Chemical Composition and Anticholinesterase Activity of the Essential Oil of Leaves and Flowers from the Ecuadorian Plant Lepechinia paniculata (Kunth) Epling.

This work aimed to study the chemical composition, cholinesterase inhibitory activity, and enantiomeric analysis of the essential oil from the aerial parts (leaves and flowers) of the plant Lepechinia paniculata (Kunth) Epling from Ecuador. The essential oil (EO) was obtained through steam distillation. The chemical composition of the oil was evaluated by gas chromatography, coupled to mass spectrometry (GC-MS) and a flame ionization detector (GC-FID). The analyses led to the identification of 69 compounds in total, of which 40 were found in the leaves and 29 were found in the flowers of the plant. The major components found in the oil were 1,8-Cineole, ß-Pinene, δ-3-Carene, α-Pinene, (E)-Caryophyllene, Guaiol, and ß-Phellandrene. Flower essential oil showed interesting selective inhibitory activity against both enzymes AChE (28.2 ± 1.8 2 µg/mL) and BuChE (28.8 ± 1.5 µg/mL). By contrast, the EO of the leaves showed moderate mean inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), with IC50 values of 38.2 ± 2.9 µg/mL and 47.4 ± 2.3 µg/mL, respectively.

Chitosan stimulates secondary metabolite production and nutrient uptake in medicinal plant Dracocephalum kotschyi.

BACKGROUND: A wide variety of secondary metabolites are synthesized from primary metabolites by plants which have a vast range in pharmaceutical, food additive and industrial applications. In recent years, the use of elicitors has opened a novel approach for the production of secondary metabolite compounds. Dracocephalum kotschyi is a valuable herb due to pharmaceutical compounds like rosmarinic acid, quercetin and apigenin. In the current study, foliar application of chitosan (0, 100, 400 mg L-1 ) as an elicitor was used. RESULTS: After chitosan treatment, the amounts of hydrogen peroxide (H2 O2 ) increased and the plant was able to increase the activities of enzymatic (guaiacol peroxidase, catalase and phenylalanine ammonium lyase) and non-enzymatic (total phenols and flavonoids) defensive metabolites. Also, foliar spray of chitosan promoted nutrient absorption which led to the accumulation of macroelements in the plant. CONCLUSIONS: Chitosan was found to be a very effective elicitor for improving rosmarinic acid and quercetin content (up to 13-fold). Also, the content of apigenin (anticancer flavonoid) showed 16-fold enhancement compared to the control. Therefore, the treatment of D. kotschyi leaves with chitosan caused a very large increase in the induction and production of important pharmaceutical compounds such as rosmarinic acid and quercetin. © 2020 Society of Chemical Industry.

The effect of purine-type cytokinin on the proliferation and production of phenolic compounds in transformed shoots of Dracocephalum forrestii.

Dracocephalum forrestii is a perennial, endemic to China plant with a number of pharmaceutical properties. Transformed shoots of the species spontaneously regenerated from hairy roots induced by Agrobacterium rhizogenes. The transgenic nature of the shoots was confirmed by polymerase chain reaction (PCR). The shoot culture was multiplied on Murashige and Skoog (MS) medium with 0.2 mg/l IAA and 0.2, 0.5, 1.0, 2.0 or 5.0 mg/l purine-type cytokinins (mT, BAR, BPA or BAP). The highest multiplication rate (about thirteen shoot or buds per explant) was obtained on MS medium with 0.2 mg/l mT after four weeks of culture. The phenolic compounds present in the hydromethanolic extracts from the D. forrestii transgenic shoots were characterized using UPLC-PDA-ESI-MS. The shoots were found to biosynthesize three phenolic acids and five flavonoid glycosides. UHPLC analysis of the hydromethanolic extracts found the predominant phenolic acid to be rosmarinic acid, with its highest content observed in shoots cultivated with 5.0 mg/l BPA. In contrast, the greatest production of flavonoid derivatives (especially acacetin derivatives) was observed in the medium supplemented with 2 mg/l BPA.

Iron oxide nanoparticles: a novel elicitor to enhance anticancer flavonoid production and gene expression in Dracocephalum kotschyi hairy-root cultures.

BACKGROUND: Dracocephalum kotschyi Boiss. is a valuable source of rosmarinic acid (RA) and methoxylated hydroxyflavones (such as xanthomicrol and cirsimaritin) with antioxidative and antiplatelet effects and with antiproliferative potential against various cancer cells. The extensive application of nanotechnology in hairy root cultures is a new sustainable production platform for producing these active constituents. In the present study, hairy roots derived from 4-week-old leaves and Agrobacterium rhizogenes strain ATCC15834 were used to investigate the impact of various concentrations of iron oxide nanoparticles (Fe NPs) in two elicitation time exposures (24 and 48 h) on growth, antioxidant enzyme activity, total phenolic and flavonoid content (TPC and TFC), and some polyphenols. Gene expression levels of phenylalanine ammonia-lyase (pal) and rosmarinic acid synthase (ras) were also analyzed. RESULTS: Iron nanoparticles enhanced biomass accumulation in hairy roots. The treatment time and Fe NP dosage largely improved the activity of antioxidant enzymes, TPC and TFC. The highest RA (1194 µg g-1 FW) content (9.7-fold), compared to controls, was detected with 24 h of exposure to 75 mg L-1 Fe NP, which was consistent with the expression of pal and ras genes under the influence of elicitation. The xanthomicrol, cirsimaritin, and isokaempferide content was increased 11.87, 3.85, and 2.27-fold, respectively. CONCLUSION: Stimulation of D. kotschyi hairy roots by Fe NPs led to a significant increase in the induction and production of important pharmaceutical compounds such as rosmarinic acid and xanthomicrol. © 2019 Society of Chemical Industry.

Phytochemical composition and biological activities of native and in vitro-propagated Micromeria croatica (Pers.) Schott (Lamiaceae).

MAIN CONCLUSION: In vitro culture conditions and kinetin induced quantitative modifications in the production of the major volatile constituents in Micromeria croatica plantlets. Antimicrobial activity of methanolic extracts obtained from micropropagated and wild-growing plants was evaluated. Micromeria spp. are aromatic plants, many of which were shown to exhibit various biological effects. The present study aimed to determine the content and the composition of the essential oil of in vitro-cultured Micromeria croatica (Pers.) Schott and to evaluate the in vitro antimicrobial activity of its methanolic extract, in order to compare its phytochemical profile and biological activity with wild-growing plants. Shoots regenerated on MS medium without plant growth regulators (PGRs) or supplemented with kinetin were used for phytochemical analysis. Essential oils from both native plant material and in vitro-cultivated M. croatica plants, with a total of 44 identified constituents, were dominated by oxygenated monoterpenes. Borneol was the main component detected in wild-growing plants (25.28%) and micropropagated plants grown on PGR-free medium (20.30%). Kinetin treatment led to increased oil yield and favored the production of oxygenated monoterpenes, dominated by geranial (33.53%) and cis-p-mentha-1(7),8-dien-ol (23.69%). The percentage of total sesquiterpenoids in micropropagated plant material was considerably lower than in wild-growing plants. In vitro culture conditions and PGRs affected the production of essential oils, inducing quantitative modifications in the production of the major volatile constituents in M. croatica plantlets. The antimicrobial activity of M. croatica methanolic extracts was investigated using the broth microdilution method. Extracts obtained from in vitro cultures generally exhibited greater antibacterial potential, compared to wild-growing plants. Among six bacterial strains tested, Bacillus cereus and Staphylococcus aureus were the most sensitive microorganisms. The present study provided evidence that in vitro culture conditions might favorably affect the antimicrobial activity of M. croatica methanolic extracts.

Modulation of callus growth and secondary metabolites in different Thymus species and Zataria multiflora micropropagated under ZnO nanoparticles stress.

