First Report of Colletotrichum gloeosporioides Causing Anthracnose on Wisteria sinensis in China.

Wisteria (Wisteria sinensis) is a well-known ornamental plant for environmental protection in the garden, which also has a high value for medicinal use. In December 2021, leaf spots were observed on W. sinensis plants growing on the campus of Jiangxi Agricultural University in Nanchang, Jiangxi Province (28.45° N, 115.49° E), with a incidence rate of 40% plants were infested (n = 100 investigated plants). Initially leaf spots were small and pale brown (Approx. 2 mm in diameter), which gradually expanded into round or irregular dark brown spots as disease progressed, and lesions developed greyish-white necrotic tissues in the center at later stages, eventually causing the leaves to rot. To isolate the pathogen, tissues (5 × 5 mm) at the margin of lesions were cut from ten symptomatic leaves, surface disinfected with 75% ethanol for 30 s followed by 2% sodium chloride (NaClO) for 1 min, rinsed three times with sterile distilled water, and the dried tissues were cultured on potato dextrose agar (PDA) at 28 ± 1℃ in darkness for 3 days. After culture purification, five isolates were obtained and the representative single spore isolate (ZTTJ1) was used for subsequent identification tests. After 10 days of incubation on PDA medium, colonies had dense aerial mycelium with a gray center and dark gray-green mycelium outward, with orange-red conidial masses distributed in a ring on the surface. The underside of the colonies was light gray to dark gray. Conidia were cylindrical, with ends obtuse-rounded, 11.83 to 20.74 × 3.34 to 5.33 µm (av=16.11 µm × 4.26 µm, n = 50) in size. These morphological characteristics were consistent with Colletotrichum gloeosporioides (Shi et al, 2019). Six conserved regions of isolate (ZTTJ1), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), ß-tublin (TUB), actin (ACT), and chitin synthase 1 (CHS1) gene regions were amplified using ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Li et al, 2018), Bt2a/Bt2b (Prihastuti et al, 2009), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999) primers, respectively. Using BLAST, ITS, GAPDH, CAL, TUB, ACT, and CHS1 gene sequences (GenBank Accession No. OP703312, OP713773, OP713775, OP713776, OP713772, OP713774, respectively) were over 99% identical to C. gloeosporioides (GenBank Accession No. MK967281, MH594288, MT449307, MN624110, MN107239 and MN908602, respectively). A maximum likelihood (ML) phylogenetic analysis based on ITS-ACT-GAPDH-CHS1-CAL-TUB2 sequences using MEGA7.0, placed isolate (ZTTJ1) within C. gloeosporioides. To complete Koch's postulates, 10 µL spore suspension (1.0 × 106 conidia/mL) of ZTTJ1 (7-day-old culture on PDA medium) was dropped onto 10 leaves wounded with a sterilized needle and 10 non-wounded leaves, respectively. Ten wounded leaves were inoculated with sterile water as controls. All leaves were incubated at 28 ± 1 °C and 90 % relative humidity (12 h/12 h light/dark). After 7 days, all wounded leaves inoculated with C. gloeosporioides developed symptoms as previously observed, while the control and non-wounded leaves remained healthy. The fungus re-isolated from the inoculated leaves were identified as C. gloeosporioides by morphological and molecular identification; the pathogen causing disease in W. sinensis was determined to be C. gloeosporioides. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. sinensis in China. This work has identified the pathogenic species of the disease, which helps to take targeted measures to control its spread, providing a basis for the prevention and treatment of the disease.

More opportunities more species: Pleistocene differentiation and northward expansion of an evergreen broad-leaved tree species Machilus thunbergii (Lauraceae) in Southeast China.

BACKGROUND: The broad continuum between tropical and temperate floras in Eastern Asia (EAS) are thought to be one of the main factors responsible for a prominent species diversity anomaly of temperate plants between EAS and eastern North America (ENS). However, how the broad continuum and niche evolution between tropical and temperate floras in EAS contributes to lineage divergence and species diversity remains largely unknown. RESULTS: Population genetic structure, demography, and determinants of genetic structure [i.e., isolation-by-distance (IBD), isolation-by-resistance (IBR), and isolation-by-environment (IBE)] of Machilus thunbergii Sieb. et Zucc. (Lauraceae) were evaluated by examining sequence variation of ten low-copy nuclear genes across 43 populations in southeast China. Climatic niche difference and potential distributions across four periods (Current, mid-Holocene, the last glacial maximum, the last interglacial) of two genetic clusters were determined by niche modelling. North and south clusters of populations in M. thunbergii were revealed and their demarcation line corresponds well with the northern boundary of tropical zone in China of Zhu & Wan. The divergence time between the clusters and demographic expansion of M. thunbergii occurred after the mid-Pleistocene climate transition (MPT, 0.8-1.2 Ma). Migration rates between clusters were asymmetrical, being much greater from north to south than the reverse. Significant effects of IBE, but non-significant effects of IBD and IBR on population genetic divergence were detected. The two clusters have different ecological niches and require different temperature regimes. CONCLUSIONS: The north-south genetic differentiation may be common across the temperate-tropical boundary in southeast China. Divergent selection under different temperature regimes (possibly above and below freezing temperature in winter) could account for this divergence pattern. The broad continuum between tropical and temperate floras in EAS may have provided ample opportunities for tropical plant lineages to acquire freezing tolerance and to colonize the temperate regions during the late-Cenozoic global cooling. Our findings shed deeper insights into the high temperate plant species diversity in EAS.

First Report of Colletotrichum karstii Causing Anthracnose of Begonia lanternaria Irmsch. in China.

Begonia lanternaria Irmsch., an ornamental plant endemic in China, which is commonly used in landscape and interior decoration. In March 2021, an estimated 30% B. lanternaria plants were observed with anthracnose-like symptoms at a botanical garden conservation greenhouse in Mengla County of Yunnan Province (21.91° N, 101.21°E). Initially, small black spots developed on the disease leaves, which gradually expanded into irregular necrotic lesions surrounded by a yellowish halo, eventually turned wilting and defoliating. Twenty diseased leaves were collected and surface-disinfested with 75% ethanol for 30 s. Small fragments (5 × 5 mm) from the margin of lesions were disinfected with 1% NaClO for 120 s, washed with sterile water three times, and cultured on potato dextrose agar (PDA) at 28 ± 1℃. After 3 days single spores from four fungal colonies with identical morphology were isolated. Colonies on PDA were 70-75 mm diam in 7 d (7.5-10.6 mm/d), with dense white to gray-white mycelia attached with brown to black-brown acervulus. The underside of the culture was yellow to yellowish-brown concentric circle. Conidia were single-celled, hyaline, straight to slightly curved, cylindrical, 12.88 to 16.66 × 6.25 to 7.97 µm (av=14.65 µm × 7.22 µm, n=50) in size. For molecular identification, genomic DNA was extracted from a representative isolate, and the internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, calmodulin gene, ß-tublin, actin, and chitin synthase 1 genes were amplified with ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Li et al, 2018), Bt2a/Bt2b (Prihastuti et al, 2009), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999) primers, respectively. The obtained DNA sequences showed over 99% homology with Colletotrichum karsti (GenBank Accession No. ITS: NR144790; GAPDH: KX578772; CAL: KY039988; TUB2: KX578804; ACT: LC412408; CHS1: KU251855), and the results of sequences were deposited into GenBank with accession No. MZ496954 (522/522 bp), MZ504978 (238/238 bp), MZ504979 (737/737 bp), MZ504982 (472/472 bp), MZ504981 (273/273 bp), MZ504980 (282/284 bp). The phylogenetic tree combined with ITS-ACT-GAPDH-CHS 1-CAL-TUB2 concatenated sequences using the maximum likelihood methods showed that the isolate was C. karsti. To confirm pathogenicity, Koch's postulates were conducted on intact plants, 10 µl spore suspension (1.0 × 106 conidia/ml) of each of four isolates (7-day-old culture on PDA) was inoculated on 15 wounded with a sterilized needle or non-wounded healthy living leaves, and 15 wounded leaves were inoculated with sterile water as controls. All leaves were incubated at 28 ± 1°C and 90% relative humidity (12 h/12 h light/dark). After 5 days, all wounded leaves inoculated with C. karsti showed symptoms similar to those previously observed, while the control and non-wounded leaves remained healthy. Colletotrichum karsti was re-isolated from inoculated leaves. C. karsti was previously reported to cause disease on Nicotiana tabacum L. (Zhao et al, 2020), Stylosanthes guianensis (Jia et al, 2017) and Fatsia japonica (Xu et al, 2020) in China. To our knowledge, this is the first report of C. karsti causing anthracnose of B. lanternaria Irmsch. in China. This disease reduces the ornamental and economic value of B. lanternaria Irmsch., and this work will provide a basis for the prevention and treatment of the disease in the future.

