Genomic distribution and context dependent functionality of novel WRKY transcription factor binding sites.

BACKGROUND: The WT-boxes NGACTTTN are novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences. Many of them are uncommon WRKY transcription factor (TF) binding sites. RESULTS: To understand their functional relevance, a genomic distribution analysis of the 16 possible WT-boxes and a functional analysis of a WT-box rich promoter was done. The genomic distribution analysis shows an enrichment of specific WT-boxes within 500 bp upstream of all Arabidopsis thaliana genes. Those that harbour a T 5' to the core sequence GACTTT can also be part of the classic WRKY binding site the W-box TTGACT/C. The MAMP-responsive gene ATEP3, a class IV chitinase, harbours seven WT-boxes within its 1000 bp upstream region. In the context of synthetic promoters, the four proximal WT-boxes confer MAMP responsivity while the three WT-boxes further upstream have no effect. Rendering the nucleotides adjacent and in the vicinity of the WT-box core sequence reveals their functional importance for gene expression. A 158 bp long ATEP3 minimal promoter harbouring the two WT-boxes CGACTTTT, confers WT-box-dependent basal and MAMP-responsive reporter gene expression. The ATEP3 gene is a proposed target of WRKY50 and WRKY70. WRKY50 negatively regulates MAMP responsivity of the two WT-boxes CGACTTTT, while WRKY70 activates gene expression in a WT-box dependent manner. Both WRKY factors bind directly to the WT-box CGACTTTT. CONCLUSION: In summary, WT-boxes are enriched in promoter regions and comprise novel and uncommon WRKY binding sites required for basal and MAMP-induced gene expression. WT-boxes not being part of a W-box may be a missing link for WRKY target gene prediction when these genes do not harbour a W-box.

Physiological Importance of Molybdate Transporter Family 1 in Feeding the Molybdenum Cofactor Biosynthesis Pathway in Arabidopsis thaliana.

Molybdate uptake and molybdenum cofactor (Moco) biosynthesis were investigated in detail in the last few decades. The present study critically reviews our present knowledge about eukaryotic molybdate transporters (MOT) and focuses on the model plant Arabidopsis thaliana, complementing it with new experiments, filling missing gaps, and clarifying contradictory results in the literature. Two molybdate transporters, MOT1.1 and MOT1.2, are known in Arabidopsis, but their importance for sufficient molybdate supply to Moco biosynthesis remains unclear. For a better understanding of their physiological functions in molybdate homeostasis, we studied the impact of mot1.1 and mot1.2 knock-out mutants, including a double knock-out on molybdate uptake and Moco-dependent enzyme activity, MOT localisation, and protein-protein interactions. The outcome illustrates different physiological roles for Moco biosynthesis: MOT1.1 is plasma membrane located and its function lies in the efficient absorption of molybdate from soil and its distribution throughout the plant. However, MOT1.1 is not involved in leaf cell imports of molybdate and has no interaction with proteins of the Moco biosynthesis complex. In contrast, the tonoplast-localised transporter MOT1.2 exports molybdate stored in the vacuole and makes it available for re-localisation during senescence. It also supplies the Moco biosynthesis complex with molybdate by direct interaction with molybdenum insertase Cnx1 for controlled and safe sequestering.

Integration of bioinformatics and synthetic promoters leads to the discovery of novel elicitor-responsive cis-regulatory sequences in Arabidopsis.

A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets. Motif similarities were determined with STAMP, classifying the 407 sequence motifs into 37 families. A comparative analysis of these 37 families with the AthaMap, PLACE, and AGRIS databases revealed similarities to known cis-elements but also led to the discovery of cis-sequences not yet implicated in pathogen response. Using a parsley (Petroselinum crispum) protoplast system and a modified reporter gene vector with an internal transformation control, 25 elicitor-responsive cis-sequences from 10 different motif families were identified. Many of the elicitor-responsive cis-sequences also drive reporter gene expression in an Agrobacterium tumefaciens infection assay in Nicotiana benthamiana. This work significantly increases the number of known elicitor-responsive cis-sequences and demonstrates the successful integration of a diverse set of bioinformatic resources combined with synthetic promoter analysis for data mining and functional screening in plant-pathogen interaction.

A novel role for Arabidopsis mitochondrial ABC transporter ATM3 in molybdenum cofactor biosynthesis.

The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to approximately 50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.

Moonlighting Arabidopsis molybdate transporter 2 family and GSH-complex formation facilitate molybdenum homeostasis.

Molybdenum (Mo) as essential micronutrient for plants, acts as active component of molybdenum cofactor (Moco). Core metabolic processes like nitrate assimilation or abscisic-acid biosynthesis rely on Moco-dependent enzymes. Although a family of molybdate transport proteins (MOT1) is known to date in Arabidopsis, molybdate homeostasis remained unclear. Here we report a second family of molybdate transporters (MOT2) playing key roles in molybdate distribution and usage. KO phenotype-analyses, cellular and organ-specific localization, and connection to Moco-biosynthesis enzymes via protein-protein interaction suggest involvement in cellular import of molybdate in leaves and reproductive organs. Furthermore, we detected a glutathione-molybdate complex, which reveals how vacuolar storage is maintained. A putative Golgi S-adenosyl-methionine transport function was reported recently for the MOT2-family. Here, we propose a moonlighting function, since clear evidence of molybdate transport was found in a yeast-system. Our characterization of the MOT2-family and the detection of a glutathione-molybdate complex unveil the plant-wide way of molybdate.

Split-HaloTag imaging assay for sophisticated microscopy of protein-protein interactions in planta.

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP-based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization. Because of their simple protocols, they have become some of the most frequently used methods. However, standard fluorescent proteins present several drawbacks for sophisticated microscopy. With the HaloTag system, these drawbacks can be overcome, as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we have established the Split-HaloTag imaging assay in plants, which is based on the reconstitution of a functional HaloTag protein upon protein-protein interaction and the subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein-protein interaction at cellular structures: the anchoring of the molybdenum cofactor biosynthesis complex to filamentous actin. In addition, a specific interaction was visualized in a more distinctive manner with subdiffractional polarization microscopy, Airyscan, and structured illumination microscopy to provide examples of sophisticated imaging. Split-GFP and Split-HaloTag can complement one another, as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interactions using specific microscopy techniques, such as 3D imaging, single-molecule tracking, and super-resolution microscopy.

Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature.

Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.

Analysis of Microbe-Associated Molecular Pattern-Responsive Synthetic Promoters with the Parsley Protoplast System.

Plants recognize pathogens by microbe-associated molecular patterns (MAMPs) and subsequently induce an immune response. The regulation of gene expression during the immune response depends largely on cis-sequences conserved in promoters of MAMP-responsive genes. These cis-sequences can be analyzed by constructing synthetic promoters linked to a reporter gene and by testing these constructs in transient expression systems. Here, the use of the parsley (Petroselinum crispum) protoplast system for analyzing MAMP-responsive synthetic promoters is described. The synthetic promoter consists of four copies of a potential MAMP-responsive cis-sequence cloned upstream of a minimal promoter and the uidA reporter gene. The reporter plasmid contains a second reporter gene, which is constitutively expressed and hence eliminates the requirement of a second plasmid used as a transformation control. The reporter plasmid is transformed into parsley protoplasts that are elicited by the MAMP Pep25. The MAMP responsiveness is validated by comparing the reporter gene activity from MAMP-treated and untreated cells and by normalizing reporter gene activity using the constitutively expressed reporter gene.

HaloTag: a new versatile reporter gene system in plant cells.

HaloTag Interchangeable Labeling Technology (HaloTag) was originally developed for mammalian cell analysis. In this report, the use of HaloTag is demonstrated in plant cells for the first time. This system allows different fluorescent colours to be used to visualize the localization of the non-fluorescent HaloTag protein within living cells. A vector was constructed which expresses the HaloTag protein under the control of the 35S promoter of cauliflower mosaic virus. The functionality of the HaloTag construct was tested in transient assays by (i) transforming tobacco protoplasts and (ii) using biolistic transformation of intact leaf cells of tobacco and poplar plants. Two to fourteen days after transformation, the plant material was incubated with ligands specific for labelling the HaloTag protein, and fluorescence was visualized by confocal laser scanning microscopy. The results demonstrate that HaloTag technology is a flexible system which generates efficient fluorescence in different types of plant cells. The ligand-specific labelling of HaloTag protein was not hampered by the plant cell wall.

Mature embryo axis-based high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (Hordeum vulgare L.).

A highly reproducible regeneration system through somatic embryogenesis from the excised mature embryos (MEs) of dry seeds of a range of European barley cultivars was developed. By minimizing the germination of plated MEs, primary callus could be obtained with high frequency which permitted efficient embryogenesis and regeneration of a large number of green plants. Different approaches were tested to reduce or prevent normal germination: (i) the use of a well defined balance of maltose and 2,4-D in the induction medium, (ii) soaking of seeds in water containing 2,4-D solution, (iii) direct culture of excised embryonic axes, (iv) longitudinally bisected MEs giving two halves, and (v) complete removal of the elongated main shoot including any roots within a week of culture initiation. Culturing of bisected MEs and whole embryonic axes gave the best responses with respect to large amounts of callus combined with minimal germination. The incorporation of BAP at low levels in the medium was found to be most effective for embryogenesis and the maintenance of long-term morphogenic capacity (more than 11 months up to now). This procedure allows the complete regeneration of plants in 16-20 weeks, from the initial isolation of MEs through all the steps to the development of plants ready to be transferred to the soil. The protocol was first developed for cv. Golden Promise and successfully applied to commercial cultivars. All cultivars tested formed embryogenic callus, with overall rates ranging from 22-55% and an average number of green plants per embryogenic callus from 1.5 to 7.5 across the genotypes.

A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos.

A fast and highly efficient short-term in vitro regeneration system was developed for barley (Hordeum vulgare L.) based on readily available explants. Clumps of multiple shoots and buds suitable for transformation were obtained 9-10 weeks after culture initiation from model and current commercial cultivars. Meristematic shoot segments (MSSs) excised from mature embryo-derived seedlings and subsequently cultured on MS-based medium containing 2 mg/l Picloram and 3 mg/l thidiazuron (TDZ) differentiated up to ten multiple shoots after 3-4 weeks with no or very little callus formation. Sectors of the already multiplied shoot clumps were further multiplied on proliferation-maintenance medium containing 2 mg/l Picloram and 2.5 mg/l TDZ. Biweekly subcultures resulted in a continuous process of multiplication of these highly differentiating green sectors without any loss of morphogenic potential. The differentiated small shoots and shoot buds gave rise to normal shoots on medium with 0.1 mg/l Picloram and 1 mg/l TDZ. After rooting on basal medium with 0.5 mg/l or 1 mg/l IBA the plants were transferred to soil and showed normal growth and fertility compared to the seed-grown plants. All of the genotypes tested formed multiple shoots. The percentage of relative MSS multiplication was 63-83%, and the average number of multiplied shoots per MSS ranged from 16 to 34 among the genotypes after 9-11 weeks.

Anaerobic induction of the maize GapC4 promoter in poplar leaves requires light and high CO2.

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 ( GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula x P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO2 atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO2 and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO2 concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.