Effects of Alternaria alternata f.sp. lycopersici toxins on pollen

Effects of the phytotoxic compounds (AAL-toxins) isolated from cell-free culture filtrates of Alternaria alternata f.sp. lycopersici on in vitro pollen development were studied. AAL-toxins inhibited both germination and tube growth of pollen from several Lycopersicon genotypes. Pollen from susceptible genotypes, however, was more sensitive for AAL-toxins than pollen from resistant plants, while pollen of species not belonging to the host range of the fungus was not significantly affected by the tested toxin concentrations. AAL-toxins elicit symptoms in detached leaf bioassays indistinguishable from those observed on leaves of fungal infected tomato plants, and toxins play a major role in the pathogenesis. Apparently, pathogenesis-related processes and mechanisms involved in disease resistance are expressed in both vegetative and generative tissues. This overlap in gene expression between the sporophytic and gametophytic level of a plant may be advantageously utilized in plant breeding programmes. Pollen may be used to distinguish susceptible and resistant plants and to select for resistances and tolerances against phytotoxins and other selective agents.

Intra- and inter-metabolite correlation spectroscopy of tomato metabolomics data obtained by liquid chromatography-mass spectrometry and nuclear magnetic resonance

Nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LCMS) are frequently used as technological platforms for metabolomics applications. In this study, the metabolic profiles of ripe fruits from 50 different tomato cultivars, including beef, cherry and round types, were recorded by both 1H NMR and accurate mass LC-quadrupole time-of-flight (QTOF) MS. Different analytical selectivities were found for these both profiling techniques. In fact, NMR and LCMS provided complementary data, as the metabolites detected belong to essentially different metabolic pathways. Yet, upon unsupervised multivariate analysis, both NMR and LCMS datasets revealed a clear segregation of, on the one hand, the cherry tomatoes and, on the other hand, the beef and round tomatoes. Intra-method (NMR¿NMR, LCMS¿LCMS) and inter-method (NMR¿LCMS) correlation analyses were performed enabling the annotation of metabolites from highly correlating metabolite signals. Signals belonging to the same metabolite or to chemically related metabolites are among the highest correlations found. Inter-method correlation analysis produced highly informative and complementary information for the identification of metabolites, even in de case of low abundant NMR signals. The applied approach appears to be a promising strategy in extending the analytical capacities of these metabolomics techniques with regard to the discovery and identification of biomarkers and yet unknown metabolites

Cytoplasmic male sterility in Petunia hybrida : a structural and histochemical analysis

