Optimizing DNA extraction protocols for bryophytes: Insights from Orthotrichaceae

Difficulties extracting high-quality RNA from recalcitrant plant tissues are often due to high levels of phenolics, carbohydrates, or other compounds that bind and/or co-precipitate with RNA. We describe here a method using soluble polyvinylpyrrolidone (PVP) and ethanol precipitation, which has been successful in several recalcitrant systems where other specialized RNA extraction methods failed to deliver suitable product. Using this method, RNA capable of reverse-transcription/PCR amplification and cDNA library construction was isolated from ripening grape berries, dry seeds of Albizia procera and radish, and leaf tissue of A. procera and Griffonia simplicifolia. This method is applicable to a variety of plant tissues.

Golden pompano (Trachinotus ovatus) is an important aquaculture species in China and broadly cultivated in Asia–Pacific region. Our previous study found that T. ovatus had a better tolerance to the high level of carbohydrate in diet (360 g/kg) compared to Micropterus salmoides. Present study investigated the tolerance mechanism of T. ovatus to high level of dietary carbohydrate (PH-360 g/kg of corn starch) on transcriptome level using Illumina sequencing compared to optimum level of dietary carbohydrate (PM-240 g/kg corn starch). A total of 208,952,492 reads were obtained and assembled into 62,377 unigenes with an average length of 822 bp and a range of 200–16,206 bp. Transcriptome annotation revealed that 27,636 unigenes with a cut-off e-value of 10−5 were functionally annotated. Specifically, 7,903 unigenes were clustered into 24 functional categories by searching against the COG database, 12,161 unigenes to 84 GO terms, 26,835 unigenes to Nr database and 23,681 unigenes to SWISS-PROT database. In addition, 13,739 unigenes were grouped into 240 KEGG pathways. There are 3,461 differentially expressed genes in PH compared to PM, in which 3,059 unigenes were significantly upregulated and 402 unigenes were significantly downregulated. Twelve key genes related to carbohydrate metabolism were further validated by quantitative real-time RT–PCR, and the results were consistent with those of transcriptome profile.Under high level of dietary carbohydrate (PH-360 g/kg), the results indicate that T. ovatus can adapt to the high level of dietary carbohydrate by enhancing glycolysis with the increased heaptic HK enzyme activity (p > 0.05), reducing gluconeogenesis with the decreased trend of PEPCK enzyme activity and maintaining the homeostasis of carbohydrate metabolism-related hormones and hormone receptor gene expressions. Histopathological examination has shown that liver cavity area was significantly increased (p > 0.05), but no visible nutritional diseases are found in fish. This study provides a valuable understanding of the molecular responses of T. ovatus fed by the high level of dietary carbohydrate (PH-360 g/kg).

We examined the changes in leaf phenolic chemistry and insect herbivory from saplings of two temperate deciduous species, Liriodendron tulipifera (tulip poplar) and Cornus florida (dogwood), planted in five microenvironments in Gilmer County, Georgia, USA. The experimental design permitted comparisons between saplings grown in an open field, under shade cloth within the field, on the edge between field and forest, in forest understory, and within canopy gaps established within the forest. Half of the trees in each microenvironment were fertilized. Leaves from each tree were sampled at the end of the growing season and 1989 and 1990 and analyzed for toughness, percent dry mass, total phenolics, hydrolyzable tannins, condensed tannins, and insect herbivory (percent leaf area damaged). The shade—tolerant dogwood saplings contained higher levels of total phenolics and hydrolyzable tannins than the shade—intolerant tulip poplar saplings. Dogwood generally had lower levels of herbivory. These results support earlier studies suggesting that slow—growing, shade—tolerant species tend to have higher levels of phenolics and experienced lower levels of herbivory than fast growing, shade—intolerant species. However, dogwood leaves contained lower levels of condensed tannins and were as tough as tulip poplar leaves. Sunlight availability had a significant positive influence on levels of phenolics in both species. Leaf phenolics generally increased with greater insolation from forest to field and when sunlight was greater within field for forest habitats. However, the levels of tannins in dogwood saplings only dropped significantly in the deep shade of the forest. The similar levels of dogwood phenolics in most microenvironments are indicative of the relatively high photosynthetic efficiency of this species in reduced light environments. Overall, these results are consistent with carbon/nutrient balance theory that predicts trade—offs in the allocation of photosynthate from defense to growth as light declines. Levels of insect herbivory and total phenolics were inversely related for dogwood. However, the relationship with tannins was less apparent. Herbivory on tulip poplar was unrelated to changes in phenolics, possibly reflecting the greater chemical diversity of that species. Fertilization increased the biomass of both species, but had no apparent influence on levels of leaf phenolics or insect hervivory. The lack of a fertilization effect was unexpected in light of previous suggestions that fertilization results in reduced phenolics and increased herbivory.

This study investigated the consequences of early season bud herbivory on host-plant phytochemistry and subsequent effects on a later mid-season leaf-feeding herbivore, to test the hypothesis that temporally segregated interguild interactions could affect herbivore success through plant-mediated responses. Our system consisted of American bass wood, Tilia americana, a bud-feeding thrips species, Thrips calcaratus, and the folivorous gypsy moth, Lymantria dispar. The impact of thrips bud-feeding on American basswood foliar chemistry and subsequent effects on gypsy moth larval preference and performance were measured. Foliar total nonstructural carbohydrates increased and phenolic levels decreased in response to bud injury, which affected larval feeding preference. In a two-choice test, gypsy moth larvae preferred leaf discs with high carbohydrate and low phenolic levels. The effects on larval performance depended on the extent of prior bud injury and were correlated with carbohydrate concentrations. In an early season assay, larval performance was lowest on moderately bud-damaged tissue, which also had the lowest total nonstructural carbohydrates. In a mid-season assay, larval performance and carbohydrate concentrations were highest in severely bud-damaged foliage. Foliar phenolics were highest in severely bud-damaged tissue in the early season assay, and in moderately damaged tissue in the mid-season assay. Gypsy moth performance was not correlated with foliar phenolic levels. Secondary (reflushed) foliage had higher carbohydrate levels than did primary (original) foliage, which correlated with increased larval performance. This study illustrates that bud-feeding herbivores can alter the phytochemistry and subsequent suitability of host-plant foliage for later folivores. The implications of these results to interactions between feeding guilds, community structure, and forest health are discussed.

Introduction . RNA isolation is a prerequisite to studying gene expression in banana and to understanding changes occurring in response to the environment. Standard extraction methods do not efficiently extract RNA from plants such as banana, with high levels of phenolics, carbohydrates, or other compounds that bind to and/or coprecipitate with RNA. Materials and methods . Five to seven RNA extraction methods were compared. Four crown-tissue storage methods were also compared. cDNA-AFLP was used to ensure that the obtained RNA was of sufficient quality for molecular applications and that RNA expression was unaltered by in situ storage. Results and discussion . The modified hot-borate method proved to be the best RNA extraction method, allowing high yields of good quality, undegraded RNA from the crown, fruit peel and pulp at all stages of ripening. The RNA obtained by this method was of sufficient quality for molecular applications such as cDNA-AFLP that give highly reproducible results. Freeze-drying of fresh tissues and tissue conservation in hot-borate buffer, two original storage methods, appear appropriate for preserving RNA in situ without ultra-low temperature. The RNA obtained was of high quality, undegraded, and useful for all downstream applications. The genome expression profile obtained by cDNA-AFLP analysis was unaltered by these methods for storing collected tissues. Conclusion . By applying all the suggested procedures in this work, it is possible to store and study gene expression in any banana fruit tissue, whatever the maturity stage, without affecting the RNA expression level.

