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Integrative studies of plant growth require spatially and temporally resolved information from high-throughput imaging systems. However, analysis and interpretation of conventional two-dimensional images is complicated by the three-dimensional nature of shoot architecture and by changes in leaf position over time, termed hyponasty. To solve this problem, Phytotyping(4D) uses a light-field camera that simultaneously provides a focus image and a depth image, which contains distance information about the object surface. Our automated pipeline segments the focus images, integrates depth information to reconstruct the three-dimensional architecture, and analyses time series to provide information about the relative expansion rate, the timing of leaf appearance, hyponastic movement, and shape for individual leaves and the whole rosette. Phytotyping(4D) was calibrated and validated using discs of known sizes, and plants tilted at various orientations. Information from this analysis was integrated into the pipeline to allow error assessment during routine operation. To illustrate the utility of Phytotyping(4D), we compare diurnal changes in Arabidopsis thaliana wild-type Col-0 and the starchless pgm mutant. Compared to Col-0, pgm showed very low relative expansion rate in the second half of the night, a transiently increased relative expansion rate at the onset of light period, and smaller hyponastic movement including delayed movement after dusk, both at the level of the rosette and individual leaves. Our study introduces light-field camera systems as a tool to accurately measure morphological and growth-related features in plants.
Significance Statement Phytotyping(4D) is a non-invasive and accurate imaging system that combines a 3D light-field camera with an automated pipeline, which provides validated measurements of growth, movement, and other morphological features at the rosette and single-leaf level. In a case study in which we investigated the link between starch and growth, we demonstrated that Phytotyping(4D) is a key step towards bridging the gap between phenotypic observations and the rich genetic and metabolic knowledge.
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.
Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desiccation but also with ability to withstand low temperatures and freezing stress. In order to identify biochemical strategies which contribute to Haberlea's remarkable stress tolerance, the metabolic reconfiguration of H. rhodopensis during low temperature (4 degrees C) and subsequent return to optimal temperatures (21 degrees C) was investigated and compared with that of the stress tolerant Thellungiella halophyla and the stress sensitive Arabidopsis thaliana. Metabolic analysis by GC-MS revealed intrinsic differences in the metabolite levels of the three species even at 21 degrees C. H. rhodopensis had significantly more raffinose, melibiose, trehalose, rhamnose, myo-inositol, sorbitol, galactinol, erythronate, threonate, 2-oxoglutarate, citrate, and glycerol than the other two species. A. thaliana had the highest levels of putrescine and fumarate, while T halophila had much higher levels of several amino acids, including alanine, asparagine, beta-alanine, histidine, isoleucine, phenylalanine, serine, threonine, and valine. In addition, the three species responded differently to the low temperature treatment and the subsequent recovery, especially with regard to the sugar metabolism. Chilling induced accumulation of maltose in H. rhodopensis and raffinose in A. thaliana but the raffinose levels in low temperature exposed Arabidopsis were still much lower than these in unstressed Haberlea. While all species accumulated sucrose during chilling, that accumulation was transient in H. rhodopensis and A. thaliana but sustained in T halophila after the return to optimal temperature. Thus, Haberlea's metabolome appeared primed for chilling stress but the low temperature acclimation induced additional stress-protective mechanisms. A diverse array of sugars, organic acids, and polyols constitute Haberlea's main metabolic defence mechanisms against chilling, while accumulation of amino acids and amino acid derivatives contribute to the low temperature acclimation in Arabidopsis and Thellungiella. Collectively, these results show inherent differences in the metabolomes under the ambient temperature and the strategies to respond to low temperature in the three species.
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.
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.
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.
The major aim of this thesis was to study the effect of nitrate on primary metabolism and in development of the model plant Arabidopsis thaliana. The present work has two separate topics. First, to investigate the GDH family, a small gene family at the interface between nitrogen and carbon metabolisms. Second, to investigate the mechanisms whereby nitrogen is regulating the transition to flowering time in Arabidopsis thaliana. To gain more insights into the regulation of primary metabolism by the functional characterization of the glutamate dehydrogenase (GDH) family, an enzyme putatively involved in the metabolism of amino acids and thus suggested to play different and essential roles in carbon and nitrogen metabolism in plants, knock out mutants and transgenic plants carrying RNA interference construct were generated and characterized. The effect of silencing GDH on carbon and nitrogen metabolisms was investigated, especially the level of carbohydrates and the amino acid pool were further analysed. It has been shown that GDH expression is regulated by light and/or sugar status therefore, phenotypic and metabolic analysis were developed in plants grown at different points of the diurnal rhythm and in response to an extended night period. In addition, we are interested in the effect of nutrient availability in the transition from vegetative growth to flowering and especially in nitrate as a metabolite that triggers widespread and coordinated changes in metabolism and development. Nutrient availability has a dramatic effect on flowering time, with a marked delay of flowering when nitrate is supplied (Stitt, 1999). The use of different mutants and transgenic plants impaired in flowering signalling pathways was crucial to evaluate the impact of different nitrate concentrations on flowering time and to better understand the interaction of nitrate-dependent signals with other main flowering signalling pathways. Plants were grown on glutamine as a constitutive source of nitrogen, and the nitrate supply varied. Low nitrate led to earlier flowering. The response to nitrate is accentuated in short days and in the CONSTANS deficient co2 mutant, whereas long days or overexpression of CONSTANS overrides the nitrate response. These results indicate that nitrates acts downstream of the known flowering signalling pathways for photoperiod, autonomy, vernalization and gibberellic acid. Global analyses of gene expression of two independent flowering systems, a light impaired mutant (co2tt4) and a constitutive over-expresser of the potent repressor of flowering (35S::FLC), were to be investigated under two different concentrations of nitrate in order to identify candidate genes that may be involved in the regulation of flowering time by nitrate.
