VISUAL-CC system uncovers the role of GSK3 as an orchestrator of vascular cell type ratio in plants.

The phloem transports photosynthetic assimilates and signalling molecules. It mainly consists of sieve elements (SEs), which act as "highways" for transport, and companion cells (CCs), which serve as "gates" to load/unload cargos. Though SEs and CCs function together, it remains unknown what determines the ratio of SE/CC in the phloem. Here we develop a new culture system for CC differentiation in Arabidopsis named VISUAL-CC, which almost mimics the process of the SE-CC complex formation. Comparative expression analysis in VISUAL-CC reveals that SE and CC differentiation tends to show negative correlation, while total phloem differentiation is unchanged. This varying SE/CC ratio is largely dependent on GSK3 kinase activity. Indeed, gsk3 hextuple mutants possess many more SEs and fewer CCs, whereas gsk3 gain-of-function mutants partially increase the CC number. Taken together, GSK3 activity appears to function as a cell-fate switch in the phloem, thereby balancing the SE/CC ratio.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium.

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

Regulation of xylem cell fate.

The vascular system is organized throughout the plant body for transporting water, nutrients, and signaling molecules. During vascular development, xylem, phloem, and procambial/cambial cells are produced in a spatiotemporally organized manner. Several key regulators for xylem cell patterning and differentiation have been discovered, including auxin, cytokinin, CLE peptides, microRNAs, HD-ZIPIIIs, VNDs, and moving transcription factors SHR and AHLs. Recent studies are identifying functional interactions among these factors that ultimately determine xylem cell fate. This review focuses on regulatory networks underlying xylem cell fate determination in root vascular development.

Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling.

During plant radial growth typically seen in trees, procambial and cambial cells act as meristematic cells in the vascular system to self-proliferate and differentiate into xylem cells. These two processes are regulated by a signalling pathway composed of a peptide ligand and its receptor; tracheary element differentiation inhibitory factor (TDIF) and TDIF RECEPTOR (TDR). Here we show that glycogen synthase kinase 3 proteins (GSK3s) are crucial downstream components of the TDIF signalling pathway suppressing xylem differentiation from procambial cells. TDR interacts with GSK3s at the plasma membrane and activates GSK3s in a TDIF-dependent fashion. Consistently, a specific inhibitor of plant GSK3s strongly induces xylem cell differentiation through BRI1-EMS SUPPRESSOR 1 (BES1), a well-known target transcription factor of GSK3s. Our findings provide insight into the regulation of cell fate determination in meristem maintenance.

SUPPRESSOR OF LLP1 1-mediated C-terminal processing is critical for CLE19 peptide activity.

Cell-to-cell communication is essential for the coordinated development of multicellular organisms. Members of the CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED (CLE) family, a group of small secretory peptides, are involved in these processes in plants. Although post-translational modifications are considered to be indispensable for their activity, the detailed mechanisms governing these modifications are not well understood. Here, we report that SUPPRESSOR OF LLP1 1 (SOL1), a putative Zn²âº carboxypeptidase previously isolated as a suppressor of the CLE19 over-expression phenotype, functions in C-terminal processing of the CLE19 proprotein to produce the functional CLE19 peptide. Newly isolated sol1 mutants are resistant to CLE19 over-expression, consistent with the previous report (Casamitjana-Martinez, E., Hofhuis, H.F., Xu, J., Liu, C.M., Heidstra, R. and Scheres, B. (2003) Curr. Biol. 13, 1435-1441). As expected, our experiment using synthetic CLE19 peptide revealed that the sol1 mutation does not compromise CLE signal transduction pathways per se. SOL1 possesses enzymatic activity to remove the C-terminal arginine residue of CLE19 proprotein in vitro, and SOL1-dependent cleavage of the C-terminal arginine residue is necessary for CLE19 activity in vivo. Additionally, the endosomal localization of SOL1 suggests that this processing occurs in endosomes in the secretory pathway. Thus, our data indicate the importance of C-terminal processing of CLE proproteins to ensure CLE activities.

RABA members act in distinct steps of subcellular trafficking of the FLAGELLIN SENSING2 receptor.

Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuing signal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. Subcellular localization of Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. However, our understanding about the mechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescent protein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PM was transported to a compartment with properties intermediate between the trans-Golgi network (TGN) and the multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the trafficking pathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, we demonstrated that transport of de novo-synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our results demonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABA members in endo- and exocytosis.

Evolution of CLE signaling: origins of the CLV1 and SOL2/CRN receptor diversity.

The shoot apical meristem is maintained by the intercellular factor, CLV3, a dodecapeptide in Arabidopsis. CLV3 belongs to the CLE family and putative CLE genes have been found in various plants, even in the moss Physcomitrella patens. Here, we report that a pteridophyte, Selaginella moelendorffii, also has 15 putative CLE genes in its genome. On the other hand, CLV1 is reported to function as a receptor for the CLV3 peptide, and other CLE peptides might be recognized by CLV1 homologues in various plants. Recent genetic studies of the crn and sol2 mutants of Arabidopsis have revealed that SOL2/CRN encodes a receptor-like kinase protein. SOL2/CRN functions together with CLV2 independently of CLV1 in the CLE signaling pathway. Phylogenetic analysis of CLV1, CLV2 and SOL2/CRN revealed that Arabidopsis, rice, Populus trichocarpa and Vitis vinifera have one copy of the SOL2/CRN and CLV2 homologues, and Selaginella moelendorffii and Physcomitrella patens have no homologues. In contrast, a number of CLV1 homologues were identified in the genomic databases of Arabidopsis, rice, Populus trichocarpa, Vitis vinifera, and even a pteridophyte, Selaginella moelendorffii, and a moss, Physcomitrella patens. These results indicate that CLV1 and its homologues play multiple roles in plant development and environmental responses, whereas SOL2/CRN and CLV2 have more specific roles in vascular plants.

Short-term biological safety of a photoelectric dye used as a component of retinal prostheses.

We have designed a new type of retinal prosthesis with a photoelectric dye that transfers photon energy to generate electric potentials. The purpose of this study was to test the safety of a photoelectric dye, 2-[2-[4-(dibutylami no)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide (NK-5962), used for retinal prostheses. The retinal cells, derived from chick neurosensory retinas at the 12-day embryonic stage, were a mixed population of retinal neurons and glial cells, and were cultured for 2 days either under protection from light or under continuous light exposure at 230 lux for 9 h daily in the presence of the photoelectric dye at varying concentrations (1.6 x 10(-5), 1.6 x 10(-6), and 1.6 x 10(-7) M) to assess cell viability by staining live cells and dead cells. Dispersed retinal pigment epithelial cells at the same embryonic stage were incubated with the photoelectric dye at varying concentrations (6.6 x 10(-5), 6.6 x 10(-6), and 6.6 x 10(-7) M) for 4 h under protection from light or under continuous light exposure at 320 lux to assess cytotoxicity by measuring the activity of lactate dehydrogenase leaking from cells. The majority of retinal cells were alive with only a small percentage of dead cells under the dark condition or the light condition in the presence or the absence of the photoelectric dye. The percentage of dead cells was significantly smaller at higher concentrations of the photoelectric dye (P = 0.0183, two-factor analysis of variance), while the percentage of dead cells was not significantly different between the dark condition and the light condition (P = 0.3102). Percent cytotoxicity values were negative, indicating protective effects in all groups of retinal pigment epithelial cells incubated with varying concentrations of the photoelectric dye. The photoelectric dye showed no cytotoxicity to chick retinal cells or retinal pigment epithelial cells on short-term exposure. In addition, this photoelectric dye might have protective effects on both types of cells.

Glial reaction to photoelectric dye-based retinal prostheses implanted in the subretinal space of rats.

We have designed a new type of retinal prosthesis using polyethylene films coupled with photoelectric dye molecules that absorb light and convert photon energy to electric potentials. An extruded-blown film of high-density polyethylene was used as the original polyethylene film. Recrystallized film was made by recrystallization from the melting of the original polyethylene film. A photoelectric dye,2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide, was coupled to the two types of polyethylene films through amide linkages. Samples of the original dye-coupled film, the dye-coupled recrystallized film, and the dye-uncoupled plain film were implanted in the subretinal space of normal adult rats. Frozen sections were cut from the eyes enucleated at 1 week or 1 month and were either stained with hematoxylin and eosin, stained immunohistochemically for glial fibrillary acidic protein (GFAP), or processed for in situ apoptosis detection. The results revealed that retinal tissue damage was negligible with no inflammatory cells and few apoptotic cells. GFAP was significantly up-regulated in retinal sites with the implantation of all types of polyethylene films at 1 week, compared with the adjacent retinal sites (P < 0.005, analysis of variance). The GFAP up-regulation was also present at 1 month for the plain film and dye-coupled recrystallized film (P < 0.05). Glial cell encirclement around the films increased significantly between 1 week and 1 month (P = 0.023, two-factor analysis of variance) but was not significantly different among the three types of polyethylene films (P = 0.4531). These results showed evidence of glial reactions to the photoelectric dye-coupled polyethylene films implanted into the subretinal space of rat eyes and also proved their basic biological safety.

Symmetric waveguides in poly(methyl methacrylate) fabricated by femtosecond laser pulses.

We report on the fabrication of symmetric waveguides in bulk poly(methyl methacrylate) (PMMA) by femtosecond laser pulses. A waveguide with a circular transverse profile can be obtained by using a slit beam shaping method. The refractive index in the core increases by up to 4.6 x 10(-4) and the waveguide works as single-mode waveguide at a wavelength of 632.8 nm. This writing technique is applied to the fabrication of a directional coupler to split a coupled beam with a 1:1 splitting ratio at 632.8 nm.

Structural modification in fused silica by a femtosecond fiber laser at 1558 nm.

We report on structural modification in fused silica by a novel commercial femtosecond fiber laser with a fundamental wavelength of 1558 nm. The refractive-index change was induced by laser pulses at a repetition rate of 173 kHz and pulse duration of 870 fs. The refractive index change with a magnitude of 1.2 x 10(-3) was estimated from the diffraction efficiencies of an internal grating.

Laser micro-welding of transparent materials by a localized heat accumulation effect using a femtosecond fiber laser at 1558 nm.

We report on laser micro-welding of materials based on a localized heat accumulation effect using an amplified femtosecond Er-fiber laser with a wavelength of 1558 nm and a repetition rate of 500 kHz. We demonstrated the welding of non-alkali alumino silicate glass substrates, resulting in a joint strength of 9.87 MPa. We also welded a non-alkali glass substrate and a silicon substrate using the 1558-nm laser pulses, resulting in a joint strength of 3.74 MPa. Our laser micro-welding technique can be extended to welding of semiconductor materials and has potential for various applications, such as three-dimensional stacks and assembly of electronic devices and microelectromechanical system devices.