Cloning and Characterization of the Gene Encoding HMGS Synthase in Polygonatum sibiricum.

The saponins of Polygonatum sibiricum had many pharmacological activities such as antitumor, antioxidation, and blood sugar lowering, which were synthesized by two pathways: mevalonate (MVA) and methylerythritol phosphate (MEP). 3-Hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) was the key enzyme in the MVA synthesis pathway, and its expression level may affect the accumulation of saponins which were the main active ingredients of P. sibiricum. In this study, we successfully cloned HMGS1 and HMGS2 from P. sibiricum and their sequence similarity was 93.71% with 89 different sites. The multiple sequence alignment results indicated that the N-terminal sequences of HMGS were conserved. Phylogenetic analysis showed that P. sibiricum, A. officinalis, N. tazetta, D. nobile, and other relatives had a common evolutionary ancestor. The expression levels of both HMGSs and the total saponin content in different tissues revealed that HMGS expression in rhizomes was positively correlated with total saponin content. Further study of the abiotic stress effect of Methyl Jasmonate (MeJA) demonstrated that the expression of HMGS1 and HMGS2 genes was induced by MeJA, peaked at 24 h, and fell by 48 h. Our present findings would provide a blueprint for future studies of HMGS and its role in triterpenoid biosynthesis in P. sibiricum.

Structure characterization and bioactivity of neutral polysaccharides from different sources of Polygonatum Mill.

Polygonati rhizoma (PR), a traditional medical and edible product, is rich in polysaccharides and exhibits physiological activity, including antioxidant, hypoglycemic and hypolipidemic properties. Neutral polysaccharides have been reported to be one of the main active ingredients of Polygonatum, with many of these fractions being responsible for the biological activity. This behavior was shown to be closely connected to the chemical structure, monosaccharide composition, and glycosidic bond type. There are few reports on the chemical constituents of the neutral polysaccharides from different sources of PR. In this study, neutral polysaccharides of PR from four different regions of China (Chun'an (Zhejiang), Xixia (Henan), Danfeng (Shanxi), and Pan'an (Zhejiang)), named CAZJ, XXHN, DFSX, and PAZJ, respectively, were isolated by anion-exchange and gel-permeation chromatography. Structures of the four polysaccharides were investigated. The results showed that all of them were mainly glucose and mannose, while the monosaccharide composition and content of polysaccharides from different sources varied. The molecular weights of CAZJ, XXHN, DFSX, and PAZJ were 14.119, 22.352, 18.127, and 15.699 kDa, respectively. Infrared spectra illustrated the existence of α-glycosidic bond and ß-glycosidic bond in the polysaccharides. CAZJ, XXHN, and DFSX possessed a pyranose ring structure, whereas PAZJ had a furanose ring structure. Congo red test indicated that XXHN, DFSX, and PAZJ had a triple-helix structure. X-ray diffraction showed that the polysaccharides consisted of crystalline and amorphous regions. All four polysaccharides exhibited different degrees of antioxidant and hypoglycemic activities with a dose-dependent manner in the 1.0-10.0 mg/mL concentration range. Correlation analysis revealed that the bioactivities of polysaccharides was significantly related to monosaccharide composition, uronic acid, and protein content. The results suggested that neutral polysaccharides could be used as potential natural antioxidants and hypoglycemic agents for functional and nutraceutical applications.

SSR Loci Analysis in Transcriptome and Molecular Marker Development in Polygonatum sibiricum.

To study the SSR loci information and develop molecular markers, a total of 435,858 unigenes in transcriptome of Polygonatum sibiricum were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MISA software, and SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 112,728 SSR loci were found in the transcriptome of Polygonatum sibiricum, which distributed in 435,858 unigenes with a distribution frequency of 25.86%. Mo-nucleotide and Di-nucleotide repeat were the main types, accounted for 83.83% of all SSRs. The repeat motifs of A/T and AC/GT were the predominant repeat types of Mo-nucleotide and Di-nucleotide, respectively. A total of 113,305 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. One hundred and fifty-four of the 500 randomly selected primers not only produced fragments with expected molecular size but also had high polymorphism, which could accurately separate the tested varieties. The gene function of unigenes containing SSR was mostly related to the molecular function of Polygonatum sibiricum. The SSR markers in transcriptome of Polygonatum sibiricum show rich type, strong specificity, and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding. The developed markers can also provide technical methods for molecular identification of intraspecific species of Polygonatum Mill. and maker-assisted breeding of superior varieties of Polygonatum Mill.

Inhibitory activities of Ranunculus japonicus Thunb. ethanol extract against hepatitis B virus.

The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract. More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that R. japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that R. japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation.