The regulation of protein function by modulating the surface charge status via sequence-locally enriched phosphorylation sites (P-sites) in so called phosphorylation "hotspots" has gained increased attention in recent years. We set out to identify P-hotspots in the model plant Arabidopsis thaliana. We analyzed the spacing of experimentally detected P-sites within peptide-covered regions along Arabidopsis protein sequences as available from the PhosPhAt database. Confirming earlier reports (Schweiger and Lanial, 2010), we found that, indeed, P-sites tend to cluster and that distributions between serine and threonine P-sites to their respected closest next P-site differ significantly from those for tyrosine P-sites. The ability to predict P-hotspots by applying available computational P-site prediction programs that focus on identifying single P-sites was observed to be severely compromised by the inevitable interference of nearby P-sites. We devised a new approach, named HotSPotter, for the prediction of phosphorylation hotspots. HotSPotter is based primarily on local amino acid compositional preferences rather than sequence position-specific motifs and uses support vector machines as the underlying classification engine. HotSPotter correctly identified experimentally determined phosphorylation hotspots in A. thaliana with high accuracy. Applied to the Arabidopsis proteome, HotSPotter-predicted 13,677 candidate P-hotspots in 9,599 proteins corresponding to 7,847 unique genes. Hotspot containing proteins are involved predominantly in signaling processes confirming the surmised modulating role of hotspots in signaling and interaction events. Our study provides new bioinformatics means to identify phosphorylation hotspots and lays the basis for further investigating novel candidate P-hotspots. All phosphorylation hotspot annotations and predictions have been made available as part of the PhosPhAt database at http://phosphat.mpimp-golm.mpg.de.
Polyadenylation is a critical 3-end processing step during maturation of pre-mRNAs, and the length of the poly(A) tail affects mRNA stability, nuclear export and translation efficiency. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A) polymerase (PAPS) isoforms fulfilling specialized functions, as reflected by their different mutant phenotypes. While PAPS1 affects several processes, such as the immune response, organ growth and male gametophyte development, the roles of PAPS2 and PAPS4 are largely unknown. Here we demonstrate that PAPS2 and PAPS4 promote flowering in a partially redundant manner. The enzymes act antagonistically to PAPS1, which delays the transition to flowering. The opposite flowering-time phenotypes in paps1 and paps2 paps4 mutants are at least partly due to decreased or increased FLC activity, respectively. In contrast to paps2 paps4 mutants, plants with increased PAPS4 activity flower earlier than the wild-type, concomitant with reduced FLC expression. Double mutant analyses suggest that PAPS2 and PAPS4 act independently of the autonomous pathway components FCA, FY and CstF64. The direct polyadenylation targets of the three PAPS isoforms that mediate their effects on flowering time do not include FLC sense mRNA and remain to be identified. Thus, our results uncover a role for canonical PAPS isoforms in flowering-time control, raising the possibility that modulating the balance of the isoform activities could be used to fine tune the transition to flowering. Significance Statement The length of the poly(A) tail affects mRNA stability, nuclear export and translation efficiency. Arabidopsis has three isoforms of nuclear poly(A) polymerase (PAPS): PAPS1 plays a major role in organ growth and plant defence. Here we show that PAPS2 and PAPS4 redundantly promote flowering and act antagonistically to PAPS1, which delays flowering. We suggest that modulating the activity of these isoforms fine-tunes the transition to flowering.
Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana
(2011)
Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (Hips) are expected to exist.
Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants.
The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2-2.5 pH units) is perceived by voltage-gated inward potassium channels (K-in) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K-in channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K-in channel KAT1, mutations in the unique histidine exposed to the solvent (His(267)) do not affect the pH dependency. We demonstrate in the present study that His(267) of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe(266) shifts its pK(a) to undetectable values through a cation-pi interaction. Instead, we show that Glu(240) placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu(240), several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel.
