Filtern
Erscheinungsjahr
Dokumenttyp
- Wissenschaftlicher Artikel (25)
- Dissertation (10)
- Rezension (2)
- Habilitation (1)
- Postprint (1)
Gehört zur Bibliographie
- ja (39)
Schlagworte
- Arabidopsis (39) (entfernen)
Institut
- Institut für Biochemie und Biologie (39) (entfernen)
Nitrogen is an essential macronutrient for plants and nitrogen fertilizers are indispensable for modern agriculture. Unfortunately, we know too little about how plants regulate their use of soil nitrogen, to maximize fertilizers-N use by crops and pastures. This project took a dual approach, involving forward and reverse genetics, to identify N-regulators in plants, which may prove useful in the future to improve nitrogen-use efficiency in agriculture. To identify nitrogen-regulated transcription factor genes in Arabidopsis that may control N-use efficiency we developed a unique resource for qRT-PCR measurements on all Arabidpsis transcription factor genes. Using closely spaced, gene-specific primer pairs and SYBR® Green to monitor amplification of double-stranded DNA, transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products, and 13% of TF transcripts were undetectable in these organs. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001-100 copies per cell. Real-time RT-PCR revealed 26 root-specific and 39 shoot-specific TF genes, most of which have not been identified as organ-specific previously. An enlarged and improved version of the TF qRT-PCR platform contains now primer pairs for 2256 Arabidopsis TF genes, representing 53 gene families and sub-families arrayed on six 384-well plates. Set-up of real-time PCR reactions is now fully robotized. One researcher is able to measure expression of all 2256 TF genes in a single biological sample in a just one working day. The Arabidopsis qRT-PCT platform was successfully used to identify 37 TF genes which transcriptionaly responded at the transcriptional level to N-deprivation or to nitrate per se. Most of these genes have not been characterized previously. Further selection of TF genes based on the responses of selected candidates to other macronutrients and abiotic stresses allowed to distinguish between TFs regulated (i) specifically by nitrogen (29 genes) (ii) regulated by general macronutrient or by salt and osmotic stress (6 genes), and (iii) responding to all major macronutrients and to abiotic stresses. Most of the N-regulated TF genes were also regulated by carbon. Further characterization of sixteen selected TF genes, revealed: (i) lack of transcriptional response to organic nitrogen, (ii) two major types of kinetics of induction by nitrate, (iii) specific responses for the majority of the genes to nitrate but not downstream products of nitrate assimilation. All sixteen TF genes were cloned into binary vectors for constitutive and ethanol inducible over expression, and the first generation of transgenic plants were obtained for almost all of them. Some of the plants constitutively over expressing TF genes under control of the 35S promoter revealed visible phenotypes in T1 generation. Homozygous T-DNA knock out lines were also obtained for many of the candidate TF genes. So far, one knock out line revealed a visible phenotype: retardation of flowering time. A forward genetic approach using an Arabidopsis ATNRT2.1 promoter : Luciferase reporter line, resulted in identification of eleven EMS mutant reporter lines affected in induction of ATNRT2.1 expression by nitrate. These lines could by divided in the following classes according to expression of other genes involved in primary nitrogen and carbon metabolism: (i) lines affected exclusively in nitrate transport, (ii) those affected in nitrate transport, acquisition, but also in glycolysis and oxidative pentose pathway, (iii) mutants affected moderately in nitrate transport, oxidative pentose pathway and glycolysis but not in primary nitrate assimilation. Thus, several different N-regulatory genes may have been mutated in this set of mutants. Map-based cloning has begun to identify the genes affected in these mutants.
