Acclimation, priming and memory in the response of Arabidopsis thaliana seedlings to cold stress

Because stress experiences are often recurrent plants have developed strategies to remember a first so-called priming stress to eventually respond more effectively to a second triggering stress. Here, we have studied the impact of discontinuous or sustained cold stress (4 degrees C) on in vitro grown Arabidopsis thaliana seedlings of different age and their ability to get primed and respond differently to a later triggering stress. Cold treatment of 7-d-old seedlings induced the expression of cold response genes but did not cause a significantly enhanced freezing resistance. The competence to increase the freezing resistance in response to cold was associated with the formation of true leaves. Discontinuous exposure to cold only during the night led to a stepwise modest increase in freezing tolerance provided that the intermittent phase at ambient temperature was less than 32h. Seedlings exposed to sustained cold treatment developed a higher freezing tolerance which was further increased in response to a triggering stress during three days after the priming treatment had ended indicating cold memory. Interestingly, in all scenarios the primed state was lost as soon as the freezing tolerance had reached the level of naive plants indicating that an effective memory was associated with an altered physiological state. Known mutants of the cold stress response (cbfs, erf105) and heat stress memory (fgt1) did not show an altered behaviour indicating that their roles do not extend to memory of cold stress in Arabidopsis seedlings

Characterisation of PDX proteins in Arabidopsis thaliana and Ginkgo biloba

Vitamin B6 übt in Form von Pyridoxal 5‘-Phosphat (PLP) als Kofaktor eine entscheidende Funktion für viele essentielle enzymatische Reaktionen in diversen Stoffwechselwegen aus und ist damit unverzichtbar für alle Organismen. Pflanzen, Pilze, Archaeen und einige Bakterien besitzen die Fähigkeit, Vitamin B6 selbst herzustellen. Tierische Organismen, der Mensch eingeschlossen, können dagegen Vitamin B6 nicht neu synthetisieren und sind auf dessen Aufnahme durch die Nahrung angewiesen. In Pflanzen wird das Vitamin durch die Aktivität der Enzyme PDX1 und PDX2 gebildet. In der vorliegenden Arbeit wurde die pflanzliche Vitamin B6 Biosynthese in Arabidopsis thaliana und Ginkgo biloba untersucht und wichtige Informationen über die Funktion der PDX Proteine erlangt. A. thaliana kodiert für drei PDX1 Proteine, AtPDX1.1, AtPDX1.2 und AtPDX1.3, sowie ein PDX2 Protein. Anhand von AtPDX Überexpressionslinien konnte gezeigt werden, daß der Gehalt an Vitamin B6 im Blattgewebe durch die ektopische Expression eines einzelnen AtPDX1 Proteins, AtPDX1.1, signifikant erhöht werden kann. Dagegen führte die ektopische Expression von AtPDX1.3 zu lichtsensiblen Pflanzen mit einem verringerten Vitamin B6 Gehalt und stark veränderten Metabolitwerten. Analysen der AtPDX1 und AtPDX2 Proteine durch Größenausschlußchromatographie und Blue Native Page zeigten, daß die Proteine in hochmolekularen Komplexen in der Pflanzenzelle vorliegen. Die Untersuchungen mit dem BIFC System wiesen zudem nach, daß die AtPDX1 Proteine in homomeren und heteromeren Komplexen in planta vorliegen können. Die Daten geben damit erstmals Hinweise auf die Bildung einer pflanzlichen PLP Synthase. Überraschenderweise konnte das biosynthetisch inaktive AtPDX1.2 Protein ebenfalls in hochmolekularen Komplexen nachgewiesen werden, was eine Bedeutung des Proteins in PLP Synthasekomplexen denkbar macht. Die weiteren Untersuchungen an AtPDX1.2 weisen zudem auf eine Beteiligung des Proteins an Vorgängen in der Embryogenese sowie bei der zellulären Stressantwort hin. Darüber hinaus konnte At5g65840 als AtPDX1 Interaktor bestätigt werden und könnte somit neue Funktionen für AtPDX1 aufzeigen. At5g65840 ist als putative Alkyl-Hydroperoxid Reduktase und Thiol- spezifisches Antioxidant beschrieben und könnte daher zusammen mit AtPDX1 Proteinen eine wichtige Rolle unter Stressbedingungen spielen. Im zweiten Teil der Arbeit wurde zum ersten Mal eine Analyse des Vitamin B6 biosynthetisch aktiven PDX1 Proteins von G. biloba durchgeführt. Die PDX1 Proteine von Ginkgo und Arabidopsis weisen einen hohen Konserviertheitsgrad auf, der durch eine phänotypische Komplementation der Vitamin B6 defizienten Arabidopsis rsr4-1 Mutante bestätigt werden konnte. Zudem zeigen die PDX1 Proteine der beiden Spezies eine hohe Übereinstimmung in ihren Bindungseigenschaften. Die subzelluläre Expression von GbPDX1 in G. biloba weist darüber hinaus auf eine Kopplung der Vitamin B6 Biosyntheseaktivität und dem 4’-O-methylpyridoxine-(Ginkgotoxin)-gehalt, was für das Verständnis der Ginkgotoxinbildung einen wichtigen Schritt darstellt. Insgesamt zeigen die in der vorgelegten Arbeit gesammelten Daten die Wichtigkeit der PDX Proteine für die Vitamin B6 Biosynthese und unterstreichen damit deren Bedeutung für den pflanzlichen Metabolismus.Vitamin B6 is an important compound for all living organisms and serves as pyridoxal 5’-phosphate (PLP) as an enzymatic cofactor for a broad range of biochemical reactions. Plants, fungi, archaea and some bacteria are able to de novo synthesize vitamin B6, whereas animals, including humans, rely on the external supply of the vitamin B6. In plants, the vitamin B6 is synthesized by the action of PDX1 and PDX2 proteins. The present work provides new information about plant PDX proteins and their function in vitamin B6 biosynthesis of Arabidopsis thaliana and Ginkgo biloba. Arabidopsis encodes for three different PDX1 proteins, named AtPDX1.1, AtPDX1.2 and AtPDX1.3 but only for one AtPDX2 protein. Analysis of plants overexpressing the different AtPDX proteins show that ectopic expression of AtPDX1.1 protein is sufficient to significantly increase the vitamin B6 content in Arabidopsis leaves. Interestingly, ectopic expression of specifically AtPDX1.3 caused a light sensitive plant phenotype, as well as reduced vitamin B6 levels and widely changed metabolite profile. Size exclusion chromatography and blue native page analysis revealed that AtPDX1 and AtPDX2 proteins assemble in high molecular weight complexes. By using BIFC technology, in planta assembly of the different AtPDX1 proteins was demonstrated. This allows postulation of homomeric and heteromeric AtPDX1 complexes. The data provide first insights into plant vitamin B6 PLP synthase complex formation. Noteworthy, AtPDX1.2 is also present in the described high molecular order complexes, although it has no vitamin B6 biosynthetic activity in vitro. This suggests a role of AtPDX1.2 in the PLP synthase complex. Further studies on AtPDX1.2 connect the function of the protein with embryo development and cellular stress response. The work on the AtPDX1 binding protein At5g65840 suggests new functions for AtPDX1 proteins. At5g65840 is described as a putative alkyl-hydroperoxide reductase und thiol-specific antioxidant, and it is potentially possible that AtPDX1 and At5g65840 proteins are connected in their function in aboitic stress response. The second part of this thesis focuses on the functional characterization of a GbPDX1 protein. Ginkgo and Arabidopsis PDX1 proteins show a high degree of functional conservation which was demonstrated by yeast-two-hybrid assays and functional complementation of the Arabidopsis PDX1.3 mutant rsr4-1. Furthermore the high expression of GbPDX1 in G. biloba tissues with increased amounts of 4’-O-methylpyridoxine (ginkgotoxin) leads to the assumption, that the formation of ginkgotoxin depends on the rate of vitamin B6 biosynthesis. Overall the work presented in this thesis underscores the significance of PDX proteins for vitamin B6 biosynthesis and whole metabolism in plants

Meeting at the DNA: Specifying Cytokinin Responses through Transcription Factor Complex Formation

Cytokinin is a plant hormone regulating numerous biological processes. Its diverse functions are realized through the expression control of specific target genes. The transcription of the immediate early cytokinin target genes is regulated by type-B response regulator proteins (RRBs), which are transcription factors (TFs) of the Myb family. RRB activity is controlled by phosphorylation and protein degradation. Here, we focus on another step of regulation, the interaction of RRBs among each other or with other TFs to form active or repressive TF complexes. Several examples in Arabidopsis thaliana illustrate that RRBs form homodimers or complexes with other TFs to specify the cytokinin response. This increases the variability of the output response and provides opportunities of crosstalk between the cytokinin signaling pathway and other cellular signaling pathways. We propose that a targeted approach is required to uncover the full extent and impact of RRB interaction with other TFs

Meeting at the DNA: Specifying Cytokinin Responses through Transcription Factor Complex Formation

Cytokinin is a plant hormone regulating numerous biological processes. Its diverse functions are realized through the expression control of specific target genes. The transcription of the immediate early cytokinin target genes is regulated by type-B response regulator proteins (RRBs), which are transcription factors (TFs) of the Myb family. RRB activity is controlled by phosphorylation and protein degradation. Here, we focus on another step of regulation, the interaction of RRBs among each other or with other TFs to form active or repressive TF complexes. Several examples in Arabidopsis thaliana illustrate that RRBs form homodimers or complexes with other TFs to specify the cytokinin response. This increases the variability of the output response and provides opportunities of crosstalk between the cytokinin signaling pathway and other cellular signaling pathways. We propose that a targeted approach is required to uncover the full extent and impact of RRB interaction with other TFs

Vitamin B6: A Long Known Compound of Surprising Complexity

In recent years vitamin B6 has become a focus of research describing the compound’s critical function in cellular metabolism and stress response. For many years the sole function of vitamin B6 was considered to be that of an enzymatic cofactor. However, recently it became clear that it is also a potent antioxidant that effectively quenches reactive oxygen species and is thus of high importance for cellular well-being. In view of the recent findings, the current review takes a look back and summarizes the discovery of vitamin B6 and the elucidation of its structure and biosynthetic pathways. It provides a detailed overview on vitamin B6 both as a cofactor and a protective compound. Besides these general characteristics of the vitamin, the review also outlines the current literature on vitamin B6 derivatives and elaborates on recent findings that provide new insights into transport and catabolism of the compound and on its impact on human health

Complex Assembly and Metabolic Profiling of Arabidopsis thaliana Plants Overexpressing Vitamin B6 Biosynthesis Proteins

In plants, vitamin B6 biosynthesis requires the activity of PDX1 and PDX2 proteins. Arabidopsis thaliana encodes for three PDX1 proteins, named PDX1.1, 1.2, and 1.3, but only one PDX2. Here, we show in planta complex assembly of PDX proteins, based on split-YFP and FPLC assays, and can demonstrate their presence in higher complexes of around 750 kDa. Metabolic profiling of plants ectopically expressing the different PDX proteins indicates a negative influence of PDX1.2 on vitamin B6 biosynthesis and a correlation between aberrant vitamin B6 content, PDX1 gene expression, and light sensitivity specifically for PDX1.3. These findings provide first insights into in planta vitamin B6 synthase complex assembly and new information on how the different PDX proteins affect plant metabolism

Root engineering in maize by increasing cytokinin degradation causes enhanced root growth and leaf mineral enrichment

Key message Root-specific expression of a cytokinin-degrading CKX gene in maize roots causes formation of a larger root system leading to higher element content in shoot organs. The size and architecture of the root system is functionally relevant for the access to water and soil nutrients. A great number of mostly unknown genes are involved in regulating root architecture complicating targeted breeding of plants with a larger root system. Here, we have explored whether root-specific degradation of the hormone cytokinin, which is a negative regulator of root growth, can be used to genetically engineer maize (Zea mays L.) plants with a larger root system. Root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene of Arabidopsis caused the formation of up to 46% more root dry weight while shoot growth of these transgenic lines was similar as in non-transgenic control plants. The concentration of several elements, in particular of those with low soil mobility (K, P, Mo, Zn), was increased in leaves of transgenic lines. In kernels, the changes in concentration of most elements were less pronounced, but the concentrations of Cu, Mn and Zn were significantly increased in at least one of the three independent lines. Our data illustrate the potential of an increased root system as part of efforts towards achieving biofortification. Taken together, this work has shown that root-specific expression of a CKX gene can be used to engineer the root system of maize and alter shoot element composition

Root engineering in maize by increasing cytokinin degradation causes enhanced root growth and leaf mineral enrichment

The size and architecture of the root system is functionally relevant for the access to water and soil nutrients. A great number of mostly unknown genes are involved in regulating root architecture complicating targeted breeding of plants with a larger root system. Here, we have explored whether root-specific degradation of the hormone cytokinin, which is a negative regulator of root growth, can be used to genetically engineer maize (Zea mays L.) plants with a larger root system. Root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene of Arabidopsis caused the formation of up to 46% more root dry weight while shoot growth of these transgenic lines was similar as in non-transgenic control plants. The concentration of several elements, in particular of those with low soil mobility (K, P, Mo, Zn), was increased in leaves of transgenic lines. In kernels, the changes in concentration of most elements were less pronounced, but the concentrations of Cu, Mn and Zn were significantly increased in at least one of the three independent lines. Our data illustrate the potential of an increased root system as part of efforts towards achieving biofortification. Taken together, this work has shown that root-specific expression of a CKX gene can be used to engineer the root system of maize and alter shoot element composition

Analysis of the Arabidopsis rsr4-1/pdx1-3 Mutant Reveals the Critical Function of the PDX1 Protein Family in Metabolism, Development, and Vitamin B6 Biosynthesis[W]

Vitamin B6 represents a highly important group of compounds ubiquitous in all living organisms. It has been demonstrated to alleviate oxidative stress and in its phosphorylated form participates as a cofactor in >100 biochemical reactions. By means of a genetic approach, we have identified a novel mutant, rsr4-1 (for reduced sugar response ), with aberrant root and leaf growth that requires supplementation of vitamin B6 for normal development. Cloning of the mutated gene revealed that rsr4-1 carries a point mutation in a member of the PDX1/SOR1/SNZ (for Pyridoxine biosynthesis protein 1/Singlet oxygen resistant 1/Snooze) family that leads to reduced vitamin B6 content. Consequently, metabolism is broadly altered, mainly affecting amino acid, raffinose, and shikimate contents and trichloroacetic acid cycle constituents. Yeast two-hybrid and pull-down analyses showed that Arabidopsis thaliana PDX1 proteins can form oligomers. Interestingly, the mutant form of PDX1 has severely reduced capability to oligomerize, potentially suggesting that oligomerization is important for function. In summary, our results demonstrate the critical function of the PDX1 protein family for metabolism, whole-plant development, and vitamin B6 biosynthesis in higher plants