Jujube or ber (Ziziphus spp.) is one of the most important fruit crops of India’s arid and semi-arid regions because of its high adaptability to resource constraints hot (semi)arid region. Jujube is a rich source of minerals, vitamins and dietary antioxidants to arid zone dwellers, where it is known as poor man’s apple. Given the present rising trends in discovering and exploiting plant-based health-promoting compounds, it is imperative to know the extent of variability with respect to fruit nutritional compositions present in the jujube germplasms. In this study, we assessed genetic variability for fruit nutritional and functional quality traits in eighteen jujube accessions belonging to three species (Z. mauritiana, Z. rotundifolia and Z. oenoplia) from an ex-situ collection from Indian arid and semi-arid regions in two consecutive fruiting seasons (2020-21 and 2021-22). Results revealed significant variability among eighteen jujube genotypes for important fruits physico-biochemical parameters. The IC 625864 (Z. oenoplia) identified as a superior accession for fruit antioxidant potential with having high levels of total phenols (256.2 mg/100 g dry weight) and total antioxidants (423 mg/100 g in FRAP). Moreover, IC 625849 (Z. mauritiana) and IC 625848 (Z. rotundifolia) were other genotypes containing high levels of phenols and total antioxidant (FRAP). Thus, while aiming for simultaneous improvement for total antioxidants with phenols, IC 625848, IC 625849 and IC 625864 genotypes can be considered as valuable resource for jujube fruit quality improvement program. Further, the high levels of phenotypic variance with high genotypic variance coupled with high heritability and genetic advance particularly for total antioxidants, total phenols, and ascorbic acid contents in fruits, indicating them to be considered as reliable biochemical markers to identify the productive genotypes having higher amounts of dietary antioxidants. Depending on the identified genotypes for their richness in the particular phytonutrients, these can be exploited either for table purpose or biofortification of other products, or using in jujube breeding program for quality improvement.

To isolate high quality total RNA from plants of purple-fleshed sweet potato (Ipomoea batatas L. Lam) is difficult because these plants contain high levels of carbohydrates and pigment compounds. In this study, we developed a quick and reliable method for total RNA extraction. Using this extraction protocol, which was derived from the traditional CTAB method with modification, we obtained high quality total RNA from all the tested tissues including leaf blade, petiole, stem, fibrous root, thick root, and storage root. Spectrophotometric analysis, together with agarose gel electrophoresis analysis of extracted total RNA, indicated that the total RNA was perfectly intact and the yields were all more than 0.2 mg/g fresh weight of different tissues. A metallothionein-like protein gene (G14) fragment was successfully amplified by reverse-transcription-PCR and cDNAs of two target genes (F3′H and DFR) were isolated by the method of 3′-RACE and 5′-RACE using the first-strand 3′-RACE-ready and 5′-RACE-ready cDNA as templates which were synthesized from the total RNA extraction, indicating that the total RNA extraction was suitable for further molecular analysis.

Long-read DNA sequencing technologies require high molecular weight (HMW) DNA of adequate purity and integrity, which can be difficult to isolate from plant material. Plant leaves usually contain high levels of carbohydrates and secondary metabolites that can impact DNA purity, affecting downstream applications. Several protocols and kits are available for HMW DNA extraction, but they usually require a high amount of input material and often lead to substantial DNA fragmentation, making sequencing suboptimal in terms of read length and data yield. We here describe a protocol for plant HMW DNA extraction from low input material (0.1 g) which is easy to follow and quick (2.5 h). This method successfully enabled us to extract HMW from four species from different families (Orchidaceae, Poaceae, Brassicaceae, Asteraceae). In the case of recalcitrant species, we show that an additional purification step is sufficient to deliver a clean DNA sample. We demonstrate the suitability of our protocol for long-read sequencing on the Oxford Nanopore Technologies PromethION® platform, with and without the use of a short fragment depletion kit.

Inputs and transport of acid mine drainage-derived heavy metals in karst areas of Southwestern China

Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device

Seaweeds are used in traditional agriculture practices because of their beneficial effects. Recently, the rising demand for organically grown foods has amplified the use of organic fertilizers such as seaweed extracts. Despite their beneficial effects, few studies have reported information about compounds in seaweed extracts that are responsible for these benefits. Thus, the aim of this study was to evaluate the potential relationships between the components and secondary metabolites in four seaweed liquid extracts from Eisenia arborea, Macrocystis pyrifera, Padina caulescens, and Sargassum horridum and their biostimulant activity through changes in the germination, growth, and protein content of tomato seedlings (Solanum lycopersicum). The E. arborea and S. horridum extracts showed similar compositions (ash, organic carbon, bicarbonates, and chlorides), minerals (Ca, Fe, and Cu) and secondary metabolites (triterpenes and saponins), albeit with different component concentrations. The chemical composition of the P. caulescens extract was significantly different from those of the other extracts; it was characterized by high levels of total nitrogen, phenols, and carbohydrates. Almost all seaweed extracts had beneficial effects on seed germination and seedling length, except the S. horridum extract that inhibits germination. The hierarchical clustering plots and principal component analysis indicated that germination and protein content are related to the presence of sterol. Shoot length was closely related to mineral levels (K, Zn, B, Na) and the C:N ratio, whereas radicle length was closely related to the content of nitrogen, carbohydrates, phenols, and flavonoids in the seaweed extracts. However, the underlying mechanisms are still unclear and require further studies.

Plant extracellular vesicles (PEVs) have attracted increasing attention due to their rich composition, good antioxidant and anti-inflammatory activity, and ability to transport drugs. As a common fruit, citrus is an ideal material for extracting PEVs because of the diversity and abundance of bioactive substances in it. In our study, citrus-derived extracellular vesicles (CEVs) were extracted from red mandarin (Citrus reticulata Blanco cv. 'Dahongpao') and it was found that they contain high levels of lipids, proteins, and carbohydrates. The high levels of total phenols and total flavonoids suggest that CEVs have good chemical antioxidant properties. We also demonstrated through cell experiments that CEVs have significant antioxidant and anti-inflammatory effects. Furthermore, we found that CEVs have an encapsulation rate of 71.5 ± 0.19% and a high drug-carrying capacity of 4.96 ± 0.22% and can enhance antioxidant and anti-inflammatory activity when loaded with tangeretin. Our results show that CEVs contain abundant bioactive components, have low toxicity, exhibit good antioxidant and anti-inflammatory properties, and can serve as drug delivery agents. This study has important implications for utilizing citrus materials and developing natural anti-oxidative and anti-inflammatory biomaterials.

Dietary β-glucan ameliorates metabolic stress caused by a high dietary carbohydrate level in Nile tilapia

ALTHOUGH HIGH YIELDS of a desired fermentation product can often be obtained by empirical manipulations of the environment in which a given organism is expected to be productive, it is more satisfying scientifically to base such control, if possible, on a closer acquaintance with the nature of the fermentation. For instance, high yields of penicillin are accompanied by characteristic metabolic changes in the medium; both can be attained by changing the physical or chemical milieu of the organism (cf. Jarvis and Johnson, 1947). Early experiments with Streptomyces griseus seemed to support the hypothesis that streptomycin production would follow a similar pattern (Garner et al., 1950); however, subsequent results showed that the relation between high streptomycin yields and gross metabolism, at least as embraced by our studies, was obscure more often than not. Waksman et al. (1946) first reported some of the changes occurring, during growth of a streptomycinproducing organism. They found a gradual rise in pH of the culture on a glucose, peptone, meat extract medium, and an increase in ammonia and amino nitrogen; accumulation of streptomycin paralleled the growth of the organism. Gottlieb and Anderson (1947) used a vegetative inoculum, which gives more rapid growth, and found that the peak of streptomycin production lagged behind the growth peak. Synthesis of streptomycin proceeded only in the presence of oxygen, and there was no correlation between pH changes and streptomycin production. Dulaney and Perlman (1947) compared the chemical changes that occurred during the submerged growth of organisms in Waksman's medium and media that contained high levels of either phosphate or carbohydrate. The growth phase was accompanied by reduction of soluble medium constituents, a high oxygen demand, and little streptomycin production. This was followed by an autolytic phase in which inorganic phosphorus and soluble nitrogen were released, the oxygen demand dropped, and streptomycin was produced. Added phosphate reduced streptomvcin yields, while high levels of carbohydrate had little effect aside from prolonging the growth phase. We have isolated a culture capable of giving relatively high yields and have attempted to analyze

Levels of soluble protein and carbohydrate (raffinose, sucrose, glucose, and fructose) in leaves from a selection of plant species were measured to determine if a relationship existed between these nutrients and infestation by Frankliniella occidentalis and Heliothrips haemorrhoidalis. Most species of host plant examined contained a higher proportion of protein than carbohydrates, and overall, leaves from species of plants that supported populations of thrips had greater levels of protein than leaves from nonhost species. New leaves and flowers that supported F. occidentalis contained high levels of carbohydrate and protein. The quantity of protein in leaves at the top of the tree, Peumus boldus, was greater than in leaves from lower levels, and the amount of feeding damage accrued by H. haemorrhoidalis was greater on the upper foliage than lower foliage. Oviposition by H. haenmorrhoidalis was positively correlated to levels of protein in host plants but not to levels of carbohydrates. Overall, levels of soluble protein in plants influenced their susceptibility to thrips more than levels of carbohydrates.