins are considered to be the regulators of specific gene expression. The aim of the present study was to elucidate whether upon infection of a plant with a virus, alterations occur in the non-histone chromatin-associated protein composition of the leaves.A survey of the literature on viral pathogenesis in plants, the properties of chromatin- associated proteins, and their possible role in the regulation of specific gene expression is given in Chapter 1.In Chapter 2 we looked for changes in the chromatin-associated protein composition of leaves from virus-infected tobacco plants. As a model system the combination 'Samsun' tobacco-tobacco mosaic virus (TMV) was used. In this combination, mosaic symptoms develop in the newly emerging leaves, the mosaic consisting of alternating patches of light-green and dark-green tissue. To ensure recovery of representative amounts of nuclei from the whole leaf and not predominantly from either light green or dark green tissue, known procedures for the isolation of nuclei had to be modified. By homogenizing the leaves in a large volume of buffer and repeated grinding of the homogenate, up to 45% of the nuclei present in the leaves were freed from the cell debris. From these nuclei chromatin was purified, and chromatin-associated proteins were dissociated from the DNA in buffer containing urea and high salt. Analysis of these proteins in SDS-containing polyacrylamide gels revealed a single consistent alteration upon TMV infection, being the induction of a new protein of about 116 kDa. The use of two-dimensional polyacrylamide gel electrophoresis showed a second alteration to occur. This second change consisted of the appearance of a new protein of about 20 kDa. This protein was serologically identified as TMV coat protein.Further investigations into the effects of both mosaic-inducing and necrotic symptoms- Producing viruses on the chromatin- associated protein constitution of different tobacco cultivars, revealed that of the viruses tested only cucumber mosaic virus (CMV) induced any specific alterations. A 'green' isolate induced a single new protein of about 28 kDa, and the 'yellow' strain P6 one of about 29 kDa. These new proteins co-migrated in SDS- containing polyacrylamide gels with the coat proteins of the respective CMV strains. CMV was also the only virus - apart from TMV - that induced mosaic symptoms. In combinations resulting in localized or systemic necrosis, no changes were observed. Thus it turned out that only if virus infection results in the development of systemic mosaic symptoms, discrete changes in the chromatinassociated protein profile occur (Chapter 3).The presence of viral coat protein associated with host chromatin represents virus specificity of the induced change. The presence of the 116 kDa protein exclusively after TMV infection was also connected to the virus. It was not induced in uninfected plants during senescence of fully-grown leaves or upon ageing of detached leaves (Chapter 3). Since a primary role for coat protein in the induction of symptoms is unlikely, we further concentrated on the TMV-induced 116 kDa protein.Whereas TMV was detected in upper leaves that become infected systemically already 96 h after inoculation of lower, fully-expanded leaves, the 116 kDa protein became discernable in the systemically-infected leaves only between 120 to 144 h after inoculation. This moment coincided with the appearance of visible symptoms (vein clearing). At that time the 116 kDa protein was also detectable, both in the soluble protein and in the sedimentable membrane fractions. However, the 116 kDa protein was found to be preferentially associated with, on the one hand, the membranous fraction and, on the other hand, the chromatin. Based on the amount of the protein in the different fractions and on morphometric analysis of tissue sections, it was calculated that the concentration in the chromatin was about eight-fold higher than in the cytoplasm. Moreover, in contrast to TMV coat protein, its dissociation from chromatin required sodium chloride. This indicates that the 116 kDa protein is bound more tightly to the chromatin than TMV coat protein. These observations strongly suggest that the 116 kDa protein may play a regulatory role in gene expression, analogous to the non-histone chromatin proteins (Chapter 4).Since TMV is known to code for a protein of a similar molecular mass, it was investigated whether the new 116 kDa chromatin-associated protein from mosaic-diseased tobacco leaves is identical with the TMV-coded 126 kDa protein. In SDS-containing polyacrylamide gels the 116 kDa protein comigrated with the 126 kDa translational product synthesized in vitro from TMV RNA. Furthermore, limited digestion of the 116 kDa polypeptide and the 126 kDa translational product with protease V8 yielded the same peptide fragments, indicating that both proteins are identical (Chapter 5).In Chapter 6 two-dimensional gel electrophoretic patterns of chromatin-associated proteins from cultivars and species of Nicotiana with different genetic constitutions were compared, using an improved protein preparation procedure. Notably the application of phenol extraction, followed by precipitation of proteins with ethanol, yielded highly concentrated protein samples. This procedure resulted in sharper and more intense protein spots on the gels than before. In all Nicotiana species and cultivars examined, 90% of the chromatin-associated proteins appeared identical. Among the 10%, differing polypeptides no specific polypeptide spot(s) could be associated with the presence of the gene N, that governs hypersensitivity towards TMV.Chapter 7 discusses the possible role of the 116 kDa protein in symptom development. It is proposed that the 116 kDa polypeptide may act as a repressor of plant genes that specifically function early in leaf development. This hypothesis is based on the observation that inhibition of DNA-dependent RNA synthesis with actinomycin D induces vein clearing and mosaic in developing leaves similar to the symptoms induced by TMV in leaves not over 15 mm in length at the moment of inoculation. Recent results by Haseloff et al . (Proc. Natl. Acad. Sci. USA 81, 4358-4363 (1984)) indicate that two similarly-sized translational products of the serologically unrelated plant viruses alfalfa mosaic virus and brome mosaic virus have approximately 20-30% amino acid homology with the TMV-coded 116 kDa polypeptide. Such observations suggest that other plant viruses may use similar strategies to interact with their hosts.