Kaliumionen (K<sup>+) sind die am häufigsten vorkommenden anorganischen Kationen in Pflanzen. Gemessen am Trockengewicht kann ihr Anteil bis zu 10% ausmachen. Kaliumionen übernehmen wichtige Funktionen in verschiedenen Prozessen in der Pflanze. So sind sie z.B. essentiell für das Wachstum und für den Stoffwechsel. Viele wichtige Enzyme arbeiten optimal bei einer K<sup>+ Konzentration im Bereich von 100 mM. Aus diesem Grund halten Pflanzenzellen in ihren Kompartimenten, die am Stoffwechsel beteiligt sind, eine kontrollierte Kaliumkonzentration von etwa 100 mM aufrecht. Die Aufnahme von Kaliumionen aus dem Erdreich und deren Transport innerhalb der Pflanze und innerhalb einer Pflanzenzelle wird durch verschiedene Kaliumtransportproteine ermöglicht. Die Aufrechterhaltung einer stabilen K<sup>+ Konzentration ist jedoch nur möglich, wenn die Aktivität dieser Transportproteine einer strikten Kontrolle unterliegt. Die Prozesse, die die Transportproteine regulieren, sind bis heute nur ansatzweise verstanden. Detailliertere Kenntnisse auf diesem Gebiet sind aber von zentraler Bedeutung für das Verständnis der Integration der Transportproteine in das komplexe System des pflanzlichen Organismus. In dieser Habilitationsschrift werden eigene Publikationen zusammenfassend dargestellt, in denen die Untersuchungen verschiedener Regulationsmechanismen pflanzlicher Kaliumkanäle beschrieben werden. Diese Untersuchungen umfassen ein Spektrum aus verschiedenen proteinbiochemischen, biophysikalischen und pflanzenphysiologischen Analysen. Um die Regulationsmechanismen grundlegend zu verstehen, werden zum einen ihre strukturellen und molekularen Besonderheiten untersucht. Zum anderen werden die biophysikalischen und reaktionskinetischen Zusammenhänge der Regulationsmechanismen analysiert. Die gewonnenen Erkenntnisse erlauben eine neue, detailliertere Interpretation der physiologischen Rolle der Kaliumtransportproteine in der Pflanze.
The multidrug and toxic compounds extrusion (MATE) family includes hundreds of functionally uncharacterised proteins from bacteria and all eukaryotic kingdoms except the animal kingdom, that function as drug/toxin::Na<sup>+ or H<sup>+ antiporters. In Arabidopsis thaliana the MATE family comprises 56 members, one of which is NIC2 (Novel Ion Carrier 2). Using heterologous expression systems including Escherichia coli and Saccharomyces cerevisiae, and the homologous expression system of Arabidopsis thaliana, the functional characterisation of NIC2 was performed. It has been demonstrated that NIC2 confers resistance of E. coli towards the chemically diverse compounds such as tetraethylammonium chloride (TEACl), tetramethylammonium chloride (TMACl) and a toxic analogue of indole-3-acetic acid, 5-fluoro-indole-acetic acid (F-IAA). Therefore, NIC2 may be able to transport a broad range of drug and toxic compounds. In wild-type yeast the expression of NIC2 increased the tolerance towards lithium and sodium, but not towards potassium and calcium. In A. thaliana, the overexpression of NIC2 led to strong phenotypic changes. Under normal growth condtions overexpression caused an extremely bushy phenotype with no apical dominance but an enhanced number of lateral flowering shoots. The amount of rossette leaves and flowers with accompanying siliques were also much higher than in wild-type plants and the senescence occurred earlier in the transgenic plants. In contrast, RNA interference (RNAi) used to silence NIC2 expression, induced early flower stalk development and flowering compared with wild-type plants. In additon, the main flower stalks were not able to grow vertically, but instead had a strong tendency to bend towards the ground. While NIC2 RNAi seedlings produced many lateral roots outgrowing from the primary root and the root-shoot junction, NIC2 overexpression seedlings displayed longer primary roots that were characterised by a 2 to 4 h delay in the gravitropic response. In addition, these lines exhibited an enhanced resistance to exogenously applied auxins, i.e. indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) when compared with the wild-type roots. Based on these results, it is suggested that the NIC2 overexpression and NIC2 RNAi phenotypes were due to decreased or increased levels of auxin, respectively. The ProNIC2:GUS fusion gene revealed that NIC2 is expressed in the stele of the elongation zone, in the lateral root cap, in new lateral root primordia, and in pericycle cells of the root system. In the vascular tissue of rosette leaves and inflorescence stems, the expression was observed in the xylem parenchyma cells, while in siliques it was also in vascular tissue, but as well in the dehiscence and abscission zones. The organ- and tissue-specific expression sites of NIC2 correlate with the sites of auxin action in mature Arabidopsis plants. Further experiments using ProNIC2:GUS indicated that NIC2 is an auxin-inducible gene. Additionally, during the gravitropic response when an endogenous auxin gradient across the root tip forms, the GUS activity pattern of the ProNIC2:GUS fusion gene markedly changed at the upper side of the root tip, while at the lower side stayed unchanged. Finally, at the subcellular level NIC2-GFP fusion protein localised in the peroxisomes of Nicotana tabacum BY2 protoplasts. Considering the experimental results, it is proposed that the hypothetical function of NIC2 is the efflux transport which takes part in the auxin homeostasis in plant tissues probably by removing auxin conjugates from the cytoplasm into peroxisomes.
