Summary The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises the questions of how auxin and cytokinin govern fern root system architecture and whether this can tell us something about the origin of that root.Using Azolla filiculoides, we characterized the influence of IAA and zeatin on adventitious fern root meristems and vasculature by Nomarski microscopy. Simultaneously, RNAseq analyses, yielding 36 091 contigs, were used to uncover how the phytohormones affect root tip gene expression.We show that auxin restricts Azolla root meristem development, while cytokinin promotes it; it is the opposite effect of what is observed in Arabidopsis. Global gene expression profiling uncovered 145 genes significantly regulated by cytokinin or auxin, including cell wall modulators, cell division regulators and lateral root formation coordinators.Our data illuminate both evolution and development of fern roots. Promotion of meristem size through cytokinin supports the idea that root meristems of euphyllophytes evolved from shoot meristems. The foundation of these roots was laid in a postembryonically branching shoot system.

No abstract

Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.Fluoroquinolone resistance in gram-positive cocci is related to mutations in the DNA gyrase and topoisomerase IV genes (8-12, 16, 23) and the active efflux of agents (1, 3, 13, 21-25, 31, 44). Because fluoroquinolones differ in both their target affinity (8,16,(33)(34)(35)(36)39) and their activation of efflux pumps (7,13,21,22,24,25,31,42), one can speculate that the phenotypic expression of quinolone resistance will also differ. Studies have shown that fluoroquinolone resistance can be selected for in pneumococci and staphylococci (5,6,37).In order to analyze the ability of newer fluoroquinolones to cause resistance development in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus, we repeatedly exposed six clinical strains of each species to ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin.Approximately 5 ϫ 10 7 CFU of each of the 18 strains was added to tubes containing 9.9 ml of appropriate broth containing antibiotic concentrations ranging from 3 doubling dilutions above to 3 doubling dilutions below the MIC of each of the six agents. The tubes were then incubated for 24 h at 37°C. Aliquots from the test tubes containing the highest drug concentration that permitted visible growth were used following a 1:100 dilution to inoculate a second set of serial drug dilutions. After overnight incubation, the bacteria were transferred again. Finally, after 10 serial transfers, the bacteria for which the MICs were the highest were collected, stored, and also subcultured on quinolone-free agar for 10 days to assess the stability of resistance.MICs were determined by the microdilution methodology according to NCCLS guidelines (29, 30). Ciprofloxacin MIC determinations were conducted in the presence and absence of reserpine (20 g/ml; tests were repeated three times) for all of the original isolates (n ϭ 18) as well as for all of the selected mutants (n ϭ 108) (7).S. pneumoniae and S. aureus isolates were analyzed before and after transfers for mutations in the quinolone-resistance determining regions (QRDRs) of parC or grlA and gyrA, respectively (19,40,41).The MIC results from subculturing as well as the mutations in the QRDRs of S. pneumoniae and S. aureus are summarized in the Tables 1 to 3. Subculturing with newer quinolones led to resistance development in all three species. This is in line with previous reports with regard to cephalosporins, macrolides, and older quinolones in pneumococci (5, 6, 37).Resistance was stable in all cases; i.e., the MICs for the 108 selected mutants remained within 1 doubling dilution after 10 tr...

No abstract

30 Membrane traffic maintains the organization of the eukaryotic cell and delivers cargo proteins to their 31 subcellular destinations such as sites of action or degradation. Membrane vesicle formation requires ARF 32 GTPase activation by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), 33 resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with 34 monomeric proteins, although ARF-GEFs have been shown to form dimers in vivo. This feature is conserved 35 across the eukaryotes, however its biological significance is unknown. Here we demonstrate ARF1 36 dimerization in vivo and we show that ARF-GEF dimers mediate ARF1 dimer formation. Mutational 37 disruption of ARF1 dimers interfered with ARF1-dependent trafficking but not coat protein recruitment in 38 Arabidopsis. Mutations disrupting simultaneous binding of two ARF1•GDPs by the two SEC7 domains of 39 GNOM ARF-GEF dimer prevented stable interaction of ARF1 with ARF-GEF and thus, efficient ARF1 40 activation. Our results suggest a model of activation-dependent dimerization of membrane-inserted 41 ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary 42 conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur 43 in eukaryotes in general. 44 45 Keywords