Effects of exogenous GA3 on stem secondary growth of Pinus massoniana seedlings.

Gibberellins (GAs) play a crucial role in regulating secondary growth in angiosperms, but their effects on the secondary growth of gymnosperms are rarely reported. In this study, we administered exogenous GA3 to two-year-old P. massoniana seedlings, and examined its effects on anatomical structure, physiological and biochemical changes, and gene expression in stems. The results showed that exogenous GA3 could enhance xylem development in P. massoniana by promoting cell division. The content of endogenous hormone (including auxins, brassinosteroids, and gibberellins) were changed and the genes related to phytohormone biosynthesis and signaling pathway, such as GID1, DELLA, TIR1, ARF, SAUR, CPD, BR6ox1, and CYCD3, were differentially expressed under GA3 treatment. Furthermore, GA3 and BR (brassinosteroid) might act synergistically in promoting secondary growth in P. massoniana. Additionally, lignin content was significantly increased after GA3 treatment accompanied by the express of lignin biosynthesis related genes. PmCAD (TRINITY_DN142116_c0_g1), a crucial gene involved in the lignin biosynthesis, was cloned and overexpressed in Nicotiana benthamiana, significantly promoting the xylem development and enhancing stem lignification. It was regarded as a key candidate gene for improving stem growth of P. massoniana. The findings of this study have demonstrated the impact of GA3 treatment on secondary growth of stems in P. massoniana, providing a foundation for understanding the molecular regulatory mechanism of stem secondary growth in Pinaceae seedlings and offering theoretical guidance for cultivating new germplasm with enhanced growth and yield.

The miRNA-mRNA Regulatory Modules of Pinus massoniana Lamb. in Response to Drought Stress.

Masson pine (Pinus massoniana Lamb.) is a major fast-growing woody tree species and pioneer species for afforestation in barren sites in southern China. However, the regulatory mechanism of gene expression in P. massoniana under drought remains unclear. To uncover candidate microRNAs, their expression profiles, and microRNA-mRNA interactions, small RNA-seq was used to investigate the transcriptome from seedling roots under drought and rewatering in P. massoniana. A total of 421 plant microRNAs were identified. Pairwise differential expression analysis between treatment and control groups unveiled 134, 156, and 96 differential expressed microRNAs at three stages. These constitute 248 unique microRNAs, which were subsequently categorized into six clusters based on their expression profiles. Degradome sequencing revealed that these 248 differentially expressed microRNAs targeted 2069 genes. Gene Ontology enrichment analysis suggested that these target genes were related to translational and posttranslational regulation, cell wall modification, and reactive oxygen species scavenging. miRNAs such as miR482, miR398, miR11571, miR396, miR166, miRN88, and miRN74, along with their target genes annotated as F-box/kelch-repeat protein, 60S ribosomal protein, copper-zinc superoxide dismutase, luminal-binding protein, S-adenosylmethionine synthase, and Early Responsive to Dehydration Stress may play critical roles in drought response. This study provides insights into microRNA responsive to drought and rewatering in Masson pine and advances the understanding of drought tolerance mechanisms in Pinus.

Effects of PmaIAA27 and PmaARF15 genes on drought stress tolerance in pinus massoniana.

BACKGROUND: Auxin plays an important role in plant resistance to abiotic stress. The modulation of gene expression by Auxin response factors (ARFs) and the inhibition of auxin/indole-3-acetic acid (Aux/IAA) proteins play crucial regulatory roles in plant auxin signal transduction. However, whether the stress resistance of Masson pine (Pinus massoniana), as a representative pioneer species, is related to Aux/IAA and ARF genes has not been thoroughly studied and explored. RESULTS: The present study provides preliminary evidence for the regulatory role of the PmaIAA27 gene in abiotic stress response in Masson pine. We investigated the effects of drought and hormone treatments on Masson pine by examining the expression patterns of PmaIAA27 and PmaARF15 genes. Subsequently, we conducted gene cloning, functional testing using transgenic tobacco, and explored gene interactions. Exogenous auxin irrigation significantly downregulated the expression of PmaIAA27 while upregulating PmaARF15 in Masson pine seedlings. Moreover, transgenic tobacco with the PmaIAA27 gene exhibited a significant decrease in auxin content compared to control plants, accompanied by an increase in proline content - a known indicator of plant drought resistance. These findings suggest that overexpression of the PmaIAA27 gene may enhance drought resistance in Masson pine. To further investigate the interaction between PmaIAA27 and PmaARF15 genes, we performed bioinformatics analysis and yeast two-hybrid experiments which revealed interactions between PB1 structural region of PmaARF15 and PmaIAA27. CONCLUSION: The present study provides new insights into the regulatory functions of Aux/IAA and ARF genes in Masson pine. Overexpression of PmaIAA gene may have negative effects on the growth of Masson pine, but may improve the drought resistance. Therefore, this study has great application prospects.

