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Starch is an essential biopolymer produced by plants. Starch can be made inside source tissue (such as leaves) and sink tissue (such as fruits and tubers). Nevertheless, understanding how starch metabolism is regulated in source and sink tissues is fundamental for improving crop production.
Despite recent advances in the understanding of starch and its metabolism, there is still a knowledge gap in the source and sink metabolism. Therefore, this study aimed to summarize the state of the art regarding starch structure and metabolism inside plants. In addition, this study aimed to elucidate the regulation of starch metabolism in the source tissue using the leaves of a model organism, Arabidopsis thaliana, and the sink tissue of oil palm (Elaeis guineensis) fruit as a commercial crop.
The research regarding the source tissue will focus on the effect of the blockage of starch degradation on the starch parameter in leaves, especially in those of A. thaliana, which lack both disproportionating enzyme 2 (DPE2) and plastidial glucan phosphorylase 1 (PHS1) (dpe2/phs1). The additional elimination of phosphoglucan water dikinase (PWD), starch excess 4 (SEX4), isoamylase 3 (ISA3), and disproportionating enzyme 1 (DPE1) in the dpe2/phs1 mutant background demonstrates the alteration of starch granule number per chloroplast. This study provides insights into the control mechanism of granule number regulation in the chloroplast.
The research regarding the sink tissue will emphasize the relationship between starch metabolism and the lipid metabolism pathway in oil palm fruits. This study was conducted to observe the alteration of starch parameters, metabolite abundance, and gene expression during oil palm fruit development with different oil yields. This study shows that starch and sucrose can be used as biomarkers for oil yield in oil palms. In addition, it is revealed that the enzyme isoforms related to starch metabolism influence the oil production in oil palm fruit.
Overall, this thesis presents novel information regarding starch metabolism in the source tissue of A.thaliana and the sink tissue of E.guineensis. The results shown in this thesis can be applied to many applications, such as modifying the starch parameter in other plants for specific needs.
Sulphur, a macronutrient essential for plant growth, is among the most versatile elements in living organisms. Unfortunately, little is known about regulation of sulphate uptake and assimilation by plants. Identification of sulphate signalling processes will allow to control sulphate acquisition and assimilation and may prove useful in the future to improve sulphur-use efficiency in agriculture. Many of genes involved in sulphate metabolism are regulated on transcriptional level by products of other genes called transcription factors (TF). Several published experiments revealed TF genes that respond to sulphate deprivation, but none of these have been so far been characterized functionally. Thus, we aimed at identifying and characterising transcription factors that control sulphate metabolism in the model plant Arabidopsis thaliana. To achieve that goal we postulated that factors regulating Arabidopsis responses to inorganic sulphate deficiency change their transcriptional levels under sulphur-limited conditions. By comparing TF transcript profiles from plants grown on different sulphate regimes, we identified TF genes that may specifically induce or repress changes in expression of genes that allow plants to adapt to changes in sulphate availability. Candidate genes obtained from this screening were tested by reverse genetics approaches. Transgenic plants constitutively overproducing selected TF genes and mutant plants, lacking functional selected TF genes (knock out), were used. By comparing metabolite and transcript profiles from transgenic and wild type plants we aimed at confirming the role of selected AP2 TF candidate genes in plant adaptation to sulphur unavailability. After preliminary characterisation of WRKY24 and MYB93 TF genes, we postulate that these factors are involved in a complex multifactorial regulatory network, in which WRKY24 and MYB93 would act as superior factors regulating other transcription factors directly involved in the regulation of S-metabolism genes. Results obtained for plants overproducing TOE1 and TOE2 TF genes suggests that these factors may be involved in a mechanism, which is promoting synthesis of an essential amino acid, methionine, over synthesis of another amino acid, cysteine. Thus, TOE1 and TOE2 genes might be a part of transcriptional regulation of methionine synthesis. Approaches creating genetically manipulated plants may produce plant phenotypes of immediate biotechnological interest, such as plants with increased sulphate or sulphate-containing amino acid content, or better adapted to the sulphate unavailability.