This thesis presents an analysis of the structural and histochemical aspects of cytoplasmic male sterility (cms) in Petuniahybrida . In petunia and in other crops, cms is the most commonly used tool for hybrid seed production. Application of the trait makes hybrid seed production possible without the need of emasculation of the maternal line. However, in spite of its economic importance, little is known on the primary causative factor and the initial step of pollen abortion in cms plants. Insights in the initial effects of cms may lead to a more comprehensive understanding of the regulation and expression of male sterility controlling genes, and, additionally, may possibly provide strategies for the introduction or induction of male sterility in crops in which cms systems are not available.In the first Chapter, some molecular aspects of cms are evaluated. Several lines of evidence indicate that the genetic determinants responsible for cms are carried by the mitochondrial genome. The mitochondrial involvement is found in a variety of plant species, including Petuniahybrida . Most of the mitochondrial encoded polypeptides are components of complexes which are responsible for key steps in the process of oxidative phosphorylation and the generation of ATP. Correspondingly, mitochondria isolated from tissues of cms plants, may code for an aberrant polypeptide composition of components of one of these complexes. Nevertheless, more information on the expression of mitochondrial genes in different anther tissues at various stages of development is necessary before we can conclude whether or not the deviations in mitochondrial DNA are functionally associated with the non-formation of viable pollen.The initial abnormalities in anther development of cms plants are generally found in the tapetal tissue. Also in the cms form of Petuniahybrida cv. Blue Bedder (BBS), the first symptoms of deviation are found in the tapetum (Chapter 2). Light microscopical analysis shows, that in BBS anthers, the tapetal breakdown begins at the prophase stage of the meiocytes. At the preceding stages of development, microsporogenesis in BBS anthers is normal and indistinguishable from the development in the male fertile counterpart (BBF). At the ultrastructural level, the initial aberration of BBS anthers is represented by the presence of large vacuoles in the cytoplasm of the tapetal cells (Chapter 3). At the leptotene stage of the meiocytes, these vacuoles are the first symptoms of degeneration. At later stages, the tapetal and sporogenous cells are highly distorted, the nucleus is disrupted and the cytoplasm disorganized. Mitochondria and plastids degenerate and many lipid droplets are present.Chapter 4 describes the way in which the biochemical and histochemical aspects of an enzyme system are influenced by the degeneration of the tapetal and sporogenous tissues. The Chapter gives information on the isoenzyme pattern, the activity, and the localization of esterases in anther tissues of cms and male fertile petunia cultivars. Esterases are rather unspecific, nuclear encoded enzymes occuring in all plant parts. The biochemical data show that, from the early meiosi S onward, esterase activity in cms-type anthers remains at a low level and hardly any new isoenzyme bands show up as compared to the situation in the male fertile counterpart. The histochemical determinations reveal, that in male fertile-type anthers, esterase activity is concentrated in the outer tapetal layer at late prophase and that it accumulates there till the early microspore stage. In anthers of cms plants, esterase accumulation in the tapetal cells ceases at the moment that tapetal breakdown becomes evident. These results suggest that the differences in total esterase activity and esterase isoenzyme patterns are an effect rather than a cause of the failing pollen formation.In cms forms of different species, aberrations in cytochrome c oxidase activity and other mitochondrial redox processes are associated with the cms plasmatype. A biochemical determination of the cytochrome c oxidase activity in anthers of Petuniahybrida and Zeamays is given in Chapter 5. The biochemical analysis is combined with a cytochemical localization of enzyme activity in mitochondria of sporogenous and tapetal tissues in both species. The data show that in anthers of different cms maize strains, the cytochrome c oxidase activity is reduced in comparison with the level found in male fertile-type anthers. Additionally, there are consistent cytochemical differences in the mitochondrial organization of cytochrome c oxidase activity between pollen of cms- S and male fertile maize plants. The aberrations in enzyme activity are observed at stages of development at which the structural aspects of degeneration are not yet evident. In fact, the deviation in cytochrome c oxidase may represent the initial symptom of male sterility in this maize type. Contrarily, in petunia, the first detectable differences in the mitochondrial enzyme activity occur only after the initial effects of tapetal degeneretion are apparent. Hence, in petunia, the decline in cytochrome c oxidase activity is the result rather than the cause of the proceeding process of degeneration.In Chapter 7 it is postulated that the cms specific deviations in the mitochondrial genome induce alterations in protein complexes which are essential for energy generating processes. Possibly, these aberrations adversely affect the energy status of cms cells. However, BBF and BBS plants possess similar growth characteristics, and, apparently, the viability of plants with cms plasmatype, is not diminished by the mitochondrial defects. In fact, abnormalities in the development of cms plants are only observed in particular anther tissues. These results may suggest that the aberrations in the mitochondrial genome are only expressed in the tapetal or sporogenous tissues at certain moments of development. However, this assumption is inconsistent with the fact that deviations in mitochondrial products are sometimes found in organelles isolated from vegetative parts of the plant. An alternative explanation for the tissue specific character of cms is, that the degeneration of the anther tissues is initiated by the specific metabolism of the cells. The adenylate energy charge ratios of petunia anther tissues is discussed in Chapter 6. As compared with petunia leaf tissue, the results give evidences for the particular metabolic state of the tapetal and sporogenous tissues. Examples of the metabolic activity in anthers of other plant species are evaluated in Chapter 7. Furthermore, the structural analyses as presented in the second and the third Chapter of this thesis, reveal that the cms petunia anther development is distorted at the moment at which there is a considerable rise in the metabolic activity of the tapetal cells of the male fertile counterpart. Possibly, during moments of energetic stress, the mitochondrial synthesis of energyrich products in tapetal cells of cms petunia is insufficient to meet the energetic demands for the normal functioning of the cells at that stage. Hence, as a result of defects in the mitochondrial genome, the tapetal, and consequently, the sporogenous tissues degenerate