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.

Transcriptome Analysis of Different Sections of Rhizome in Polygonatum sibiricum Red. and Mining Putative Genes Participate in Polysaccharide Biosynthesis.

As a traditional Chinese herb, the rhizomes of Polygonatum sibiricum Red. are rich in various compounds which have plenty of pharmacological applications and biological activities. Among them, Polygonatum sibiricum polysaccharides (PSP) are the main active ingredients and exhibit a broad range of pharmacological. Based on previous researches, identifying genes involved in PSP biosynthesis will help delineate such pathway at the molecular level. In that case, we performed RNA sequencing analysis for two sections of P. sibiricum Red.'s rhizomes significantly different in PSP content. A total of 435,858 unigenes were obtained by assembling transcripts from both sections and 29,548 (6.77%) ones were annotated in all seven public databases. Analyzing count data of RNA-seq, 13,460 differential expression genes (DEGs) between two sections of rhizomes were acquired. After DEGs were mapped to KEGG databases, twelve represented KEGG pathways related to PSP biosynthesis were summed up. And most DEGs were assigned to the pathway of "Starch and sucrose metabolism". Finally, seventeen candidate genes whose expression levels were related to the polysaccharide content, were considered involving PSP biosynthesis in P. sibiricum Red. The present study lays a foundation of researching the molecular mechanisms of PSP biosynthesis.

Genome-Wide Characterization and Expression Analysis of bZIP Gene Family Under Abiotic Stress in Glycyrrhiza uralensis.

bZIP gene family is one of the largest transcription factor families. It plays an important role in plant growth, metabolic, and environmental response. However, complete genome-wide investigation of bZIP gene family in Glycyrrhiza uralensis remains unexplained. In this study, 66 putative bZIP genes in the genome of G. uralensis were identified. And their evolutionary classification, physicochemical properties, conserved domain, functional differentiation, and the expression level under different stress conditions were further analyzed. All the members were clustered into 13 subfamilies (A-K, M, and S). A total of 10 conserved motifs were found in GubZIP proteins. Members from the same subfamily shared highly similar gene structures and conserved domains. Tandem duplication events acted as a major driving force for the evolution of bZIP gene family in G. uralensis. Cis-acting elements and protein-protein interaction networks showed that GubZIPs in one subfamily are involved in multiple functions, while some GubZIPs from different subfamilies may share the same functional category. The miRNA network targeting GubZIPs showed that the regulation at the transcriptional level may affect protein-protein interaction networks. We suspected that domain-mediated interactions may categorize a protein family into subfamilies in G. uralensis. Furthermore, the tissue-specific gene expression patterns of GubZIPs were analyzed using the public RNA-seq data. Moreover, gene expression level of 66 bZIP family members under abiotic stress treatments was quantified by using qRT-PCR. The results of this study may serve as potential candidates for functional characterization in the future.

SmKFB5 protein regulates phenolic acid biosynthesis by controlling the degradation of phenylalanine ammonia-lyase in Salvia miltiorrhiza.

Phenolic acids are the major secondary metabolites and significant bioactive constituents of the medicinal plant Salvia miltiorrhiza. Many enzyme-encoding genes and transcription factors involved in the biosynthesis of phenolic acids have been identified, but the underlying post-translational regulatory mechanisms are poorly understood. Here, we demonstrate that the S. miltiorrhiza Kelch repeat F-box protein SmKFB5 physically interacts with three phenylalanine ammonia-lyase (PAL) isozymes and mediates their proteolytic turnover via the ubiquitin-26S proteasome pathway. Disturbing the expression of SmKFB5 reciprocally affected the abundance of SmPAL protein and the accumulation of phenolic acids, suggesting that SmKFB5 is a post-translational regulator responsible for the turnover of PAL and negatively controlling phenolic acids. Furthermore, we discovered that treatment of the hairy root of S. miltiorrhiza with methyl jasmonate suppressed the expression of SmKFB5 while inducing the transcription of SmPAL1 and SmPAL3. These data suggested that methyl jasmonate consolidated both transcriptional and post-translational regulation mechanisms to enhance phenolic acid biosynthesis. Taken together, our results provide insights into the molecular mechanisms by which SmKFB5 mediates the regulation of phenolic acid biosynthesis by jasmonic acid, and suggest valuable targets for plant breeders in tailoring new cultivars.

Antioxidant and Antidiabetic Activity of Proanthocyanidins from Fagopyrum dibotrys.