Thymus species are aromatic plants with diverse applications in food industries and medicine. This study was conducted to evaluate the potential effect of ZnO nanoparticles (NPs) on callus proliferation and thymol and carvacrol production in three Thymus species, that is, T. vulgaris, T. daenensis, and T. kotschyanus, and Zataria multiflora. For this purpose, callus induction was performed on Murashige and Skoog (MS) medium containing different plant growth regulators (PGRs). After optimization of callus growth, the effects of different concentrations of ZnO NPs (100 and 150 mg L-1 ) were investigated. MS containing 2 mg L-1 of 2, 4-dichlorophenoxy acetic acid (2,4-D) and 1 mg L-1 of kinetin (Kin) revealed significantly highest fresh weight (0.18 g) of callus in T. kotschyanus. Callus growth rate (0.079 mm day-1 ) was found highest in T. vulgaris under similar conditions. Moreover, highest callus induction (92.50%) was achieved by T. kotschyanus in MS containing 2.5 mg L-1 of 2,4-D. Regarding the highest content of thymol (22.8 mg L-1 ) and carvacrol (0.68 mg L-1 ) evaluated by high-performance liquid chromatography, best results were achieved under 150 mg L-1 of ZnO NPs in T. kotschyanus and T. daenesis, respectively. This is simple and cost-effective method to be applied on industrial level for production of enhanced secondary metabolites content.

Enhanced accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures analyzed by UPLC-MS/MS.

Harpagide and its derivatives have valuable medicinal properties, such as anti-inflammatory, analgesic and potential antirheumatic effects. There is the demand for searching plant species containing these iridoids or developing biotechnological methods to obtain the compounds. The present study investigated the effects of methyl jasmonate (MeJa, 50 µM), ethephon (Eth, 50 µM) and L-phenylalanine (L-Phe, 2.4 g/L of medium), added to previously selected variant of Murashige and Skoog medium (supplemented with plant growth regulators: 6-benzylaminopurine 1.0 mg/L, α-naphthaleneacetic acid 0.5 mg/L, gibberellic acid 0.25 mg/L) on the accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures. Plant material was harvested 2 and 8 days after the supplementation. Iridoids were quantitatively analyzed by the UPLC-MS/MS method in extracts from the biomass and the culture medium. It was found that all of the variants caused an increase in the accumulation of harpagide. In the biomass harvested after 2 days, the highest harpagide content of 247.3 mg/100 g DW was found for variant F (L-Phe and Eth), and the highest 8-O-acetyl-harpagide content of 138 mg/100 g DW for variant E (L-Phe and MeJa). After 8 days, in some variants, a portion of the metabolites was released into the culture medium. Considering the total amount of the compounds (in the biomass and medium), the highest accumulation of harpagide, amounting to 619 mg/100 g DW, was found in variant F, and the highest amount of 8-O-acetyl-harpagide, of 255.4 mg/100 g DW, was found in variant H (L-Phe, MeJa, Eth) when harvested on the 8th day. These amounts were, respectively, 24.7 and 4.8 times higher than in the control culture, and were, respectively, 15 and 6.7 times higher than in the leaves of the soil-grown plant. The total amount of the two iridoids was highest for variant F (0.78% DW) and variant H (0.68% DW) when harvested on the 8th day. The results indicate that the agitated shoot cultures of M. melissophyllum can be a rich source of harpagide and 8-O-acetyl-harpagide, having a potential practical application. To the best of our knowledge we present for the first time the results of the quantitative UPLC-MS/MS analysis of harpagide and 8-O-acetyl-harpagide in M. melissophyllum shoot cultures and the enhancement of their accumulation by means of medium supplementation with elicitors and precursor.

Effects of surface sterilisation with green synthesised silver nanoparticles on Lamiaceae seeds.