Evolutionary history of a relict conifer, Pseudotaxus chienii (Taxaceae), in south-east China during the late Neogene: old lineage, young populations.

BACKGROUND AND AIMS: Many monotypic gymnosperm lineages in south-east China paradoxically remain in relict status despite long evolutionary histories and ample opportunities for allopatric speciation, but this paradox has received little attention and has yet to be resolved. Here, we address this issue by investigating the evolutionary history of a relict conifer, Pseudotaxus chienii (Taxaceae). METHODS: DNA sequences from two chloroplast regions and 14 nuclear loci were obtained for 134 samples. The demographic history was inferred and the contribution of isolation by environment (IBE) in patterning genetic divergence was compared with that of isolation by distance (IBD). KEY RESULTS: Three genetic clusters were identified. Approximate Bayesian computation analyses showed that the three clusters diverged in the late Pliocene (~3.68 Ma) and two admixture events were detected. Asymmetric gene flow and similar population divergence times (~ 3.74 Ma) were characterized using the isolation with migration model. Neither IBD nor IBE contributed significantly to genetic divergence, and the contribution of IBE was much smaller than that of IBD. CONCLUSIONS: These results suggest that several monotypic relict gymnosperm lineages like P. chienii in south-east China did not remain in situ and undiversified for millions of years. On the contrary, they have been evolving and the extant populations have become established more recently, having insufficient time to speciate. Our findings provide a new perspective for understanding the formation and evolution of the relict gymnosperm flora of China as well as of the Sino-Japanese Flora.

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN-truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment.

Phylogeography of Sophora moorcroftiana Supports Wu's Hypothesis on the Origin of Tibetan Alpine Flora.

Wu hypothesized that the Tibetan flora originated mostly from the paleotropical Tertiary flora in the Hengduan Mountains by adapting to the cold and arid environments associated with the strong uplift of the Qinghai-Tibet Plateau (QTP). Here, we combine the phylogeographic history of Sophora moorcroftiana with that of Sophora davidii to explore the speciation of S. moorcroftiana to test this hypothesis. We collected 151 individuals from 17 populations and sequenced 2 chloroplast fragments and the internal transcribed spacer of rDNA. Five chlorotypes and 9 ribotypes were detected but no significant phylogeographic structure was revealed. The integrated results of phylogeographic studies of these 2 species clearly support the progenitor-derivative relationship between them. We infer that the western peripheral population of S. davidii migrated westwards from the Hengduan Mountains to the middle reaches of the Yarlung Zangbo River and differentiated from its ancestor in the process of adaptation to increasingly cold and arid environments with the uplift of the QTP and finally evolved into S. moorcroftiana during the Late Pliocene. In addition, our findings shed light on the idea that natural selection, as imposed by climate differentiation (especially mean diurnal range and precipitation seasonality), directly drove this peripatric speciation event after geographic isolation. The speciation of S. moorcroftiana is a strong case supporting Wu's hypothesis about the origin of Tibet's flora.