In silico identification of genes regulated by abscisic acid in Arabidopsis thaliana (L.) Heynh.
(2005)
Abscisic acid (ABA) is a major plant hormone that plays an important role during plant growth and development. During vegetative growth ABA mediates (in part) responses to various environmental stresses such as cold, drought and high salinity. The response triggered by ABA includes changes in the transcript level of genes involved in stress tolerance. The aim of this project was the In silico identification of genes putatively regulated by ABA in A. thaliana. In silico predictions were combined with experimental data in order to evaluate the reliability of computational predictions. Taking advantage of the genome sequence of A. thaliana publicly available since 2000, 1 kb upstream sequences were screened for combinations of cis-elements known to be involved in the regulation of ABA-responsive genes. It was found that around 10 to 20 percent of the genes of A. thaliana might be regulated by ABA. Further analyses of the predictions revealed that certain combinations of cis-elements that confer ABA-responsiveness were significantly over-represented compared with results in random sequences and with random expectations. In addition, it was observed that other combinations that confer ABA-responsiveness in monocotyledonous species might not be functional in A. thaliana. It is proposed that ABA-responsive genes in A. thaliana show pairs of ABRE (abscisic acid responsive element) with MYB binding sites, DRE (dehydration responsive element) or with itself. The analysis of the distances between pairs of cis-elements suggested that pairs of ABREs are bound by homodimers of ABRE binding proteins. In contrast, pairs between MYB binding sites and ABRE, or DRE and ABRE showed a distance between cis-elements that suggested that the binding proteins interact through protein complexes and not directly. The comparison of computational predictions with experimental data confirmed that the regulatory mechanisms leading to the induction or repression of genes by ABA is very incompletely understood. It became evident that besides the cis-elements proposed in this study to be present in ABA-responsive genes, other known and unknown cis-elements might play an important role in the transcriptional regulation of ABA-responsive genes. For example, auxin-related cis elements, or the cis-elements recognized by the NAM-family of transcription factors (Non-Apical meristem). This work documents the use of computational and experimental approaches to analyse possible interactions between cis-elements involved in the regulation of ABA-responsive genes. The computational predictions allowed the distinction between putatively relevant combinations of cis-elements from irrelevant combinations of cis-elements in ABA-responsive genes. The comparison with experimental data allowed to identify certain cis-elements that have not been previously associated to the ABA-mediated transcriptional regulation, but that might be present in ABA-responsive genes (e.g. auxin responsive elements). Moreover, the efforts to unravel the gene regulatory network associated with the ABA-signalling pathway revealed that NAM-transcription factors and their corresponding binding sequences are important components of this network.
‘Heterosis’ is a term used in genetics and breeding referring to hybrid vigour or the superiority of hybrids over their parents in terms of traits such as size, growth rate, biomass, fertility, yield, nutrient content, disease resistance or tolerance to abiotic and abiotic stress. Parental plants which are two different inbred (pure) lines that have desired traits are crossed to obtain hybrids. Maximum heterosis is observed in the first generation (F1) of crosses. Heterosis has been utilised in plant and animal breeding programs for at least 90 years: by the end of the 21st century, 65% of worldwide maize production was hybrid-based. Generally, it is believed that an understanding of the molecular basis of heterosis will allow the creation of new superior genotypes which could either be used directly as F1 hybrids or form the basis for the future breeding selection programmes. Two selected accessions of a research model plant Arabidopsis thaliana (thale cress) were crossed to obtain hybrids. These typically exhibited a 60-80% increase of biomass when compared to the average weight of both parents. This PhD project focused on investigating the role of selected regulatory genes given their potentially key involvement in heterosis. In the first part of the project, the most appropriate developmental stage for this heterosis study was determined by metabolite level measurements and growth observations in parents and hybrids. At the selected stage, around 60 candidate regulatory genes (i.e. differentially expressed in hybrids when compared to parents) were identified. Of these, the majority were transcription factors, genes that coordinate the expression of other genes. Subsequent expression analyses of the candidate genes in biomass-heterotic hybrids of other Arabidopsis accessions revealed a differential expression in a gene subset, highlighting their relevance for heterosis. Moreover, a fraction of the candidate regulatory genes were found within DNA regions closely linked to the genes that underlie the biomass or growth heterosis. Additional analyses to validate the role of selected candidate regulatory genes in heterosis appeared insufficient to establish their role in heterosis. This uncovered a need for using novel approaches as discussed in the thesis. Taken together, the work provided an insight into studies on the molecular mechanisms underlying heterosis. Although studies on heterosis date back to more than one hundred years, this project as many others revealed that more investigations will be needed to uncover this phenomenon.