Proanthocyanidins are natural glycosidase inhibitors with excellent antioxidant activity. This study aims to search for a new source of proanthocyanidins for the prevention and treatment of type 2 diabetes with higher content and better activity and get their structure elucidated. First, the total proanthocyanidins contents (TOPCs), antioxidant activity, antidiabetic activity of seven common Polygonaceae plants were analyzed and compared. Then proanthocyanidins from the rhizome of Fagopyrum dibotrys were purified, and the detailed structure was comprehensively analyzed by ultraviolet visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), 13C nuclear magnetic resonance spectroscopy (13C NMR), reversed-phase high-performance liquid chromatography-electrospray mass spectrometry (RP-HPLC-ESI-MS), and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The rhizome of F. dibotrys showed the highest TOPCs, the strongest antioxidant, and antidiabetic activities; the TOPCs, antioxidant and antidiabetic activities were all very significantly positively correlated. Proanthocyanidins purified from the rhizome of F. dibotrys showed better antidiabetic activity than grape seed proanthocyanidins (GsPs). Seventy-two proanthocyanidins from trimer to undecamer with a mean degree of polymerization (mDP) of about 5.02 ± 0.21 were identified with catechin and epicatechin as the dominant monomers. Conclusion: Proanthocyanidins are the main antioxidant and antidiabetic active substances of F. dibotrys and are expected to be developed into potential antioxidant and hypoglycemic products.

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.

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.

Comprehensive evaluation of wild Cordyceps cicadae from different geographical origins by TOPSIS method based on the macroscopic infrared spectroscopy (IR) fingerprint.

Cordyceps cicadae is an entomogenous fungus that has been used as a valuable traditional Chinese herbal tonic, however, it can be difficult to discern the false from the genuine. In this study, the macroscopic IR fingerprint methods containing Fourier transform infrared spectroscopy (FT-IR) and second derivative infrared spectroscopy (SD-IR) were used to elucidate wild C. cicadae. The TOPSIS (Technique for Order Preference by Similarity to Ideal Solution) method was used to comprehensively evaluate C. cicadae from different geographical origins based on the macroscopic infrared spectroscopy (IR) fingerprint. The FT-IR spectra of C. cicadae exhibited the major characteristics of the absorptive peaks of carbohydrates, lipids and nucleosides at the position of 3291, 2925, 2845, 1651, 1547, 1455, 1080 and 950 cm-1. The high resolution of SD-IR further amplified the difference and revealed the potentially characteristic IR absorption spectrum. TOPSIS evaluation showed that C. cicadae from Anhui possess the strongest intensity of absorption bands among all the samples. Notably, FT-IR combined with SD-IR can effectively reveal the overall chemical components without damaging medicinal materials, and TOPSIS methods can provide a novel scientific evidence for comprehensively assessing different origins of wild C. cicadae.

Overexpression of SmbHLH10 enhances tanshinones biosynthesis in Salvia miltiorrhiza hairy roots.

The bHLH transcription factors have important role in regulation of plant growth, development, and secondary metabolism. Tanshinones are the major pharmaceutical components present in Salvia miltiorrhiza Bunge. It has been reported that bHLHs have functions in terpenoids biosynthesis. Here, we got a bHLH family member named SmbHLH10 which could positively regulate tanshinones biosynthesis in S. miltiorrhiza hairy roots. In the SmbHLH10-overexpressing line 6, four major tanshinones contents were reaching 2.51-fold (dihydrotanshinone I), 2.84-fold (cryptotanshinone), 2.89- fold (tanshinone I), 2.68-fold (tanshinone II A) of WT, respectively. The variation in tanshinones biosynthetic pathway gene transcription was generally consistent with tanshinones content. DXS2, DXS3 and DXR of MEP pathway were induced substantially, reaching 10-fold, 3-fold, 5.74-fold higher of the WT, respectively. The downstream pathway genes CPS1, CPS5 and CYP76AH1 were highest in line OE-SmbHLH10-6, reached 4.93, 16.29 and 3.27-fold of the WT, respectively, while KSL1's expression was highest in line OE-SmbHLH10-4, 4.64-fold of WT. Yeast one-hybrid assays results showed that SmbHLH10 could binds the predicted G-box motifs within the promoters of DXS2, CPS1 and CPS5. These findings indicated that SmbHLH10 could directly binds to G-box in the pathway genes' promotor, activate their expression and then upregulate tanshinones biosynthesis.

Identification of seeds based on molecular markers and secondary metabolites in Senna obtusifolia and Senna occidentalis.