Drivers of intra-seasonal δ13 C signal in tree-rings of Pinus sylvestris as indicated by compound-specific and laser ablation isotope analysis.

Carbon isotope composition of tree-ring (δ13 CRing ) is a commonly used proxy for environmental change and ecophysiology. δ13 CRing reconstructions are based on a solid knowledge of isotope fractionations during formation of primary photosynthates (δ13 CP ), such as sucrose. However, δ13 CRing is not merely a record of δ13 CP . Isotope fractionation processes, which are not yet fully understood, modify δ13 CP during sucrose transport. We traced, how the environmental intra-seasonal δ13 CP signal changes from leaves to phloem, tree-ring and roots, for 7 year old Pinus sylvestris, using δ13 C analysis of individual carbohydrates, δ13 CRing laser ablation, leaf gas exchange and enzyme activity measurements. The intra-seasonal δ13 CP dynamics was clearly reflected by δ13 CRing , suggesting negligible impact of reserve use on δ13 CRing . However, δ13 CP became increasingly 13 C-enriched during down-stem transport, probably due to post-photosynthetic fractionations such as sink organ catabolism. In contrast, δ13 C of water-soluble carbohydrates, analysed for the same extracts, did not reflect the same isotope dynamics and fractionations as δ13 CP , but recorded intra-seasonal δ13 CP variability. The impact of environmental signals on δ13 CRing , and the 0.5 and 1.7‰ depletion in photosynthates compared ring organic matter and tree-ring cellulose, respectively, are useful pieces of information for studies exploiting δ13 CRing .

Glutathione Plays a Positive Role in the Proliferation of Pinus koraiensis Embryogenic Cells.

In the large-scale breeding of conifers, cultivating embryogenic cells with good proliferative capacity is crucial in the process of somatic embryogenesis. In the same cultural environment, the proliferative capacity of different cell lines is significantly different. To reveal the regulatory mechanism of proliferation in woody plant cell lines with different proliferative potential, we used Korean pine cell lines with high proliferative potential 001#-001 (Fast) and low proliferative potential 001#-010 (Slow) for analysis. A total of 17 glutathione-related differentially expressed genes was identified between F and S cell lines. A total of 893 metabolites was obtained from the two cell lines in the metabolomic studies. A total of nine metabolites related to glutathione was significantly upregulated in the F cell line compared with the S cell line. The combined analyses revealed that intracellular glutathione might be the key positive regulator mediating the difference in proliferative capacity between F and S cell lines. The qRT-PCR assay validated 11 differentially expressed genes related to glutathione metabolism. Exogenous glutathione and its synthase inhibitor L-buthionine-sulfoximine treatment assay demonstrated the positive role of glutathione in the proliferation of Korean pine embryogenic cells.

Identification of WRKY transcription factor family genes in Pinus massoniana Lamb. and their expression patterns and functions in response to drought stress.

BACKGROUND: Pinus massoniana Lamb. is the timber species with the widest distribution and the largest afforestation area in China, providing a large amount of timber, turpentine and ecological products. Seasonal drought caused by climate warming severely constrains the quality and growth of P. massoniana forests. WRKY transcription factors play an important role in plant responses to abiotic stress. In this study, the molecular mechanisms by which P. massoniana responds to drought stress were analysed based on the P. massoniana WRKY (PmWRKY) family of genes. RESULTS: Forty-three PmWRKYs are divided into three major families, 7 sub-families, and the conserved motifs are essentially the same. Among these 43 PmWRKYs express under drought stress but with different expression patterns in response to stress. PmWRKYs respond to drought stress induced by exogenous hormones of SA, ABA, and MeJA. The expression of PmWRKY6, PmWRKY10, and PmWRKY30 up-regulate in different families and tissues under drought stress, while PmWRKY22 down-regulate. Transgenetic tobaccos of PmWRKY31 are with lower malondialdehyde (MDA) content and higher proline (Pro) content than wild type (WT) tobaccos. In transgenic tobaccos of PmWRKY31, expression levels of related genes significantly improve, and drought tolerance enhance. CONCLUSIONS: This study analysed the molecular biological characteristics of PmWRKYs and investigated the expression patterns and functions of PmWRKYs in response to drought stress in P. massoniana. The results of this study provide a basis for in-depth research of the molecular functions of PmWRKYs in response to drought stress.