In a very simplified view, the plant leaf growth can be reduced to two processes, cell division and cell expansion, accompanied by expansion of their surrounding cell walls. The vacuole, as being the largest compartment of the plant cell, plays a major role in controlling the water balance of the plant. This is achieved by regulating the osmotic pressure, through import and export of solutes over the vacuolar membrane (the tonoplast) and by controlling the water channels, the aquaporins. Together with the control of cell wall relaxation, vacuolar osmotic pressure regulation is thought to play an important role in cell expansion, directly by providing cell volume and indirectly by providing ion and pH homestasis for the cytosoplasm. In this thesis the role of tonoplast protein coding genes in cell expansion in the model plant Arabidopsis thaliana is studied and genes which play a putative role in growth are identified. Since there is, to date, no clearly identified protein localization signal for the tonoplast, there is no possibility to perform genome-wide prediction of proteins localized to this compartment. Thus, a series of recent proteomic studies of the tonoplast were used to compile a list of cross-membrane tonoplast protein coding genes (117 genes), and other growth-related genes from notably the growth regulating factor (GRF) and expansin families were included (26 genes). For these genes a platform for high-throughput reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) was developed by selecting specific primer pairs. To this end, a software tool (called QuantPrime, see http://www.quantprime.de) was developed that automatically designs such primers and tests their specificity in silico against whole transcriptomes and genomes, to avoid cross-hybridizations causing unspecific amplification. The RT-qPCR platform was used in an expression study in order to identify candidate growth related genes. Here, a growth-associative spatio-temporal leaf sampling strategy was used, targeting growing regions at high expansion developmental stages and comparing them to samples taken from non-expanding regions or stages of low expansion. Candidate growth related genes were identified after applying a template-based scoring analysis on the expression data, ranking the genes according to their association with leaf expansion. To analyze the functional involvement of these genes in leaf growth on a macroscopic scale, knockout mutants of the candidate growth related genes were screened for growth phenotypes. To this end, a system for non-invasive automated leaf growth phenotyping was established, based on a commercially available image capture and analysis system. A software package was developed for detailed developmental stage annotation of the images captured with the system, and an analysis pipeline was constructed for automated data pre-processing and statistical testing, including modeling and graph generation, for various growth-related phenotypes. Using this system, 24 knockout mutant lines were analyzed, and significant growth phenotypes were found for five different genes.
