CYP710A genes encoding sterol C22-desaturase in <Emphasis Type="Italic">Physcomitrella patens</Emphasis> as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants

We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC–MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56–60% of total sterol) and sitosterol to a lesser extent (8–12%); this sterol profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14 proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with β-sitosterol to produce stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K _m values for β-sitosterol of CYP710A13 (1.0 ± 0.043 μM) and CYP710A14 (2.1 ± 0.17 μM) were at comparable levels of those reported with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Alternative complement pathway of channel catfish (Ictalurus punctatus): molecular characterization, mapping and expression analysis of factors Bf/C2 and Df

The complement system is important in both innate and adaptive host defense against microbial infection in vertebrates. It contains three pathways: the classical, alternative, and lectin pathways. Complement component factors B and D are two crucial proteases in the alternative pathway. In this study, the genes of complement factors Bf/C2 and Df from channel catfish, Ictalurus punctatus were identified and characterized. Two complement factor B-related genes, Bf/C2A and Bf/C2B, and factor D gene Df were identified. Phylogenetic analysis suggested that Bf/C2A and Bf/C2B is likely orthologous to factor B and factor C2, respectively. Southern blot results suggested that these three genes are all single-copy genes in the catfish genome. The catfish Bf/C2A, Bf/C2B and Df genes were genetically mapped on linkage group 3, 20 and 29, respectively. Bf/C2A and Bf/C2B are highly expressed in liver and kidney, while Df is highly expressed in gill and spleen. After infection with Edwardsiella ictaluri, the expression of Bf/C2A, Bf/C2B and Df genes were found to be remarkably induced in the gill, liver, spleen and kidney at some sampling times, indicating that these three complement factors play a pivotal role in immune responses after the bacterial infection in catfish.     

The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway

The C2 domain is a targeting domain that responds to intracellular Ca²⁺ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P₂ specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca²⁺ specifically binds to the Ca²⁺-binding region, facilitating PtdIns(4,5)P₂ access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P₂ binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P₂ binding are essential for the plasma membrane localization of PKCα when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P₂ and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P₂ is hydrolyzed to generate diacylglycerol and IP₃, an important amount still remains in the membrane where it is available to activate PKCα. These findings entail revision of the currently accepted model of PKCα recruitment to the membrane and its activation.     

Decomposition of soil and plant carbon from pasture systems after 9 years of exposure to elevated CO2: impact on C cycling and modeling

Elevated atmospheric CO₂ may alter decomposition rates through changes in plant material quality and through its impact on soil microbial activity. This study examines whether plant material produced under elevated CO₂ decomposes differently from plant material produced under ambient CO₂. Moreover, a long‐term experiment offered a unique opportunity to evaluate assumptions about C cycling under elevated CO₂ made in coupled climate–soil organic matter (SOM) models. Trifolium repens and Lolium perenne plant materials, produced under elevated (60 Pa) and ambient CO₂ at two levels of N fertilizer (140 vs. 560 kg ha^?1 yr^?1), were incubated in soil for 90 days. Soils and plant materials used for the incubation had been exposed to ambient and elevated CO₂ under free air carbon dioxide enrichment conditions and had received the N fertilizer for 9 years. The rate of decomposition of L. perenne and T. repens plant materials was unaffected by elevated atmospheric CO₂ and rate of N fertilization. Increases in L. perenne plant material C : N ratio under elevated CO₂ did not affect decomposition rates of the plant material. If under prolonged elevated CO₂ changes in soil microbial dynamics had occurred, they were not reflected in the rate of decomposition of the plant material. Only soil respiration under L. perenne, with or without incorporation of plant material, from the low‐N fertilization treatment was enhanced after exposure to elevated CO₂. This increase in soil respiration was not reflected in an increase in the microbial biomass of the L. perenne soil. The contribution of old and newly sequestered C to soil respiration, as revealed by the ¹³C‐CO₂ signature, reflected the turnover times of SOM–C pools as described by multipool SOM models. The results do not confirm the assumption of a negative feedback induced in the C cycle following an increase in CO₂, as used in coupled climate–SOM models. Moreover, this study showed no evidence for a positive feedback in the C cycle following additional N fertilization.     

Effects of climate and atmospheric CO2 partial pressure on the global distribution of C4 grasses: present, past, and future

C₄ photosynthetic physiologies exhibit fundamentally different responses to temperature and atmospheric CO₂ partial pressures (pCO₂) compared to the evolutionarily more primitive C₃ type. All else being equal, C₄ plants tend to be favored over C₃ plants in warm humid climates and, conversely, C₃ plants tend to be favored over C₄ plants in cool climates. Empirical observations supported by a photosynthesis model predict the existence of a climatological crossover temperature above which C₄ species have a carbon gain advantage and below which C₃ species are favored. Model calculations and analysis of current plant distribution suggest that this pCO₂-dependent crossover temperature is approximated by a mean temperature of 22°C for the warmest month at the current pCO₂ (35 Pa). In addition to favorable temperatures, C₄ plants require sufficient precipitation during the warm growing season. C₄ plants which are predominantly graminoids of short stature can be competitively excluded by trees (nearly all C₃ plants) – regardless of the photosynthetic superiority of the C₄ pathway – in regions otherwise favorable for C₄. To construct global maps of the distribution of C₄ grasses for current, past and future climate scenarios, we make use of climatological data sets which provide estimates of the mean monthly temperature to classify the globe into areas which should favor C₄ photosynthesis during at least 1 month of the year. This area is further screened by excluding areas where precipitation is <25 mm per month during the warm season and by selecting areas classified as grasslands (i.e., excluding areas dominated by woody vegetation) according to a global vegetation map. Using this approach, grasslands of the world are designated as C₃, C₄, and mixed under current climate and pCO₂. Published floristic studies were used to test the accuracy of these predictions in many regions of the world, and agreement with observations was generally good. We then make use of this protocol to examine changes in the global abundance of C₄ grasses in the past and the future using plausible estimates for the climates and pCO₂. When pCO₂ is lowered to pre-industrial levels, C₄ grasses expanded their range into large areas now classified as C₃ grasslands, especially in North America and Eurasia. During the last glacial maximum (∼18 ka BP) when the climate was cooler and pCO₂ was about 20 Pa, our analysis predicts substantial expansion of C₄ vegetation – particularly in Asia, despite cooler temperatures. Continued use of fossil fuels is expected to result in double the current pCO₂ by sometime in the next century, with some associated climate warming. Our analysis predicts a substantial reduction in the area of C₄ grasses under these conditions. These reductions from the past and into the future are based on greater stimulation of C₃ photosynthetic efficiency by higher pCO₂ than inhibition by higher temperatures. The predictions are testable through large-scale controlled growth studies and analysis of stable isotopes and other data from regions where large changes are predicted to have occurred. Received: 3 July 1997 / Accepted: 3 December 1997