570 Biowissenschaften; Biologie
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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.
Plants encounter biotic and abiotic stresses many times during their life cycle and this limits their productivity. Moderate heat stress (HS) primes a plant to survive higher temperatures that are lethal in the naïve state. Once temperature stress subsides, the memory of the priming event is actively retained for several days preparing the plant to better cope with recurring HS. Recently, chromatin regulation at different levels has been implicated in HS memory. Here, we report that the chromatin protein BRUSHY1 (BRU1)/TONSOKU/MGOUN3 plays a role in the HS memory in Arabidopsis thaliana. BRU1 is also involved in transcriptional gene silencing and DNA damage repair. This corresponds with the functions of its mammalian orthologue TONSOKU‐LIKE/NFΚBIL2. During HS memory, BRU1 is required to maintain sustained induction of HS memory‐associated genes, whereas it is dispensable for the acquisition of thermotolerance. In summary, we report that BRU1 is required for HS memory in A. thaliana, and propose a model where BRU1 mediates the epigenetic inheritance of chromatin states across DNA replication and cell division.
Plants can be primed by a stress cue to mount a faster or stronger activation of defense mechanisms upon subsequent stress. A crucial component of such stress priming is the modified reactivation of genes upon recurring stress; however, the underlying mechanisms of this are poorly understood. Here, we report that dozens of Arabidopsis thaliana genes display transcriptional memory, i.e. stronger upregulation after a recurring heat stress, that lasts for at least 3 days. We define a set of transcription factors involved in this memory response and show that the transcriptional memory results in enhanced transcriptional activation within minutes of the onset of a heat stress cue. Further, we show that the transcriptional memory is active in all tissues. It may last for up to a week, and is associated during this time with histone H3 lysine 4 hypermethylation. This transcriptional memory is cis-encoded, as we identify a promoter fragment that confers memory onto a heterologous gene. In summary, heat-induced transcriptional memory is a widespread and sustained response, and our study provides a framework for future mechanistic studies of somatic stress memory in higher plants.
EARLY STARVATION1 specifically affects the phosphorylation action of starch-related dikinases
(2018)
Starch phosphorylation by starch-related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50-kDa starch-binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various invitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, -glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.
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.
Recent advances in gene function prediction rely on ensemble approaches that integrate results from multiple inference methods to produce superior predictions. Yet, these developments remain largely unexplored in plants. We have explored and compared two methods to integrate 10 gene co-function networks for Arabidopsis thaliana and demonstrate how the integration of these networks produces more accurate gene function predictions for a larger fraction of genes with unknown function. These predictions were used to identify genes involved in mitochondrial complex I formation, and for five of them, we confirmed the predictions experimentally. The ensemble predictions are provided as a user-friendly online database, EnsembleNet. The methods presented here demonstrate that ensemble gene function prediction is a powerful method to boost prediction performance, whereas the EnsembleNet database provides a cutting-edge community tool to guide experimentalists.
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.