Depth of dormancy in tomato (Lycopersicon esculentum Mill.) seeds is related to the progression of the cell cycle prior to the induction of dormancy

Cell cycle activities are initiated following imbibition of non-dormant seeds. However, it is not known whether cell cycle related events other than DNA replication also remain suppressed in imbibed dormant seeds. The objective of this study was to demonstrate that the transitions between the non-dormant and dormant (both primary and secondary) states are reflected in cell cycle events, such as DNA replication and the changing patterns of the microtubular cytoskeleton involved in the processes of growth and development. The present studies were conducted on seeds from tomato (Lycopersicon esculentum cv. Moneymaker) that possessed primary dormancy or were manipulated to attain secondary dormancy. In addition, a non-dormant abscisic acid (ABA)-deficient mutant, sitw, was used. DNA replication, as measured by flow cytometry, and -tubulin accumulation, analysed by immunoblotting, were compared with immunocytological studies of active DNA synthesis and microtubular cytoskeleton formation. It is shown that the depth of dormancy, which distinguishes primary and secondary dormancy, may depend on the progression of the cell cycle prior to the induction of dormancy

A non-directed approach to the differential analysis of multiple LC-MS-derived metabolic profiles

An essential element of any strategy for non-targeted metabolomics analysis of complex biological extracts is the capacity to perform comparisons between large numbers of samples. As the most widely used technologies are all based on mass spectrometry (e.g. GCMS, LCMS), this entails that we must be able to compare reliably and (semi)automatically large series of chromatographic mass spectra from which compositional differences are to be extracted in a statistically justifiable manner. In this paper we describe a novel approach for the extraction of relevant information from multiple full-scan metabolic profiles derived from LC–MS analyses. Specifically-designed software has made it possible to combine all mass peaks on the basis of retention time and m/z values only, without prior identification, to produce a data matrix output which can then be used for multivariate statistical analysis. To demonstrate the capacity of this approach, aqueous methanol extracts from potato tuber tissues of eight contrasting genotypes, harvested at two developmental stages have been used. Our results showed that it is possible to discover reproducibly discriminatory mass peaks related both to the genetic origin of the material as well as the developmental stage at which it was harvested. In addition the limitations of the approach are explored by a careful evaluation of the alignment quality

Genetic mapping of semi-polar metabolites in pepper fruits (Capsicum sp.): towards unravelling the molecular regulation of flavonoid quantitative trait loci

Untargeted LCMS profiling of semi-polar metabolites followed by metabolite quantitative trait locus (mQTL) analysis was performed in ripe pepper fruits of 113 F2 plants derived from a cross between Capsicum annuum AC1979 (no. 19) and Capsicum chinense No. 4661 Selection (no. 18). The parental accessions were selected based on their variation in fruit morphological characteristics and fruit content of some target phytonutrients. Clear segregation of fruit colour and fruit metabolite profiles was observed in the F2 population. The F2 plants formed three clusters based on their metabolite profiles. Of the total of 542 metabolites, 52 could be annotated, including a range of flavonoids, such as flavone C-glycosides, flavonol O-glycosides and naringenin chalcone, as well as several phenylpropanoids, a capsaicin analogue, fatty acid derivatives and amino acid derivatives. Interval mapping revealed 279 mQTLs in total. Two mQTL hotspots were found on chromosome 9. These two chromosomal regions regulated the relative levels of 35 and 103 metabolites, respectively. Analysis also revealed an mQTL for a capsaicin analogue, located on chromosome 7. Confirmation of flavonoid mQTLs using a set of six flavonoid candidate gene markers and their corresponding expression data (expression QTLs) indicated the Ca-MYB12 transcription factor gene on chromosome 1 and the gene encoding flavone synthase (FS-2) on chromosome 6 as likely causative genes determining the variation in naringenin chalcone and flavone C-glycosides, respectively, in this population. The combination of large-scale metabolite profiling and QTL analysis provided valuable insight into the genomic regions and genes important for the production of (secondary) metabolites in pepper fruit. This will impact breeding strategies aimed at optimising the content of specific metabolites in pepper frui