Exogenous ethephon treatment on the biosynthesis and accumulation of astragaloside IV in Astragalus membranaceus Bge. Var. Mongholicus (Bge.) Hsiao.

BACKGROUND: Astragaloside IV is a main medicinal active ingredient in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao, which is also the key biomarker of A. membranaceus quality. Ethylene has been well-documented to involve in secondary metabolites biosynthesis in plants. Nevertheless, how ethylene regulates astragaloside IV biosynthesis in A. membranaceus is still unclear. Therefore, in the present study different dosages and time-dependent exogenous application of ethephon (Eth) were employed to analyze astragaloside IV accumulation and its biosynthesis genes expression level in hydroponically A. membranaceus. RESULTS: Exogenous 200 µmol·L- 1Eth supply is most significantly increased astragaloside IV contents in A. membranaceus when compared with non-Eth supply. After 12 h 200 µmol·L- 1 Eth treatment, the astragaloside IV contents reaching the highest content at 3 d Eth treatment(P ≤ 0.05). Moreover, After Eth treatment, all detected key genes involved in astragaloside IV synthesis were significant decrease at 3rd day(P ≤ 0.05). However, SE displayed a significant increase at the 3rd day under Eth treatment(P ≤ 0.05). Under Eth treatment, the expression level of FPS, HMGR, IDI, SS, and CYP93E3 exhibited significant negative correlations with astragaloside IV content, while expression level of SE displayed a significant positive correlation. CONCLUSIONS: These findings suggest that exogenous Eth treatment can influence the synthesis of astragaloside IV by regulating the expression of FPS, HMGR, IDI, SS, CYP93E3 and SE. This study provides a theoretical basis for utilizing molecular strategies to enhance the quality of A. membranaceus.

The SmERF1b-like regulates tanshinone biosynthesis in Salvia miltiorrhiza hairy root.

The ethylene response factor family genes are involved in the regulation of secondary metabolism in Salvia miltiorrhiza, but the mechanism underlying this regulation remains elusive. In the present study, based on the cDNA library of S. miltiorrhiza, an AP2/ERF gene was cloned and named SmERF1b-like. This gene exhibited a significant response to exogenous ethylene supply, such that ethylene remarkably upregulated SmERF1b-like expression levels in the leaves of S. miltiorrhiza. Subcellular localization showed that SmERF1b-like is located in the nucleus. Furthermore, SmERF1b-like showed a binding affinity with a GCC-box motif in the promoter region of genes associated with tanshinone biosynthesis in S. miltiorrhiza. Overexpression of SmERF1b-like in hairy roots of S. miltiorrhiza substantially upregulated SmCPS1 and SmKSL1 expression levels, resulting in increased biosynthesis of tanshinone I and cryptotanshinone contents. This finding provides valuable theoretical support for the utilization of a plant genetic engineering strategy to enhance S. miltiorrhiza resources.

Nitrate enhances cadmium accumulation through modulating sulfur metabolism in sweet sorghum.

Sweet sorghum deploys tremendous potential for phytoremediation of cadmium (Cd)-polluted soils. Nitrate increases Cd accumulation in sweet sorghum, but the mechanism underlying this is still elusive. Sulfur-containing metabolites have been corroborated to play important roles in Cd tolerance in plants. Thus, whether sulfur metabolism contributed to nitrate-increased Cd accumulation in sweet sorghum was investigated in the present study. Two-way ANOVA analysis showed that most sulfur-containing metabolites concentrations and relevant enzymes activities were regulated by nitrate, Cd and interplay of nitrate and Cd. By using grey correlation analysis and Pearson correlation coefficient, Cd accumulation in shoots as affected by nitrate was also mainly ascribed to sulfur metabolism. ATP sulfurylase (ATPS) activities and non-protein thiol (NPT) concentrations in leaves were the two prominent factors that positively correlated with Cd accumulation in shoots. Excess nitrate elevated ATPS activities in leaves which contributed to increased NPT and phytochelatins (PCs) concentrations in leaves. Nitrate enhanced Cd accumulation in shoots of sweet sorghum under a low level of Cd treatment. Intriguingly, Cd accumulation in shoots of sweet sorghum was similar between a low level and a high level of Cd treatment. Principal Components Analysis (PCA) based on 34 parameters failed to separate the low Cd treatment from the high Cd treatment either, suggesting sweet sorghum is exclusively suitable for phytoremediation of slight Cd-polluted arable lands. Taken together, enhanced Cd accumulation in shoots of sweet sorghum by excess nitrate application is closely correlated with sulfur metabolism containing elevated ATPS activities, NPT and PCs concentrations in leaves.

