Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth.

Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth.

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange between plants and the atmosphere in response to light, CO2, and the plant hormone abscisic acid. Light-induced stomatal opening is mediated by at least three key components: the blue light receptor phototropin (phot1 and phot2), plasma membrane H(+)-ATPase, and plasma membrane inward-rectifying K(+) channels. Very few attempts have been made to enhance stomatal opening with the goal of increasing photosynthesis and plant growth, even though stomatal resistance is thought to be the major limiting factor for CO2 uptake by plants. Here, we show that transgenic Arabidopsis plants overexpressing H(+)-ATPase using the strong guard cell promoter GC1 showed enhanced light-induced stomatal opening, photosynthesis, and plant growth. The transgenic plants produced larger and increased numbers of rosette leaves, with ∼42-63% greater fresh and dry weights than the wild type in the first 25 d of growth. The dry weights of total flowering stems of 45-d-old transgenic plants, including seeds, siliques, and flowers, were ∼36-41% greater than those of the wild type. In addition, stomata in the transgenic plants closed normally in response to darkness and abscisic acid. In contrast, the overexpression of phototropin or inward-rectifying K(+) channels in guard cells had no effect on these phenotypes. These results demonstrate that stomatal aperture is a limiting factor in photosynthesis and plant growth, and that manipulation of stomatal opening by overexpressing H(+)-ATPase in guard cells is useful for the promotion of plant growth.

Purification and characterization of ß-xylosidase that is active for plant complex type N-glycans from tomato (Solanum lycopersicum): removal of core α1-3 mannosyl residue is prerequisite for hydrolysis of ß1-2 xylosyl residue.

In this study, we purified and characterized the ß-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified ß-xylosidase (ß-Xyl'ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that ß-Xyl'ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, ß-Xyl'ase Le-1 showed optimum activity at about pH 5 at 40 °C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. ß-Xyl'ase Le-1 hydrolyzed the ß1-2 xylosyl residue from Man1Xyl1GlcNAc2-PA, Man1Xyl1Fuc1GlcNAc2-PA, and Man2Xyl1Fuc1GlcNAc2-PA, but not that from Man3Xyl1GlcNAc2-PA or Man3Xyl1Fuc1GlcNAc2-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of ß-Xyl'ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the ß1-2 xylosyl residue by ß-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.

Characterization of shade avoidance responses in Lotus japonicus.

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.

FLOWERING LOCUS T regulates stomatal opening.

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.

Genomewide characterization of the light-responsive and clock-controlled output pathways in Lotus japonicus with special emphasis of its uniqueness.

During the last decade, tremendous progress has been made in understanding the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana, mainly taking advantage of the availability of its entire genomic sequence. It is also well understood how the clock controls the photomorphogenesis of seedlings, including the shade avoidance response, and how the clock controls the photoperiodic flowering time in the spring annual long-days herb A. thaliana. Based on this, here we attempt to shed light on these clock-controlled fundamental and physiological events in Lotus japonicus, which is a perennial temperate legume with a morphological nature quite different from Arabidopsis. In the Lotus database, we first compiled as many clock-, light-, and flowering-associated coding sequences as possible, which appear to be orthologous or homologous to the Arabidopsis counterparts. Then we focused on the PHYTOCHROME INTERACTING FACTOR4 (PIF4)-mediated photomorphogenic pathway and the FLOWERING LOCUS T (FT)-mediated photoperiodic flowering pathway. It was shown in L. japonicus that the putative LjPIF4 homologue is expressed in a manner dependent on the circadian clock, and the putative LjFT orthologue is expressed coincidentally and especially in the long-days conditions, as in the case of A. thaliana. LjFT is capable of promoting flowering in A. thaliana, whereas the function of LjPIF4 seems to be divergent to a certain extent from that of AtPIF4. These results are discussed with emphasis on the intriguing differences between these model plant species.