Throughout their lifetime plants need to adapt to temperature changes. Plants adapt to nonfreezing cold temperatures in a process called cold priming (cold acclimation) and lose the acquired freezing tolerance during warmer temperatures through deacclimation. The alternation of both processes is essential for plants to achieve optimal fitness in response to different temperature conditions. Cold acclimation has been extensively studied, however, little is known about the regulation of deacclimation. This thesis elucidates the process of deacclimation on a physiological and molecular level in Arabidopsis thaliana. Electrolyte leakage measurements during cold acclimation and up to four days of deacclimation enabled the identification of four knockout mutants (hra1, lbd41, mbf1c and jub1) with a slower rate of deacclimation compared to the wild type. A transcriptomic study using RNA-Sequencing in A. thaliana Col-0, jub1 and mbf1c identified the importance of the inhibition of stress responsive and Jasmonate-ZIM-domain genes as well as the regulation of cell wall modifications during deacclimation. Moreover, measurements of alcohol dehydrogenase activity and gene expression changes of hypoxia markers during the first four days of deacclimation evidently showed that a hypoxia response is activated during deacclimation. Epigenetic regulation was observed to be extensively involved during cold acclimation and 24 h of deacclimation in A. thaliana. Further, both deacclimation studies showed that the previous hypothesis that heat stress might play a role in early deacclimation, is not likely. A number of DNA- and histone demethylases as well as histone variants were upregulated during deacclimation suggesting a role in plant memory. Recently, multiple studies have shown that plants are able to retain memory of a previous cold stress even after a week of deacclimation. In this work, transcriptomic and metabolomic analyses of Arabidopsis during 24 h of priming (cold acclimation) and triggering (recurring cold stress after deacclimation) revealed a uniquely significant and transient induction of DREB1D, DREB1E and DREB1F transcription factors during triggering contributing to fine-tuning of the second cold stress response. Furthermore, genes encoding Late Embryogenesis Abundant (LEA) and antifreeze proteins and proteins detoxifying reactive oxygen species were higher induced during late triggering (24 h) compared to primed samples, while cell wall remodelers of the class xyloglucan endotransglucosylase/hydrolase were early responders of triggering. The high induction of cell wall remodelers during deacclimation as well as triggering proposes that these proteins play an essential role in the stabilization of the cells during growth as well as the response to recurring stresses. Collectively this work gives new insights on the regulation of deacclimation and cold stress memory in A. thaliana and opens the door to future targeted studies of essential genes in this process.
Elucidating the molecular basis of enhanced growth in the Arabidopsis thaliana accession Bur-0
(2021)
The life cycle of flowering plants is a dynamic process that involves successful passing through several developmental phases and tremendous progress has been made to reveal cellular and molecular regulatory mechanisms underlying these phases, morphogenesis, and growth. Although several key regulators of plant growth or developmental phase transitions have been identified in Arabidopsis, little is known about factors that become active during embryogenesis, seed development and also during further postembryonic growth. Much less is known about accession-specific factors that determine plant architecture and organ size. Bur-0 has been reported as a natural Arabidopsis thaliana accession with exceptionally big seeds and a large rosette; its phenotype makes it an interesting candidate to study growth and developmental aspects in plants, however, the molecular basis underlying this big phenotype remains to be elucidated. Thus, the general aim of this PhD project was to investigate and unravel the molecular mechanisms underlying the big phenotype in Bur-0.
Several natural Arabidopsis accessions and late flowering mutant lines were analysed in this study, including Bur-0. Phenotypes were characterized by determining rosette size, seed size, flowering time, SAM size and growth in different photoperiods, during embryonic and postembryonic development. Our results demonstrate that Bur-0 stands out as an interesting accession with simultaneously larger rosettes, larger SAM, later flowering phenotype and larger seeds, but also larger embryos. Interestingly, inter-accession crosses (F1) resulted in bigger seeds than the parental self-crossed accessions, particularly when Bur-0 was used as the female parental genotype, suggesting parental effects on seed size that might be maternally controlled. Furthermore, developmental stage-based comparisons revealed that the large embryo size of Bur-0 is achieved during late embryogenesis and the large rosette size is achieved during late postembryonic growth. Interestingly, developmental phase progression analyses revealed that from germination onwards, the length of developmental phases during postembryonic growth is delayed in Bur-0, suggesting that in general, the mechanisms that regulate developmental phase progression are shared across developmental phases.
On the other hand, a detailed physiological characterization in different tissues at different developmental stages revealed accession-specific physiological and metabolic traits that underlie accession-specific phenotypes and in particular, more carbon resources during embryonic and postembryonic development were found in Bur-0, suggesting an important role of carbohydrates in determination of the bigger Bur-0 phenotype. Additionally, differences in the cellular organization, nuclei DNA content, as well as ploidy level were analyzed in different tissues/cell types and we found that the large organ size in Bur-0 can be mainly attributed to its larger cells and also to higher cell proliferation in the SAM, but not to a different ploidy level.
Furthermore, RNA-seq analysis of embryos at torpedo and mature stage, as well as SAMs at vegetative and floral transition stage from Bur-0 and Col-0 was conducted to identify accession-specific genetic determinants of plant phenotypes, shared across tissues and developmental stages during embryonic and postembryonic growth. Potential candidate genes were identified and further validation of transcriptome data by expression analyses of candidate genes as well as known key regulators of organ size and growth during embryonic and postembryonic development confirmed that the high confidence transcriptome datasets generated in this study are reliable for elucidation of molecular mechanisms regulating plant growth and accession-specific phenotypes in Arabidopsis.
Taken together, this PhD project contributes to the plant development research field providing a detailed analysis of mechanisms underlying plant growth and development at different levels of biological organization, focusing on Arabidopsis accessions with remarkable phenotypical differences. For this, the natural accession Bur-0 was an ideal outlier candidate and different mechanisms at organ and tissue level, cell level, metabolism, transcript and gene expression level were identified, providing a better understanding of different factors involved in plant growth regulation and mechanisms underlying different growth patterns in nature.
Due to global climate change providing food security for an increasing world population is a big challenge. Especially abiotic stressors have a strong negative effect on crop yield. To develop climate-adapted crops a comprehensive understanding of molecular alterations in the response of varying levels of environmental stresses is required. High throughput or ‘omics’ technologies can help to identify key-regulators and pathways of abiotic stress responses. In addition to obtain omics data also tools and statistical analyses need to be designed and evaluated to get reliable biological results.
To address these issues, I have conducted three different studies covering two omics technologies. In the first study, I used transcriptomic data from the two polymorphic Arabidopsis thaliana accessions, namely Col-0 and N14, to evaluate seven computational tools for their ability to map and quantify Illumina single-end reads. Between 92% and 99% of the reads were mapped against the reference sequence. The raw count distributions obtained from the different tools were highly correlated. Performing a differential gene expression analysis between plants exposed to 20 °C or 4°C (cold acclimation), a large pairwise overlap between the mappers was obtained. In the second study, I obtained transcript data from ten different Oryza sativa (rice) cultivars by PacBio Isoform sequencing that can capture full-length transcripts. De novo reference transcriptomes were reconstructed resulting in 38,900 to 54,500 high-quality isoforms per cultivar. Isoforms were collapsed to reduce sequence redundancy and evaluated, e.g. for protein completeness level (BUSCO), transcript length, and number of unique transcripts per gene loci. For the heat and drought tolerant aus cultivar N22, I identified around 650 unique and novel transcripts of which 56 were significantly differentially expressed in developing seeds during combined drought and heat stress. In the last study, I measured and analyzed the changes in metabolite profiles of eight rice cultivars exposed to high night temperature (HNT) stress and grown during the dry and wet season on the field in the Philippines. Season-specific changes in metabolite levels, as well as for agronomic parameters, were identified and metabolic pathways causing a yield decline at HNT conditions suggested.
In conclusion, the comparison of mapper performances can help plant scientists to decide on the right tool for their data. The de novo reconstruction of rice cultivars without a genome sequence provides a targeted, cost-efficient approach to identify novel genes responding to stress conditions for any organism. With the metabolomics approach for HNT stress in rice, I identified stress and season-specific metabolites which might be used as molecular markers for crop improvement in the future.