Transcriptome and HPLC Analysis Reveal the Regulatory Mechanisms of Aurantio-Obtusin in Space Environment-Induced Senna obtusifolia Lines.

Senna obtusifolia is a famous medicinal plant that is widely used in Asian countries. Its seed plays an important role in the treatment of many diseases because it contains various anthraquinones and flavonoids. Our previous studies have indicated that three space environment-induced S. obtusifolia lines (SP-lines) i.e., QC10, QC29, and QC46, have higher seed yield and aurantio-obtusin (AO) content. However, the underlying mechanism of higher AO content in SP-lines is still unknown. Herein, transcriptome sequencing and HPLC were employed to analyze the differences between SP-lines and ground control (GC3) and elucidate the regulatory mechanisms of AO accumulation in SP-lines. The results show that 4002 differentially expressed genes (DEGs) were identified in SP-lines versus (vs.) GC3. DEGs in the QC10 vs. GC3, QC29 vs. GC3, and QC46 vs. GC3 comparisons were classified into 28, 36, and 81 GO terms and involved in 63, 74, and 107 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG pathway and gene expression analysis revealed that DEGs involved in anthraquinone pathways were significantly elevated in QC10 and QC46. Integrating the results of GO annotation, KEGG enrichment, and gene expression analysis, we propose that the elevated genes such as DAHPS, DHQS, and MenB enhance the metabolic flux in the anthraquinone pathway and promote AO content in QC10 and QC46. Taken together, this study elucidated the mechanism of AO content in SP-lines and provides valuable genetic information for S. obtusifolia. In addition, to the best of our knowledge, this study presents the first transcriptome analysis of environment-induced medicinal plants and paves the way to select elite S. obtusifolia varieties in the future.

Sustainable management of cadmium-contaminated soils as affected by exogenous application of nutrients: A review.

Cadmium (Cd) pollution in arable land is of great concern as it impairs plant growth and further threats human health via food-chain. Exogenous supplementation of nutrients is an environmentally-friendly, cost-effective, convenient and feasible strategy for regulating Cd uptake, transport and accumulation in plants. To sustain Cd-contaminated soils management, on the one hand, a low level of the Cd-contaminated soil is expected to cultivate crops with decreased Cd accumulation as affected by exogenous nutrients application, on another hand, a high level of the Cd-contaminated soil is suggested to cultivate phytoextraction plants with increased Cd accumulation as affected by exogenous nutrients application. Nevertheless, effects of nutrients on Cd accumulation in plants are still ambiguous. Thus, data of Cd accumulation in shoots of plants as affected by exogenous application of nutrients were collected from previously published articles between 2005 and 2021 in the present study. According to the data, exogenous supply of calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn) and silicon (Si) to a larger extent decrease Cd amounts in shoots of plants. By contrast, exogenous nitrogen (N), and deficient Ca, Mg and Fe supply have a great possibility to increase Cd amounts in shoots of plants. Although exogenous application of phosphorus (P), sulfur (S), potassium (K), zinc (Zn) and selenium (Se) have a great opportunity to increase biomass, they show different effects on Cd concentrations. As a result, the odds are even for increasing and decreasing Cd amounts in shoots of plants. Taken together, exogenous application of Ca, Mg, Fe, Mn and Si might decrease Cd accumulation in plants that are recommended for crops production. Exogenous N and deficient Ca, Mg and Fe supply might increase Cd accumulation in plants that are recommended for phytoextraction plants. Exogenous application of P, S, K, Zn and Se have half a chance to increase or decrease Cd accumulation in plants. Therefore, dosages, forms and species should be taken into account when exogenous P, S, K, Zn and Se are added.

