Adverse effects of instrumentation in incubating Adélie penguins (Pygoscelis adeliae)

The use of data-loggers has permitted to explore the biology of free-ranging animals. However, this method has also been reported to reduce reproductive success while the reasons of this deleterious effect remain poorly documented. In this study, we aimed to identify critical periods of the breeding cycle of Adélie penguins (Pygoscelis adeliae) when the reproductive success may decrease because of instrumentation. For this purpose, we monitored 40 pairs, where one parent was instrumented before egg laying and 30 pairs without devices (controls). These pairs were followed at least during the incubation period but the majority was monitored during the entire breeding season. Reproductive success was affected in pairs where males were instrumented. This was not due to extra chick mortality during chick rearing but to a significantly lower hatching success. Moreover, the use of artificial eggs recording incubation temperatures and egg rotation indicated that in instrumented incubating males, eggs spent as much time at optimal incubation temperatures as control eggs but were rotated at a higher frequency. In Adélie penguins, males initiate incubation and it has been established that the early stage of incubation is one of the most critical periods for embryonic development. The low hatching rate observed in instrumented males was associated with a higher egg rotation rate, perhaps as a stress response to the presence of the instrument. Even though the causal effects remain unclear, instrumentation severely affected hatching success. For these reasons, we recommend equipping birds after the early incubation.     

Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: A study involving site-directed mutagenesis,HPLC, and real-time ESI-MS

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5–10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of + 1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC₅₀ value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from − 3 to − 1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.     

26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

A 26 kDa endochitinase from barley seeds was enzymatically characterized exclusively by electrospray ionization mass spectrometry (ESI-MS). At first, oligosaccharide hydrolysis catalyzed by the barley chitinase was monitored in real-time by ESI-MS. The reaction time-course obtained by ESI-MS monitoring was found to be consistent with the data obtained earlier by HPLC, and the quantitative profile was successfully simulated by kinetic modeling of the enzymatic hydrolysis. It is obvious that the real-time monitoring method by ESI-MS allows a faster and cheaper determination of the chitinase activity with unlabeled substrate. Further, the enzymatic activity of the E67Q mutant of the barley chitinase was analyzed and the role of Glu67 was discussed comparing the mass spectra of enzyme protein obtained in native and in denatured conditions. Then it was determined that the observed loss of the enzymatic activity in E67Q is definitely caused by a point mutation of Glu67 but not due to partial unfolding of the mutated enzyme. Finally, association constants of enzyme–oligosaccharide complexes were calculated from Scatchard plots obtained by mass spectra. The binding free energy values obtained for E67Q were found to be comparable to those previously obtained in liquid phase, but less dependent upon the chain length of the oligosaccharides. To our knowledge, this study is the first enzymatic characterization of chitinase exclusively by such an innovative ESI-MS system.     

Backbone 1H, 13C,and 15N assignments of yeast Ump1, an intrinsically disordered protein that functions as a proteasome assembly chaperone

Eukaryotic proteasome assembly is a highly organized process mediated by several proteasome-specific chaperones, which interact with proteasome assembly intermediates. In yeast, Ump1 and Pba1-4 have been identified as assembly chaperones that are dedicated to the formation of the proteasome 20S catalytic core complex. The crystal structures of Pba chaperones have been reported previously, but no detailed information has been provided for the structure of Ump1. Thus, to better understand the mechanisms underlying Ump1-mediated proteasome assembly, we characterized the conformation of Ump1 in solution using NMR. Backbone chemical shift data indicated that Ump1 is an intrinsically unstructured protein and largely devoid of secondary structural elements.     

Identification and Expression Analysis of Cytokinin Metabolic Genes in Soybean under Normal and Drought Conditions in Relation to Cytokinin Levels

Cytokinins (CKs) mediate cellular responses to drought stress and targeted control of CK metabolism can be used to develop drought-tolerant plants. Aiming to manipulate CK levels to improve drought tolerance of soybean cultivars through genetic engineering of CK metabolic genes, we surveyed the soybean genome and identified 14 CK biosynthetic (isopentenyltransferase, GmIPT) and 17 CK degradative (CK dehydrogenase, GmCKX) genes. Comparative analyses of GmIPTs and GmCKXs with Arabidopsis counterparts revealed their similar architecture. The average numbers of abiotic stress-inducible cis-elements per promoter were 0.4 and 1.2 for GmIPT and GmCKX genes, respectively, suggesting that upregulation of GmCKXs, thereby reduction of CK levels, maybe the major events under abiotic stresses. Indeed, the expression of 12 GmCKX genes was upregulated by dehydration in R2 roots. Overall, the expressions of soybean CK metabolic genes in various tissues at various stages were highly responsive to drought. CK contents in various organs at the reproductive (R2) stage were also determined under well-watered and drought stress conditions. Although tRNA-type GmIPT genes were highly expressed in soybean, cis-zeatin and its derivatives were found at low concentrations. Moreover, reduction of total CK content in R2 leaves under drought was attributable to the decrease in dihydrozeatin levels, suggesting a role of this molecule in regulating soybean's responses to drought stress. Our systematic analysis of the GmIPT and GmCKX families has provided an insight into CK metabolism in soybean under drought stress and a solid foundation for in-depth characterization and future development of improved drought-tolerant soybean cultivars by manipulation of CK levels via biotechnological approach.     

King penguin demography since the last glaciation inferred from genome-wide data

How natural climate cycles, such as past glacial/interglacial patterns, have shaped species distributions at the high-latitude regions of the Southern Hemisphere is still largely unclear. Here, we show how the post-glacial warming following the Last Glacial Maximum (ca 18 000 years ago), allowed the (re)colonization of the fragmented sub-Antarctic habitat by an upper-level marine predator, the king penguin Aptenodytes patagonicus. Using restriction site-associated DNA sequencing and standard mitochondrial data, we tested the behaviour of subsets of anonymous nuclear loci in inferring past demography through coalescent-based and allele frequency spectrum analyses. Our results show that the king penguin population breeding on Crozet archipelago steeply increased in size, closely following the Holocene warming recorded in the Epica Dome C ice core. The following population growth can be explained by a threshold model in which the ecological requirements of this species (year-round ice-free habitat for breeding and access to a major source of food such as the Antarctic Polar Front) were met on Crozet soon after the Pleistocene/Holocene climatic transition.