Overexpression of <i>SmANS</i> Enhances Anthocyanin Accumulation and Alters Phenolic Acids Content in <i>Salvia miltiorrhiza</i> and <i>Salvia miltiorrhiza</i> Bge f. <i>alba</i> Plantlets

Flavonoids play multiple roles in plant coloration and stress resistance and are closely associated with human health. Flavonoids and non-flavonoids (such as phenolic acids) are produced via the phenylpropanoid-derived pathway. Anthocyanidin synthase (ANS) catalyzes the synthesis of anthocyanins from leucoanthocyanidin in the flavonoids branched pathway. In this study, SmANS from Salvia miltiorrhiza was cloned and mainly localized in the endoplasmic reticulum (ER), plastids, Golgi, plasma membrane, and nucleus of tobacco epidermal cells, and was most highly expressed in purple petals in S. miltiorrhiza, whereas it showed almost no expression in white petals, green calyxes, and pistils in S. miltiorrhiza Bge f. alba. Overexpressed SmANS enhanced anthocyanin accumulation but reduced salvianolic acid B (SAB) and rosmarinic acid (RA) biosynthesis in S. miltiorrhiza and S. miltiorrhiza Bge f. alba plantlets, meanwhile, it restored the purple-red phenotype in S. miltiorrhiza Bge f. alba. These changes were due to reallocation of the metabolic flow, which was influenced by the SmANS gene. These findings indicate that SmANS not only plays a key role in anthocyanin accumulation in S. miltiorrhiza, but also acts as a &#8220;switch&#8221; for the coloration of S. miltiorrhiza Bge f. alba. This study provides baseline information for further research on flavonoids metabolism and improvement of anthocyanin or phenolic acid production by genetic engineering

Bluethunder: A 2-level Directional Predictor Based Side-Channel Attack against SGX

Software Guard Extension (SGX) is a hardware-based trusted execution environment (TEE) implemented in recent Intel commodity processors. By isolating the memory of security-critical applications from untrusted software, this mechanism provides users with a strongly shielded environment called enclave for executing programs safely. However, recent studies have demonstrated that SGX enclaves are vulnerable to side-channel attacks. In order to deal with these attacks, several protection techniques have been studied and utilized.In this paper, we explore a new pattern history table (PHT) based side-channel attack against SGX named Bluethunder, which can bypass existing protection techniques and reveal the secret information inside an enclave. Comparing to existing PHT-based attacks (such as Branchscope [ERAG+18]), Bluethunder abuses the 2-level directional predictor in the branch prediction unit, on top of which we develop an exploitation methodology to disclose the input-dependent control flow in an enclave. Since the cost of training the 2-level predictor is pretty low, Bluethunder can achieve a high bandwidth during the attack. We evaluate our attacks on two case studies: extracting the format string information in the vfprintf function in the Intel SGX SDK and attacking the implementation of RSA decryption algorithm in mbed TLS. Both attacks show that Bluethunder can recover fine-grained information inside an enclave with low training overhead, which outperforms the latest PHT-based side channel attack (Branchscope) by 52×. Specifically, in the second attack, Bluethunder can recover the RSA private key with 96.76% accuracy in a single run

The Protein Kinase SmSnRK2.6 Positively Regulates Phenolic Acid Biosynthesis in Salvia miltiorrhiza by Interacting with SmAREB1

Subclass III members of the sucrose non-fermenting-1-related protein kinase 2 (SnRK2) play essential roles in both the abscisic acid signaling and abiotic stress responses of plants by phosphorylating the downstream ABA-responsive element (ABRE)-binding proteins (AREB/ABFs). This comprehensive study investigated the function of new candidate genes, namely SmSnRK2.3, SmSnRK2.6, and SmAREB1, with a view to breeding novel varieties of Salvia miltiorrhiza with improved stress tolerance stresses and more content of bioactive ingredients. Exogenous ABA strongly induced the expression of these genes. PlantCARE predicted several hormones and stress response cis-elements in their promoters. SmSnRK2.6 and SmAREB1 showed the highest expression levels in the leaves of S. miltiorrhiza seedlings, while SmSnRK2.3 exhibited a steady expression in their roots, stems, and leaves. A subcellular localization assay revealed that both SmSnRK2.3 and SmSnRK2.6 were located in the cell membrane, cytoplasm, and nucleus, whereas SmAREB1 was exclusive to the nucleus. Overexpressing SmSnRK2.3 did not significantly promote the accumulation of rosmarinic acid (RA) and salvianolic acid B (Sal B) in the transgenic S. miltiorrhiza hairy roots. However, overexpressing SmSnRK2.6 and SmAREB1 increased the contents of RA and Sal B, and regulated the expression levels of structural genes participating in the phenolic acid-branched and side-branched pathways, including SmPAL1, SmC4H, Sm4CL1, SmTAT, SmHPPR, SmRAS, SmCHS, SmCCR, SmCOMT, and SmHPPD. Furthermore, SmSnRK2.3 and SmSnRK2.6 interacted physically with SmAREB1. In summary, our results indicate that SmSnRK2.6 is involved in stress responses and can regulate structural gene transcripts to promote greater metabolic flux to the phenolic acid-branched pathway, via its interaction with SmAREB1, a transcription factor. In this way, SmSnRK2.6 contributes to the positive regulation of phenolic acids in S. miltiorrhiza hairy roots

SmGRAS1 and SmGRAS2 Regulate the Biosynthesis of Tanshinones and Phenolic Acids in Salvia miltiorrhiza

Salvia miltiorrhiza is one of the most widely used traditional Chinese medicinal plants because of its excellent performance in treating heart diseases. Tanshinones and phenolic acids are two important classes of effective metabolites, and their biosynthesis has attracted widespread interest. Here, we functionally characterized SmGRAS1 and SmGRAS2, two GRAS family transcription factors from S. miltiorrhiza. SmGRAS1/2 were highly expressed in the root periderm, where tanshinones mainly accumulated in S. miltiorrhiza. Overexpression of SmGRAS1/2 upregulated tanshinones accumulation and downregulated GA, phenolic acids contents, and root biomass. However, antisense expression of SmGRAS1/2 reduced the tanshinones accumulation and increased the GA, phenolic acids contents, and root biomass. The expression patterns of biosynthesis genes were consistent with the changes in compounds accumulation. GA treatment increased tanshinones, phenolic acids, and GA contents in the overexpression lines, and restored the root growth inhibited by overexpressing SmGRAS1/2. Subsequently, yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays (EMSA) showed SmGRAS1 promoted tanshinones biosynthesis by directly binding to the GARE motif in the SmKSL1 promoter and activating its expression. Yeast two-hybrid assays showed SmGRAS1 interacted physically with SmGRAS2. Taken together, the results revealed that SmGRAS1/2 acted as repressors in root growth and phenolic acids biosynthesis but as positive regulators in tanshinones biosynthesis. Overall, our findings revealed the potential value of SmGRAS1/2 in genetically engineering changes in secondary metabolism. &nbsp;</p

Amplification of black carbon light absorption induced by atmospheric aging: temporal variation at seasonal and diel scales in urban Guangzhou

Black carbon (BC) aerosols have been widely recognized as a vital climate forcer in the atmosphere. Amplification of light absorption can occur due to coatings on BC during atmospheric aging, an effect that remains uncertain in accessing the radiative forcing of BC. Existing studies on the absorption enhancement factor (E-abs) have poor coverage on both seasonal and diurnal scales. In this study, we applied a recently developed minimum R squared (MRS) method, which can cover both seasonal and diurnal scales, for E-abs quantification. Using field measurement data in Guangzhou, the aims of this study are to explore (1) the temporal dynamics of BC optical properties at seasonal (wet season, 31 July-10 September; dry season, 15 November 2017-15 January 2018) and did scales (1 h time resolution) in the typical urban environment and (2) the influencing factors on E-abs temporal variability. Mass absorption efficiency at 520 nm by primary aerosols (MAE(p520)) determined by the MRS method exhibited a strong seasonality (8.6 m(2) g(-1) in the wet season and 16.8 m(2) g(-1) in the dry season). E-abs520 was higher in the wet season (1.51 +/- 0.50) and lower in the dry season (1.29 +/- 0.28). Absorption Angstrom exponent (AAE(470-660)) in the dry season (1.46 +/- 0.12) was higher than that in the wet season (1.37 +/- 0.10). Collective evidence showed that the active biomass burning (BB) in the dry season effectively altered the optical properties of BC, leading to elevated MAE, MAE(p) and AAE in the dry season compared to those in the wet season. Diurnal E(abs520 )was positively correlated with AAE470-660 (R-2 = 0.71) and negatively correlated with the AE33 aerosol loading compensation parameter (k) (R-2 = 0.74) in the wet season, but these correlations were significantly weaker in the dry season, which may be related to the impact of BB. This result suggests that during the wet season, the lensing effect was more likely dominating the AAE diurnal variability rather than the contribution from brown carbon (BrC). Secondary processing can affect Eabs diurnal dynamics The E-abs520 exhibited a clear dependency on the ratio of secondary organic carbon to organic carbon (SOC/OC), confirming the contribution of secondary organic aerosols to E-abs; Ea(bs520) correlated well with nitrate and showed a clear dependence on temperature. This new finding implies that gas-particle partitioning of semivolatile compounds may potentially play an important role in steering the diurnal fluctuation of E-abs520. In the dry season, the diurnal variability in E-abs520 was associated with photochemical aging as evidenced by the good correlation (R-2 =0.69) between oxidant concentrations (O-x = O-3 + NO2) and E-abs520

Molecular cloning and characterisation of two enzymes involved in the rosmarinic acid biosynthesis pathway of Prunella vulgaris L.

Prunella vulgaris, a widely-used medicinal perennial herb, contains various active compounds and has multifaceted medicinal activities. As the index component in P. vulgaris, rosmarinic acid (RA) is a typical phenolic acid, with significant anti-inflammatory, anti-oxidant, anti-tumour, anti-viral, and anti-microbial properties. To better understand the RA biosynthetic pathway in P. vulgaris, we isolated and cloned the cDNA sequences of putative RA synthase (PvRAS) and cytochrome P450 monooxygenase (PvCYP98A101), two important enzymes catalysing RA biosynthesis. Sequence analysis revealed that PvRAS contained an open reading frame (ORF) of 1305 bp encoding a 435-amino-acid residue belonging to the BAHD acyltransferase family, and PvCYP98A101 contained an ORF of 1530 bp encoding a 510-amino-acid residue, as a member of the CYP98A family. The deduced PvRAS and PvCYP98A101 amino acid sequences shared high similarity with other putative/known RASs and CYPs, respectively. Quantitative real-time PCR analysis showed that constitutive expression of PvRAS and PvCYP98A101 was much higher in roots than in leaves, stems, or spikes. Further analysis indicated that PvRAS was localised in the cytosol and nucleus, whilst PvCYP98A101 existed as a membrane protein in the endoplasmic reticulum. These results highlight the RA biosynthesis pathway in P. vulgaris and, provide useful information to engineer natural products