Plant growth and survival depend on photosynthesis in the leaves. This involves the uptake of carbon dioxide from the atmosphere and the simultaneous capture of light energy to produce organic molecules, which enter metabolism and are converted to many other compounds which then serve as building blocks for biomass growth. Leaves are organs specialised for photosynthetic carbon dioxide fixation. The function of leaves involves many trade-offs which must be optimised in order to achieve effective use of resources and maximum photosynthesis. It is known that the morphology of leaves adjusts to the growth environment of plants and this is important for optimising their function for photosynthesis. However, it is unclear how this adjustment is regulated. The general aim of the work presented in this thesis is to understand how leaf growth and morphology are regulated in the model species Arabidopsis thaliana. Special attention was dedicated to the possibility that there might be internal metabolic signals within the plant which affect the growth and development of leaves. In order to investigate this question, leaf growth and development must be considered beyond the level of the single organ and in the context of the whole plant because leaves do not grow autonomously but depend on resources and regulatory influences delivered by the rest of the plant. Due to the complexity of this question, three complementary approaches were taken. In the first and most specific approach it was asked whether a proposed down-stream component of sucrose signalling, trehalose-6-phosphate (Tre-6-P), might influence leaf development and growth. To investigate this question, transgenic Arabidopsis lines with perturbed levels of Tre-6-P were generated using the constitutive 35S promoter to express bacterial enzymes involved in trehalose metabolism. These experiments also led to an unanticipated project concerning a possible role for Tre-6-P in stomatal function, which is another very important function in leaves. In a second and more general approach it was investigated whether changes in sugar levels in plants affect the morphogenesis of leaves in response to light. For this, a series of metabolic mutants impaired in central metabolism were grown in one light environment and their leaf morphology was analysed. In a third and even more general approach the natural variation in leaf and rosette morphological traits was investigated in a panel of wild Arabidopsis accessions with the aim of understanding how leaf morphology affects leaf function and whole plant growth and how different traits relate to each other. The analysis included measurements of leaf morphological traits as well as the number of leaves in the plant to put leaf morphology in a whole plant context. The variance in plant growth could not be explained by variation in photosynthetic rates and only to a small degree by variation in rates of dark respiration. There were four key axes of variation in rosette and leaf morphology – leaf area growth, leaf thickness, cell expansion and leaf number. These four processes were integrated in the context of whole plant growth by models that employed a multiple linear regression approach. This then led to a theoretical approach in which a simple allometric mathematical model was constructed, linking leaf number, leaf size and plant growth rate together in a whole plant context in Arabidopsis.
Large-scale co-expression approach to dissect secondary cell wall formation across plant species
(2011)
Plant cell walls are complex composites largely consisting of carbohydrate-based polymers, and are generally divided into primary and secondary walls based on content and characteristics. Cellulose microfibrils constitute a major component of both primary and secondary cell walls and are synthesized at the plasma membrane by cellulose synthase (CESA) complexes. Several studies in Arabidopsis have demonstrated the power of co-expression analyses to identify new genes associated with secondary wall cellulose biosynthesis. However, across-species comparative co-expression analyses remain largely unexplored. Here, we compared co-expressed gene vicinity networks of primary and secondary wall CESAsin Arabidopsis, barley, rice, poplar, soybean, Medicago, and wheat, and identified gene families that are consistently co-regulated with cellulose biosynthesis. In addition to the expected polysaccharide acting enzymes, we also found many gene families associated with cytoskeleton, signaling, transcriptional regulation, oxidation, and protein degradation. Based on these analyses, we selected and biochemically analyzed T-DNA insertion lines corresponding to approximately twenty genes from gene families that re-occur in the co-expressed gene vicinity networks of secondary wall CESAs across the seven species. We developed a statistical pipeline using principal component analysis and optimal clustering based on silhouette width to analyze sugar profiles. One of the mutants, corresponding to a pinoresinol reductase gene, displayed disturbed xylem morphology and held lower levels of lignin molecules. We propose that this type of large-scale co-expression approach, coupled with statistical analysis of the cell wall contents, will be useful to facilitate rapid knowledge transfer across plant species.
The permeation pore of K+ channels is formed by four copies of the pore domain. AtKCO3 is the only putative voltage-independent K+ channel subunit of Arabidopsis thaliana with a single pore domain. KCO3-like proteins recently emerged in evolution and, to date, have been found only in the genus Arabidopsis (A. thaliana and A. lyrata). We show that the absence of KCO3 does not cause marked changes in growth under various conditions. Only under osmotic stress we observed reduced root growth of the kco3-1 null-allele line. This phenotype was complemented by expressing a KCO3 mutant with an inactive pore, indicating that the function of KCO3 under osmotic stress does not depend on its direct ability to transport ions. Constitutively overexpressed AtKCO3 or AtKCO3::G FP are efficiently sorted to the tonoplast indicating that the protein is approved by the endoplasmic reticulum quality control. However, vacuoles isolated from transgenic plants do not have significant alterations in current density. Consistently, both AtKCO3 and AtKCO3::GFP are detected as homodimers upon velocity gradient centrifugation, an assembly state that would not allow for activity. We conclude that if AtKCO3 ever functions as a K+ channel, active tetramers are held by particularly weak interactions, are formed only in unknown specific conditions and may require partner proteins.
Membranes of eukaryotic cells contain high lipid-order sterol-rich domains that are thought to mediate temporal and spatial organization of cellular processes. Sterols are crucial for execution of cytokinesis, the last stage of cell division, in diverse eukaryotes. The cell plate of higher-plant cells is the membrane structure that separates daughter cells during somatic cytokinesis. Cell-plate formation in Arabidopsis relies on sterol- and DYNAMIN-RELATED PROTEIN1A (DRP1A)-dependent endocytosis. However, functional relationships between lipid membrane order or lipid packing and endocytic machinery components during eukaryotic cytokinesis have not been elucidated. Using ratiometric live imaging of lipid order-sensitive fluorescent probes, we show that the cell plate of Arabidopsis thaliana represents a dynamic, high lipid-order membrane domain. The cell-plate lipid order was found to be sensitive to pharmacological and genetic alterations of sterol composition. Sterols co-localize with DRP1A at the cell plate, and DRP1A accumulates in detergent-resistant membrane fractions. Modifications of sterol concentration or composition reduce cell-plate membrane order and affect DRP1A localization. Strikingly, DRP1A function itself is essential for high lipid order at the cell plate. Our findings provide evidence that the cell plate represents a high lipid-order domain, and pave the way to explore potential feedback between lipid order and function of dynamin-related proteins during cytokinesis.
Background: The identification of brassinosteroid (BR) deficient and BR insensitive mutants provided conclusive evidence that BR is a potent growth-promoting phytohormone. Arabidopsis mutants are characterized by a compact rosette structure, decreased plant height and reduced root system, delayed development, and reduced fertility. Cell expansion, cell division, and multiple developmental processes depend on BR. The molecular and physiological basis of BR action is diverse. The BR signalling pathway controls the activity of transcription factors, and numerous BR responsive genes have been identified. The analysis of dwarf mutants, however, may to some extent reveal phenotypic changes that are an effect of the altered morphology and physiology. This restriction holds particularly true for the analysis of established organs such as rosette leaves. Results: In this study, the mode of BR action was analysed in established leaves by means of two approaches. First, an inhibitor of BR biosynthesis (brassinazole) was applied to 21-day-old wild-type plants. Secondly, BR complementation of BR deficient plants, namely CPD (constitutive photomorphogenic dwarf)-antisense and cbb1 (cabbage1) mutant plants was stopped after 21 days. BR action in established leaves is associated with stimulated cell expansion, an increase in leaf index, starch accumulation, enhanced CO2 release by the tricarboxylic acid cycle, and increased biomass production. Cell number and protein content were barely affected. Conclusion: Previous analysis of BR promoted growth focused on genomic effects. However, the link between growth and changes in gene expression patterns barely provided clues to the physiological and metabolic basis of growth. Our study analysed comprehensive metabolic data sets of leaves with altered BR levels. The data suggest that BR promoted growth may depend on the increased provision and use of carbohydrates and energy. BR may stimulate both anabolic and catabolic pathways.
Leaf senescence is an active process with a pivotal impact on plant productivity. It results from extensive signalling cross-talk coordinating environmental factors with intrinsic age-related mechanisms. Although many studies have shown that leaf senescence is affected by a range of external parameters, knowledge about the regulatory systems that govern the interplay between developmental programmes and environmental stress is still vague. Salinity is one of the most important environmental stresses that promote leaf senescence and thus affect crop yield. Improving salt tolerance by avoiding or delaying senescence under stress will therefore play an important role in maintaining high agricultural productivity. Experimental evidence suggests that hydrogen peroxide (H2O2) functions as a common signalling molecule in both developmental and salt-induced leaf senescence. In this study, microarray-based gene expression profiling on Arabidopsis thaliana plants subjected to long-term salinity stress to induce leaf senescence was performed, together with co-expression network analysis for H2O2-responsive genes that are mutually up-regulated by salt induced-and developmental leaf senescence. Promoter analysis of tightly co-expressed genes led to the identification of seven cis-regulatory motifs, three of which were known previously, namely CACGTGT and AAGTCAA, which are associated with reactive oxygen species (ROS)-responsive genes, and CCGCGT, described as a stress-responsive regulatory motif, while the others, namely ACGCGGT, AGCMGNC, GMCACGT, and TCSTYGACG were not characterized previously. These motifs are proposed to be novel elements involved in the H2O2-mediated control of gene expression during salinity stress-triggered and developmental senescence, acting through upstream transcription factors that bind to these sites.