Functional analysis of selected DOF transcription factors in the model plant Arabidopsis thaliana
(2007)
Transcription factors (TFs) are global regulators of gene expression playing essential roles in almost all biological processes, and are therefore of great scientific and biotechnological interest. This project focused on functional characterisation of three DNA-binding-with-one-zinc-finger (DOF) TFs from the genetic model plant Arabidopsis thaliana, namely OBP1, OBP2 and AtDOF4;2. These genes were selected due to severe growth phenotypes conferred upon their constitutive over-expression. To identify biological processes regulated by OBP1, OBP2 and AtDOF4;2 in detail molecular and physiological characterization of transgenic plants with modified levels of OBP1, OBP2 and AtDOF4;2 expression (constitutive and inducible over-expression, RNAi) was performed using both targeted and profiling technologies. Additionally expression patterns of studied TFs and their target genes were analyzed using promoter-GUS lines and publicly available microarray data. Finally selected target genes were confirmed by chromatin immuno-precipitation and electrophoretic-mobility shift assays. This combinatorial approach revealed distinct biological functions of OBP1, OBP2 and AtDOF4;2. Specifically OBP2 controls indole glucosinolate / auxin homeostasis by directly regulating the enzyme at the branch of these pathways; CYP83B1 (Skirycz et al., 2006). Glucosinolates are secondary compounds important for defence against herbivores and pathogens in the plants order Caparales (e.g. Arabidopsis, canola and broccoli) whilst auxin is an essential plant hormone. Hence OBP2 is important for both response to biotic stress and plant growth. Similarly to OBP2 also AtDOF4;2 is involved in the regulation of plant secondary metabolism and affects production of various phenylpropanoid compounds in a tissue and environmental specific manner. It was found that under certain stress conditions AtDOF4;2 negatively regulates flavonoid biosynthetic genes whilst in certain tissues it activates hydroxycinnamic acid production. It was hypothesized that this dual function is most likely related to specific interactions with other proteins; perhaps other TFs (Skirycz et al., 2007). Finally OBP1 regulates both cell proliferation and cell expansion. It was shown that OBP1 controls cell cycle activity by directly targeting the expression of core cell cycle genes (CYCD3;3 and KRP7), other TFs and components of the replication machinery. Evidence for OBP1 mediated activation of cell cycle during embryogenesis and germination will be presented. Additionally and independently on its effects on cell proliferation OBP1 negatively affects cell expansion via reduced expression of cell wall loosening enzymes. Summing up this work provides an important input into our knowledge on DOF TFs function. Future work will concentrate on establishing exact regulatory networks of OBP1, OBP2 and AtDOF4;2 and their possible biotechnological applications.
Sucrose synthase (Susy) is a key enzyme of sucrose metabolism, catalysing the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Therefore, its activity, localization and function have been studied in various plant species. It has been shown that Susy can play a role in supplying energy in companion cells for phloem loading (Fu and Park, 1995), provides substrates for starch synthesis (Zrenner et al., 1995), and supplies UDP-glucose for cell wall synthesis (Haigler et al., 2001). Analysis of the Arabidopsis genome identifies six Susy isoforms. The expression of these isoforms was investigated using promoter-reporter gene constructs (GUS) and real time RT-PCR. Although these isoforms are closely related at the protein level they have radically different spatial and temporal patterns of expression in the plant with no two isoforms showing the same distribution. More than one isoform is expressed in all organs examined. Some of them have high but specific expression in particular organs or developmental stages whilst others are constantly expressed throughout the whole plant and across various stages of development. The in planta function of the six Susy isoforms were explored through analysis of T-DNA insertion mutants and RNAi lines. Plants without the expression of individual isoforms show no differences in growth and development, and are not significantly different from wild type plants in soluble sugars, starch and cellulose contents under all growth conditions investigated. Analysis of T-DNA insertion mutant lacking Sus3 isoform that was exclusively expressed in stomata cells only had a minor influence on guard cell osmoregulation and/or bioenergetics. Although none of the sucrose synthases appear to be essential for normal growth under our standard growth conditions, they may be necessary for growth under stress conditions. Different isoforms of sucrose synthase respond differently to various abiotic stresses. It has been shown that oxygen deprivation up regulates Sus1 and Sus4 and increases total Susy activity. However, the analysis of the plants with reduced expression of both Sus1 and Sus4 revealed no obvious effects on plant performance under oxygen deprivation. Low temperature up regulates Sus1 expression but the loss of this isoform has no effect on the freezing tolerance of non acclimated and cold acclimated plants. These data provide a comprehensive overview of the expression of this gene family which supports some of the previously reported roles for Susy and indicates the involvement of specific isoforms in metabolism and/or signalling.