Pinus mugo Essential Oil Impairs STAT3 Activation through Oxidative Stress and Induces Apoptosis in Prostate Cancer Cells.

Essential oils (EOs) and their components have been reported to possess anticancer properties and to increase the sensitivity of cancer cells to chemotherapy. The aim of this work was to select EOs able to downregulate STAT3 signaling using Western blot and RT-PCR analyses. The molecular mechanism of anti-STAT3 activity was evaluated through spectrophotometric and fluorometric analyses, and the biological effect of STAT3 inhibition was analyzed by flow cytometry and wound healing assay. Herein, Pinus mugo EO (PMEO) is identified as an inhibitor of constitutive STAT3 phosphorylation in human prostate cancer cells, DU145. The down-modulation of the STAT3 signaling cascade decreased the expression of anti-proliferative as well as anti-apoptotic genes and proteins, leading to the inhibition of cell migration and apoptotic cell death. PMEO treatment induced a rapid drop in glutathione (GSH) levels and an increase in reactive oxygen species (ROS) concentration, resulting in mild oxidative stress. Pretreatment of cells with N-acetyl-cysteine (NAC), a cell-permeable ROS scavenger, reverted the inhibitory action of PMEO on STAT3 phosphorylation. Moreover, combination therapy revealed that PMEO treatment displayed synergism with cisplatin in inducing the cytotoxic effect. Overall, our data highlight the importance of STAT3 signaling in PMEO cytotoxic activity, as well as the possibility of developing adjuvant therapy or sensitizing cancer cells to conventional chemotherapy.

Some Minor Characteristics of Spectrophotometric Determination of Antioxidant System and Phenolic Metabolism Enzyme Activity in Wood Plant Tissues of Pinus sylvestris L.

INTRODUCTION: A comprehensive study of enzymes of the antioxidant system (AOS) and phenolic metabolism is an actual subject of biochemical research; changes in the activity of these enzymes can be used as a diagnostic sign. At the same time, practically little attention has been paid to describing the regularities of these enzymatic reactions. The article presents the chemical kinetics study of reactions catalyzed by superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in Scots pine trunk tissues (Pinus sylvestris L.). The dependence of the enzyme reaction rate on the enzyme concentration and the substrate concentration is presented, and the pH-optimum for each reaction is established. BACKGROUND: Determination of AOS enzyme activity and PAL activity in woody plants has many difficulties. The chemical composition of pine trunk tissues affects determining AOS enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL activity gives perfect results when considering all additional controls by taking into account minor characteristics. OBJECTIVE: This study aimed at determining the AOS enzyme activity in 40-year-old Scots pine (Pinus sylvestris L.) plants growing in the Karelian (Russia) forest seed plantation. METHODS: Plant tissues were ground in liquid nitrogen to a uniform mass and homogenized at 4 °C in the buffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM MgCl2, and 0.5 mM PMSF. After 15-min of extraction, the homogenate was centrifuged at 12000 g for 10 min (MPW-351R centrifuge, Poland). The supernatant was purified on 20 cm3 columns with Sephadex G-250. Aliquots with the highest protein amount were collected. In tissues, the protein concentration was 10-50 µg/ml. Proteins in the extracts were quantified by a Bradford assay. The enzyme activity was determined spectrophotometrically on a SpectroStar Nano plate spectrophotometer (BMG Labtech, Germany). RESULTS: Our study made it possible to modify the methods for determining the activity of superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in Scots pine trunk tissues. The enzymatic reaction rate dependence on the enzyme concentration and the substrate concentration was determined, and pH-optimum was also noted. This methodological article also provides formulas for calculating the activities of the enzymes. CONCLUSION: We found that determining AOS enzyme activity and PAL activity in woody plants is challenging. The chemical composition of the xylem and phloem of pine affects determining AOS enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL activity gives perfect results when considering all additional controls by taking into account minor characteristics.

UV-B and UV-C radiation trigger both common and distinctive signal perceptions and transmissions in Pinus tabuliformis Carr.

In plants, ultraviolet (UV)-light is an important driver for growth and natural distribution, and is also a valuable tool for manipulating productivity as well as biotic interactions. Understanding of plant responses to different UV radiation is sparse, especially from a systems biology perspective and particularly for conifers. Here, we evaluated the physiological and transcriptomic responses to the short-term application of high-irradiance UV-B and UV-C waves on Pinus tabuliformis Carr., a major conifer in Northern China. By undertaking time-ordered gene coexpression network analyses and network comparisons incorporating physiological traits and gene expression variation, we uncovered communalities but also differences in P. tabuliformis responses to UV-B and UV-C. Both types of spectral bands caused a significant inhibition of photosynthesis, and conversely, the improvement of antioxidant capacity, flavonoid production and signaling pathways related to stress resistance, indicating a clear switch from predominantly primary metabolism to enhanced defensive metabolism in pine. We isolated distinct subnetworks for photoreceptor-mediated signal transduction, maximum quantum efficiency of photosystem II (Fv/Fm) regulation and flavonoid biosynthesis in response to UV-B and UV-C radiation. From these subnetworks, we further identified phototropins as potentially important elements in both UV-B and UV-C signaling and, for the first time, suggesting peptide hormones to be involved in promoting flavonoid biosynthesis against UV-B, while these hormones seem not to be implicated in the defense against UV-C exposure. The present study employed an effective strategy for disentangling the complex physiological and genetic regulatory mechanisms in a nonmodel plant species, and thus, provides a suitable reference for future functional evaluations and artificial UV-light mediated growing strategies in plant production.

Does oxygen affect ageing mechanisms of Pinus densiflora seeds? A matter of cytoplasmic physical state.

During desiccation, the cytoplasm of orthodox seeds solidifies into an intracellular glass with highly restricted diffusion and molecular mobility. Temperature and water content govern seed ageing rates, while oxygen (O2) can promote deteriorative reactions. However, whether the cytoplasmic physical state affects involvement of O2 in seed ageing remains unresolved. We aged Pinus densiflora seeds by controlled deterioration (CD) at 45 °C and distinct relative humidity (RH), resulting in cells with a glassy (11% and 30% RH) or fluid (60% and 80% RH) cytoplasm. Hypoxic conditions (0.4% O2) during CD delayed seed deterioration, lipid peroxidation, and decline of antioxidants (glutathione, α-tocopherol, and γ-tocopherol), but only when the cytoplasm was glassy. In contrast, when the cytoplasm was fluid, seeds deteriorated at the same rate regardless of O2 availability, while being associated with limited lipid peroxidation, detoxification of lipid peroxide products, substantial loss of glutathione, and resumption of glutathione synthesis. Changes in metabolite profiles provided evidence of other O2-independent enzymatic reactions in a fluid cytoplasm, including aldo-keto reductase and glutamate decarboxylase activities. Biochemical profiles of seeds stored under seed bank conditions resembled those obtained after CD regimes that maintained a glassy cytoplasm. Overall, O2 contributed more to seed ageing when the cytoplasm was glassy, rather than fluid.

Overexpression of geranyl diphosphate synthase (PmGPPS1) boosts monoterpene and diterpene production involved in the response to pine wood nematode invasion.

Outbreaks of pine wood nematode (PWN; Bursaphelenchus xylophilus) represent a severe biotic epidemic for the Pinus massoniana in China. When invaded by the PWN, the resistant P. massoniana might secret abundant oleoresin terpenoid to form certain defensive fronts for survival. However, the regulatory mechanisms of this process remain unclear. Here, the geranyl diphosphate synthase (PmGPPS1) gene was identified from resistant P. massoniana. Tissue-specific expression patterns of PmGPPS1 at transcript and protein level in resistant P. massoniana were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Functional characteristics analysis of PmGPPS1 was performed on transgenic Nicotiana benthamiana by overexpression, as genetic transformation of P. massoniana is, so far, not possible. In summary, we identified and functionally characterized PmGPPS1 from the resistant P. massoniana following PWN inoculation. Tissue-specific expression patterns and localization of PmGPPS1 indicated that it may play a positive role involved in the metabolic and defensive processes of oleoresin terpenes production in response to PWN attack. Furthermore, overexpression of PmGPPS1 may enhance the production of monoterpene, among which limonene reduced the survival of PWN in vitro. In addition, PmGPPS1 upregulated the expression level of key genes involved in mevalonic acid (MVA) pathway, the methylerythritol phosphate (MEP) pathway and gibberellins (GAs) biosynthesis to boost the growth and development of tobacco through a feedback regulation mechanism. Our results offered new insights into the pivotal role of the PmGPPS1 involved in terpene-based defense mechanisms responding to the PWN invasion in resistant P. massoniana and provided a new metabolic engineering scenario to improve monoterpene production in tobacco.

Biomediated synthesis, characterization, and biological applications of nickel oxide nanoparticles derived from Toona ciliata, Ficus carica and Pinus roxburghii.

Biomediated ecofriendly method for the synthesis of nickel oxide nanoparticles using plants extracts (Toona ciliata, Ficus carica and Pinus roxburghii) has been reported. The nanoparticles so obtained were characterized by various techniques such as ultraviolet-visible, powder X-ray diffraction, Fourier transform infrared spectroscopy, attenuated total reflectance spectroscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, thermogravimetric analysis and fluorescence spectroscopy. Formation of nickel oxide nanoparticles was confirmed by Fourier transform infrared spectroscopy and X-ray diffraction where the former technique ascertains the formation of bond between nickel and oxygen. The nickel oxide nanoparticles were found to be crystalline cubic face centered and show intense photoluminescence emission at 416, 414 and 413 nm, respectively. The antibacterial activity was studied against gram positive and gram negative bacterial species by agar well diffusion method. The nickel oxide nanoparticles show better activity against some bacterial strains with reference to the standard drugs Ciprofloxacin and Gentamicin. The anthelmintic activity against Pheretima posthuma of nanomaterials obtained from Pinus roxburghii was found to be greater than that derived from Toona ciliata and Ficus carica using the standard drug Albendazole. This method takes the advantage of the sustainable and economic approach for the synthesis of metal oxide nanoparticles.

Wood transcriptome analysis of Pinus densiflora identifies genes critical for secondary cell wall formation and NAC transcription factors involved in tracheid formation.

Although conifers have significant ecological and economic value, information on transcriptional regulation of wood formation in conifers is still limited. Here, to gain insight into secondary cell wall (SCW) biosynthesis and tracheid formation in conifers, we performed wood tissue-specific transcriptome analyses of Pinus densiflora (Korean red pine) using RNA sequencing. In addition, to obtain full-length transcriptome information, PacBio single molecule real-time iso-sequencing was carried out using RNAs from 28 tissues of P. densiflora. Subsequent comparative tissue-specific transcriptome analysis successfully pinpointed critical genes encoding key proteins involved in biosynthesis of the major secondary wall components (cellulose, galactoglucomannan, xylan and lignin). Furthermore, we predicted a total of 62 NAC (NAM, ATAF1/2 and CUC2) family transcription factor members and identified seven PdeNAC genes preferentially expressed in developing xylem tissues in P. densiflora. Protoplast-based transcriptional activation analysis found that four PdeNAC genes, homologous to VND, NST and SND/ANAC075, upregulated GUS activity driven by an SCW-specific cellulose synthase promoter. Consistently, transient overexpression of the four PdeNACs induced xylem vessel cell-like SCW deposition in both tobacco (Nicotiana benthamiana) and Arabidopsis leaves. Taken together, our data provide a foundation for further research to unravel transcriptional regulation of wood formation in conifers, especially SCW formation and tracheid differentiation.

Use of a combination of the MD simulations and NMR spectroscopy to determine the regulatory mechanism of pulsed electric field (PEF) targeting at C-terminal histidine of VNAVLH.

In this study, the targeted regulatory mechanism of pulsed electric field (PEF) was explored for antioxidant activity improvement in four peptides, RGAVIH, RGAVLH, VNAVIH, and VNAVLH, of the pine nut (Pinus koraiensis Sieb. et Zucc). The VNAVLH peptide exhibited the best antioxidant activity and the ß-sheet content decreased to a minimum value at 40 kV/cm. Moreover, the chemical shifts of hydrogen atoms of 2-H Asn and 6-H His shifted to a higher magnetic field. The connectivity between NαH (3.62 ppm) and CαH (8.10 ppm) of 6-His residue disappeared in PEF-treated peptide. Molecule dynamics (MD) simulation verified that the distances of Nα(H78)-Cα(H80) and H82-O94 increased, whereas -OH and -Cß(H83) got closer in histidine residue after applying the electric field force. Therefore, the antioxidant activity enhancement of VNAVLH might due to the targeted regulation of PEF treatment on NαH-CαH and imidazole group in histidine.

Purification and Identification of Pine Nut (Pinus yunnanensis Franch.) Protein Hydrolysate and Its Antioxidant Activity in Vitro and in Vivo.

In this study, the pine nut (Pinus yunnanensis Franch.) protein was hydrolyzed by alkaline protease and trypsin to prepare pine nut protein hydrolysate (PNPH). The chemical, intracellular and in vivo antioxidant capacity of PNPH were evaluated. PNPH owned the ability of scavenging free radicals, and it could protect the HepG2 cells from oxidative damage by preserving cell viability. Moreover, PNPH could reduce the malondialdehyde (MDA) content and improved the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in serum, heart and liver of aging mice induced by D-galactose. Further, the PNPH was stepwise purified and identified, and 15 peptides were identified from purified fraction in PNPH. The three-dimension structures of identified peptides were predicted. Among all identified peptides, peptide 3, 7, 8 and 11 were presumed to possess good antioxidant activity. Overall, PNPH and purified peptides isolated from PNPH have potential application prospects in the field of natural antioxidants and anti-aging functional foods.

Growth dynamics of galls and chemical defence response of Pinus thunbergii Parl. to the pine needle gall midge, Thecodiplosis japonensis Uchida & Inouye (Diptera: Cecidomyiidae).

The pine needle gall midge, Thecodiplosis japonensis Uchida et Inouye, is a newly invasive pest in China that mainly harms Pinus thunbergii and P. densiflora. The occurrence and damage caused by T. japonensis in pure stands of P. thunbergii were investigated, and the needle growth and needle compound content were measured. Based on the above steps, the growth dynamics of galls and chemical defense response of P. thunbergii to attack by the gall midge were revealed. The results showed that the adults of T. japonensis in Qingdao city, China, emerged from the end of May to late July, with a peak in mid-June. Needles of P. thunbergii began to differentiate in late June and stopped growing in mid-September. The length of infested needles was 60.17% less than that of healthy needles. On average, there were 9 ± 4 larvae in each gall, 22 at most and 1 at least. The number of larvae within a gall had no significant effect on the size of the gall or larvae. Compared with that in the ungalled tissues, the content of amino acids in the galled pine needle tissues increased by 40.83%, while the content of total polyphenols, tannins, carotenoids, total triterpenes, total alkaloids and other secondary substances decreased to varying degrees, which was favourable for the growth and development of the T. japonensis larvae.

A traceless clean-up method coupled with gas chromatography and mass spectrometry for analyzing polycyclic aromatic hydrocarbons in complex plant leaf matrices.

This study developed a traceless clean-up method by combining solid phase extraction (SPE) with gas purge-microsyringe extraction (GP-MSE) to purify sample extracts for the determination of polycyclic aromatic hydrocarbons (PAHs) in plant leaves. SPE exhibited good purification performance for the removal of polar lipids, while the GP-MSE technique effectively eliminated less-volatile lipids hence realizing zero damage to the instrument, and significantly improved the peak tailings. After ultrasonic extraction, the combined two-step clean-up procedure successfully removed over 99% of lipids from nineteen types of tree leaves, and PAHs in tree leaves were determined by GC-MS. The relative standard deviations (RSDs) for intra-day (n = 3) and inter-day (n = 3) analyses of PAHs in spiked willow samples were in the range of 0.8%-12.1% and 4.7%-15.3%, respectively. The recoveries of PAHs from spiked willow extracts ranged from 74 to 90%, with an average of 86%. The method detection limit (MDL) of PAHs in tree leaves ranged from 0.1 to 4.9 ng g-1 dry weight. In conclusion, the clean-up method in this study realized the analysis of PAHs in plant leaves with high accuracy, sensitivity and reproducibility. Most importantly, the two-step purification method significantly minimizes damage to the GC-MS system particularly to the column and ion source, which is beneficial to ensure continuous analysis of a large number of samples with good performance.

Hormonal and gene dynamics in de novo shoot meristem formation during adventitious caulogenesis in cotyledons of Pinus pinea.

KEY MESSAGE: Several members of WOX and KNOX gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer Pinus pinea. Similar to Arabidopsis thaliana, Pinus pinea shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in P. pinea cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in P. pinea. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2-6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes PpWUS, PpWOX5, PpKN2, PpKN3 and PipiRR1. WUS is functional in pines and has an important role in caulogenesis. Interestingly, WOX5 also seems to participate in the process, although its specific role has not been determined.

Apoptosis induction in lung and prostate cancer cells through silver nanoparticles synthesized from Pinus roxburghii bioactive fraction.

The current study was carried out to synthesize silver nanoparticles (AgNPs) via bioactive fraction of Pinus roxburghii needles using a simple, cost-effective, and eco-friendly green chemistry method. As butanol fraction of P. roxburghii exhibited maximum anticancer activity on lung adenocarcinomas (A549) as compared to other fractions therefore, butanol fraction was used to synthesize silver nanoparticles (PNb-AgNPs). The characterization studies by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS) and selected area electron diffraction (SAED) confirmed the synthesis of the nanoparticles. The field emission scanning electron microscopy (FESEM) and high-resolution transmission electron microscopy (HRTEM) analysis showed the spherical structure of nanoparticles with an average diameter of approximately 80 nm. Interestingly, PNb-AgNPs exhibited significant cytotoxicity towards both A549 and prostatic small cell carcinomas (PC-3) with IC50 values of 11.28 ± 1.28 µg/ml and 56.27 ± 1.17 µg/ml, respectively, while lacking toxicity against normal human breast epithelial cells (fR2) and human peripheral blood lymphocytes (PBL). Further, enhanced reactive oxygen species generation, mitochondrial depolarization, apoptotic cell population (sub-G1) and DNA fragmentation observed in cancer cells were treated with PNb-AgNPs. Apoptosis was demonstrated by caspase-3 and PARP-1 activation in PNb-AgNPs-pretreated cancer cells. These results strongly suggest that PNb-AgNPs are capable of inducing cancer cell death and could act as a therapeutic nanoformulation for cancer.

Amano Lipase PS-catalyzed Hydrolysis of Pine Nut Oil for the Fatty Acids Production Using Deep Eutectic Solvent as Co-solvent.

Free fatty acids (FFAs) are the important material used in food, personal care, emulsifiers, adhesives and surfactants. In order to enhance the preparation of FFAs, the effects of reaction variables, optimization, thermodynamic property for the Amano lipase PS catalyzed hydrolysis of pine nut oil (PNO) using deep eutectic solvents (DESs) as co-solvents were studied. The results showed that FFAs could be successfully prepared from pine nut oil through Amano lipase PS catalyzed hydrolysis using Choline chloride:Urea (ChCl:U, 1:2, mol/mol) as co-solvent. Under the optimal conditions (reaction temperature 46°C, water amount 38%, DES addition 43%, lipase dosage 7.6%, reaction time 13 h), the maximum content of FFAs in the products and degree of hydrolysis (DH) of oil were up to 89.1 ± 1.9% and 92.7 ± 2.2%, respectively. The effects of reaction variables on the hydrolysis increased in the order of DES addition < reaction temperature < reaction time < lipase dosage < water amount. The thermodynamics (Arrhenius equation) for the triglycerides hydrolysis was V = 4289.39·exp(-22942.09/RT) with the activation energy (Ea) of 22.94 kJ/mol. The Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were 81.50 ± 2.64 kJ/mol, 20.18 ± 0.12 kJ/mol and -184.59 ± 0.36 J/mol/K, respectively. The lipase in the aqueous DES could be directly re-used for 3 times.