Contrasting biomass allocations explain adaptations to cold and drought in the world's highest-growing angiosperms.

BACKGROUND AND AIMS: Understanding biomass allocation among plant organs is crucial for comprehending plant growth optimization, survival and responses to global change drivers. Yet, mechanisms governing mass allocation in vascular plants from extreme elevations exposed to cold and drought stresses remain poorly understood. METHODOLOGY: We analyzed organ mass weights and fractions in 258 Himalayan herbaceous species across diverse habitats (wetland, steppe, alpine), growth forms (annual, perennial taprooted, rhizomatous, cushiony), and climatic gradients (3500-6150 m elevation) to explore whether biomass distribution adhered to fixed allometric or optimal partitioning rules, and how variation in size, phylogeny, and ecological preferences influence their strategies for resource allocation. KEY FINDINGS: Following the optimal partitioning theory, Himalayan plants distribute more biomass to key organs vital for acquiring and preserving limited resources necessary for their growth and survival. Allocation strategies are mainly influenced by plant growth forms and habitat conditions, notably temperature, water availability, and evaporative demands. Alpine plants primarily invest in belowground stem bases for storage and regeneration, reducing aboveground stems while increasing leaf mass fraction to maximize carbon assimilation in their short growing season. Conversely, arid steppe plants prioritize deep roots over leaves to secure water and minimize transpiration. Wetland plants allocate resources to aboveground stems and belowground rhizomes, enabling them to resist competition and grazing in fertile environments. CONCLUSIONS: Himalayan plants from extreme elevations optimize their allocation strategies to acquire scarce resources under specific conditions, efficiently investing carbon from supportive to acquisitive and protective functions with increasing cold and drought. Intraspecific variation and shared ancestry did not significantly alter Himalayan plants' biomass allocation strategies. Despite diverse evolutionary histories, plants from similar habitats have developed comparable phenotypic structures to adapt to their specific environments. This study offers new insights into plant adaptations in diverse Himalayan environments and underscores the importance of efficient resource allocation for survival and growth in challenging conditions.

A Low-Cost Method of Measuring the In Situ Primary Productivity of Periphyton Communities of Lentic Waters.

Measuring the in situ primary productivity of periphyton during the growing season gradient can elucidate the quantitative effect of environmental drivers (mainly phosphorus concentration and light intensity) and species composition on primary productivity. Primary productivity is mainly driven by light intensity, temperature, availability of nutrients, and distribution of the ionic species of the carbonate system in the respective depths of the euphotic zone. It is a complex system that is very difficult to simulate in the laboratory. This cheap, transportable, and easy-to-build floating barge allows measuring the primary productivity accurately-directly under the actual natural conditions. The methodology is based on measuring the primary productivity in real time using noninvasive oxygen sensors integrated into tightly sealed glass jars, enabling online oxygen flux monitoring and providing new insights into metabolic activities. Detailed seasonal in situ measurements of gross primary productivity of microbial mats (or other benthic organisms) can improve current knowledge of the processes controlling primary productivity dynamics in lentic waters.

Low Specific Phosphorus Uptake Affinity of Epilithon in Three Oligo- to Mesotrophic Post-mining Lakes.

Epilithon contributes to phosphorus (P) cycling in lakes, but its P uptake traits have been rarely studied. We measured the chemical composition of epilithon and its inorganic P uptake kinetics using isotope 33P in three deep oligo- to mesotrophic post-mining lakes in April, July, and October 2019. Over the sampling period, epilithon biomass doubled, while the P content in biomass dropped to 60% of the April values, and the seasonal changes in P content expressed per epilithon area were only marginal and statistically not significant. High epilithic C:P molar ratios (677 on average) suggested strong P deficiency in all investigated lakes. Regarding the kinetic parameters of phosphorus uptake, maximum uptake velocity (V max , seasonal range 1.9-129 mg P g OM-1 h-1) decreased by an order of magnitude from April to October, while half-saturation constant (K S , seasonal range 3.9-135 mg P L-1) did not show any consistent temporal trend. Values of epilithic specific P uptake affinity (SPUA E , seasonal range 0.08-3.1 L g OM-1 h-1) decreased from spring to autumn and were two to four orders of magnitude lower than the corresponding values for seston (SPUA sest ), which showed an opposite trend. Considering our results, we suggest a possible mechanism underlying a stable coexistence of planktonic and epilithic microorganisms, with plankton prospering mostly in summer and autumn and epilithon in winter and spring season. Additionally, a phenomenon of reversible abiotic P adsorption on epilithon was observed.

Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides.

Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes.IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.

Functionally distinct assembly of vascular plants colonizing alpine cushions suggests their vulnerability to climate change.

BACKGROUND AND AIMS: Alpine cushion plants can initially facilitate other species during ecological succession, but later on can be negatively affected by their development, especially when beneficiaries possess traits allowing them to overrun their host. This can be reinforced by accelerated warming favouring competitively strong species over cold-adapted cushion specialists. However, little empirical research has addressed the trait-based mechanisms of these interactions. The ecological strategies of plants colonizing the cushion plant Thylacospermum caespitosum (Caryophyllaceae), a dominant pioneer of subnival zones, were studied in the Western Himalayas. METHODS: To assess whether the cushion colonizers are phylogenetically and functionally distinct, 1668 vegetation samples were collected, both in open ground outside the cushions and inside their live and dead canopies, in two mountain ranges, Karakoram and Little Tibet. More than 50 plant traits related to growth, biomass allocation and resource acquisition were measured for target species, and the phylogenetic relationships of these species were studied [or determined]. KEY RESULTS: Species-based trait-environment analysis with phylogenetic correction showed that in both mountain ranges Thylacospermum colonizers are phylogenetically diverse but functionally similar and are functionally different from species preferring bare soil outside cushions. Successful colonizers are fast-growing, clonal graminoids and forbs, penetrating the cushion by rhizomes and stolons. They have higher root-to-shoot ratios, leaf nitrogen and phosphorus concentrations, and soil moisture and nutrient demands, sharing the syndrome of competitive species with broad elevation ranges typical of the late stages of primary succession. In contrast, the species from open ground have traits typical of stress-tolerant specialists from high and dry environments. CONCLUSION: Species colonizing tight cushions of T. caespitosum are competitively strong graminoids and herbaceous perennials from alpine grasslands. Since climate change in the Himalayas favours these species, highly specialized subnival cushion plants may face intense competition and a greater risk of decline in the future.

A new microcystin producing Nostoc strain discovered in broad toxicological screening of non-planktic Nostocaceae (cyanobacteria).

Benthic cyanobacteria recognized as producers of natural products, including cyanotoxins, have been neglected for systematic toxicological studies. Thus, we have performed a broad study investigating cyanotoxin potential of 311 non-planktic nostocacean representatives combining molecular and chemical analyses. Out of these, a single strain Nostoc sp. Treb K1/5, was identified as a new microcystin producer. Microcystins [Asp3]MC-YR, [Asp3]MC-FR, [Asp3]MC-HtyR and Ala-Leu/Ile-Asp-Arg-Adda-Glu-Mdha are reported for the first time from the genus Nostoc. All the studied strains were also analyzed for the occurrence of nodularins, cylindrospermopsin and (homo)anatoxin-a, yet no novel producer has been discovered. Our findings indicate rare occurrence of the common cyanotoxins in non-planktic nostocaceae which is in contrast with frequent reports of cyanotoxin producers among phylogenetically closely related planktic cyanobacteria.

Macrochaete gen. nov. (Nostocales, Cyanobacteria), a taxon morphologically and molecularly distinct from Calothrix.

Historically, the genus Calothrix included all noncolonial, tapered, heterocytous filaments within the cyanobacteria. However, recent molecular phylogenies show that "Calothrix" defined in this sense represents five distinct clades. The type species of Calothrix is marine, with solitary basal heterocytes, no akinetes, and distal ends tapering abruptly into short hairs. We examined the morphology and phylogeny of 45 tapering cyanobacteria in the Rivulariaceae, including freshwater and marine representatives of both Calothrix (35 strains) and its sister taxon Rivularia (10 strains). The marine Calothrix fall into two lineages, but we lack the generitype and so cannot identify the clade corresponding to the type species. The freshwater and soil Calothrix fall into the C. parietina clade and are characterized by having a basal heterocyte, no akinetes, and gradual tapering-but not into a long hyaline hair. Macrochaete gen. nov. is a freshwater taxon sister to the Calothrix lineages but clearly separated from Rivularia. The species in this genus differ morphologically from Calothrix by their ability to produce two heteromorphic basal heterocytes and specific secondary structures of the 16S-23S ITS. An additional feature present in most species is the presence of a distal, long hyaline hair, but this character has incomplete penetrance due to its expression only under specific environmental conditions (low phosphate), and in one species appears to be lost. We recognize three species: M. psychrophila (type species) from cold environments (high mountains, Antarctica), M. santannae from wet walls of subtropical South America, and M. lichenoides, a phycobiont of lichens from Europe.

Vegetation dynamics at the upper elevational limit of vascular plants in Himalaya.

A rapid warming in Himalayas is predicted to increase plant upper distributional limits, vegetation cover and abundance of species adapted to warmer climate. We explored these predictions in NW Himalayas, by revisiting uppermost plant populations after ten years (2003-2013), detailed monitoring of vegetation changes in permanent plots (2009-2012), and age analysis of plants growing from 5500 to 6150 m. Plant traits and microclimate variables were recorded to explain observed vegetation changes. The elevation limits of several species shifted up to 6150 m, about 150 vertical meters above the limit of continuous plant distribution. The plant age analysis corroborated the hypothesis of warming-driven uphill migration. However, the impact of warming interacts with increasing precipitation and physical disturbance. The extreme summer snowfall event in 2010 is likely responsible for substantial decrease in plant cover in both alpine and subnival vegetation and compositional shift towards species preferring wetter habitats. Simultaneous increase in summer temperature and precipitation caused rapid snow melt and, coupled with frequent night frosts, generated multiple freeze-thaw cycles detrimental to subnival plants. Our results suggest that plant species responses to ongoing climate change will not be unidirectional upward range shifts but rather multi-dimensional, species-specific and spatially variable.

Gardening in the zone of death: an experimental assessment of the absolute elevation limit of vascular plants.

Vascular plants in the western Tibetan Plateau reach 6000 m--the highest elevation on Earth. Due to the significant warming of the region, plant ranges are expected to shift upwards. However, factors governing maximum elevational limits of plant are unclear. To experimentally assess these factors, we transplanted 12 species from 5750 m to 5900 m (upper edge of vegetation) and 6100 m (beyond range) and monitored their survival for six years. In the first three years (2009-2012), there were plants surviving beyond the regional upper limit of vegetation. This supports the hypothesis of dispersal and/or recruitment limitation. Substantial warming, recorded in-situ during this period, very likely facilitated the survival. The survival was ecologically a non-random process, species better adapted to repeated soil freezing and thawing survived significantly better. No species have survived at 6100 m since 2013, probably due to the extreme snowfall in 2013. In conclusion, apart from the minimum heat requirements, our results show that episodic climatic events are decisive determinants of upper elevational limits of vascular plants.

Some Like it High! Phylogenetic Diversity of High-Elevation Cyanobacterial Community from Biological Soil Crusts of Western Himalaya.

The environment of high-altitudinal cold deserts of Western Himalaya is characterized by extensive development of biological soil crusts, with cyanobacteria as dominant component. The knowledge of their taxonomic composition and dependency on soil chemistry and elevation is still fragmentary. We studied the abundance and the phylogenetic diversity of the culturable cyanobacteria and eukaryotic microalgae in soil crusts along altitudinal gradients (4600-5900 m) at two sites in the dry mountains of Ladakh (SW Tibetan Plateau and Eastern Karakoram), using both microscopic and molecular approaches. The effects of environmental factors (altitude, mountain range, and soil physico-chemical parameters) on the composition and biovolume of phototrophs were tested by multivariate redundancy analysis and variance partitioning. Both phylogenetic diversity and composition of morphotypes were similar between Karakorum and Tibetan Plateau. Phylogenetic analysis of 16S rRNA gene revealed strains belonging to at least five genera. Besides clusters of common soil genera, e.g., Microcoleus, Nodosilinea, or Nostoc, two distinct clades of simple trichal taxa were newly discovered. The most abundant cyanobacterial orders were Oscillatoriales and Nostacales, whose biovolume increased with increasing elevation, while that of Chroococales decreased. Cyanobacterial species richness was low in that only 15 morphotypes were detected. The environmental factors accounted for 52 % of the total variability in microbial data, 38.7 % of which was explained solely by soil chemical properties, 14.5 % by altitude, and 8.4 % by mountain range. The elevation, soil phosphate, and magnesium were the most important predictors of soil phototrophic communities in both mountain ranges despite their different bedrocks and origin. The present investigation represents a first record on phylogenetic diversity of the cyanobacterial community of biological soil crusts from Western Himalayas and first record from altitudes over 5000 m.