Each organ of a multicellular organism is unique at the level of its tissues and cells. Furthermore, responses to environmental stimuli or developmental signals occur differentially at the single cell or tissue level. This underlines the necessity of precise investigation of the “building block of life” -the individual cell. Although recently large amount of data concerning different aspects of single cell performance was accumulated, our knowledge about development and differentiation of individual cell within specialized tissue are still far from being complete. To get more insight into processes that occur in certain individual cell during its development and differentiation changes in gene expression during life cycle of A. thaliana leaf hair cell (trichome) were explored in this work. After onset of trichome development this cell changes its cell cycle: it starts endoreduplication (a modified cell cycle in which DNA replication continues in the absence of mitosis and cytokinesis). This makes trichomes a suitable model for studying cell cycle regulation, regulation of cell development and differentiation. Cells of interest were sampled by puncturing them with glass microcapillaries. Each sample contained as few as ten single cells. At first time trichomes in initial stage of trichome development were investigated. To allow their sampling they were specifically labelled by green fluorescent protein (GFP). In total three cell types were explored: pavement cells, trichome initials and mature trichomes. Comparison of gene expression profiles of these cells allowed identification of the genes differentially expressed in subsequent stages of trichome development. Bioinformatic analysis of genes preferentially expressed in trichome initials showed their involvement in hormonal, metal, sulphur response and cell-cycle regulation. Expression pattern of three selected candidate genes, involved in hormonal response and early developmental processes was confirmed by independent method. Effects of mutations in these genes on both trichome and plant development as well as on plant metabolism were analysed. As an outcome of this work novel components in the sophisticated machinery of trichome development and cell cycle progression were identified. These factors could integrate hormone stimuli and network interactions between characterized and as yet unknown members of this machinery. I expect findings presented in this work to enhance and complement our current knowledge about cell cycle progression and trichome development, as well as about performance of the individual cell in general.
Plants are the primary producers of biomass and thereby the basis of all life. Many varieties are cultivated, mainly to produce food, but to an increasing amount as a source of renewable energy. Because of the limited acreage available, further improvements of cultivated species both with respect to yield and composition are inevitable. One approach to further progress in developing improved plant cultivars is a systems biology oriented approach. This work aimed to investigate the primary metabolism of the model plant A.thaliana and its relation to plant growth using quantitative genetics methods. A special focus was set on the characterization of heterosis, the deviation of hybrids from their parental means for certain traits, on a metabolic level. More than 2000 samples of recombinant inbred lines (RILs) and introgression lines (ILs) developed from the two accessions Col-0 and C24 were analyzed for 181 metabolic traces using gas-chromatography/ mass-spectrometry (GC-MS). The observed variance allowed the detection of 157 metabolic quantitative trait loci (mQTL), genetic regions carrying genes, which are relevant for metabolite abundance. By analyzing several hundred test crosses of RILs and ILs it was further possible to identify 385 heterotic metabolic QTL (hmQTL). Within the scope of this work a robust method for large scale GC-MS analyses was developed. A highly significant canonical correlation between biomass and metabolic profiles (r = 0.73) was found. A comparable analysis of the results of the two independent experiments using RILs and ILs showed a large agreement. The confirmation rate for RIL QTL in ILs was 56 % and 23 % for mQTL and hmQTL respectively. Candidate genes from available databases could be identified for 67 % of the mQTL. To validate some of these candidates, eight genes were re-sequenced and in total 23 polymorphisms could be found. In the hybrids, heterosis is small for most metabolites (< 20%). Heterotic QTL gave rise to less candidate genes and a lower overlap between both populations than was determined for mQTL. This hints that regulatory loci and epistatic effects contribute to metabolite heterosis. The data described in this thesis present a rich source for further investigation and annotation of relevant genes and may pave the way towards a better understanding of plant biology on a system level.