BACKGROUND: Senna obtusifolia and Senna occidentalis (Leguminosae), whose seeds have similar appearance and chemical constituents, are easily confused in using their seeds. To elucidate the similarities and differences between S. obtusifolia seeds and S. occidentalis seeds, three molecular markers and high performance liquid chromatography (HPLC) were employed to evaluate the seeds characteristics of these two medicinal herbs. RESULTS: The results showed that selected 3 ISSR and 7 SCoT primers could distinguish S. obtusifolia seeds from S. occidentalis seeds based on the specific band and UPGMA dendrogram. ITS2 sequence indicated that the intra-specific similarity of 20 S. obtusifolia and 16 S. occidentalis was 99.79 and 100.0%, respectively, while the inter-specific similarity between S . obtusifolia and S. occidentalis was 89.58%. Although phylogenetic analysis revealed that these two species had a close relationship, they were assigned to different branches. HPLC fingerprint results showed that seeds of S. obtusifolia and S. occidentalis shared some secondary metabolites, but aurantio-obtusin was not detected in S. occidentalis seeds which could differentiate S. obtusifolia seeds from S. occidentalis seeds. CONCLUSIONS: The present study not only compared the seeds characters of S. obtusifolia and S. occidentalis from molecular and secondary metabolites levels, but also provided a convenient method to identify S. obtusifolia seeds and S. occidentalis seeds effectively.

Isaria cicadae conidia possess antiproliferative and inducing apoptosis properties in gynaecological carcinoma cells.

Isaria cicadae is an entomogenous fungus that has been used as a traditional Chinese medicinal materials to treat different diseases, including cancer. However, Isaria cicadae conidia for inhibitory activity against breast cancer cells growth are still not systematically studied. The present aim was to elucidate the phytochemical composition of Isaria cicadae conidia and to explore relevant anti-cancer potential in gynaecological carcinoma MCF-7 and Hela cells. Isaria cicadae conidia were identified by UPLC-ESI-Q-TOF-MS: high performance liquid chromatography-electrospray/quadrupole time of flight tandem mass spectrometry technology. Eight main compounds were identified which are nucleosides, cordycepic acid, cordycepin, beauvericin and myriocin by MS fragmentation ions. The nuclear morphology indicated the typical characteristics of apoptosis by Hoechst staining. Annexin V/PI staining revealed that the number of apoptotic cells was increased by Isaria cicadae conidia treatment. Furthermore, Isaria cicadae conidia also induced the caspase-mediated mitochondrial apoptosis pathway. The findings suggest that the full-scale active ingredients highlight the significance of Isaria cicadae conidia as potential anti-cancer agent in China.

Artemisia dominant species succession relating to the soil moisture decrease in abandoned land of the Loess Plateau (China): comparative study of drought-adaptive characteristics.

Artemisia scoparia, Artemisia sacrorum and Artemisia giraldii were three dominant Artemisia species which successive grew in the secondary succession on abandoned land of the Loess Plateau. The succession accompanied the soil moisture steady decrease with field age after their abandonment. To elucidate the relationship between the Artemisia species succession and their drought-adaptation, three dominant species and a contrastive species Artemisia annua (mesophyte), were selected to compare their drought-resistant characteristics, including morphological and anatomical traits of leaf and root. Then physiological responses were investigated in mature plants after drought treatment. The results indicated that three dominant species leaf presented drought-adaptive structures, such as bushy trichomes, transitional or isolateral leaf cells, thick cuticles and epidermal cells. However, A. annua had no leaf traits involved in drought-adaptation. In addition, A. sacrorum and A. giraldii contained large root systems, while A. scoparia and A. annua utilized succulent roots. The physiological responses to drought suggested that A. giraldii had strong regulation in water using strategy, growth, as well as superoxide dismutase and catalase activity. A. sacrorum and A. giraldii could maintain high ascorbate peroxidase activity and malondialdehyde content, while A. scoparia and A. giraldii presented higher peroxidase activity, ascorbate and soluble sugar content. A. annua exhibited high proline and carotenoid contents under drought. The drought-resistant of the four Artemisia species presented the order of A. giraldii > A. sacrorum > A. scoparia > A. annua, which was consistent with their succession on abandoned land.

Spaceflight-induced variation on biological traits and effective components of Cassia obtusifolia.

The dry seeds of Cassia obtusifolia were carried by the "ShenZhou 8" satellite and sowed after landing. Based on our pri- or study on SP1, the characteristics of plants growth, physiological index and content of effective components were examined. The results showed that the QC10, QC29 strains matured 5 d earlier compared with control. The plant height, across diameter and ground diameter of QC10, QC29, QC46 strains was superior to the control at whole growth period. The branch number increased ranging from 4 to 11 and the number of pods reached 321, 313,281, respectively, which was dramatically higher than the control (246). The yield of QC10, QC29, QC46 strains increased noticeably from 31.4 to 63.2 g. The 1000-seed-weight of QC10, QC29, QC46 strains was 25.86, 25.88, 24.06 g, while the control was 23.69 g. Compared to the control, the mass fraction of chlorophyll was enhanced 1.098, 1.016, 0.297 mg. There was no significant difference in aurantio-obtusin and chrysophanol content of seeds. Through two years research, three high-yield mutant strains were obtained. This study indicates that spaceflight-induced mutants could provide new germplasm for C. obtusifolia breeding and offers the theoretical basis for further utilization of spaceflight-induced mutation to breed high-quality C. obtusifolia strains.