Nanoparticles have been used in many areas of biotechnology. In this study, an alternative surface sterilisation method was established for plant tissue cultures. Silver nanoparticles synthesised via green synthesis were used for the surface sterilisation of Lamiaceae seeds (Salvia farinecae, Ocimum basilicum - Large Leaf Italian, Thymus vulgaris, Ocimum basilicum var. purpurascens). Water extracts of dried Alkanna tinctorum rhizomes and Syzygium aromaticum flowers were utilised in the bioreduction of silver ions. The seeds were exposed to 0, 1, 7, 14 and 28 day-old colloidal solutions of silver nanoparticles and their effects on germination and surface sterilisation were determined. Fresh (0 and 1 day-old) colloidal solutions of silver nanoparticles were found very effective on surface sterilisation (100%). Moreover, they showed no negative effect on both germination and morphology of plantlets. It was shown that silver nanoparticles can be used as a surface sterilisation agent and they have no adverse effects on seed germination and in vitro plantlet growth.

Comparative metabolomic responses to gibberellic acid and 6-benzylaminopurine in Cunila menthoides Benth. (Lamiaceae): a contribution to understand the metabolic pathways.

KEY MESSAGE: Gibberellic acid elicited synthesis of many phenols from different classes and enhanced production of sesquiterpenoids, polyterpenoids, steroids and monoterpenoids compared to control and 6-benzylaminopurine. Little is known about the effects of 6-benzylaminopurine (BA) and gibberellic acid (GA3) on the synthesis of secondary metabolites in species of Lamiaceae. In this study, for the first time, the profile of secondary metabolites in plantlets of Cunila menthoides was characterized, using UPLC-ESI-Qq-oaTOF-MS. Ninety metabolites were identified, including polyphenols and terpenes. BA down-regulated most of the identified molecules in relation to GA3 and MS0 (control). The results showed that GA3 elicited synthesis of many phenols from different classes, and seemed to play a major role in the shikimate pathway in relation to BA. GA3 enhanced production of sesquiterpenoids, polyterpenoids, steroids and monoterpenoids compared to MS0 and BA, and also seemed to positively influence the MEP/DOXP and MVA pathways. These data show the most comprehensive metabolomic profile of Cunila menthoides to date, and the effects of BA and GA3 on the synthesis of secondary metabolites, modulating quantitative aspects of metabolism in Lamiaceae.

Smelling the metal: Volatile organic compound emission under Zn excess in the mint Tetradenia riparia.

This work investigated the effect of Zn excess on growth, metal accumulation and photosynthetic changes in Tetradenia riparia, in relation to possible variations in the composition of the plant volatilome. Experiments were carried out in hydroponics exposing plants to a range of Zn concentrations. Zinc excess negatively affected plant growth in a dose-dependent manner. The metal was accumulated proportionally to its concentration in the medium and preferentially allocated to roots. All the photosynthetic parameters and the concentration of some photosynthetic pigments were negatively affected by Zn, whereas the level of leaf total soluble sugars remained unchanged. Twenty-three different VOCs were identified in the plant volatilome. Each compound was emitted at a different level and intensity of emission was manifold increased by the presence of Zn in the growth medium. The Zn-induced compounds could represent both an adaptive response (f.i. methanol, acetylene, C6-aldehydes, isoprene, terpenes) and a damage by-product (f.i. propanal, acetaldehyde, alkyl fragments) of the metal presence in the culture medium. Given that the Zn-mediated induction of those VOCs, considered protective, occurred even under a Zn-limited photosynthetic capacity, our work supports the hypothesis of an active role of such molecules in an adaptive plant response to trace metal stress.

Fullerene C60 for enhancing phytoremediation of urea plant wastewater by timber plants.

Phytoremediation has been applied as a promising and cost-effective technique for removing nutrient pollutants from wastewater. In this study, the effect of fullerene C60 was assessed on enhancing the phytoremediation efficiency of teak plants over a period of 1 month. Teak plants were supplied with fullerene C60 (0, 25, or 50 mg L-1) and fed daily with two types of urea plant wastewater (with and without adding optimum ratio of phosphorus and potassium). The required volume of wastewater by the teak plants, nitrogen removal percentage, plant growth parameters (plant height, number of leaves, leaf surface area, and dry biomass), and nutrient content was recorded throughout the study. The results showed that addition of 25 mg L-1 fullerene C60 to urea plant wastewater could increase water uptake and nitrogen recovery of the teak plants. Plant growth and nutrient contents of teak plants were also increased in the presence of 25 mg L-1 fullerene C60. However, addition of 50 mg L-1 fullerene C60 to the wastewater decreased the values for water uptake and nitrogen recovery. The findings indicated that addition of proper amount of fullerene C60 to the teak-based remediation system can increase the efficiency of the plants for nitrogen removal.

