PHYSIOLOGICAL SNAPSHOTS REFLECT ECOLOGICAL PERFORMANCE OF THE SEA PALM,POSTELSIA PALMAEFORMIS (PHAEOPHYCAEA) ACROSS INTERTIDAL ELEVATION AND EXPOSURE GRADIENTS1

Postelsia palmaeformis Ruprecht is an intertidal kelp found only on very wave‐exposed rocky shores of the northeast Pacific. In areas dominated by mussels, Postelsia depends on wave‐induced disturbances to complete its life‐history cycle. Postelsia also recruits where mussels are absent, but not at less wave‐exposed shores. Thus, physical conditions related to wave exposure limit its horizontal distribution. It is not clear what limits the vertical distribution of Postelsia. We investigated factors contributing to Postelsia's limited distribution using transplant experiments, demographic monitoring, and field fluorometry to evaluate growth and performance across gradients of tidal elevation and wave exposure. Survivorship and growth were sharply reduced at upper and wave‐protected edges relative to mid‐level, wave‐exposed sporophytes. Reproductive output was reduced at upper and lower levels, and growth but not survivorship was lower at the lower level. Effects were independent of population of origin and were a manifestation of the environment. Maximum electron transport rates (ETR_m), light saturation parameters (E_k), and maximum quantum yields (ΔF/F_m) provided insight into physiological dynamics; all were lowest at the high edge, but increased when desiccation stress was alleviated by a mock sea‐spray treatment. The ETR_m and E_k values of low sporophytes were not as high as the values for mid‐sporophytes, despite higher or equivalent nitrogen content, chl a, and absorptance, suggesting a trade‐off between light‐capturing and carbon‐fixation capacity. Physiological limitations at upper and lower levels and deleterious desiccation effects at wave‐protected sites prevent establishment, thus constraining Postelsia to a mid‐zone, wave‐exposed distribution. Physical conditions related to wave exposure may limit the horizontal distribution of Postelsia because this kelp is also found in areas where mussels are lacking but not on less wave‐exposed shores.     

Chromatic photoacclimation, photosynthetic electron transport and oxygen evolution in the Chlorophyll d-containing oxyphotobacterium Acaryochloris marina

Changes in photosynthetic pigment ratios showed that the Chlorophyll d-dominated oxyphotobacterium Acaryochloris marina was able to photoacclimate to different light regimes. Chl d per cell were higher in cultures grown under low irradiance and red or green light compared to those found when grown under high white light, but phycocyanin/Chl d and carotenoid/Chl d indices under the corresponding conditions were lower. Chl a, considered an accessory pigment in this organism, decreased respective to Chl d in low irradiance and low intensity non-white light sources. Blue diode PAM (Pulse Amplitude Modulation) fluorometry was able to be used to measure photosynthesis in Acaryochloris. Light response curves for Acaryochloris were created using both PAM and O₂ electrode. A linear relationship was found between electron transport rate (ETR), measured using a PAM fluorometer, and oxygen evolution (net and gross photosynthesis). Gross photosynthesis and ETR were directly proportional to one another. The optimum light for white light (quartz halogen) was about 206 ± 51 μmol m^− 2 s^− 1 (PAR) (Photosynthetically Active Radiation), whereas for red light (red diodes) the optimum light was lower (109 ± 27 μmol m^− 2 s^− 1 (PAR)). The maximum mean gross photosynthetic rate of Acaryochloris was 73 ± 7 μmol mg Chl d^− 1 h^− 1. The gross photosynthesis/respiration ratio (P_g/R) of Acaryochloris under optimum conditions was about 4.02 ± 1.69. The implications of our findings will be discussed in relation to how photosynthesis is regulated in Acaryochloris.     

Influence of UV radiation and visible light on Porphyra umbilicalis: Photoinhibition and MAA concentration

Porphyra umbilicalis was cultured under constant light conditions but showed a diurnal pattern in chlorophyll fluorescence. Photoinhibition after light treatment was determined by PAM fluorescence measurements. Treatment with only UV irradiation caused a slow but steady decline in the effective photosynthetic quantum yield from which there was no recovery. Solar simulated irradiation led to a large decrease in quantum yield after short periods of irradiation; partial recovery occurred after shading the samples. No significant difference was found between samples exposed to PAR only or to PAR + UV-A and/or UV-B irradiation. Determination of mycosporine-like amino acids (MAAs)before and during exposure to solar simulated irradiation showed a high initial concentration of MAAs but no increase due to the irradiance treatment. This revised version was published online in August 2006 with corrections to the Cover Date.     

An HPLC‐ESMS study on the solid‐phase assembly of C‐terminal proline peptides

DKP formation is a serious side reaction during the solid‐phase synthesis of peptide acids containing either Pro or Gly at the C‐terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt‐Cl‐resin with a limited incorporation of the C‐terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC‐ESMS is a useful method for analysing complex samples, such as those formed when C‐terminal Pro peptides are prepared by non‐optimized solid‐phase strategies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes

This Letter details our efforts to develop new M₄ PAM scaffolds with improved pharmacological properties. This endeavor involved replacing the 3,4-dimethylpyridazine core with two novel cores: a 2,3-dimethyl-2H-indazole-5-carboxamide core or a 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide core. Due to shallow SAR, these cores were further evolved into two unique tricyclic cores: an 8,9-dimethyl-8H-pyrazolo[3,4-h]quinazoline core and an 1-methyl-1H-[1,2,3]triazolo[4,5-h]quinazoline core. Both tricyclic cores displayed low nanomolar potency against both human and rat M₄.     