Leaf senescence is a developmentally controlled process, which is additionally modulated by a number of adverse environmental conditions. Nitrogen shortage is a well-known trigger of precocious senescence in many plant species including crops, generally limiting biomass and seed yield. However, leaf senescence induced by nitrogen starvation may be reversed when nitrogen is resupplied at the onset of senescence. Here, the transcriptomic, hormonal, and global metabolic rearrangements occurring during nitrogen resupply-induced reversal of senescence in Arabidopsis thaliana were analysed. The changes induced by senescence were essentially in keeping with those previously described; however, these could, by and large, be reversed. The data thus indicate that plants undergoing senescence retain the capacity to sense and respond to the availability of nitrogen nutrition. The combined data are discussed in the context of the reversibility of the senescence programme and the evolutionary benefit afforded thereby. Future prospects for understanding and manipulating this process in both Arabidopsis and crop plants are postulated.
Polyadenylation is a decisive 3’ end processing step during the maturation of pre-mRNAs. The length of the poly(A) tail has an impact on mRNA stability, localization and translatability. Accordingly, many eukaryotic organisms encode several copies of canonical poly(A) polymerases (cPAPs). The disruption of cPAPs in mammals results in lethality. In plants, reduced cPAP activity is non-lethal. Arabidopsis encodes three nuclear cPAPs, PAPS1, PAPS2 and PAPS4, which are constitutively expressed throughout the plant. Recently, the detailed analysis of Arabidopsis paps1 mutants revealed a subset of genes that is preferentially polyadenylated by the cPAP isoform PAPS1 (Vi et al. 2013). Thus, the specialization of cPAPs might allow the regulation of different sets of genes in order to optimally face developmental or environmental challenges.
To gain insights into the cPAP-based gene regulation in plants, the phenotypes of Arabidopsis cPAPs mutants under different conditions are characterized in detail in the following work. An involvement of all three cPAPs in flowering time regulation and stress response regulation is shown. While paps1 knockdown mutants flower early, paps4 and paps2 paps4 knockout mutants exhibit a moderate late-flowering phenotype. PAPS1 promotes the expression of the major flowering inhibitor FLC, supposedly by specific polyadenylation of an FLC activator. PAPS2 and PAPS4 exhibit partially overlapping functions and ensure timely flowering by repressing FLC and at least one other unidentified flowering inhibitor. The latter two cPAPs act in a novel regulatory pathway downstream of the autonomous pathway component FCA and act independently from the polyadenylation factors and flowering time regulators CstF64 and FY. Moreover, PAPS1 and PAPS2/PAPS4 are implicated in different stress response pathways in Arabidopsis. Reduced activity of the poly(A) polymerase PAPS1 results in enhanced resistance to osmotic and oxidative stress. Simultaneously, paps1 mutants are cold-sensitive. In contrast, PAPS2/PAPS4 are not involved in the regulation of osmotic or cold stress, but paps2 paps4 loss-of-function mutants exhibit enhanced sensitivity to oxidative stress provoked in the chloroplast. Thus, both PAPS1 and PAPS2/PAPS4 are required to maintain a balanced redox state in plants. PAPS1 seems to fulfil this function in concert with CPSF30, a polyadenylation factor that regulates alternative polyadenylation and tolerance to oxidative stress.
The individual paps mutant phenotypes and the cPAP-specific genetic interactions support the model of cPAP-dependent polyadenylation of selected mRNAs. The high similarity of the polyadenylation machineries in yeast, mammals and plants suggests that similar regulatory mechanisms might be present in other organism groups. The cPAP-dependent developmental and physiological pathways identified in this work allow the design of targeted experiments to better understand the ecological and molecular context underlying cPAP-specialization.
In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic alpha subunit associates with a regulatory beta subunit and an activating gamma subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1 alpha subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.
The coordination of cell polarity within the plane of the tissue layer (planar polarity) is crucial for the development of diverse multicellular organisms. Small Rac/Rho-family GTPases and the actin cytoskeleton contribute to planar polarity formation at sites of polarity establishment in animals and plants. Yet, upstream pathways coordinating planar polarity differ strikingly between kingdoms. In the root of Arabidopsis thaliana, a concentration gradient of the phytohormone auxin coordinates polar recruitment of Rho-of-plant (ROP) to sites of polar epidermal hair initiation. However, little is known about cytoskeletal components and interactions that contribute to this planar polarity or about their relation to the patterning machinery. Here, we show that ACTIN7 (ACT7) represents a main actin isoform required for planar polarity of root hair positioning, interacting with the negative modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2). ACT7, AIP1-2 and their genetic interaction are required for coordinated planar polarity of ROP downstream of ethylene signalling. Strikingly, AIP1-2 displays hair cell file-enriched expression, restricted by WEREWOLF (WER)-dependent patterning and modified by ethylene and auxin action. Hence, our findings reveal AIP1-2, expressed under control of the WER-dependent patterning machinery and the ethylene signalling pathway, as a modulator of actin-mediated planar polarity.
The coordination of cell polarity within the plane of the tissue layer (planar polarity) is crucial for the development of diverse multicellular organisms. Small Rac/Rho-family GTPases and the actin cytoskeleton contribute to planar polarity formation at sites of polarity establishment in animals and plants. Yet, upstream pathways coordinating planar polarity differ strikingly between kingdoms. In the root of Arabidopsis thaliana, a concentration gradient of the phytohormone auxin coordinates polar recruitment of Rho-of-plant (ROP) to sites of polar epidermal hair initiation. However, little is known about cytoskeletal components and interactions that contribute to this planar polarity or about their relation to the patterning machinery. Here, we show that ACTIN7 (ACT7) represents a main actin isoform required for planar polarity of root hair positioning, interacting with the negative modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2). ACT7, AIP1-2 and their genetic interaction are required for coordinated planar polarity of ROP downstream of ethylene signalling. Strikingly, AIP1-2 displays hair cell file-enriched expression, restricted by WEREWOLF (WER)-dependent patterning and modified by ethylene and auxin action. Hence, our findings reveal AIP1-2, expressed under control of the WER-dependent patterning machinery and the ethylene signalling pathway, as a modulator of actin-mediated planar polarity.
Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5-untranslated region (5-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress.
The study illustrates the response of different Arabidopsis thaliana leaf cells and tissues to oxidative stress at the translational level, an aspect of reactive oxygen species (ROS) biology that has been little studied in the past. Our data reveal insights into how translational regulation of ROS-responsive genes is fine-tuned at the cellular level, a phenomenon contributing to the integrated physiological response of leaves to stresses involving changes in ROS levels.
Responses to pathogens, including host transcriptional reprogramming, require partially antagonistic signalling pathways dependent on the phytohormones salicylic (SA) and jasmonic (JA) acids. However, upstream factors modulating the interplay of these pathways are not well characterized. Here, we identify the transcription factor ANAC032 from Arabidopsis thaliana as one such regulator in response to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst). ANAC032 directly represses MYC2 activation upon Pst attack, resulting in blockage of coronatine-mediated stomatal reopening which restricts entry of bacteria into plant tissue. Furthermore, ANAC032 activates SA signalling by repressing NIMIN1, a key negative regulator of SA-dependent defence. Finally, ANAC032 reduces expression of JA-responsive genes, including PDF1.2A. Thus, ANAC032 enhances resistance to Pst by generating an orchestrated transcriptional output towards key SA- and JA-signalling genes coordinated through direct binding of ANAC032 to the MYC2, NIMIN1 and PDF1.2A promoters.
Phytohormones act in concert to coordinate plant growth and the response to environmental cues. Gibberellins (GAs) are growth-promoting hormones that recently emerged as modulators of plant immune signaling. By regulating the stability of DELLA proteins, GAs intersect with the signaling pathways of the classical primary defense hormones, salicylic acid (SA) and jasmonic acid (JA), thereby altering the final outcome of the immune response. DELLA proteins confer resistance to necrotrophic pathogens by potentiating JA signaling and raise the susceptibility to biotrophic pathogens by attenuating the SA pathway. Here, we show that JUB1, a core element of the GA - brassinosteroid (BR) - DELLA regulatory module, functions as a negative regulator of defense responses against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and mediates the crosstalk between growth and immunity.