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.
About 2,000 of the more than 27,000 genes of the genetic model plant Arabidopsis thaliana encode for transcription factors (TFs), proteins that bind DNA in the promoter region of their target genes and thus act as transcriptional activators and repressors. Since TFs play essential roles in nearly all biological processes, they are of great scientific and biotechnological interest. This thesis concentrated on the functional characterisation of four selected members of the Arabidopsis DOF-family, namely DOF1.2, DOF3.1, DOF3.5 and DOF5.2, which were selected because of their specific expression pattern in the root tip, a region that comprises the stem cell niche and cells for the perception of environmental stimuli. DOF1.2, DOF3.1 and DOF3.5 are previously uncharacterized members of the Arabidopsis DOF-family, while DOF5.2 has been shown to be involved in the phototrophic flowering response. However, its role in root development has not been described so far. To identify biological processes regulated by the four DOF proteins in detail, molecular and physiological characterization of transgenic plants with modified levels of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 expression (constitutive and inducible over-expression, artificial microRNA) was performed. Additionally expression patterns of the TFs and their target genes were analyzed using promoter-GUS lines and publicly available microarray data. Finally putative protein-protein interaction partners and upstream regulating TFs were identified using the yeast two-hybrid and one-hybrid system. This combinatorial approach revealed distinct biological functions of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 in the context of root development. DOF1.2 and DOF3.5 are specifically and exclusively expressed in the root cap, including the central root cap (columella) and the lateral root cap, organs which are essential to direct oriented root growth. It could be demonstrated that both genes work in the plant hormone auxin signaling pathway and have an impact on distal cell differentiation. Altered levels of gene expression lead to changes in auxin distribution, abnormal cell division patterns and altered root growth orientation. DOF3.1 and DOF5.2 share a specific expression pattern in the organizing centre of the root stem cell niche, called the quiescent centre. Both genes redundantly control cell differentiation in the root´s proximal meristem and unravel a novel transcriptional regulation pathway for genes enriched in the QC cells. Furthermore this work revealed a novel bipartite nuclear localisation signal being present in the protein sequence of the DOF TF family from all sequenced plant species. Summing up, this work provides an important input into our knowledge about the role of DOF TFs during root development. Future work will concentrate on revealing the exact regulatory networks of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 and their possible biotechnological applications.
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.
Characterization of maximal enzyme catalytic rates in central metabolism of Arabidopsis thaliana
(2020)
Availability of plant-specific enzyme kinetic data is scarce, limiting the predictive power of metabolic models and precluding identification of genetic factors of enzyme properties. Enzyme kinetic data are measuredin vitro, often under non-physiological conditions, and conclusions elicited from modeling warrant caution. Here we estimate maximalin vivocatalytic rates for 168 plant enzymes, including photosystems I and II, cytochrome-b6f complex, ATP-citrate synthase, sucrose-phosphate synthase as well as enzymes from amino acid synthesis with previously undocumented enzyme kinetic data in BRENDA. The estimations are obtained by integrating condition-specific quantitative proteomics data, maximal rates of selected enzymes, growth measurements fromArabidopsis thalianarosette with and fluxes through canonical pathways in a constraint-based model of leaf metabolism. In comparison to findings inEscherichia coli, we demonstrate weaker concordance between the plant-specificin vitroandin vivoenzyme catalytic rates due to a low degree of enzyme saturation. This is supported by the finding that concentrations of nicotinamide adenine dinucleotide (phosphate), adenosine triphosphate and uridine triphosphate, calculated based on our maximalin vivocatalytic rates, and available quantitative metabolomics data are below reportedKMvalues and, therefore, indicate undersaturation of respective enzymes. Our findings show that genome-wide profiling of enzyme kinetic properties is feasible in plants, paving the way for understanding resource allocation.
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.
The AtNFXL1 gene encodes a NF-X1 type zinc finger protein required for growth under salt stress
(2006)
The human NF-X1 protein and homologous proteins in eukaryotes represent a class of transcription factors which are characterised. by NF-X1 type zinc finger motifs. The Arabidopsis genome encodes two NF-X1 homologs, which we termed AtNFXL1 and AtNFXL2. Growth and survival was impaired in atnfxl1 knock-out mutants and AtNFXL1-antisense plants under salt stress in comparison to wild-type plants. In contrast, 35S: :AtNFXL1 plants showed higher survival rates. The AtNFXL2 protein potentially plays an antagonistic role. The Arabidopsis NF-X1 type zinc finger proteins likely are part of regulatory mechanisms, which protect major processes such as photosynthesis.
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.
Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.
Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.