The role of electron transport in determining the temperature dependence of the photosynthetic rate in spinach leaves grown at contrasting temperatures

The temperature response of the uncoupled whole-chain electron transport rate (ETR) in thylakoid membranes differs depending on the growth temperature. However, the steps that limit whole-chain ETR are still unclear and the question of whether the temperature dependence of whole-chain ETR reflects that of the photosynthetic rate remains unresolved. Here, we determined the whole-chain, PSI and PSII ETR in thylakoid membranes isolated from spinach leaves grown at 30 degrees C [high temperature (HT)] and 15 degrees C [low temperature (LT)]. We measured temperature dependencies of the light-saturated photosynthetic rate at 360 microl l(-1) CO2 (A360) in HT and LT leaves. Both of the temperature dependences of whole-chain ETR and of A360 were different depending on the growth temperature. Whole-chain ETR was less than the rates of PSI ETR and PSII ETR in the broad temperature range, indicating that the process was limited by diffusion processes between the PSI and PSII. However, at high temperatures, whole-chain ETR appeared to be limited by not only the diffusion processes but also PSII ETR. The C3 photosynthesis model was used to evaluate the limitations of A360 by whole-chain ETR (Pr) and ribulose bisphosphate carboxylation (Pc). In HT leaves, A360 was co-limited by Pc and Pr at low temperatures, whereas at high temperatures, A360 was limited by Pc. On the other hand, in LT leaves, A360 was solely limited by Pc over the entire temperature range. The optimum temperature for A360 was determined by Pc in both HT and LT leaves. Thus, this study showed that, at low temperatures, the limiting step of A360 was different depending on the growth temperature, but was limited by Pc at high temperatures regardless of the growth temperatures.     

slr1923 of Synechocystis sp. PCC6803 is essential for conversion of 3,8-divinyl(proto)chlorophyll(ide) to 3-monovinyl(proto)chlorophyll(ide)

The deduced amino acid sequence of an slr1923 gene of Synechocystis sp. PCC6803 is homologous to archaean F(420)H(2) dehydrogenase, which acts as a soluble subcomplex of reduced nicotinamide adenine dinucleotide dehydrogenase complex I. In this study, the gene was inactivated and characteristics of the mutant were analyzed. The mutant grew slower than the wild type under 100 microE m(-2) s(-1) but did not grow under high light intensity (300 microE m(-2) s(-1)). The cellular content of chlorophyll was lower in the mutant, and the absorption spectrum showed a shift in the absorption peak of the Soret band to a longer wavelength by about 10 nm compared with the wild type. It was found, by high-performance liquid chromatography analysis, that the retention time of chlorophyll of the mutant is shorter than that of the wild type and that the peak wavelength of the Soret band was also shifted to a longer wavelength by 11 nm. Proton nuclear magnetic resonance analysis of the chlorophyll of the mutant revealed that the ethyl group of position 8 of ring B is replaced with a vinyl group. The spectrum indicates that the chlorophyll of the mutant is not a normal (3-vinyl)chlorophyll a but a 3,8-divinylchlorophyll a. These results strongly suggest that the Slr1923 protein is essential for the conversion from divinylchlorophyll(ide) to normal chlorophyll(ide). We thus designate this gene cvrA (a gene indispensable for cyanobacterial vinyl reductase).     

Chondroitin 4-O-sulfotransferase-1 modulates Wnt-3a signaling through control of E disaccharide expression of chondroitin sulfate

Wnt-3a is a ligand that activates the beta-catenin-dependent pathway in Wnt signaling, which is implicated in numerous physiological events such as morphogenesis. So far, heparan sulfate (HS) proteoglycans have been highlighted as a low affinity receptor for morphogens containing Wnts. Here we show the importance of chondroitin sulfate (CS) proteoglycans in the efficient signaling of Wnt-3a and the structural features of CS required for the regulation of Wnt-3a signaling. Wnt-3a signaling was depressed in a mouse L cell mutant, called sog9, which is defective in the EXT1 gene encoding the HS-synthesizing enzyme and the chondroitin 4-O-sulfotransferase (C4ST-1) gene compared with parental L cells. The transfection of sog9 cells with C4ST-1 resulted in the recovery of Wnt-3a signaling, whereas the expression of EXT1 in sog9 cells could not restore Wnt-3a signaling. In addition, the expression level of introduced C4ST-1 correlated with the recovery of Wnt-3a signaling accompanied by the increased expression of the E disaccharide unit of CS. Interestingly, molecular interaction analyses using Biacore revealed that squid CS-E (rich in the E disaccharide unit) bound strongly to Wnt-3a (K(d)=13.2 nm) to the same extent as heparin from bovine lung (K(d)=8.43 nm). In contrast, other CS isoforms as well as HS isolated from bovine kidney showed little binding activity to Wnt-3a. Moreover, exogenously added CS-E potently inhibited the accumulation of beta-catenin induced by Wnt-3a. These results suggest that CS-E-like structures synthesized by C4ST-1 participate in Wnt-3a signaling and modulate the physiological events caused by Wnt-3a signals.     

Engineering of an <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolic pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.     

Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF ^317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF ^317A –bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l⁻¹ d-glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.     

Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and NAD⁺) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and NAD⁺ were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.     

Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid

Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.