Nitrate Increases Cadmium Accumulation in Sweet Sorghum for Improving Phytoextraction Efficiency Rather Than Ammonium.

Sweet sorghum has potential for phytoextraction of cadmium (Cd) owning to its large biomass and relatively high Cd tolerance. Nitrogen affects both growth and Cd concentrations in plants. However, different forms of nitrogen effects on Cd accumulation in sweet sorghum to improve efficiency of Cd phytoremediation is still elusive. In this study, nitrate substantially promoted both dry weight and Cd concentrations in leaves, stems + sheaths and roots of sweet sorghum when compared with ammonium. As a result, Cd accumulation in nitrate-supplied sweet sorghum was around 3.7-fold of that in ammonium-supplied plants under unbuffered pH condition, while the fold was about 2.2 under buffered pH condition. We speculated pH values and Cd species in the growth medium to some extent contributed to increased Cd accumulation as affected by nitrate. Net photosynthesis rate and Fv/Fm of nitrate-treated plants under Cd stress were higher than that of ammonium-treated plants when the pH was unbuffered. Responses of antioxidant capacity in roots to Cd stress with nitrate application were stronger than that with ammonium supplementation. Taken together, nitrate is more suitable than ammonium for Cd phytoextraction by using sweet sorghum, which is able to enhance at least double efficiency of phytoextraction.

Effect of K-/S- segments on subcellular localization and dimerization of wheat dehydrin WZY1-2.

Dehydrins (DHNs) belong to group Ⅱ late embryogenesis abundant (LEA) proteins which perform multiple functions in plants during stress conditions. Both K- and S-segments are conserved domains in the dehydrin protein family; however, there are only a few in vivo functional studies for these two conserved segments. In this study, the DHN gene wzy1-2 was isolated from Triticum aestivum and its K-/S-segment-truncated derivatives were generated. In order to explore the biological function of these two conserved fragments, subcellular localization and dimerization detection assays were performed for the K-/S-segment-truncated derivatives. Results of GFP fusion and bimolecular fluorescence complementation (BiFC) assays indicated that WZY1-2 localized to nucleus as a homologous dimer. The S-segment partially regulated the nuclear localization of WZY1-2 but did not affect its dimerization, while the K-segment influenced neither the dimer formation nor the subcellular localization.

Molecular role of ethylene in fruit r​ipening of Ziziphus jujube Mill.

The fruit of Chinese jujube (Ziziphus jujube) is widely consumed by human beings due to its high proteins, vitamins, and mineral nutrients. The harvest time of Chinese jujube fruit determines its quality, while ethylene plays a pivotal role in fruit ripening. Nevertheless, the relationship between ethylene biosynthesis/signal transduction and fruit ripening of Chinese jujube is still elusive. Here, the Chinese jujube fruit ripening with its fruit peel color change from cyan to dark red at seven different ripening stages (stage I-VII) and expression levels of genes related to ethylene synthesis and signal transduction were determined. Results showed that expression levels of ZjACO1-3, ZjETR2, ZjERF1, and ZjERF4 were increasingly upregulated, whereas the expression levels of ZjERS1, ZjETI, ZjERF2, and ZjERF3 were downregulated from green to red fruit ripening stages. Among them, ZjACO1-3 promoters contain ethylene response element. Taken together, Chinese jujube fruit ripening might be affected by the ethylene signaling which was mainly regulated by ZjACO, a gene involved in ethylene biosynthesis. This research supports theories and techniques for the storage, preservation and molecular breeding of Z. jujube.

Transcriptomic analysis reveals the GRAS family genes respond to gibberellin in Salvia miltiorrhiza hairy roots.

BACKGROUND: Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants with high medicinal value. Gibberellins are growth-promoting phytohormones that regulate numerous growth and developmental processes in plants. However, their role on the secondary metabolism regulation has not been investigated. RESULTS: In this study, we found that gibberellic acid (GA) can promote hairy roots growth and increase the contents of tanshinones and phenolic acids. Transcriptomic sequencing revealed that many genes involved in the secondary metabolism pathway were the GA-responsive. After further analysis of GA signaling pathway genes, which their expression profiles have significantly changed, it was found that the GRAS transcription factor family had a significant response to GA. We identified 35 SmGRAS genes in S. miltiorrhiza, which can be divided into 10 subfamilies. Thereafter, members of the same subfamily showed similar conserved motifs and gene structures, suggesting possible conserved functions. CONCLUSIONS: Most SmGRAS genes were significantly responsive to GA, indicating that they may play an important role in the GA signaling pathway, also participating in the GA regulation of root growth and secondary metabolism in S. miltiorrhiza.

The ethylene response factor SmERF8 regulates the expression of SmKSL1 and is involved in tanshinone biosynthesis in Saliva miltiorrhiza hairy roots.

Saliva miltiorrhiza ethylene response factor (SmERF), predicted to be expressed genome-wide, is the potential regulator of tanshinone biosynthesis. However, few studies have investigated its transcriptional regulation pathways in tanshinone biosynthesis. Here, we report an ethylene response factor (SmERF8), which was screened by the SmKSL1 (a key gene in tanshinone biosynthesis) promoter from the S. miltiorrhiza cDNA library. The SmERF8, highly expressed in S. miltiorrhiza root head, is sensitive to Eth stress, and its protein was enriched in the nucleus. The SmERF8 recognizes the GCC-box in the SmKSL1 promoter. Overexpression and RNAi of SmERF8 in S. miltiorrhiza transgenic hairy roots showed that the tanshinone contents were significantly increased in the overexpression transgenic lines and decreased in RNAi lines. These results suggest that the SmERF8 may be a central activator that regulates the expression of SmKSL1 by binding the GCC-box and then promoting tanshinone biosynthesis. Thus, the SmERF8 may functionally accelerate tanshinone biosynthesis by the transcriptional regulation of its key gene.

[Cloning,subcellular localization and expression pattern analysis of transcription factor SmGRAS3 from Salvia miltiorrhiza].

GRAS transcription factors play important roles in the regulation of plant root growth and GA signaling. In this study,SmGRAS3 gene was cloned,which open reading frame was 2 247 bp,and encoding 748 amino acids. The physicochemical properties and structure of SmGRAS3 and its encoded protein were analyzed by bioinformatics software. This gene belongs to the SCL9 subfamily of the GRAS family,and its promoter sequence mainly contains the light response,stress response,and hormone response elements. It may interact with the GA signal pathway and anti-stress related proteins. The subcellular localization showed that SmGRAS3 protein was mainly located in the nucleus. The expression pattern analysis showed that the expression of Sm GRAS3 was the highest in the root and the lowest in the stem,and both light and low temperature could induce the high expression level of SmGRAS3. This study provides a foundation for further study on the roles of SmGRAS3 gene in the root growth and stress tolerance of Salvia miltiorrhiza.

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.

Overexpression of SmANS Enhances Anthocyanin Accumulation and Alters Phenolic Acids Content in Salvia miltiorrhiza and Salvia miltiorrhiza Bge f. alba 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 "switch" 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.

Physiological, transcriptional, and metabolic alterations in spaceflight-subjected Senna obtusifolia.

Senna obtusifolia is a widely used medicinal herb in Asian countries. To select elite cultivars, S. obtusifolia seeds were carried by "ShenZhou Ⅷ" recoverable satellite to space. Three spaceflight-subjected lines (SP-lines), namely QC10, QC29, QC46, and their ground control line (GC-line) were cultivated on the ground. Previous studies demonstrated that biological traits and secondary metabolites are different between SP-lines and GC-line. Here, we combined physiological, transcriptional, and metabolic studies to compare the differences between SP-lines and GC-line. The results showed that activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and monodehydroascorbate reductase (MDHAR) were dramatically increased in SP-lines as compared to that of GC-line. Transcript levels of SOD, POD, CAT, APX, and MDHAR were significantly up-regulated in SP-lines. Malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents decreased in SP-lines. Seed yields of QC29 and QC46 were considerably higher than that of GC-line. Besides, QC29 had significantly higher aurantio-obtusin content. Pearson correlation coefficient analysis revealed positive relationships between POD and aurantio-obtusin, as well as APX and aurantio-obtusin. In conclusion, SP-lines have higher antioxidant gene expression level and antioxidant enzyme activity as compared to that of GC-line. With higher seed yield and aurantio-obtusin content, QC29 can be used to breed elite S. obtusifolia cultivars. This study provides a new insight in SP-lines and paves the way to breed elite S. obtusifolia cultivars in the future.

Distribution and chemical forms of cadmium in Coptis chinensis Franch. determined by laser ablation ICP-MS, cell fractionation, and sequential extraction.

Coptis chinensis Franch., is a widely used medicinal plant in China. This plant is often contaminated by cadmium (Cd) and render health risk to human consumers. Understanding distribution of Cd and its chemical forms is important to evaluate accumulation of the metal and its detoxification mechanisms in this plant. Since few studies have focused on this aspect, we used laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to spatially locate Cd in rhizome cross-sections, and ICP-MS to analyze the Cd subcellular distribution and the chemical forms of Cd in different tissues. Rhizome bioimaging results showed that Cd was distributed predominantly within the periderm, cortex, pith, and root trace vascular bundle. The LA-ICP-MS results suggested that Ca2+ channels might be a pathway for Cd entry into the plant. Subcellular distribution data indicated that most of Cd was associated with the cell wall (41.8-77.1%) and the soluble fraction (14.4-52.7%) in all tissues. Analysis of chemical forms revealed that majority Cd existed in less mobile and less toxic forms in all tissues, and P could convert to insoluble phosphate with Cd to moderate Cd toxicity. The new understanding of Cd accumulation and detoxification might provide novel strategies for reducing the levels of Cd in C. chinensis Franch., thereby mitigating its potential transfer to humans and providing a theoretical basis for evaluating the Cd status in other medicinal plants. Further, our findings might provide a basis for establishing a reasonable Cd limit level of traditional Chinese medicinal materials.

DNA barcoding for the efficient and accurate identification of medicinal polygonati rhizoma in China.

Polygonati rhizoma (PR), a traditional medicinal and edible product with various bioactive components (Polygonatum polysaccharides, saponins, phenols, and flavonoids), is widely consumed in China. However, other species with morphological characteristics similar to those of the actual components are being used to replace or adulterate PR, causing issues with quality control and product safety. The morphological similarity of PR and its substitutes makes classic morphological identification challenging. To address this issue, DNA barcoding-based identification using ITS2 and psbA-trnH sequences was applied in this study to evaluate the efficiency and accuracy of this approach in identifying PR samples collected from 39 different regions in China. The identification of PR by this method was confirmed by other methods (phylogeny-based and character-based methods), and all the samples were clearly and accurately distinguished. This study highlights the efficient and accurate nature of DNA barcoding in PR identification. Applying this technique will provide a means to differentiate PR from other altered formulations, thus improving product quality and safety for consumers of PR and its products.

Localization and Speciation of Chromium in Coptis chinensis Franch. using Synchrotron Radiation X-ray Technology and Laser Ablation ICP-MS.

Coptis chinensis Franch. is one of the most important medicinal plants globally. However, this species contains relatively high concentrations of chromium (Cr) which potentially detrimental to human health. It is important to understand Cr localization and speciation in order to evaluate its accumulation and transportation mechanisms and minimize Cr transfer to humans. As little previous work in this area has been carried out, we utilized synchrotron radiation microscopic X-ray fluorescence (SR-µXRF) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to spatially locate Cr, X-ray absorption near-edge spectroscopy (XANES) to analyze Cr speciation, and inductively coupled plasma mass spectrometry (ICP-MS) to detect Cr subcellular concentration. Micromapping results showed that Cr was distributed predominantly within the vascular cylinder, the periderm and some outer cortex, and the cortex and some vascular bundles in root, rhizome, and petiole, respectively. XANES data showed that Cr(VI) can be reduced to Cr(III) when grown with Cr(VI), and yielded a novel conclusion that this plant contain elemental chromium. ICP-MS data showed that Cr was primarily compartmentalized in cell walls in all tissues. The new insights on Cr accumulation in C. chinensis Franch. provide a theoretical basis for the evaluation of Cr in other medicinal plants.

The ethylene response factor SmERF6 co-regulates the transcription of SmCPS1 and SmKSL1 and is involved in tanshinone biosynthesis in Salvia miltiorrhiza hairy roots.

MAIN CONCLUSION: The SmERF6, which recognizes the GCC-box of SmCPS1 and SmKSL1 promoter in nucleus, regulates the tanshinone biosynthesis in Salvia miltiorrhiza hairy roots. Tanshinone, an important medicinal ingredient in Salvia miltiorrhiza, is best known for its use in medicine. However, the transcription factor regulation of tanshinone biosynthesis is unclear. Here, we isolated and identified a transcription factor in the ERF family of S. miltiorrhiza, SmERF6, which was screened from an S. miltiorrhiza cDNA library by the promoters of two key tanshinone synthesis genes (SmKSL1 and SmCPS1); this factor regulated tanshinone biosynthesis. The gene was highly expressed in the root and responded to ethylene treatment. SmERF6 modulated tanshinone biosynthesis by directly binding to an ethylene-responsive element (GCC-box) of the SmKSL1 and SmCPS1 promoters and activating their transcription. Overexpression of SmERF6 in the hairy roots increased their tanshinone accumulation, and SmERF6 silencing by RNAi led to a lower tanshinone content. Furthermore, tanshinone accumulation maintained homeostasis with the total phenolic acid and flavonoid contents in S. miltiorrhiza. These findings elucidated how SmERF6 directly co-regulates the transcription of SmCPS1 and SmKSL1 and modulates tanshinone synthesis to accelerate the metabolic flux of tanshinone accumulation in S. miltiorrhiza.

Molecular cloning and characterization of five SmGRAS genes associated with tanshinone biosynthesis in Salvia miltiorrhiza hairy roots.

The gibberellin-responsive element binding factor (GRAS) family of proteins plays an important role in the transcriptional regulation of plant development and hormone signaling. To date, there are no reports on GRAS family proteins expressed in Salvia miltiorrhiza. In this study, 28 ESTs that contained the GRAS domain were identified from a S. miltiorrhiza cDNA library. Of these, full-length sequences of five genes were cloned and sequence analysis indicated that all five proteins contain only one GRAS domain and therefore, belong to the GRAS family. The five genes were designated S. miltiorrhiza GRAS1-5 (SmGRAS1-5), which belong to groups I (SmGRAS2 and SmGRAS4), II (SmGRAS3), III (SmGRAS1), and VIII (SmGRAS5) respectively. Additionally, SmGRAS1-5 have different expression patterns in the reed head, stems, leaves, flowers, and roots of S. miltiorrhiza. In this study, the expression of SmGRAS1-5 was sensitive to Gibberellin (GA) stress and that of SmGRAS1, SmGRAS4 and SmGRAS5 was sensitive to Ethephon (Eth) stress respectively. Moreover, S. miltiorrhiza copalyl diphosphate synthases 1 (SmCPS1) and S. miltiorrhiza kaurene synthase like 1 (SmKSL1), which are two key enzymes gene in the diterpenoid biosynthesis pathway, were also response to GA and Eth stress. In addition, Dihydrotanshinone (DT-I) and Tanshinone I (T-I) content were enhanced by GA and Eth stress, Tanshinone IIA (T-IIA) content was increased by GA stress, and the accumulation of Cryptotanshinone (CT) was insensitive to both GA and Eth stress. Together, these results provide insights into functional conservation and diversification of SmGRASs and are useful information for further elucidating SmGRAS functions.

The interaction between endogenous 30S ribosomal subunit protein S11 and Cucumber mosaic virus LS2b protein affects viral replication, infection and gene silencing suppressor activity.

Cucumber mosaic virus (CMV) is a model virus for plant-virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11) was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC) assays observed by confocal laser microscopy and Glutathione S-transferase (GST) pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein.