The Minus-End-Directed Kinesin OsDLK Shuttles to the Nucleus and Modulates the Expression of Cold-Box Factor 4.

The transition to terrestrial plants was accompanied by a progressive loss of microtubule minus-end-directed dynein motors. Instead, the minus-end-directed class-XIV kinesins expanded considerably, likely related to novel functions. One of these motors, OsDLK (Dual Localisation Kinesin from rice), decorates cortical microtubules but moves into the nucleus in response to cold stress. This analysis of loss-of-function mutants in rice indicates that OsDLK participates in cell elongation during development. Since OsDLK harbours both a nuclear localisation signal and a putative leucin zipper, we asked whether the cold-induced import of OsDLK into the nucleus might correlate with specific DNA binding. Conducting a DPI-ELISA screen with recombinant OsDLKT (lacking the motor domain), we identified the Opaque2 motif as the most promising candidate. This motif is present in the promoter of NtAvr9/Cf9, the tobacco homologue of Cold-Box Factor 4, a transcription factor involved in cold adaptation. A comparative study revealed that the cold-induced accumulation of NtAvr9/Cfp9 was specifically quelled in transgenic BY-2 cells overexpressing OsDLK-GFP. These findings are discussed as a working model, where, in response to cold stress, OsDLK partitions from cortical microtubules at the plasma membrane into the nucleus and specifically modulates the expression of genes involved in cold adaptation.

Analysis of Mechanical Properties Related to Fiber Length of Closed-Loop-Recycled Offcuts of a Thermoplastic Fiber Composites (Organo Sheets).

Increasing demand for energy-efficient means of transport has steadily intensified the trend towards lightweight components. Thermoplastic glass fiber composites (organo sheets) play a major role in the production of functional automotive components. Organo sheets are cut, shaped and functionalized by injection molding to produce hybrid components, such as those used in car door modules. The cutting process produces a considerable amount of production waste, which has thus far been thermally recycled. This study develops a closed mechanical recycling process and analyzes the different steps of the process. The offcuts were shredded using two shredding methods and implemented directly in the injection-molding process. Using tensile tests and impact bending tests, the material properties of the recycled materials were compared with the virgin material. In addition, fiber length degradation via the injection-molding process and the influence of the waterjet-cutting process on the mechanical properties are investigated. Recycled offcuts are both comparable to new material in terms of mechanical properties and usability, and are also economically and ecologically advantageous. Recycling polypropylene waste with glass fiber reinforcement in a closed loop is an effective way to reduce industrial waste in a sustainable and economical production process.

The Striking Flower-in-Flower Phenotype of Arabidopsis thaliana Nossen (No-0) is Caused by a Novel LEAFY Allele.

The transition to reproduction is a crucial step in the life cycle of any organism. In Arabidopsis thaliana the establishment of reproductive growth can be divided into two phases: Firstly, cauline leaves with axillary meristems are formed and internode elongation begins. Secondly, lateral meristems develop into flowers with defined organs. Floral shoots are usually determinate and suppress the development of lateral shoots. Here, we describe a transposon insertion mutant in the Nossen accession with defects in floral development and growth. Most strikingly is the outgrowth of stems from the axillary bracts of the primary flower carrying secondary flowers. Therefore, we named this mutant flower-in-flower (fif). However, the transposon insertion in the annotated gene is not the cause for the fif phenotype. By means of classical and genome sequencing-based mapping, the mutation responsible for the fif phenotype was found to be in the LEAFY gene. The mutation, a G-to-A exchange in the second exon of LEAFY, creates a novel lfy allele and results in a cysteine-to-tyrosine exchange in the α1-helix of LEAFY's DNA-binding domain. This exchange abolishes target DNA-binding, whereas subcellular localization and homomerization are not affected. To explain the strong fif phenotype against these molecular findings, several hypotheses are discussed.

AtDAT1 Is a Key Enzyme of D-Amino Acid Stimulated Ethylene Production in Arabidopsis thaliana.

D-Enantiomers of proteinogenic amino acids (D-AAs) are found ubiquitously, but the knowledge about their metabolism and functions in plants is scarce. A long forgotten phenomenon in this regard is the D-AA-stimulated ethylene production in plants. As a starting point to investigate this effect, the Arabidopsis accession Landsberg erecta (Ler) got into focus as it was found defective in metabolizing D-AAs. Combining genetics and molecular biology of T-DNA insertion lines and natural variants together with biochemical and physiological approaches, we could identify AtDAT1 as a major D-AA transaminase in Arabidopsis. Atdat1 loss-of-function mutants and Arabidopsis accessions with defective AtDAT1 alleles were unable to produce the metabolites of D-Met, D-Ala, D-Glu, and L-Met. This result corroborates the biochemical characterization, which showed highest activity of AtDAT1 using D-Met as a substrate. Germination of seedlings in light and dark led to enhanced growth inhibition of atdat1 mutants on D-Met. Ethylene measurements revealed an increased D-AA stimulated ethylene production in these mutants. According to initial working models of this phenomenon, D-Met is preferentially malonylated instead of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This decrease of ACC degradation should then lead to the increase of ethylene production. We could observe a reciprocal relation of malonylated methionine and ACC upon D-Met application and significantly more malonyl-methionine in atdat1 mutants. Unexpectedly, the malonyl-ACC levels did not differ between mutants and wild type. With AtDAT1, the first central enzyme of plant D-AA metabolism was characterized biochemically and physiologically. The specific effects of D-Met on ACC metabolism, ethylene production, and plant development of dat1 mutants unraveled the impact of AtDAT1 on these processes; however, they are not in full accordance to previous working models. Instead, our results imply the influence of additional factors or processes on D-AA-stimulated ethylene production, which await to be uncovered.

Salt-inducible expression of OsJAZ8 improves resilience against salt-stress.

BACKGROUND: Productivity of important crop rice is greatly affected by salinity. The plant hormone jasmonate plays a vital role in salt stress adaptation, but also evokes detrimental side effects if not timely shut down again. As novel strategy to avoid such side effects, OsJAZ8, a negative regulator of jasmonate signalling, is expressed under control of the salt-inducible promoter of the transcription factor ZOS3-11, to obtain a transient jasmonate signature in response to salt stress. To modulate the time course of jasmonate signalling, either a full-length or a dominant negative C-terminally truncated version of OsJAZ8 driven by the ZOS3-11 promoter were expressed in a stable manner either in tobacco BY-2 cells, or in japonica rice. RESULTS: The transgenic tobacco cells showed reduced mortality and efficient cycling under salt stress adaptation. This was accompanied by reduced sensitivity to Methyl jasmonate and increased responsiveness to auxin. In the case of transgenic rice, the steady-state levels of OsJAZ8 transcripts were more efficiently induced under salt stress compared to the wild type, this induction was more pronounced in the dominant-negative OsJAZ8 variant. CONCLUSIONS: The result concluded that, more efficient activation of OsJAZ8 was accompanied by improved salt tolerance of the transgenic seedlings and demonstrates the impact of temporal signatures of jasmonate signalling for stress tolerance.

d-Amino Acids Are Exuded by Arabidopsis thaliana Roots to the Rhizosphere.

Proteinogenic l-amino acids (l-AAs) are essential in all kingdoms as building blocks of proteins. Their d-enantiomers are also known to fulfill important functions in microbes, fungi, and animals, but information about these molecules in plants is still sparse. Previously, it was shown that d-amino acids (d-AAs) are taken up and utilized by plants, but their ways to reduce excessive amounts of them still remained unclear. Analyses of plant d-AA content after d-Ala and d-Glu feeding opened the question if exudation of d-AAs into the rhizosphere takes place and plays a role in the reduction of d-AA content in plants. The exudation of d-Ala and d-Glu could be confirmed by amino acid analyses of growth media from plants treated with these d-AAs. Further tests revealed that other d-AAs were also secreted. Nevertheless, treatments with d-Ala and d-Glu showed that plants are still able to reduce their contents within the plant without exudation. Further exudation experiments with transport inhibitors revealed that d-AA root exudation is rather passive and comparable to the secretion of l-AAs. Altogether, these observations argued against a dominant role of exudation in the regulation of plant d-AA content, but may influence the composition of the rhizosphere.

Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus.

GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.

DPI-ELISA: a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro.

BACKGROUND: About 10% of all genes in eukaryote genomes are predicted to encode transcription factors. The specific binding of transcription factors to short DNA-motifs influences the expression of neighbouring genes. However, little is known about the DNA-protein interaction itself. To date there are only a few suitable methods to characterise DNA-protein-interactions, among which the EMSA is the method most frequently used in laboratories. Besides EMSA, several protocols describe the effective use of an ELISA-based transcription factor binding assay e.g. for the analysis of human NFκB binding to specific DNA sequences. RESULTS: We provide a unified protocol for this type of ELISA analysis, termed DNA-Protein-Interaction (DPI)-ELISA. Qualitative analyses with His-epitope tagged plant transcription factors expressed in E. coli revealed that EMSA and DPI-ELISA result in comparable and reproducible data. The binding of AtbZIP63 to the C-box and AtWRKY11 to the W2-box could be reproduced and validated by both methods. We next examined the physical binding of the C-terminal DNA-binding domains of AtWRKY33, AtWRKY50 and AtWRKY75 to the W2-box. Although the DNA-binding domain is highly conserved among the WRKY proteins tested, the use of the DPI-ELISA discloses differences in W2-box binding properties between these proteins. In addition to these well-studied transcription factor families, we applied our protocol to AtBPC2, a member of the so far uncharacterised plant specific Basic Pentacysteine transcription factor family. We could demonstrate binding to GA/TC-dinucleotide repeat motifs by our DPI-ELISA protocol. Different buffers and reaction conditions were examined. CONCLUSIONS: We successfully applied our DPI-ELISA protocol to investigate the DNA-binding specificities of three different classes of transcription factors from Arabidopsis thaliana. However, the analysis of the binding affinity of any DNA-binding protein to any given DNA sequence can be performed via this method. The DPI-ELISA is cost efficient, less time-consuming than other methods and provides a qualitative and quantitative readout. The presented DPI-ELISA protocol is accompanied by advice on trouble-shooting, which will enable scientists to rapidly establish this versatile and easy to use method in their laboratories.