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Quinoa (Chenopodium quinoa Willd.) is an herbaceous annual crop of the amaranth family (Amaranthaceae). It is increasingly cultivated for its nutritious grains, which are rich in protein and essential amino acids, lipids, and minerals. Quinoa exhibits a high tolerance towards various abiotic stresses including drought and salinity, which supports its agricultural cultivation under climate change conditions. The use of quinoa grains is compromised by anti-nutritional saponins, a terpenoid class of secondary metabolites deposited in the seed coat; their removal before consumption requires extensive washing, an economically and environmentally unfavorable process; or their accumulation can be reduced through breeding. In this study, we analyzed the seed metabolomes, including amino acids, fatty acids, and saponins, from 471 quinoa cultivars, including two related species, by liquid chromatography - mass spectrometry. Additionally, we determined a large number of agronomic traits including biomass, flowering time, and seed yield. The results revealed considerable diversity between genotypes and provide a knowledge base for future breeding or genome editing of quinoa.
The Arabidopsis thaliana NAC transcription factor JUNGBRUNNEN1 (AtJUB1) regulates growth by directly repressing GA3ox1 and DWF4, two key genes involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis, respectively, leading to GA and BR deficiency phenotypes. AtJUB1 also reduces the expression of PIF4, a bHLH transcription factor that positively controls cell elongation, while it stimulates the expression of DELLA genes, which are important repressors of growth. Here, we extend our previous findings by demonstrating that AtJUB1 induces similar GA and BR deficiency phenotypes and changes in gene expression when overexpressed in tomato (Solanum lycopersicum). Importantly, and in accordance with the growth phenotypes observed, AtJUB1 inhibits the expression of growth-supporting genes, namely the tomato orthologs of GA3ox1, DWF4 and PIF4, but activates the expression of DELLA orthologs, by directly binding to their promoters. Overexpression of AtJUB1 in tomato delays fruit ripening, which is accompanied by reduced expression of several ripeningrelated genes, and leads to an increase in the levels of various amino acids (mostly proline, beta-alanine, and phenylalanine), gamma-aminobutyric acid (GABA), and major organic acids including glutamic acid and aspartic acid. The fact that AtJUB1 exerts an inhibitory effect on the GA/BR biosynthesis and PIF4 genes but acts as a direct activator of DELLA genes in both, Arabidopsis and tomato, strongly supports the model that the molecular constituents of the JUNGBRUNNEN1 growth control module are considerably conserved across species.
Leaf senescence is an essential developmental process that involves diverse metabolic changes associated with degradation of macromolecules allowing nutrient recycling and remobilization. In contrast to the significant progress in transcriptomic analysis of leaf senescence, metabolomics analyses have been relatively limited. A broad overview of metabolic changes during leaf senescence including the interactions between various metabolic pathways is required to gain a better understanding of the leaf senescence allowing to link transcriptomics with metabolomics and physiology. In this chapter, we describe how to obtain comprehensive metabolite profiles and how to dissect metabolic shifts during leaf senescence in the model plant Arabidopsis thaliana. Unlike nucleic acid analysis for transcriptomics, a comprehensive metabolite profile can only be achieved by combining a suite of analytic tools. Here, information is provided for measurements of the contents of chlorophyll, soluble proteins, and starch by spectrophotometric methods, ions by ion chromatography, thiols and amino acids by HPLC, primary metabolites by GC/TOF-MS, and secondary metabolites and lipophilic metabolites by LC/ESI-MS. These metabolite profiles provide a rich catalogue of metabolic changes during leaf senescence, which is a helpful database and blueprint to be correlated to future studies such as transcriptome and proteome analyses, forward and reverse genetic studies, or stress-induced senescence studies.
Sucrose nonfermenting related kinase1 (SnRK1) is a conserved energy sensor kinase that regulates cellular adaptation to energy deficit in plants. Activation of SnRK1 leads to the down-regulation of ATP-consuming biosynthetic processes and the stimulation of energy-generating catabolic reactions by transcriptional reprogramming and posttranslational modifications. Although considerable progress has been made during the last years in understanding the SnRK1 signaling pathway, many of its components remain unidentified. Here, we show that the catalytic alpha-subunits KIN10 and KIN11 of the Arabidopsis (Arabidopsis thaliana) SnRK1 complex interact with the STOREKEEPER RELATED1/G-Element Binding Protein (STKR1) inside the plant cell nucleus. Overexpression of STKR1 in transgenic Arabidopsis plants led to reduced growth, a delay in flowering, and strongly attenuated senescence. Metabolite profiling revealed that the transgenic lines exhausted their carbohydrates during the dark period to a greater extent than the wild type and accumulated a range of amino acids. At the global transcriptome level, genes affected by STKR1 overexpression were broadly associated with systemic acquired resistance, and transgenic plants showed enhanced resistance toward a virulent strain of the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis Noco2. We discuss a possible connection of STKR1 function, SnRK1 signaling, and plant immunity.
