Physiology and transcriptome analysis of Artemisia argyi adaptation and accumulation to soil cadmium.

The soil pollution caused by cadmium (Cd) poses a significant threat to the environment. Therefore, identifying plants that can effectively remediate Cd-contaminated soils is urgently needed. In this study, physiological, cytological, and transcriptome analyses were performed to comprehensively understand the changes in Artemisia argyi under Cd stress. Physiological and cytological analyses indicated that A. argyi maintained normal growth with intact cell structure under Cd stress levels up to 10 mg/kg. Cytological analysis showed that Cd precipitation in leaf cells occurred in the cytoplasm and intercellular spaces. As the levels of Cd stress increased, proline accumulation in leaves increased, whereas soluble protein and soluble sugar initially increased, followed by a subsequent decline. The translocation factor was above 1 under 0.6 mg/kg Cd stress but decreased when it exceeded this concentration. Transcriptome analyses revealed several crucial Cd-influenced pathways, including amino acid, terpenoid, flavonoid, and sugar metabolisms. This study not only proved that A. argyi could enrich Cd in soil but also revealed the response of A. argyi to Cd and its resistance mechanisms, which provided insight into the cleaner production of A. argyi and the remediation of Cd-contaminated soil.

Chlorogenic acid inhibits proliferation in human hepatoma cells by suppressing noncanonical NF-κB signaling pathway and triggering mitochondrial apoptosis.

Chlorogenic acid (CGA), a phenylpropanoid derived from Eucommia ulmoides Oliver, has been shown to exhibit potent cytotoxic and anti-proliferative activities against several human cancers. However, the effects of CGA on hepatocellular carcinoma (HCC) and the underlying mechanisms have not been intensively studied. In this study, the CGA treatment effects on the viability of human hepatoma cells were investigated by MTT assay. Our data showed that CGA could dose-dependently inhibit the activity of human hepatoma cells Hep-G2 and Huh-7, but did not affect the activity and growth of normal human hepatocyte QSG-7701. The genes and pathways influenced by CGA treatment were explored by RNA sequencing and bioinformatics analysis, which identified 323 differentially expressed genes (DEGs) involved in multiple pharmacological signaling pathways such as MAPK, NF-κB, apoptosis and TGF-ß signaling pathways. Further analyses by real-time quantitative PCR, Western blot and flow cytometry revealed that CGA effectually suppressed the noncanonical NF-κB signaling pathway, meanwhile it activated the mitochondrial apoptosis of HCC by upregulation of the BH3-only protein Bcl-2 binding component 3 (BBC3). Our findings demonstrated the potential of CGA in suppressing human hepatoma cells and provided a new insight into the anti-cancer mechanism of CGA.

Expression of Two α-Type Expansins from Ammopiptanthus nanus in Arabidopsis thaliana Enhance Tolerance to Cold and Drought Stresses.

Expansins, cell-wall loosening proteins, play an important role in plant growth and development and abiotic stress tolerance. Ammopiptanthus nanus (A. nanus) is an important plant to study to understand stress resistance in forestry. In our previous study, two α-type expansins from A. nanus were cloned and named AnEXPA1 and AnEXPA2. In this study, we found that they responded to different abiotic stress and hormone signals. It suggests that they may play different roles in response to abiotic stress. Their promoters show some of the same element responses to abiotic stress and hormones, but some special elements were identified between the expansins that could be essential for their expression. In order to further testify the reliability of the above results, we conducted an analysis of ß-glucuronidase (GUS) dyeing. The analysis showed that AnEXPA1 was only induced by cold stress, whereas AnEXPA2 responded to hormone induction. AnEXPA1 and AnEXPA2 transgenic Arabidopsis plants showed better tolerance to cold and drought stresses. Moreover, the ability to scavenge reactive oxygen species (ROS) was significantly improved in the transgenic plants, and expansin activity was enhanced. These results suggested that AnEXPA1 and AnEXPA2 play an important role in the response to abiotic stress. Our research contributes to a better understanding of the regulatory network of expansins and may benefit agricultural production.

[Response of volatiles of Artemisiae Argyi Folium to climatic factors].

