Drought, temperature, and moisture availability: understanding the drivers of isotopic decoupling in native pine species of the Nepalese Himalaya.

The Himalayas experienced long-term climate changes and recent extreme weather events that affected plant growth and the physiology of tree species at high-elevation sites. This study presents the first statistically robust δ18OTR chronologies for two native pine species, Pinus roxburghii, and Pinus wallichiana, in the lower Nepalese Himalaya. The isotope chronologies exhibited 0.88‰ differences in overall mean isotope values attributed to varying elevations (460-2000 m asl). Comparative analysis of climate response using data sets from different sources and resolutions revealed the superiority of the APHRODITE (Asian Precipitation - Highly-Resolved Observational Data Integration Towards Evaluation) data set calibrated for the South Asian Summer Monsoon (SASM)-dominated region. Both species exhibited negative correlations with monsoon precipitation and positive correlations with temperature. However, during the peak monsoon season (July-August), daily resolved climate data disentangled statistically insignificant relationships, and revealed that δ18OTR is influenced by atmospheric moisture. Both congeneric species showed a decoupling between the chronologies after 1995. However, no significant change in air moisture origin and monsoon regime between the study sites was observed, indicating a consistent dominant moisture source during different monsoon seasons. Besides, we also observed the decreased inter-series correlation of both δ18OTR chronologies after 1995, with P. wallichiana experiencing a steeper decrease than P. roxburghii. The weakening correlations between and within the chronologies coincided with a regional drought during 1993-1995 in both sites, highlighting the strong regulation of local climate on the impact of regional extreme climate events. Our findings emphasise the importance of employing climate data with optimal spatial and temporal resolution for improved δ18OTR-climate relationships at the intra-annual scale while considering the influence of site-specific local environmental conditions. Assessing climate data sets with station data is vital for accurately interpreting climate change's impact on forest response and long-term climate reconstructions.

INTRAGRO: A machine learning approach to predict future growth of trees under climate change.

The escalating impact of climate change on global terrestrial ecosystems demands a robust prediction of the trees' growth patterns and physiological adaptation for sustainable forestry and successful conservation efforts. Understanding these dynamics at an intra-annual resolution can offer deeper insights into tree responses under various future climate scenarios. However, the existing approaches to infer cambial or leaf phenological change are mainly focused on certain climatic zones (such as higher latitudes) or species with foliage discolouration during the fall season. In this study, we demonstrated a novel approach (INTRAGRO) to combine intra-annual circumference records generated by dendrometers coupled to the output of climate models to predict future tree growth at intra-annual resolution using a series of supervised and unsupervised machine learning algorithms. INTRAGRO performed well using our dataset, that is dendrometer data of P. roxburghii Sarg. from the subtropical mid-elevation belt of Nepal, with robust test statistics. Our growth prediction shows enhanced tree growth at our study site for the middle and end of the 21st century. This result is remarkable since the predicted growing season by INTRAGRO is expected to shorten due to changes in seasonal precipitation. INTRAGRO's key advantage is the opportunity to analyse changes in trees' intra-annual growth dynamics on a global scale, regardless of the investigated tree species, regional climate and geographical conditions. Such information is important to assess tree species' growth performance and physiological adaptation to growing season change under different climate scenarios.

Whole-Genome Sequence Data Analysis of Anoxybacillus kamchatkensis NASTPD13 Isolated from Hot Spring of Myagdi, Nepal.

Anoxybacillus kamchatkensis NASTPD13 isolated from Paudwar hot spring of Myagdi, Nepal, upon morphological and biochemical analysis revealed to be Gram-positive, straight or slightly curved, rod-shaped, spore-forming, catalase, and oxidase-positive facultative anaerobes. It grows over a wide range of pH (5.0-11) and temperature (37-75°C), which showed growth in different reduced carbon sources such as starch raffinose, glucose, fructose, inositol, trehalose, sorbitol, mellobiose, and mannitol in aerobic conditions. Furthermore, the partial sequence obtained upon sequencing showed 99% sequence similarity in 16S rRNA gene sequence with A. kamchatkensis JW/VK-KG4 and was suggested to be Anoxybacillus kamchatkensis. Moreover, whole-genome analysis of NASTPD13 revealed 2,866,796 bp genome with a G+C content of 41.6%. Analysis of the genome revealed the presence of 102 RNA genes, which includes sequences coding for 19 rRNA and 79 tRNA genes. While the 16S rRNA gene sequence of strain NASTPD13 showed high similarity (>99%) to those of A. kamchatkensis JW/VK-KG4, RAST analysis of NASTPD13 genome suggested that A. kamchatkensis G10 is actually the closest neighbor in terms of sequence similarity. The genome annotation by RAST revealed various genes encoding glycoside hydrolases supporting that it can utilize several reduced carbon sources as observed and these genes could be important for carbohydrate-related industries. Xylanase pathway, particularly the genomic region encoding key enzymes for xylan depolymerization and xylose metabolism, further confirmed the presence of the complete gene in xylan metabolism. In addition, the complete xylose utilization gene locus analysis of NASTPD13 genome revealed all including D-xylose transport ATP-binding protein XylG and XylF, the xylose isomerase encoding gene XylA, and the gene XylB coding for a xylulokinase supported the fact that the isolate contains a complete set of genes related to xylan degradation, pentose transport, and metabolism. The results of the present study suggest that the isolated A. kamchatkensis NASTPD13 containing xylanase-producing genes could be useful in lignocellulosic biomass-utilizing industries where pentose polymers could also be utilized along with the hexose polymers.

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13.

Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6-9) and temperature (30-65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 µM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications.

Characterization of the Genetic Diversity of Acid Lime (Citrus aurantifolia (Christm.) Swingle) Cultivars of Eastern Nepal Using Inter-Simple Sequence Repeat Markers.

Acid lime (Citrus aurantifolia (Christm.) Swingle) is an important fruit crop, which has high commercial value and is cultivated in 60 out of the 77 districts representing all geographical landscapes of Nepal. A lack of improved high-yielding varieties, infestation with various diseases, and pests, as well as poor management practices might have contributed to its extremely reduced productivity, which necessitates a reliable understanding of genetic diversity in existing cultivars. Hereby, we aim to characterize the genetic diversity of acid lime cultivars cultivated at three different agro-ecological gradients of eastern Nepal, employing PCR-based inter-simple sequence repeat (ISSR) markers. Altogether, 21 polymorphic ISSR markers were used to assess the genetic diversity in 60 acid lime cultivars sampled from different geographical locations. Analysis of binary data matrix was performed on the basis of bands obtained, and principal coordinate analysis and phenogram construction were performed using different computer algorithms. ISSR profiling yielded 234 amplicons, of which 87.18% were polymorphic. The number of amplified fragments ranged from 7­18, with amplicon size ranging from ca. 250­3200 bp. The Numerical Taxonomy and Multivariate System (NTSYS)-based cluster analysis using the unweighted pair group method of arithmetic averages (UPGMA) algorithm and Dice similarity coefficient separated 60 cultivars into two major and three minor clusters. Genetic diversity analysis using Popgene ver. 1.32 revealed the highest percentage of polymorphic bands (PPB), Nei's genetic diversity (H), and Shannon's information index (I) for the Terai zone (PPB = 69.66%; H = 0.215; I = 0.325), and the lowest of all three for the high hill zone (PPB = 55.13%; H = 0.173; I = 0.262). Thus, our data indicate that the ISSR marker has been successfully employed for evaluating the genetic diversity of Nepalese acid lime cultivars and has furnished valuable information on intrinsic genetic diversity and the relationship between cultivars that might be useful in acid lime breeding and conservation programs in Nepal.

Investigation of antioxidant, antimicrobial and toxicity activities of lichens from high altitude regions of Nepal.

BACKGROUND: Several lichen species are reported to be used tradiationally in many theraupatic practices. Many lichen species are reported as sources of several bioactive natural compounds. Several lichen species of Nepal are so far chemically unexplored. METHODS: The morphological, anatomical and phytochemical characteristics of lichens were compared for the taxonomic identification of the species. Methanol- water extract of lichens were sub fractionated into hexane, dichloromethane and methanol fractions for bioactivity assays. Antimicrobial activities of extracts were evaluated agaisnt pathogenic bacteria and fungal species. DPPH test was used for antioxidant potential evaluation. Brineshrimp test was perfermed to evaluate toxicity of the extracts. RESULTS: A total of 84 lichen specimens were collected and identified from Annapurna Conservation Area (ACA) Nepal. The specimens were identified as belonging to 19 genera and 47 species. Methanol fractions of 16 specimens and dichloromethane (DCM) fractions of 21 lichens specimens showed antioxidant activities comparable with commercial standards (BHA, Butylated hydroxyanisole, IC50=4.9±0.9 µg/mL) even at crude extract level. Similarly, the DCM fraction of 17 lichens showed potential antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus KCTC3881) and DCM fractions of 45 lichens showed antimicrobial activity against a Gram-negative bacterium (Klebsiella pneumoniae KCTC2242). DCM fractions of three lichens showed antifungal activity against the yeast, Candida albicans KCTC 7965. Likewise, methanol fractions of 39 lichens and DCM fractions of 74 lichens showed strong toxicity against brine shrimp nauplii with more than 80% mortality. CONCLUSION: Such biological activity-rich lichen specimens warrant further research on exploration of natural products with antioxidant, antimicrobial and anti cancer (toxic) potential.

Aldehyde dehydrogenase expression in Metaphire posthuma as a bioindicator to monitor heavy metal pollution in soil.

BACKGROUND: Soil contamination and associated pollution plays a detrimental role in soil flora and fauna. Soil is processed and remodeled by subterranean earthworms, accordingly are referred to as soil chemical engineers. These worms, besides processing carbon and nitrogen, serve as minors for processing metals. In heavy metal contaminated soils, they accumulate heavy metals, which in turn cause altered gene expression, including aldehyde dehydrogenase (ALDH) enzymes. This study explores the possibility of ALDH expression in earthworms as a novel biomarker for the heavy metal contamination of soil. RESULTS: Earthworms cultured in contaminated soils accumulated significantly higher levels of Pb and Cd. Similarly, significantly higher levels of ALDH enzyme activities were observed in earthworms cultured in soils contaminated with Pb and Cd. The ALDH activity was found to be highest in worms cultured in 5 ppm heavy metal contaminated soils. Although, ALDH activities decreased as the heavy metal concentration in soil increased, they were significantly higher when compared to control worms cultured in uncontaminated soils. The accumulation of heavy metal in earthworms measured after 28 days decreased as the heavy metal concentration in soil increased. CONCLUSIONS: Levels of ALDH expression correlated with total Pb and Cd concentration in the earthworm tissue. This study showed that the ALDH activity in earthworms could potentially be used as a biomarker to show heavy metal pollution in soil.

Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30-70 °C) and pH (6.2-9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

Specific and unspecific responses of plants to cold and drought stress.

Different environmental stresses to a plant may result in similar responses at the cellular and molecular level. This is due to the fact that the impacts of the stressors trigger similar strains and downstream signal transduction chains. A good example for an unspecific response is the reaction to stressors which induce water deficiency e.g.drought, salinity and cold, especially frost. The stabilizing effect of liquid water on the membrane bilayer can be supported by compatible solutes and special proteins. At the metabolic level, osmotic adjustment by synthesis of low-molecular osmolytes (carbohydrates, betains, proline) can counteract cellular dehydration and turgor loss. Taking the example of Pinus sylvestris, changes at the level of membrane composition, and concomitantly of photosynthetic capacity during frost hardening is shown. Additionally the effect of photoperiod as measured via the phytochrome system and the effect of subfreezing temperatures on the incidence of frost hardening is discussed. Extremely hydrophilic proteins such as dehydrins are common products protecting not only the biomembranes in ripening seeds (late embryogenesis abundant proteins)but accumulate also in the shoots and roots during cold adaptation, especially in drought tolerant plants. Dehydrins are characterized by conserved amino acid motifs, called the K-,Y-or S-segments. Accumulation of dehydrins can be induced not only by drought, but also by cold,salinity,treatment with abscisic acid and methyl jasmonate. Positive effects of the overexpression of a wild chickpea (Cicer pinnatifidum) dehydrin in tobacco plants on the dehydration tolerance is shown. The presentation discusses the perception of cold and drought,the subsequent signal transduction and expression of genes and their products. Differences and similarities between the plant responses to both stressors are also discussed.