Computational Studies on Acetylcholinesterases.

Functions of biomolecules, in particular enzymes, are usually modulated by structural fluctuations. This is especially the case in a gated diffusion-controlled reaction catalyzed by an enzyme such as acetylcholinesterase. The catalytic triad of acetylcholinesterase is located at the bottom of a long and narrow gorge, but it catalyzes the extremely rapid hydrolysis of the neurotransmitter, acetylcholine, with a reaction rate close to the diffusion-controlled limit. Computational modeling and simulation have produced considerable advances in exploring the dynamical and conformational properties of biomolecules, not only aiding in interpreting the experimental data, but also providing insights into the internal motions of the biomolecule at the atomic level. Given the remarkably high catalytic efficiency and the importance of acetylcholinesterase in drug development, great efforts have been made to understand the dynamics associated with its functions by use of various computational methods. Here, we present a comprehensive overview of recent computational studies on acetylcholinesterase, expanding our views of the enzyme from a microstate of a single structure to conformational ensembles, strengthening our understanding of the integration of structure, dynamics and function associated with the enzyme, and promoting the structure-based and/or mechanism-based design of new inhibitors for it.

Grain yield and quality performances of different late-season rice cultivars in response to experimental warming in subtropical China.

Introduction: Climate warming has pronounced effects on rice production in China. However, late-seasons rice cultivars are diverse in double rice cropping systems, and the actual responses in grain yield and quality of different late-season rice cultivars to climate warming are still unclear. Methods: A two-year field warming experiment was conducted by using free-air temperature increase facilities with three widely-planted late-season rice cultivars, including Taiyou398 (TY, short growth duration indica hybrid rice), Jiuxiangnian (JXN, long growth duration indica inbred rice), and Yongyou1538 (YY, long growth duration indica-japonica hybrid rice) in a double rice cropping system in subtropical China. Results: Warming (1.9-2.0°C) had no significant effects on the grain yields of TY and JXN, but significantly decreased that of YY by 4.8% relative to ambient treatment due to a reduction of spikelet number. Compared to ambient treatment, the head rice yields of TY and YY did not change while that of JXN increased by 6.3% under warming conditions. Warming significantly increased the head rice rates of JXN and YY by 6.6% and 7.8%, and the chalky grain rates of TY, JXN, and YY by 79.1%, 21.6%, and 7.6%, respectively. Under warming conditions, the amylose content of JXN and YY decreased significantly by 7.5% and 8.8%, and the setback of three cultivars decreased significantly by an average of 41.5%. Conclusion: Warming could improve the milling and eating qualities of long growth duration late-season rice (JXN and YY) and increase or maintain their head rice yield, even though decreased the grain yield of indica-japonica hybrid rice (YY). These results will provide a better understanding for the selection of suitable late-season rice cultivars under future climate warming conditions.

Peripatric speciation within Torreya fargesii (Taxaceae) in the Hengduan Mountains inferred from multi-loci phylogeography.

BACKGROUND: The Hengduan Mountains (HDM) are one of the major global biodiversity hotspots in the world. Several evolutionary scenarios, especially in-situ diversification, have been proposed to account for the high species richness of temperate plants. However, peripatric speciation, an important mode of allopatric speciation, has seldom been reported in this region. RESULTS: Here, two chloroplast DNA regions and 14 nuclear loci were sequenced for 112 individuals from 10 populations of Torreya fargesii var. fargesii and 63 individuals from 6 populations of T. fargesii var. yunnanensis. Population genetic analyses revealed that the two varieties are well differentiated genetically (FST, 0.5765) and have uneven genetic diversity (π, 0.00221 vs. 0.00073 on an average of nuclear loci). The gene genealogical relationship showed that T. fargesii var. yunnanensis is inferred as derived from T. fargesii var. fargesii, which was further supported by the coalescent simulations (DIYABC, fastsimcoal2 and IMa2). By the coalescent simulations, the divergence time (~ 2.50-3.65 Ma) and the weak gene flow between the two varieties were detected. The gene flow was asymmetrical and only occurred in later stages of divergence, which is caused by second contact due to the population expansion (~ 0.61 Ma) in T. fargesii var. fargesii. In addition, niche modeling indicated that the two varieties are differentiated geographically and ecologically and have unbalanced distribution range. CONCLUSIONS: Overall, T. fargesii var. fargesii is always parapatric with respect to T. fargesii var. yunnanensis, and the latter derived from the former in peripatry of the HDM following a colonization from central China during the late Pliocene. Our findings demonstrate that peripatric speciation following dispersal events may be an important evolutionary scenario for the formation of biodiversity hotspot of the HDM.

Climate heterogeneity shapes phylogeographic pattern of Hippophae gyantsensis (Elaeagnaceae) in the east Himalaya-Hengduan Mountains.

The interaction of recent orographic uplift and climate heterogeneity acted as a key role in the East Himalaya-Hengduan Mountains (EHHM) has been reported in many studies. However, how exactly the interaction promotes clade diversification remains poorly understood. In this study, we both used the chloroplast trnT-trnF region and 11 nuclear microsatellite loci to investigate the phylogeographic structure and population dynamics of Hippophae gyantsensis and estimate what role geological barriers or ecological factors play in the spatial genetic structure. The results showed that this species had a strong east-west phylogeographic structure, with several mixed populations identified from microsatellite data in central location. The intraspecies divergence time was estimated to be about 3.59 Ma, corresponding well with the recent uplift of the Tibetan Plateau. Between the two lineages, there was significant climatic differentiation without geographic barriers. High consistency between lineage divergence, climatic heterogeneity, and Qingzang Movement demonstrated that climatic heterogeneity but not geographic isolation drives the divergence of H. gyantsensis, and the recent regional uplift of the QTP, as the Himalayas, creates heterogeneous climates by affecting the flow of the Indian monsoon. The east group of H. gyantsensis experienced population expansion c. 0.12 Ma, closely associated with the last interglacial interval. Subsequently, a genetic admixture event between east and west groups happened at 26.90 ka, a period corresponding to the warm inter-glaciation again. These findings highlight the importance of the Quaternary climatic fluctuations in the recent evolutionary history of H. gyantsensis. Our study will improve the understanding of the history and mechanisms of biodiversity accumulation in the EHHM region.

Differentiation of Morphological Traits and Genome-Wide Expression Patterns between Rice Subspecies Indica and Japonica.

Changes in gene expression patterns can lead to the variation of morphological traits. This phenomenon is particularly evident in recent evolution events such as crop domestication and responses to environmental stress, where alterations in expression levels can efficiently give rise to domesticated syndromes and adaptive phenotypes. Rice (Oryza sativa L.), one of the world's most crucial cereal crops, comprises two morphologically distinct subspecies, Indica and Japonica. To investigate the morphological divergence between these two rice subspecies, this study planted a total of 315 landrace individuals of both Indica and Japonica under identical cultivation conditions. Out of the 16 quantitative traits measured in this study, 12 exhibited significant differences between the subspecies. To determine the genetic divergence between Indica and Japonica at the whole-genome sequence level, we constructed a phylogenetic tree using a resequencing dataset encompassing 95 rice landrace accessions. The samples formed two major groups that neatly corresponded to the two subspecies, Indica and Japonica. Furthermore, neighbor-joining (NJ) trees based on the expression quantity of effectively expressed genes (EEGs) across five different tissues categorized 12 representative samples into two major clades aligning with the two subspecies. These results imply that divergence in genome-wide expression levels undergoes stabilizing selection under non-stressful conditions, with evolutionary trends in expression levels mirroring sequence variation levels. This study further supports the pivotal role of changes in genome-wide expression regulation in the divergence of the two rice subspecies, Indica and Japonica.

Species Delimitation of Asteropyrum (Ranunculaceae) Based on Morphological, Molecular, and Ecological Variation.

Objectively evaluating different lines of evidence within a formalized framework is the most efficient and theoretically grounded approach for defining robust species hypotheses. Asteropyrum Drumm. et Hutch. is a small genus of perennial herb containing two species, A. cavaleriei and A. peltatum. The distinction of these two species mainly lies in the shape and size of leaf blades. However, these characters have been considered labile and could not differentiate the two species reliably. In this study, we investigated the variation of the leaf blades of 28 populations across the whole range of Asteropyrum using the landmark-based geometric morphometrics (GMM), sought genetic gaps within this genus using DNA barcoding, phylogenetic reconstruction and population genetic methods, and compared the predicted ecological niches of the two species. The results showed that the leaf form (shape and size) was overlapped between the two species; barcode gap was not detected within the genus Asteropyrum; and little ecological and geographical differentiation was found between the two taxa. Two genetic clusters detected by population genetic analysis did not match the two morphospecies. The results suggest that there are no distinct boundaries between the two species of Asteropyrum in terms of morphology, genetics and ecology and this present classification should be abandoned. We anticipate that range-wide population genomic studies would properly delineate the species boundaries and help to understand the evolution and speciation within Asteropyrum.

How sequence determines elasticity of disordered proteins.

How nature tunes sequences of disordered protein to yield the desired coiling properties is not yet well understood. To shed light on the relationship between protein sequence and elasticity, we here investigate four different natural disordered proteins with elastomeric function, namely: FG repeats in the nucleoporins; resilin in the wing tendon of dragonfly; PPAK in the muscle protein titin; and spider silk. We obtain force-extension curves for these proteins from extensive explicit solvent molecular dynamics simulations, which we compare to purely entropic coiling by modeling the four proteins as entropic chains. Although proline and glycine content are in general indicators for the entropic elasticity as expected, divergence from simple additivity is observed. Namely, coiling propensities correlate with polyproline II content more strongly than with proline content, and given a preponderance of glycines for sufficient backbone flexibility, nonlocal interactions such as electrostatic forces can result in strongly enhanced coiling, which results for the case of resilin in a distinct hump in the force-extension curve. Our results, which are directly testable by force spectroscopy experiments, shed light on how evolution has designed unfolded elastomeric proteins for different functions.

Glycosylation enhances peptide hydrophobic collapse by impairing solvation.

Post-translational N-glycosylation of proteins is ubiquitous in eukaryotic cells, and has been shown to influence the thermodynamics of protein collapse and folding. However, the mechanism for this influence is not well understood. All-atom molecular dynamics simulations are carried out to study the collapse of a peptide linked to a single N-glycan. The glycan is shown to perturb the local water hydrogen-bonding network, rendering it less able to solvate the peptide and thus enhancing the hydrophobic contribution to the free energy of collapse. The enhancement of the hydrophobic collapse compensates for the weakened entropic coiling due to the bulky glycan chain and leads to a stronger burial of hydrophobic surface, presumably enhancing folding. This conclusion is reinforced by comparison with coarse-grained simulations, which contain no explicit solvent and correspondingly exhibit no significant thermodynamic changes on glycosylation.

Evolutionary Hotspots of Seed Plants in Subtropical China: A Comparison With Species Diversity Hotspots of Woody Seed Plants.

Genetic diversity is a fundamental level of biodiversity. However, it is frequently neglected in conservation prioritization because intraspecific genetic diversity is difficult to measure at large scales. In this study, we synthesized population genetic or phylogeographic datasets of 33 seed plants in subtropical China into multi-species genetic landscapes. The genetic landscapes identified 18 evolutionary hotspots with high within-population genetic diversity (WGD), and among-population genetic diversity (AGD), or both. The western subtropical China is rich in AGD (possessing four major AGD hotspots), deserving a high conservation priority. We found that WGD was positively correlated with longitude, with most WGD hotspots locating in east subtropical China. The results showed that the locations of 12 of 18 evolutionary hotspots corresponded approximately to those of previously identified species diversity (SD) hotspots, however, a positive and significant correlation existed only between AGD and SD, not between WGD and SD. Therefore, spatial patterns of species richness in plants in subtropical China cannot generally be used as surrogate for their intraspecific diversity. This study identified multi-species evolutionary hotspots and correlated multi-species genetic diversity with SD across subtropical China for the first time, providing profound implications for the conservation of biodiversity in this important ecoregion.

Gorge Motions of Acetylcholinesterase Revealed by Microsecond Molecular Dynamics Simulations.

Acetylcholinesterase, with a deep, narrow active-site gorge, attracts enormous interest due to its particularly high catalytic efficiency and its inhibitors used for treatment of Alzheimer's disease. To facilitate the massive pass-through of the substrate and inhibitors, "breathing" motions to modulate the size of the gorge are an important prerequisite. However, the molecular mechanism that governs such motions is not well explored. Here, to systematically investigate intrinsic motions of the enzyme, we performed microsecond molecular dynamics simulations on the monomer and dimer of Torpedo californica acetylcholinesterase (TcAChE) as well as the complex of TcAChE bound with the drug E2020. It has been revealed that protein-ligand interactions and dimerization both keep the gorge in bulk, and opening events of the gorge increase dramatically compared to the monomer. Dynamics of three subdomains, S3, S4 and the Ω-loop, are tightly associated with variations of the gorge size while the dynamics can be changed by ligand binding or protein dimerization. Moreover, high correlations among these subdomains provide a basis for remote residues allosterically modulating the gorge motions. These observations are propitious to expand our understanding of protein structure and function as well as providing clues for performing structure-based drug design.

Transition state ensemble for the folding of B domain of protein A: a comparison of distributed molecular dynamics simulations with experiments.

Folding pathways of the B domain of staphylococcal protein A have been sampled with a distributed computing approach. Starting from an extended structure, the method employs an index measuring topological similarity to the native structure to selectively sample trajectory branches leading to the native fold. Unperturbed and continuous folding trajectories are drawn on a physics-based atomic potential energy surface with an implicit solvent. The sampled folding trajectories demonstrate a similar sequence of events: the earlier stage involves a partial formation of helix 2 and to a less extent of helix 1 at their N terminals, followed by the hydrophobic collapse between residues F14, I17, and L18 on helix 1 and residues R28, F31, and I32 on helix 2, which results in the rigidification of the helix turn from R28 to I32. Helix 2 is then able to extend, allowing for the formation to turn 2. The above description explains one experimental result why a G30A mutant of the protein was observed to be the fastest folder among proteins of its size. And the ensemble of structures right before the final collapse is in good agreement with the transition state ensemble mapped by another recent experiment with Fersht Phi values. We emphasize that because the approach here does not provide quantifications of the free energy landscape, our model of the transition state ensemble emerges from comparisons of simulations and previous experimental results rather from the simulation results alone. On the other hand, as our approach does not rely on a low-dimensional free energy surface, it can complement methods based on the construction of free energy surfaces.

Base-catalyzed peptide hydrolysis is insensitive to mechanical stress.

Biochemical reactions can be guided by mechanical stress. An external force has been previously shown both experimentally and theoretically to act as a catalyst for the scission of a disulfide bond in thiol/disulfide exchange reactions. How the dynamics of peptide hydrolysis, one of the most prevalent biochemical reactions, is influenced by a stretching force was investigated here using combined quantum and molecular mechanical (QM/MM) simulations together with transition path sampling. Our simulations predict mechanical force to only marginally enhance the reactivity of the rate-limiting step, the nucleophilic attack of hydroxide to the peptide moiety, and not to alter the reaction mechanism, even though the peptide bond and its π-electron conjugation is weakened by force. We describe a previously unidentified hydrogen bonded intermediate state, which is likely to play a role in general in base-catalyzed and analogous enzymatic reactions. Our predictions can be directly tested by single molecule stretching experiments.