Combined headspace solid-phase microextraction with gas chromatography-mass spectrometry (HS-SPME-GC-MS), this paper studied the volatiles of Artemisiae Argyi Folium that respectively collected from Kongdong, Huanglong, Ankang, Qichun and Tongbai，and then explored the influence of climatic factors on the main components of the volatiles. The results showed that 54, 52, 57, 47 and 55 compounds were respectively identified from the leaves from five regions, mainly consist of monoterpenes, sesquiterpenoids and their oxygen-containing derivatives, a few aldehydes, alkanes and benzenes. It is different that the volatiles of Artemisiae Argyi Folium that collected in different habitats, neither composition nor content. There are total 18 kinds of compounds that exist in all five leaves. Eucalyptol, camphor, terpinen-4-ol, eugenol and caryophyllene could be considered to be the main conponents because of their efficacy or toxicity and relatively high content. Grey correlation analysis shows that the annual lowest temperature and annual sunshine duration have a great influence on the content of eucalyptol and camphor; the effect of annual precipitation and annual accumulated temperature on the content of terpinen-4-ol and caryophyllene was relatively significant; while the annual sunshine duration and the annual highest temperature are the main influencing factors for eugenol. The research suggests that long-day and extreme temperature may be more conductive to the accumulation of volatiles. According to the laws of effect of climatic factors on volatiles, we can optimize the cultivation conditions and select suitable locality to improve the quality of Artemisiae Argyi Folium.

Hydrogen peroxide is involved in salicylic acid-elicited rosmarinic acid production in Salvia miltiorrhiza cell cultures.

Salicylic acid (SA) is an elicitor to induce the biosynthesis of secondary metabolites in plant cells. Hydrogen peroxide (H2O2) plays an important role as a key signaling molecule in response to various stimuli and is involved in the accumulation of secondary metabolites. However, the relationship between them is unclear and their synergetic functions on accumulation of secondary metabolites are unknown. In this paper, the roles of SA and H2O2 in rosmarinic acid (RA) production in Salvia miltiorrhiza cell cultures were investigated. The results showed that SA significantly enhanced H2O2 production, phenylalanine ammonia-lyase (PAL) activity, and RA accumulation. Exogenous H2O2 could also promote PAL activity and enhance RA production. If H2O2 production was inhibited by NADPH oxidase inhibitor (IMD) or scavenged by quencher (DMTU), RA accumulation would be blocked. These results indicated that H2O2 is secondary messenger for signal transduction, which can be induced by SA, significantly and promotes RA accumulation.

Genome-wide exploration of the GDSL-type esterase/lipase gene family in rapeseed reveals several BnGELP proteins active during early seedling development.

The Gly-Asp-Ser-Leu (GDSL)-type esterase/lipase proteins (GELP) are one of the most important families of lipolytic enzymes and play prominent roles in seed germination and early seedling establishment through mobilizing the lipids stored in seeds. However, there are no comprehensive studies systematically investigating the GELP gene family in Brassica napus (BnGELP), and their biological significance to these physiological processes are far from understood. In the present study, a total of 240 BnGELP genes were identified in B. napus cultivar "Zhongshuang 11" (ZS11), which is nearly 2.3-fold more GELP genes than in Arabidopsis thaliana. The BnGELP genes clustered into 5 clades based on phylogenetic analysis. Ten BnGELPs were identified through zymogram analysis of esterase activity followed by mass spectrometry, among which five clustered into the clade 5. Gene and protein architecture, gene expression, and cis-element analyses of BnGELP genes in clade 5 suggested that they may play different roles in different tissues and in response to different abiotic stresses. BnGELP99 and BnGELP159 were slightly induced by cold, which may be attributed to two low-temperature responsive cis-acting regulatory elements present in their promoters. An increased activity of esterase isozymes by cold was also observed, which may reflect other cold inducible esterases/lipases in addition to the ten identified BnGELPs. This study provides a systemic view of the BnGELP gene family and offers a strategy for researchers to identify candidate esterase/lipase genes responsible for lipid mobilization during seed germination and early seedling establishment.

Genome-Wide Identification of the Cytochrome P450 Superfamily Genes and Targeted Editing of BnCYP704B1 Confers Male Sterility in Rapeseed.

The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar "Zhongshuang 11" (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes.

Genome-Wide Analysis of MYB Transcription Factor Gene Superfamily Reveals BjPHL2a Involved in Modulating the Expression of BjCHI1 in Brassica juncea.

Brassica juncea is an economically important vegetable and oilseed crop. The MYB transcription factor superfamily is one of the largest transcription factor families in plants, and plays crucial roles in regulating the expression of key genes involved in a variety of physiological processes. However, a systematic analysis of the MYB transcription factor genes in Brassica juncea (BjMYB) has not been performed. In this study, a total of 502 BjMYB superfamily transcription factor genes were identified, including 23 1R-MYBs, 388 R2R3-MYBs, 16 3R-MYBs, 4 4R-MYBs, 7 atypical MYBs, and 64 MYB-CCs, which is approximately 2.4-fold larger than that of AtMYBs. Phylogenetic relationship analysis revealed that the MYB-CC subfamily consists of 64 BjMYB-CC genes. The expression pattern of members of PHL2 subclade homologous genes in Brassica juncea (BjPHL2) after Botrytis cinerea infection were determined, and BjPHL2a was isolated from a yeast one-hybrid screen with the promoter of BjCHI1 as bait. BjPHL2a was found to localize mainly in the nucleus of plant cells. An EMSA assay confirmed that BjPHL2a binds to the Wbl-4 element of BjCHI1. Transiently expressed BjPHL2a activates expression of the GUS reporter system driven by a BjCHI1 mini-promoter in tobacco (Nicotiana benthamiana) leaves. Taken together, our data provide a comprehensive evaluation of BjMYBs and show that BjPHL2a, one of the members of BjMYB-CCs, functions as a transcription activator by interacting with the Wbl-4 element in the promoter of BjCHI1 for targeted gene-inducible expression.

[Response of understory species distribution of Robinia pseudoacacia plantation to environmental factors in loess hilly region, China.] / é»„åœŸä¸˜é™µåŒºåˆºæ§ç¾¤è½æž—ä¸‹ç‰©ç§åˆ†å¸ƒå¯¹çŽ¯å¢ƒå› å­çš„å“åº”.

We explored the distribution characteristics and influencing factors of understory species in Robinia pseudoacacia plantation in Wuqi, Ansai, Mizhi, Yichuan and other places, in combination with niche breadth, soil, altitude and other environmental factors. We analyzed the response mechanism of species distribution to environmental factor to provide a scientific basis for the mana-gement of R. pseudoacacia plantation in loess hilly region. The results showed that the broadly distributed understory species in R. pseudoacacia plantation were Setaria viridis, Heteropappus altaicus, Artemisia scoparia, Poa sphondylodes, Artemisia leucophylla, Ixeris sonchifolia, and Incarvillea sinensis. With the increasing rehabilitationage, the dominant understory species distribution followed the order: Artemisia capillaries → P. sphondylodes → A. scoparia → others (Rubia cordifolia, Rosa rubus and so on) → A. leucophylla → S. viridis. Results from principal component analysis showed that soil total phosphorus (25.6%), altitude (20.3%) and soil total nitrogen (19.3%) were the key factors influencing understory species distribution in R. pseudoacacia plantation. Soil organic carbon content, soil total nitrogen content, soil total phosphorus content, soil water content and the number of understory species distribution in R. pseudoacacia plantation were generally positively correlated with the degree of correlation varying across different species. There was no correlation between the slope aspect and the understory species distribution in R. pseudoacacia plantation. In conclusion, topography and soil factors played important roles in driving understory species distribution. The steeper the slope, the higher the altitude, the fewer understory species in R. pseudoacacia plantation. Understory species distribution was mainly affected by soil total phosphorus content and altitude. The understory species distribution reflected the differences of soil nutrient status, which had a certain guiding role in the management of R. pseudoacacia plantation.

[Effects of salicylic acid on synthesis of rosmarinic acid and related enzymes in the suspension cultures of Salvia miltiorrhiza].

Rosmarinic acid (RA), a phenolic acid, is one of the important secondary metabolites produced in Salvia miltiorrhiza. To observe the influence of salicylic acid (SA), an elicitor, on the synthesis of RA and related enzymes, we treated the cell suspension cultures of S. miltiorrhiza with SA and L-a-aminooxy-beta-phenylpropionic acid (AOPP), a competitive inhibitor of tyrosine aminotransferase (TAT). Under this condition, the activities of related enzymes, such as phenylalanine ammonia-lyase and TAT were traced and assayed; the accumulative amount of RA was measured. The results showed that the PAL activity reached the peak at 4 h, 124% higher than that of the control, and the content of RA reached its maximum ((5.914 +/- 0.296) mg/g dry weight) at 8 h, after treated by 6.25 mg/L SA on day 6 of the suspension culture. The results of treatment with 0.1 micromol/L AOPP showed that AOPP affected little on the TAT activity, while the PAL activity was significantly influenced, with 44% lower than that of the control at 6 h. Meanwhile, the reduced accumulation of RA ((4.709 +/- 0.204) mg/g dry weight) paralleled with the decrease in PAL activity. The co-treatment by 0.1 micromol/L AOPP and 6.25 mg/L SA relieved the restriction imposed by AOPP on PAL, and made the cell cultures accumulate more RA than sole treatment with AOPP, indicated that SA induced the accumulation of RA in suspension cell culture of S. miltiorrhiza, and the rate-limiting effect of PAL was stronger than TAT.

The protein kinase MEK1/2 participates in the regulation of ascorbate and glutathione content by jasmonic acid in Agropyron cristatum leaves.

This study investigated the role of the protein kinase MEK1/2 in the regulation of ascorbate and glutathione content by jasmonic acid in Agropyron cristatum leaves. The results showed that JA induced increases in the transcript levels and activities of APX, GR, MDHAR, DHAR, GalLDH and γ-ECS, the contents of AsA, GSH, total ascorbate and total glutathione, and the ratios of AsA/DHA and GSH/GSSG, and reduced the E(GSSG/2GSH). The increases, except for the transcript level and activity of γ-ECS and the reduction in E(GSSG/2GSH,) were all suppressed by pre-treatment with the MEK1/2 inhibitors PD98059 and U0126. The results of western blot analyses showed that JA induced increases in the phosphorylation level of MEK1/2. Our results suggest that JA could induce the activation of MEK1/2 by increasing the phosphorylation level, which, in turn, resulted in the up-regulation of ascorbate and glutathione content in A. cristatum leaves.

[Effects of drying and re-watering on the photosynthesis and active oxygen metabolism of Periploca sepium seedlings].

Taking two-year-old Periploca sepium seedlings as test materials, an experiment with controlled soil water contents was conducted to study the effects of repeated drying and re-watering on the leaf photosynthetic characteristics and the lipid peroxidation and antioxidant system in young leaves, mature leaves, old leaves, new stems, and fine roots. The seedlings were subjected to three cycles of drying and re-watering, with regular irrigation to maintain the soil water content at around 80% of field capacity as the control (CK). Under drying, the leaf relative water content (RWC) and net photosynthesis rate (Pn) decreased significantly, while the leaf photosynthetic pigments content increased. When the seedlings were re-watered, their leaf RWC recovered to the CK level, showing a strong repair capacity after drying. Both the leaf chlorophyll content and the Pn after repeated drying and re-watering presented a higher level than those of the CK, indicating a compensatory effect appeared and an appropriate drought stress being able to induce the adaptability of P. sepium to drought stress. Stomatal closure was the main factor limiting P. sepium photosynthesis under drought stress, while non-stomatal limitation only worked at noon. Under drying, the superoxide anion radical (O2-*) production rate in young leaves, new stems, and fine roots increased while the malondialdehyde (MDA) contents decreased, suggesting that these young tissues were not suffered from the oxidative stress. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in different organs had different variation trends, with those in fine roots changed actively, suggesting the important role of fine roots in the acclimation of P. sepium to drought environment. It was the cooperation and coordination among plant organs that made P. sepium more adaptive to the repeated drying and wetting conditions in drought-prone regions.

[Characteristics of Robinia pseudoacacia water physiological ecology under different habitats in North Shaanxi gully areas of Loess Plateau].

With locust (Robinia pseudoacacia), the main tree species in afforestation, as test material, this paper studied the characteristics of its water physiological ecology and productivity under four habitats, i. e. , sunny, shady, semi-sunny and semi-shady hillsides, in the North Shaanxi gully areas of Loess Plateau. The mean water content in 0 - 500 cm soil layer was 8.87% (shady), 8.06% (semi-shady), 7.62% (semi-sunny), and 6.96% (sunny), respectively. There was a significant discrepancy between shady, semi-shady, semi-sunny and sunnyhilisides (a 0.01), and the difference between shady, semi-shady and semi-sunny, as well as between semi-sunny and sunny hillsides was also significant (a 0.05) . Remarkable relationships were found between leaf RWC, locus WSD and soil water content (SWC) . The leaf RWC and locust water potential under sunny hillside were lower, but those under shady hillside were higher. The daily mean transpiration was in order of shady (4.07 microg x cm(-2) x s(-1)) > semi-shady (3.89 microg x cm(-2) x s(-1)) > semi-sunny (3.05 microg x cm(-2) x s(-1)) > sunny (2.70 microg x cm(-2)s(-1)) hillside. The remarkable difference of transpiration appeared at 11:00 and 13:00, and there existed a remarkable relationship between transpiration and light intensity, RH and soil water content. All of these resulted in a diversity of locust biomass under different habitats, the highest under shady hillside, and the lowest under sunny hillside, and the differences between different habitats were all significant. It could be concluded that soil water content was the main factor affecting locust growth.