Cas9蛋白变体VQR高效识别水稻NGAC前间区序列邻近基序

成簇的规律间隔短回文重复序列及CRISPR相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated 9, CRISPR/Cas9)系统是近年来发展起来并被广泛应用的第三代基因组编辑工具。但是,该系统的酿脓链球菌Cas9(Streptococcus pyogenes, SpCas9)仅能识别NGG前间区序列邻近基序(protospacer adjacent motif, PAM),极大地限制了基因组编辑的范围。SpCas9变体VQR(D1135V/R1335Q/T1337R)在水稻中可识别NGAA、NGAG和NGAT PAM,但尚不清楚是否能识别NGAC PAM。本研究利用改进后的CRISPR/VQR系统对水稻中3个相对低效的VQR靶位点NAL1-Q1、NAL1-Q2和LPA1-Q进行了编辑,结果表明改进后的CRISPR/VQR系统可以高效编辑这3个靶位点,编辑效率分别为9.75%、43.90%和29.26%。为了明确改进后的CRISPR/VQR系统对NGAC PAM的识别情况,本研究选择水稻叶片宽度调控基因NARROW LEAF 1 (NAL1)中的NAL-C位点和蜡质合成基因GLOSSY1 (GL1)中的GL1-C位点进行基因编辑,并获得57株转基因水稻。靶位点PCR扩增及测序结果表明,NAL1-C和GL1-C靶标位点突变的植株分别为27株和44株,突变率分别为47.36%和77.19%;其中NAL1-C/GL1-C双突变植株为26株,双突变率为45.61%。进一步分析表明,CRISPR/VQR系统造成的突变有4种类型,分别为杂合突变、双等位突变、嵌合体突变和纯合突变,其中以杂合突变和双等位突变为主。这些结果表明,改进的CRISPR/VQR系统可以高效编辑水稻NGAC PAM位点,并产生丰富的突变类型。本研究为水稻及其他植物相关基因NGAC PAM位点的编辑提供了理论依据。     

Submerged versus air-exposed intertidal macrophyte productivity: from physiological to community-level assessments

The photosynthetic productivity of the intertidal communities dominated by the seagrass Zostera noltii and the cordgrass Spartina maritima was assessed in two contrasting situations during a tidal cycle, i.e., air exposure and water immersion. Two complementary methods were used: infra red gas analysis of CO₂ flux measurements in whole communities and chlorophyll a fluorescence measurements of individual plants photosynthetic activity. Higher photosynthetic rates of Z. noltii in air were observed both at the individual plants response level determined by chlorophyll fluorescence and at the community level measured as gas exchange (CO₂ uptake). S. maritima plants consistently showed low photosynthetic response when immersed. Gross community production (GCP) measured as carbon dioxide uptake was always higher in air than in water for both communities. When immersed, the GCP of both communities was similar. However, when exposed to the air, the GCP of the S. maritima community was higher than the one of Z. noltii's. The key factor in CO₂ assimilation by air-exposed Z. noltii was the retention of water in sediment microdepressions. During low tide, depressions in the sediment retain a considerable amount of water, enough to maintain leaf hydration. In these conditions, rapid air-water CO₂ diffusion occurs, making it readily available to plants. The community gas exchange measurements compared well with the fluorescence indications. Both Z. noltii and S. maritima were shown to be responsible for the overall pattern of photosynthetic carbon fixation within their respective communities, both during submersion and emersion periods. The short-term incubations method described in this report proved to be a valuable tool for field measurements of intertidal lagoon productivity. It provides fast and precise values of carbon dioxide fixation, both in submerged and air-exposed communities.     

The role of niche measures in explaining the abundance–distribution relationship in tropical lotic chironomids

In situ measurements of both community metabolism (primary production and respiration) and PAM fluorometry were conducted during emersion on intertidal sediments in the Mont Saint-Michel Bay, in areas where oysters and mussels were cultivated. Results highlighted a low benthic metabolism compared to other intertidal areas previously investigated with the same methods. Comparisons between gross community primary production and relative electron transport rates confirmed this statement. More specifically, primary productivity remained very low all over the year, whereas the associated microalgal biomass was estimated to be high. We suggest that the microphytobenthic community studied was characterized by a self-limitation of its primary productivity by its own biomass, as previously shown in Marennes-Oléron Bay for example. The almost permanent high biomass would represent a limiting factor for micromigration processes within the first millimetres of the sediment. This could be explained by very low resuspension processes occurring in the western part of the bay, enhanced by the occurrence of numerous aquaculture structures that could decrease tidal currents in the benthic boundary layer. Handling editor: N. Desroy     

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

In Synechocystis sp. PCC 6803, the loop domain (aa 1–70) of the phycobilisome core-membrane linker, L_CM, was found to interact with the glycosyl transferase homolog, Sll1466. Growth of a Sll1466 knock-out mutant was slightly faster in low light, but strongly inhibited in high light; the phenotype is discussed in relation to the regulation of light energy transfer to photosystem II. At the molecular level, the mutant shows the following changes compared to the wild type: (1) a smaller size and higher mobility of phycobilisomes on the thylakoid membrane, and (2) a changed lipid composition of the thylakoid membrane, especially decreased amounts of digalactosyl diacylglycerol. These results indicate a profound regulatory role for Sll1466 in regulating photosynthetic energy transfer.