Antioxidative responses of axenic protonema cultures of the moss Physcomitrella patens exposed to 10 μM Cd over 40 d were studied. Cd treatment suppressed growth by ca. 75% with concomitant browning of some filaments and suppression of chlorophyll autofluorescence but had no impact on tissue water content. Despite this negative growth responses which could be related to enhanced ROS formation (as detected using fluorescence staining reagents for total ROS, hydroperoxides and lipid peroxidation), some metabolites revealed strong elevation by Cd which could contribute to attenuation of long-term Cd stress (elevation of ascorbic, malic and citric acids). Molar ratio of malate to Cd was 12.7 and citrate to Cd 2.5, thus potentially contributing to Cd chelation. Interestingly, GSH/GSSG pool and nitric oxide formation remained unaltered by Cd. Accumulation of Cd reached 82 μg/g DW with bioaccumulation factor of 73. Data indicate that Cd induces elevation of potentially protective metabolites even after prolonged exposure though they do not prevent oxidative stress sufficiently.

• MeSH
• Antioxidants metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Cadmium analysis   toxicity   MeSH
• Bryopsida drug effects   metabolism   MeSH
• Oxidative Stress drug effects   MeSH
• Hydrogen Peroxide metabolism   MeSH
• Lipid Peroxidation drug effects   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Publication type
• Journal Article MeSH

Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.

• MeSH
• Arabidopsis drug effects   growth & development   MeSH
• Cinnamates chemistry   metabolism   pharmacology   MeSH
• Cyclin B genetics   metabolism   MeSH
• Plants, Genetically Modified MeSH
• Isomerism MeSH
• Plant Roots drug effects   growth & development   metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Bryopsida drug effects   growth & development   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Qa-SNARE Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Selaginellaceae drug effects   growth & development   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH

While the molecular basis for cytokinin action is quite well understood in flowering plants, little is known about the cytokinin signal transduction in early diverging land plants. The genome of the bryophyte Physcomitrella patens (Hedw.) B.S. encodes three classical cytokinin receptors, the CHASE domain-containing histidine kinases, CHK1, CHK2, and CHK3. In a complementation assay with protoplasts of receptor-deficient Arabidopsis thaliana as well as in cytokinin binding assays, we found evidence that CHK1 and CHK2 receptors can function in cytokinin perception. Using gene targeting, we generated a collection of CHK knockout mutants comprising single (Δchk1, Δchk2, Δchk3), double (Δchk1,2, Δchk1,3, Δchk2,3), and triple (Δchk1,2,3) mutants. Mutants were characterized for their cytokinin response and differentiation capacities. While the wild type did not grow on high doses of cytokinin (1 µM benzyladenine), the Δchk1,2,3 mutant exhibited normal protonema growth. Bud induction assays showed that all three cytokinin receptors contribute to the triggering of budding, albeit to different extents. Furthermore, while the triple mutant showed no response in this bioassay, the remaining mutants displayed budding responses in a diverse manner to different types and concentrations of cytokinins. Determination of cytokinin levels in mutants showed no drastic changes for any of the cytokinins; thus, in contrast to Arabidopsis, revealing only small impacts of cytokinin signaling on homeostasis. In summary, our study provides a first insight into the molecular action of cytokinin in an early diverging land plant and demonstrates that CHK receptors play an essential role in bud induction and gametophore development.

• MeSH
• Biological Assay MeSH
• Butadienes pharmacology   MeSH
• Cytokinins pharmacology   MeSH
• Phenotype MeSH
• Adaptation, Physiological MeSH
• Gene Knockout Techniques MeSH
• Hemiterpenes pharmacology   MeSH
• Bryopsida drug effects   genetics   metabolism   MeSH
• Mutation genetics   MeSH
• Pentanes pharmacology   MeSH
• Receptors, Cell Surface chemistry   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Proteins chemistry   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Shoot branching is a primary contributor to plant architecture, evolving independently in flowering plant sporophytes and moss gametophytes. Mechanistic understanding of branching is largely limited to flowering plants such as Arabidopsis, which have a recent evolutionary origin. We show that in gametophytic shoots of Physcomitrella, lateral branches arise by re-specification of epidermal cells into branch initials. A simple model co-ordinating the activity of leafy shoot tips can account for branching patterns, and three known and ancient hormonal regulators of sporophytic branching interact to generate the branching pattern- auxin, cytokinin and strigolactone. The mode of auxin transport required in branch patterning is a key divergence point from known sporophytic pathways. Although PIN-mediated basipetal auxin transport regulates branching patterns in flowering plants, this is not so in Physcomitrella, where bi-directional transport is required to generate realistic branching patterns. Experiments with callose synthesis inhibitors suggest plasmodesmal connectivity as a potential mechanism for transport.

• MeSH
• Models, Biological MeSH
• Biological Transport drug effects   MeSH
• Cytokinins biosynthesis   MeSH
• Plant Epidermis cytology   growth & development   MeSH
• Plants, Genetically Modified MeSH
• Indoleacetic Acids metabolism   pharmacology   MeSH
• Lactones pharmacology   MeSH
• Bryopsida drug effects   growth & development   MeSH
• Morphogenesis drug effects   MeSH
• Mutation genetics   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Plant Growth Regulators pharmacology   MeSH
• Plant Proteins metabolism   MeSH
• Body Patterning drug effects   MeSH
• Plant Shoots drug effects   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.

• MeSH
• Biocatalysis * drug effects   MeSH
• Cytokinins chemistry   metabolism   MeSH
• Nitrogen pharmacology   MeSH
• Phenotype MeSH
• Phylogeny MeSH
• Gene Knockout Techniques MeSH
• Hydrolysis drug effects   MeSH
• Kinetics MeSH
• Crystallography, X-Ray MeSH
• Zea mays drug effects   enzymology   genetics   MeSH
• Bryopsida drug effects   enzymology   genetics   growth & development   MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Mutagenesis, Site-Directed MeSH
• Mutant Proteins chemistry   metabolism   MeSH
• N-Glycosyl Hydrolases chemistry   genetics   metabolism   MeSH
• Pyrimidines chemistry   metabolism   MeSH
• Ribonucleosides chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Substrate Specificity drug effects   MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH