Diversity of Retinal Ganglion Cells Identified by Transient GFP Transfection in Organotypic Tissue Culture of Adult Marmoset Monkey Retina

The mammalian retina has more diversity of neurons than scientists had once believed in order to establish complicated vision processing. In the monkey retina, morphological diversity of retinal ganglion cells (RGCs) besides dominant midget and parasol cells has been suggested. However, characteristic subtypes of RGCs in other species such as bistratified direction-selective ganglion cells (DSGC) have not yet been identified. Increasing interest has been shown in the common marmoset (Callithrix jacchus) monkey as a “super-model” of neuroscientific research. Here, we established organotypic tissue culture of the adult marmoset monkey retina with particle-mediated gene transfer of GFP to survey the morphological diversity of RGCs. We successfully incubated adult marmoset monkey retinas for 2 to 4 days ex vivo for transient expression of GFP. We morphologically examined 121 RGCs out of more than 3240 GFP-transfected cells in 5 retinas. Among them, we identified monostratified or broadly stratified ganglion cells (midget, parasol, sparse, recursive, thorny, and broad thorny ganglion cells), and bistratified ganglion cells (recursive, large, and small bistratified ganglion cells [blue-ON/yellow-OFF-like]). By this survey, we also found a candidate for bistratified DSGC whose dendrites were well cofasciculated with ChAT-positive starburst dendrites, costratified with ON and OFF ChAT bands, and had honeycomb-shaped dendritic arbors morphologically similar to those in rabbits. Our genetic engineering method provides a new approach to future investigation for morphological and functional diversity of RGCs in the monkey retina.     

Post‐illumination transient O2‐uptake is driven by photorespiration in tobacco leaves

This study aims to elucidate the molecular mechanism for the transient increase in the O₂‐uptake rate in tobacco (Nicotiana tabacum cv Xanthi) leaves after turning off actinic lights (ALs). The photosynthetic O₂ evolution rate reaches a maximum shortly after the onset of illumination with ALs and then decreases to zero in atmospheric CO₂/O₂ conditions. After turning off the ALs, tobacco leaves show a transient increase in the O₂‐uptake rate, the post‐illumination transient O₂‐uptake, and thereafter, the O₂‐uptake rate decreases to the level of the dark‐respiration rate. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], maintained a steady‐state value distinct from the photosynthetic O₂‐evolution rate. In high‐[CO₂] conditions, the photosynthetic O₂‐evolution rate and Y(II) showed a parallel behavior, and the post‐illumination transient O₂‐uptake was suppressed. On the other hand, in maize leaves (a C4 plant), even in atmospheric CO₂/O₂ conditions, Y(II) paralleled the photosynthetic O₂‐evolution rate and the post‐illumination transient O₂‐uptake was suppressed. Hypothesizing that the post‐illumination transient O₂‐uptake is driven by C3 plant photorespiration in tobacco leaves, we calculated both the ribulose 1,5‐bisphosphate carboxylase‐ and oxygenase‐rates (Vc and Vo) from photosynthetic O₂‐evolution and the post‐illumination transient O₂‐uptake rates. These values corresponded to those estimated from simultaneous chlorophyll fluorescence/O₂‐exchange analysis. Furthermore, the H⁺‐consumption rate for ATP synthesis in both photosynthesis and photorespiration, calculated from both Vc and Vo that were estimated from chlorophyll fluorescence/CO₂‐exchange analysis, showed a positive linear relationship with the dissipation rate of the electrochromic shift signal. Thus, these findings support our hypothesis.     

The rate-limiting step for CO(2) assimilation at different temperatures is influenced by the leaf nitrogen content in several C(3) crop species

Effects of nitrogen (N) supply on the limiting step of CO(2) assimilation rate (A) at 380 μmol mol(-1) CO(2) concentration (A(380) ) at several leaf temperatures were studied in several crops, since N nutrition alters N allocation between photosynthetic components. Contents of leaf N, ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) increased with increasing N supply, but the cyt f/Rubisco ratio decreased. Large leaf N content was linked to a high stomatal (g(s) ) and mesophyll conductance (g(m) ), but resulted in a lower intercellular (C(i) ) and chloroplast CO(2) concentration (C(c) ) because the increase in g(s) and g(m) was insufficient to compensate for change in A(380) . The A-C(c) response was used to estimate the maximum rate of RuBP carboxylation (V(cmax) ) and chloroplast electron transport (J(max) ). The J(max) /V(cmax) ratio decreased with reductions in leaf N content, which was consistent with the results of the cyt f/Rubisco ratio. Analysis using the C(3) photosynthesis model indicated that A(380) tended to be limited by RuBP carboxylation in plants grown at low N concentration, whereas it was limited by RuBP regeneration in plants grown at high N concentration. We conclude that the limiting step of A(380) depends on leaf N content and is mainly determined by N partitioning between Rubisco and electron transport components.     

Overproduction of PGR5 enhances the electron sink downstream of photosystem I in a C4 plant,Flaveria bidentis

C₄ plants can fix CO₂ efficiently using CO₂‐concentrating mechanisms (CCMs), but they require additional ATP. To supply the additional ATP, C₄ plants operate at higher rates of cyclic electron transport around photosystem I (PSI), in which electrons are transferred from ferredoxin to plastoquinone. Recently, it has been reported that the NAD(P)H dehydrogenase‐like complex (NDH) accumulated in the thylakoid membrane in leaves of C₄ plants, making it a candidate for the additional synthesis of ATP used in the CCM. In addition, C₄ plants have higher levels of PROTON GRADIENT REGULATION 5 (PGR5) expression, but it has been unknown how PGR5 functions in C₄ photosynthesis. In this study, PGR5 was overexpressed in a C₄ dicot, Flaveria bidentis. In PGR5‐overproducing (OP) lines, PGR5 levels were 2.3‐ to 3.0‐fold greater compared with wild‐type plants. PGR5‐like PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1), which cooperates with PGR5, increased with PGR5. A spectroscopic analysis indicated that in the PGR5‐OP lines, the acceptor side limitation of PSI was reduced in response to a rapid increase in photon flux density. Although it did not affect CO₂ assimilation, the overproduction of PGR5 contributed to an enhanced electron sink downstream of PSI.     

Rubisco activase is a key regulator of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature

The role of Rubisco activase in steady-state and non-steady-state photosynthesis was analyzed in wild-type (Oryza sativa) and transgenic rice that expressed different amounts of Rubisco activase. Below 25°C, the Rubisco activation state and steady-state photosynthesis were only affected when Rubisco activase was reduced by more than 70%. However, at 40°C, smaller reductions in Rubisco activase content were linked to a reduced Rubisco activation state and steady-state photosynthesis. As a result, overexpression of maize Rubisco activase in rice did not lead to an increase of the Rubisco activation state, nor to an increase in photosynthetic rate below 25°C, but had a small stimulatory effect at 40°C. On the other hand, the rate at which photosynthesis approached the steady state following an increase in light intensity was rapid in Rubisco activase-overexpressing plants, intermediate in the wild-type, and slowest in antisense plants at any leaf temperature. In Rubisco activase-overexpressing plants, Rubisco activation state at low light was maintained at higher levels than in the wild-type. Thus, rapid regulation by Rubisco activase following an increase in light intensity and/or maintenance of a high Rubisco activation state at low light would result in a rapid increase in Rubisco activation state and photosynthetic rate following an increase in light intensity. It is concluded that Rubisco activase plays an important role in the regulation of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature.     

PILRalpha is a herpes simplex virus-1 entry coreceptor that associates with glycoprotein B

Glycoprotein B (gB) is one of the essential components for infection by herpes simplex virus-1 (HSV-1). Although several cellular receptors that associate with glycoprotein D (gD), such as herpes virus entry mediator (HVEM) and Nectin-1, have been identified, specific molecules that mediate HSV-1 infection by associating with gB have not been elucidated. Here, we found that paired immunoglobulin-like type 2 receptor (PILR) alpha associates with gB, and cells transduced with PILRalpha become susceptible to HSV-1 infection. Furthermore, HSV-1 infection of human primary cells expressing both HVEM and PILRalpha was blocked by either anti-PILRalpha or anti-HVEM antibody. Our results demonstrate that cellular receptors for both gB and gD are required for HSV-1 infection and that PILRalpha plays an important role in HSV-1 infection as a coreceptor that associates with gB. These findings uncover a crucial aspect of the mechanism underlying HSV-1 infection.     

Responses of the chloroplast glyoxalase system to high CO2 concentrations

ABSTRACT

Sugar metabolism pathways such as photosynthesis produce dicarbonyls, e.g. methylglyoxal (MG), which can cause cellular damage. The glyoxalase (GLX) system comprises two enzymes GLX1 and GLX2, and detoxifies MG; however, this system is poorly understood in the chloroplast, compared with the cytosol. In the present study, we determined GLX1 and GLX2 activities in spinach chloroplasts, which constituted 40% and 10%, respectively, of the total leaf glyoxalase activity. In Arabidopsis thaliana, five GFP-fusion GLXs were present in the chloroplasts. Under high CO₂ concentrations, where increased photosynthesis promotes the MG production, GLX1 and GLX2 activities in A. thaliana increased and the expression of AtGLX1-2 and AtGLX2-5 was enhanced. On the basis of these findings and the phylogeny of GLX in oxygenic phototrophs, we propose that the GLX system scavenges MG produced in chloroplasts during photosynthesis.