Effect of different ethanolic concentrations on antioxidant properties and cytoprotective activities of Platostoma palustre Blume.

The objective of this study was to evaluate the antioxidant activities of Mesona procumbens ethanolic extracts (MPEEs) which displayed variable antioxidant levels with different ethanolic concentrations. Compared to MPEEs with 20, 40 and 80% ethanol, the 60% MPEE exhibited a higher total phenol content (TPC), total flavonoid content (TFC), 1,1-diphenyl-2-picryl hydrazyl (DPPH)- and 2,2-azino-bis-(3-ethyl benzothiazoline-6-sulphonic acid (ABTS)-scavenging activities, reducing power, protection of Raw 264.7 mouse macrophages against H2O2-induced damage, and inhibition of reactive oxygen species (ROS) induced by 2,2-azobis(2-amidino propane) dihidrochloride (AAPH). IC50 values of DPPH and ABTS radicals by MPEEs were highly and significantly associated with TPCs and TFCs. The most significant protective effect against oxidative DNA damage was also found to be the 60% MPEE at a concentration of 3.6 mg/mL. In addition, the cell viability test showed that none of the MPEEs had any cell toxicity up to a concentration of 250 µg/mL. The 60% MPEE exhibited higher in vitro and ex vivo antioxidant activities, possessed a protective capability for the biological membrane system, and can be used as a functional ingredient representing a potential source of natural antioxidants to prevent and treat oxidative stress-related disorders.

Dissecting the proteome dynamics of the salt stress induced changes in the leaf of diploid and autotetraploid Paulownia fortunei.

Exposure to high salinity can trigger acclimation in many plants. Such an adaptative response is greatly advantageous for plants and involves extensive reprogramming at the molecular level. Acclimation allows plants to survive in environments that are prone to increasing salinity. In this study, diploid and autotetraploid Paulownia fortunei seedlings were used to detect alterations in leaf proteins in plants under salt stress. Up to 152 differentially abundant proteins were identified by Multiplex run iTRAQ-based quantitative proteomic and LC-MS/MS methods. Bioinformatics analysis suggested that P. fortunei leaves reacted to salt stress through a combination of common responses, such as induced metabolism, signal transduction, and regulation of transcription. This study offers a better understanding of the mechanisms of salt tolerance in P. fortunei and provides a list of potential target genes that could be engineered for salt acclimation in plants, especially trees.

Copper-induced alteration in sucrose partitioning and its relationship to the root growth of two Elsholtzia haichowensis Sun populations.

Hydroponic culture was used to comparatively investigate the copper (Cu)-induced alteration to sucrose metabolism and biomass allocation in two Elsholtzia haichowensis Sun populations with one from a Cu-contaminated site (CS) and the other from a non-contaminated site (NCS). Experimental results revealed that biomass allocation preferred roots over shoots in CS population, and shoots over roots in NCS population under Cu exposure. The difference in biomass allocation was correlated with the difference in sucrose partitioning between the two populations. Cu treatment (45 µM) significantly decreased leaf sucrose content and increased root sucrose content in CS population as a result of the increased activities of leaf sucrose synthesis enzymes (sucrose phosphate synthetase and sucrose synthase) and root sucrose cleavage enzyme (vacuolar invertase), which led to increased sucrose transport from leaves to roots. In contrast, higher Cu treatment increased sucrose content in leaves and decreased sucrose content in roots in NCS population as a result of the decreased activities of root sucrose cleavage enzymes (vacuolar and cell wall invertases) that led to less sucrose transport from leaves to roots. These results provide important insights into carbon resource partitioning and biomass allocation strategies in metallophytes and are beneficial for the implementation of phytoremediation techniques.