Bacterial community of cushion plant Thylacospermum ceaspitosum on elevational gradient in the Himalayan cold desert.

Although bacterial assemblages are important components of soils in arid ecosystems, the knowledge about composition, life-strategies, and environmental drivers is still fragmentary, especially in remote high-elevation mountains. We compared the quality and quantity of heterotrophic bacterial assemblages between the rhizosphere of the dominant cushion-forming plant Thylacospermum ceaspitosum and its surrounding bulk soil in two mountain ranges (East Karakoram: 4850-5250 m and Little Tibet: 5350-5850 m), in communities from cold steppes to the subnival zone in Ladakh, arid Trans-Himalaya, northwest India. Bacterial communities were characterized by molecular fingerprinting in combination with culture-dependent methods. The effects of environmental factors (elevation, mountain range, and soil physico-chemical parameters) on the bacterial community composition and structure were tested by multivariate redundancy analysis and conditional inference trees. Actinobacteria dominate the cultivable part of community and represent a major bacterial lineage of cold desert soils. The most abundant genera were Streptomyces, Arthrobacter, and Paenibacillus, representing both r- and K-strategists. The soil texture is the most important factor for the community structure and the total bacteria counts. Less abundant and diverse assemblages are found in East Karakoram with coarser soils derived from leucogranite bedrock, while more diverse assemblages in Little Tibet are associated with finer soils derived from easily weathering gneisses. Cushion rhizosphere is in general less diverse than bulk soil, and contains more r-strategists. K-strategists are more associated with the extremes of the gradient, with drought at lowest elevations (4850-5000 m) and frost at the highest elevations (5750-5850 m). The present study illuminates the composition of soil bacterial assemblages in relation to the cushion plant T. ceaspitosum in a xeric environment and brings important information about heterotrophic bacteria in Himalayan soil.

Evaluation of adamantane hydroxamates as botulinum neurotoxin inhibitors: synthesis, crystallography, modeling, kinetic and cellular based studies.

Botulinum neurotoxins (BoNTs) are the most lethal biotoxins known to mankind and are responsible for the neuroparalytic disease botulism. Current treatments for botulinum poisoning are all protein based and thus have a limited window of treatment opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a therapeutic strategy for the treatment of botulism as it may provide an effective post exposure remedy. Using a combination of crystallographic and modeling studies a series of hydroxamates derived from 1-adamantylacetohydroxamic acid (3a) were prepared. From this group of compounds, an improved potency of about 17-fold was observed for two derivatives. Detailed mechanistic studies on these structures revealed a competitive inhibition model, with a K(i)=27 nM, which makes these compounds some of the most potent small molecule, non-peptidic BoNT/A LC inhibitors reported to date.

Tissue factor-activated coagulation cascade in the tumor microenvironment is critical for tumor progression and an effective target for therapy.

Tissue factor (TF), a rate-limiting enzyme cofactor in activating coagulation, is highly expressed in a wide spectrum of human tumor and tumor stromal cells. Using TF-deficient cancer cells and a conditional TF-knockout mouse model, we show that TF expressed by cancer cells, but not by the host stromal cells, plays a critical role in tumor growth. In the tumor microenvironment, serum coagulation factors are readily extravasated and therefore lead to continuous TF-mediated activation of coagulation proteases. To target this highly specific cascade of serine proteases, we used both a TF:VIIa inhibitor and doxorubicin-based prodrugs that are selectively activated by TF:FVIIa, FXa, and thrombin. Treatment with the TF:FVIIa inhibitor led to growth retardation in breast tumor models. In contrast, treatment with the prodrug eliminated primary tumor cells and lung metastases without apparent toxicity. Our findings offer preclinical proof of principle that targeting the coagulation cascade that is activated in the tumor microenvironment can be a highly effective approach for cancer therapy.

Synthesis and evaluation of library of betulin derivatives against the botulinum neurotoxin A protease.

Botulinum neurotoxins (BoNTs) are the most toxic proteins currently known. Current treatments for botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a new therapeutic strategy for the treatment of botulism as it may provide an effective post-exposure remedy. As such, a small library of 40 betulin derivatives was synthesized and screened against the light chain of BoNT serotype A (LC/A); five positive hits (IC(50) <100 µM) were uncovered. Detailed evaluation of inhibition mechanism of three most active compounds revealed a competitive model, with sub-micromolar K(i) value for the best inhibitor (7). Unfortunately, an in vitro cell-based assay did not show any protection of rat cerebellar neurons against BoNT/A intoxication by 7.

Effects of hydroxamate metalloendoprotease inhibitors on botulinum neurotoxin A poisoned mouse neuromuscular junctions.

Currently the only therapy for botulinum neurotoxin A (BoNT/A) poisoning is antitoxin. Antidotes that are effective after BoNT/A has entered the motor nerve terminals would dramatically benefit BoNT/A therapy. Inhibition of proteolytic activity of BoNT/A light chain by metalloendoprotease inhibitors (MEIs) is under development. We tested the effects of MEIs on in vitro as well as in vivo BoNT/A poisoned mouse nerve-muscle preparations (NMPs). The K(i) for inhibition of BoNT/A metalloendoprotease was 0.40 and 0.36 muM, respectively, for 2,4-dichlorocinnamic acid hydroxamate (DCH) and its methyl derivative, ABS 130. Acute treatment of nerve-muscle preparations with 10 pM BoNT/A inhibited nerve-evoked muscle twitches, reduced mean quantal content, and induced failures of endplate currents (EPCs). Bath application of 10 muM DCH or 5 muM ABS 130 reduced failures, increased the quantal content of EPCs, and partially restored muscle twitches after a delay of 40-90 min. The restorative effects of DCH and ABS 130, as well as 3,4 diaminopyridine (DAP) on twitch tension were greater at 22 degrees C compared to 37 degrees C. Unlike DAP, neither DCH nor ABS 130 increased Ca(2+) levels in cholinergic Neuro 2a cells. Injection of MEIs into mouse hind limbs before or after BoNT/A injection neither prevented the toe spread reflex inhibition nor improved muscle functions. We suggest that hydroxamate MEIs partially restore neurotransmission of acutely BoNT/A poisoned nerve-muscle preparations in vitro in a temperature dependent manner without increasing the Ca(2+) levels within motor nerve endings.

Botulinum neurotoxin A protease: discovery of natural product exosite inhibitors.

A new mechanistic class of BoNT/A zinc metalloprotease inhibitors, from Echinacea, exemplified by the natural product d-chicoric acid (I1) is disclosed. A detailed evaluation of chicoric acid's mechanism of inhibition reveals that the inhibitor binds to an exosite, displays noncompetitive partial inhibition, and is synergistic with a competitive active site inhibitor when used in combination. Other components found in Echinacea, I3 and I4, were also inhibitors of the protease.

Benzylidene cyclopentenediones: First irreversible inhibitors against botulinum neurotoxin A's zinc endopeptidase.

A series of benzylidene cyclopentenedione-based inhibitors, acting through covalent modification of the active site of botulinum neurotoxin A light chain metalloprotease, are reported.

Investigations into small molecule non-peptidic inhibitors of the botulinum neurotoxins.

Botulinum neurotoxins (BoNTs), proteins secreted by the bacteria genus Clostridium, represent a group of extremely lethal toxins and a potential bioterrorism threat. As the current therapeutic options are of a predominantly prophylactic nature and cannot be used en masse, new strategies and ultimately potential treatments are desperately needed to combat any widespread release of these neurotoxins. In these regards, our laboratory has been working on developing new alternatives to treat botulinum intoxication through the development of inhibitors of the light chain proteases, the etiological agent which causes BoNT intoxication. Such a strategy has required the construction of two high-throughput screens and small molecule non-peptidic libraries; excitingly, inhibitors of the BoNT/A protease have been uncovered and are being optimized via structure activity relationship studies.

Toward the discovery of potent inhibitors of botulinum neurotoxin A: development of a robust LC MS based assay operational from low to subnanomolar enzyme concentrations.

The development of a sensitive, yet reliable assay for the analysis of botulinum neurotoxin A (BoNT/A) inhibitors is described; using this assay a new protease inhibitor was characterized and found to be one of the most potent inhibitors reported to date.

A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy.

Tumor targeting peptides are promising vehicles for site-directed cancer therapy. Pep42, a cyclic 13-mer oligopeptide that specifically binds to glucose-regulated protein 78 (GRP78) and internalized into cancer cells, represents an excellent vehicle for tumor cell-specific chemotherapy. Here, we report the synthesis and evaluation of Pep42-prodrug conjugates that contain a cathepsin B-cleavable linker, resulting in the traceless release of drug inside the cancer cells.