The desiccation-tolerant plant Haberlea rhodopensis can withstand months of darkness without any visible senescence. Here, we investigated the molecular mechanisms of this adaptation to prolonged (30 d) darkness and subsequent return to light. H. rhodopensis plants remained green and viable throughout the dark treatment. Transcriptomic analysis revealed that darkness regulated several transcription factor (TF) genes. Stress-and autophagy-related TFs such as ERF8, HSFA2b, RD26, TGA1, and WRKY33 were up-regulated, while chloroplast-and flowering-related TFs such as ATH1, COL2, COL4, RL1, and PTAC7 were repressed. PHYTOCHROME INTERACTING FACTOR4, a negative regulator of photomorphogenesis and promoter of senescence, also was down-regulated. In response to darkness, most of the photosynthesis-and photorespiratory-related genes were strongly down-regulated, while genes related to autophagy were up-regulated. This occurred concomitant with the induction of SUCROSE NON-FERMENTING1-RELATED PROTEIN KINASES (SnRK1) signaling pathway genes, which regulate responses to stress-induced starvation and autophagy. Most of the genes associated with chlorophyll catabolism, which are induced by darkness in dark-senescing species, were either unregulated (PHEOPHORBIDE A OXYGENASE, PAO; RED CHLOROPHYLL CATABOLITE REDUCTASE, RCCR) or repressed (STAY GREEN-LIKE, PHEOPHYTINASE, and NON-YELLOW COLORING1). Metabolite profiling revealed increases in the levels of many amino acids in darkness, suggesting increased protein degradation. In darkness, levels of the chloroplastic lipids digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylglycerol, and sulfoquinovosyldiacylglycerol decreased, while those of storage triacylglycerols increased, suggesting degradation of chloroplast membrane lipids and their conversion to triacylglycerols for use as energy and carbon sources. Collectively, these data show a coordinated response to darkness, including repression of photosynthetic, photorespiratory, flowering, and chlorophyll catabolic genes, induction of autophagy and SnRK1 pathways, and metabolic reconfigurations that enable survival under prolonged darkness.
Metabolism is a key determinant of plant growth and modulates plant adaptive responses. Increased metabolic variation due to heterozygosity may be beneficial for highly homozygous plants if their progeny is to respond to sudden changes in the habitat. Here, we investigate the extent to which heterozygosity contributes to the variation in metabolism and size of hybrids of Arabidopsis thaliana whose parents are from a single growth habitat. We created full diallel crosses among seven parents, originating from Southern Germany, and analysed the inheritance patterns in primary and secondary metabolism as well as in rosette size in situ. In comparison to primary metabolites, compounds from secondary metabolism were more variable and showed more pronounced non-additive inheritance patterns which could be attributed to epistasis. In addition, we showed that glucosinolates, among other secondary metabolites, were positively correlated with a proxy for plant size. Therefore, our study demonstrates that heterozygosity in local A. thaliana population generates metabolic variation and may impact several tasks directly linked to metabolism.
Leaf senescence is a key process in plants that culminates in the degradation of cellular constituents and massive reprogramming of metabolism for the recovery of nutrients from aged leaves for their reuse in newly developing sinks. We used molecular-biological and metabolomics approaches to identify NAC transcription factor (TF) RD26 as an important regulator of metabolic reprogramming in Arabidopsis thaliana. RD26 directly activates CHLOROPLAST VESICULATION (CV), encoding a protein crucial for chloroplast protein degradation, concomitant with an enhanced protein loss in RD26 over-expressors during senescence, but a reduced decline of protein in rd26 knockout mutants. RD26 also directly activates LKR/SDH involved in lysine catabolism, and PES1 important for phytol degradation. Metabolic profiling revealed reduced c-aminobutyric acid (GABA) in RD26 overexpressors, accompanied by the induction of respective catabolic genes. Degradation of lysine, phytol and GABA is instrumental for maintaining mitochondrial respiration in carbon-limiting conditions during senescence. RD26 also supports the degradation of starch and the accumulation of mono-and disaccharides during senescence by directly enhancing the expression of AMY1, SFP1 and SWEET15 involved in carbohydrate metabolism and transport. Collectively, during senescence RD26 acts by controlling the expression of genes across the entire spectrum of the cellular degradation hierarchy.
Leaf senescence is an essential physiological process in plants that supports the recycling of nitrogen and other nutrients to support the growth of developing organs, including young leaves, seeds, and fruits. Thus, the regulation of senescence is crucial for evolutionary success in wild populations and for increasing yield in crops. Here, we describe the influence of a NAC transcription factor, SlNAP2 (Solanum lycopersicum NAC-like, activated by Apetala3/Pistillata), that controls both leaf senescence and fruit yield in tomato (S. lycopersicum). SlNAP2 expression increases during age-dependent and dark-induced leaf senescence. We demonstrate that SlNAP2 activates SlSAG113 (S. lycopersicum SENESCENCE-ASSOCIATED GENE113), a homolog of Arabidopsis (Arabidopsis thaliana) SAG113, chlorophyll degradation genes such as SlSGR1 (S. lycopersicum senescence-inducible chloroplast stay-green protein 1) and SlPAO (S. lycopersicum pheide a oxygenase), and other downstream targets by directly binding to their promoters, thereby promoting leaf senescence. Furthermore, SlNAP2 directly controls the expression of genes important for abscisic acid (ABA) biosynthesis, S. lycopersicum 9-cis-epoxycarotenoid dioxygenase 1 (SlNCED1); transport, S. lycopersicum ABC transporter G family member 40 (SlABCG40); and degradation, S. lycopersicum ABA 8′-hydroxylase (SlCYP707A2), indicating that SlNAP2 has a complex role in establishing ABA homeostasis during leaf senescence. Inhibiting SlNAP2 expression in transgenic tomato plants impedes leaf senescence but enhances fruit yield and sugar content likely due to prolonged leaf photosynthesis in aging tomato plants. Our data indicate that SlNAP2 has a central role in controlling leaf senescence and fruit yield in tomato.
Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.
Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.