Interactive Effects of Arbuscular Mycorrhizal Fungi and Copper Stress on Flowering Phenology and Reproduction of Elsholtzia splendens.

Plant responses to heavy metal contamination may depend on the presence of arbuscular mycorrhizal fungi (AMF). Elsholtzia splendens is an indicator species for the presence of copper (Cu) mines because both its flowering phenology and reproduction are tolerant to heavy metals. To test whether effects of Cu on the flowering phenology and reproduction of E. splendens depend on the presence of AMF, we conducted a factorial experiment with two Cu treatments (with or without Cu addition) crossed with two AMF treatments (with or without AMF inoculation). Without AMF, Cu addition significantly delayed the onset dates, ending dates and peak dates of flowering and decreased flowering duration. However, AMF inoculation reversed the effects of Cu stress, with recovered flowering onset and ending dates and increased the flowering duration. Cu addition significantly decreased inflorescence width and number, inflorescence biomass, vegetative biomass and total seed number, but significantly increased 1000-seed weight. AMF inoculation significantly increased vegetative biomass. Two-way ANOVA results showed that the interactive effects between Cu addition and AMF inoculation were significant on the inflorescence number, vegetative biomass and total seed number. These results indicate that AMF can alleviate the Cu stress on the flowering phenology and reproduction of E. splendens.

Heterologous expression and comparative characterization of vacuolar invertases from Cu-tolerant and non-tolerant populations of Elsholtzia haichowensis.

KEY MESSAGE: Vacuolar invertases (VINs) from Cu-tolerant and non-tolerant populations of Elsholtzia haichowensis have similar enzyme properties, and the enzyme protein divergences contribute little to the varied VIN activities between the contrasting populations. In our previous studies of Elsholtzia haichowensis, vacuolar invertase (VIN) activity in roots of a Cu-tolerant population was found to be significantly higher than that of a non-tolerant population under Cu stress. Divergences of amino acid residues in a sucrose-binding box and other regions of the VINs were detected. To test whether the amino acid divergences influence the enzyme properties of VINs, and thus are relevant to the differences in enzyme activities between the contrasting populations of E. haichowensis, two VIN genes from the Cu-tolerant population (EhCvINV) and non-tolerant population (EhNvINV) were heterologously expressed in Pichia pastoris, and the enzyme properties of the recombinants were characterized and compared. Both of the recombinant enzymes showed temperature optima of 70 °C and pH optima of 4.5-5.5. Copper as well as other heavy metals caused almost the same inhibition to EhNvINV and EhCvINV. No statistically significant differences were observed between EhNvINV and EhCvINV in K m and k cat values for sucrose. The results provided evidence that the observed residue divergences had little influence on the enzyme properties of VIN in E. haichowensis, and the varied VIN activities between the contrasting populations under Cu stress were not relevant to the amino acid divergences in the proteins. Also, some other possible reasons accounting for this difference in invertase activities were discussed, such as up-regulation of expression of the EhCvINV gene under Cu stress, as Cu tolerance mechanisms in Cu-mine plants.

Molecular cloning and expression profiling of a chalcone synthase gene from Lamiophlomis rotata.

Lamiophlomis rotata is a renowned Chinese medicinal plant. Chalcone synthase (CHS) is important in flavonoid and isoflavonoid biosynthesis, catalysing the formation of naringenin chalcone in plants. A full-length cDNA encoding the CHS gene was cloned from L. rotata based on the highly conserved CHS gene sequences of Labiatae plants. A blast search showed its homology (named LrCHS) with other CHS genes of Labiate plants. The full-length genomic DNA of LrCHS was 2026 bp with one intron of 651 bp, two exons of 178 bp and 998 bp, flanked by a 73 bp 5'-UTR and a 126 bp 3'-UTR. The cDNA sequence of the LrCHS gene had an 1176 bp open reading frame encoding a 391 amino acid protein of 42,798 Da. The CHS protein predicted from L. rotata showed 79-86% identity with CHS of other plant species. We conducted a phylogenetic analysis of nine families containing 48 plants and L. rotata based on the full amino acid sequences of CHS proteins. Consequently, LrCHS was located in the Labiatae branch. Additionally, we examined LrCHS gene expression patterns in different tissues by quantitative real-time PCR with specific primers. The expression analysis showed preferential expression of LrCHS in flowers and leaves during the flowering stage. Total flavonoid content and CHS gene expression exhibited similar patterns during L. rotata organ development. In agreement with its function as an elicitor-responsive gene, LrCHS expression was coordinated by methyl jasmonate and UV light, and induced between 6 and 18 h. These results provide a molecular basis for additional functional studies of LrCHS in L. rotata.