Starch is an insoluble polyglucan, comprises of two polymers, namely, the branched α-1,4: α-1,6-D-glucan amylopectin and the almost unbranched α-1,4-D-glucan amylose. The growth of all plants is directly dependent on the accumulation of transitory starch during the daytime when photosynthesis takes place and subsequently starch degradation during the night. Starch phosphorylation takes place by starch-related dikinases called α-glucan, water dikinase (GWD), and phosphoglucan, water dikinase (PWD), and is a very important step in starch degradation. The biochemical mechanisms of phosphorylation of starch are not properly understood. Recent studies have found that there are two starch binding proteins namely, Early Starvation1 (ESV1) and Like Early Starvation1 (LESV), which play an important role in starch metabolism. It has been shown that ESV1 and LESV proteins affect the starch phosphorylation activity of GWD and PWD enzymes, which control the rate of degradation of starch granules. In this thesis, various in vitro assays were performed to identify and understand the mechanism of recombinant proteins; ESV1 and LESV on the starch degradation. The starch degradation was performed by phosphorylation enzymes, GWD and PWD separately. In various enzymatic assays, the influence of the ESV1 and LESV on the actions of GWD and PWD on the surfaces of different native starch granules were analysed. Furthermore, ESV1 and LESV have specifically shown influences on the phosphorylation activities of GWD and PWD on the starch granule surfaces in an antagonistic pattern in such a way that, the GWD mediated phosphorylation were significantly reduced while PWD mediated phosphorylation were significantly increased respectively. In another set of experiments, ISA and BAM hydrolyzing enzymes were used to alter the structure of starch, and then determine the effect of both dikinases mediated phosphorylation in the presence of ESV1 and LESV on the altered starch granules surfaces. In these results, significant decreases in both GWD and PWD mediated phosphorylation were observed in all the treatments containing either ESV1 or LESV proteins only or both ESV1 and LESV. It was also found that LESV preferentially binds to both amylose and amylopectin, while ESV1 binds to highly ordered glucans such as maltodextrins and amylopectin, which are crystalline in structure. Both ESV1 or LESV proteins either individually or in combination have shown influence on the activity of GWD and PWD phosphate incorporation into the starch granules via reduction even though at different percentages depending on the sources of starch, therefore it is difficult to distinguish the specific function between them. The biochemical studies have shown that protein-glucan interaction specifically between ESV1 or LESV or in combination with different species of starch granules has very strong surface binding, or it might be possible that both the proteins not only bind to the surface of the starch granules but also have entered deep inside the glucan structure of the starch granules. However, the results also revealed that ESV1 and LESV did not alter the autophosphorylation of the dikinases. Also, the chain length distribution pattern of the released glucan chains after treatment of starch with ISA enzyme was evaluated with respect to the degree of polymerization (DP) of the different starch granules. Capillary electrophoresis was employed to study the effect of LESV and ESV1 on the chain length distribution. In summary, this study confirms that ESV1 and LESV play an important role in organizing and regulating the starch metabolism process. In the later half, studies were performed to monitor whether the metabolism of carbohydrates and partitioning, contribute to the higher salt tolerance of the facultative halophyte Hordeum marinum when compared to glycophyte Hordeum vulgare. Seedlings with the same size from both species were hydroponically grown at 0, 150, and 300 mM of NaCl for 3 weeks. H. marinum maintained a high relative growth rate, which was found concomitant in higher aptitude plants to maintain efficient shoot tissue hydration and integrity of membrane under salt conditions when compared to H. vulgare. Hence, our data suggested that the change in the starch storage, distribution of soluble sugar concentrations between source and sink organs, and also changes in the level of enzymes involved in the starch metabolism was significant to give insights into the importance of carbohydrate metabolism in barley species with regards to the salt tolerance. Although these results are still in their nascent state, it could be vital for other researchers to formulate future studies. The preliminary results which were studies about the carbohydrate metabolism and partitioning in salt responses in the halophyte H. marinum and the glycophyte H. vulgare revealed that salt tolerance in barley species is not due to osmotic adjustments, but due to other reasons that were not explored in the past studies. However, the activity of DPE2 in H. vulgare was not hampered by the presence of NaCl as observed. While Pho1 and Pho2, activities were highly increased in cultivated barley. These findings could be suggestive of a possible role of these enzymes in the responses of carbohydrate metabolism to salinity. When sea and cultivated barley species were compared, it was discovered that the former had more versatility in carbohydrate metabolism and distribution.