Isolation and Analysis of Cell Wall Proteome in Elsholtzia splendens Roots Using ITRAQ with LC-ESI-MS/MS.

Cell wall proteins (CWPs) are a prime site for signal perception and defense responses to environmental stresses. To gain further insights into CWPs and their molecular function, traditional techniques (e.g., two-dimensional gel electrophoresis) may be ineffective for special proteins. Elsholtzia splendens is a copper-tolerant plant species that grow on copper deposits. In this study, a fourplex isobaric tag was used for relative and absolute quantitation with liquid chromatography-tandem mass spectrometry approach to analyze the root CWPs of E. splendens. A total of 479 unique proteins were identified, including 121 novel proteins. Approximately 80.79 % of the proteins were extracted in the CaCl2 fraction, 16.08 % were detected in the NaCl fraction, and 3.13 % were identified in both fractions. The identified proteins have been involved in various processes, including cell wall remodeling, signal transduction, defense, and carbohydrate metabolism, thereby indicating a complex regulatory network in the apoplast of E. splendens roots. This study presents the first large-scale analysis of CWPs in metal-tolerant plants, which may be of paramount importance to understand the molecular functions and metabolic pathways in the root cell wall of copper-tolerant plants.

Roles of H2S in adaptation of alpine plants Lamiophlomis rotata to altitude gradients.

Hydrogen sulfide (H2S) is an important gaseous transmitter in organisims. It widespreads in the organs and tissues of animals and participates in the biological process of cardiovascular relaxation, cell apoptosis and protection, inflammation and neuromodulation. H2S also can be synthesized in plants system and is involved in stress responses and the biological process of growth and development. This review describes the synthesis and biological function of H2S in plants. Based on our research for the adaptation of Lamiophlomis rotata to different altitude gradients, we firstly proposed H2S plays an important role in the adaptation of Lamiophlomis rotata to alpine environment.

Comparative proteomic analysis reveals the role of hydrogen sulfide in the adaptation of the alpine plant Lamiophlomis rotata to altitude gradient in the Northern Tibetan Plateau.

MAIN CONCLUSION: We found the novel role of hydrogen sulfide in the adaptation of the alpine plant to altitude gradient in the Northern Tibetan Plateau. Alpine plants have developed strategies to survive the extremely cold conditions prevailing at high altitudes; however, the mechanism underlying the evolution of these strategies remains unknown. Hydrogen sulfide (H2S) is an essential messenger that enhances plant tolerance to environmental stress; however, its role in alpine plant adaptation to environmental stress has not been reported until now. In this work, we conducted a comparative proteomics analysis to investigate the dynamic patterns of protein expression in Lamiophlomis rotata plants grown at three different altitudes. We identified and annotated 83 differentially expressed proteins. We found that the levels and enzyme activities of proteins involved in H2S biosynthesis markedly increased at higher altitudes, and that H2S accumulation increased. Exogenous H2S application increased antioxidant enzyme activity, which reduced ROS (reactive oxygen species) damage, and GSNOR (S-nitrosoglutathione reductase) activity, which reduced RNS (reactive nitrogen species) damage, and activated the downstream defense response, resulting in protein degradation and proline and sugar accumulation. However, such defense responses could be reversed by applying H2S biosynthesis inhibitors. Based on these findings, we conclude that L. rotata uses multiple strategies to adapt to the alpine stress environment and that H2S plays a central role during this process.