Assessment of the soil organic carbon sink in a project for the conversion of farmland to forestland: a case study in Zichang county, Shaanxi, China.

The conversion of farmland to forestland not only changes the ecological environment but also enriches the soil with organic matter and affects the global carbon cycle. This paper reviews the influence of land use changes on the soil organic carbon sink to determine whether the Chinese "Grain-for-Green" (conversion of farmland to forestland) project increased the rate of SOC content during its implementation between 1999 and 2010 in the hilly and gully areas of the Loess Plateau in north-central China. The carbon sink was quantified, and the effects of the main species were assessed. The carbon sink increased from 2.26×106 kg in 1999 to 8.32×106 kg in 2010 with the sustainable growth of the converted areas. The black locust (Robinia pseudoacacia L.) and alfalfa (Medicago sativa L.) soil increased SOC content in the top soil (0-100 cm) in the initial 7-yr period, while the sequestration occurred later (>7 yr) in the 100-120 cm layer after the "Grain-for-Green" project was implemented. The carbon sink function measured for the afforested land provides evidence that the Grain-for-Green project has successfully excavated the carbon sink potential of the Shaanxi province and served as an important milestone for establishing an effective organic carbon management program.

Proline accumulation in leaves of Periploca sepium via both biosynthesis up-regulation and transport during recovery from severe drought.

Drought resistance and recovery ability are two important requisites for plant adaptation to drought environments. Proline (Pro) metabolism has been a major concern in plant drought tolerance. However, roles of Pro metabolism in plant recovery ability from severe drought stress are largely unexplored. Periploca sepium Bunge has gained increasing attention for its adaptation to dry environments. Here, we investigated Pro metabolism in different tissues of P. sepium seedlings in the course of drought stress and recovery. We found that leaf Pro metabolism response during post-drought recovery was dependant on drought severity. Pro biosynthesis was down-regulated during recovery from -0.4 MPa but increased continually and notably during recovery from -1.0 MPa. Significant correlation between Pro concentration and Δ1-pyrroline-5-carboxylate synthetase activity indicates that Glutamate pathway is the predominant synthesis route during both drought and re-watering periods. Ornithine δ-aminotransferase activity was up-regulated significantly only during recovery from -1.0 MPa, suggesting positive contribution of ornithine pathway to improving plant recovery capacity from severe drought. In addition to up-regulation of biosynthesis, Pro transport from stems and roots also contributed to high Pro accumulation in leaves and new buds during recovery from -1.0 MPa, as indicated by the combined analysis of Pro concentration and its biosynthesis in stems, roots and new buds. Except its known roles as energy, carbon and nitrogen sources for plant rapid recovery, significant positive correlation between Pro concentration and total antioxidant activity indicates that Pro accumulation can also promote plant damage repair ability by up-regulating antioxidant activity during recovery from severe drought stress.

Different roles of the mevalonate and methylerythritol phosphate pathways in cell growth and tanshinone production of Salvia miltiorrhiza hairy roots.

Salvia miltiorrhiza has been widely used in the treatment of coronary heart disease. Tanshinones, a group of diterpenoids are the main active ingredients in S. miltiorrhiza. Two biosynthetic pathways were involved in tanshinone biosynthesis in plants: the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme of the MVA pathway. The 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) are the key enzymes of the MEP pathway. In this study, to reveal roles of the MVA and the MEP pathways in cell growth and tanshinone production of S. miltiorrhiza hairy roots, specific inhibitors of the two pathways were used to perturb metabolic flux. The results showed that the MVA pathway inhibitor (mevinolin, MEV) was more powerful to inhibit the hairy root growth than the MEP pathway inhibitor (fosmidomycin, FOS). Both MEV and FOS could significantly inhibit tanshinone production, and FOS was more powerful than MEV. An inhibitor (D, L-glyceraldehyde, DLG) of IPP translocation strengthened the inhibitory effects of MEV and FOS on cell growth and tanshinone production. Application of MEV resulted in a significant increase of expression and activity of HMGR at 6 h, and a sharp decrease at 24 h. FOS treatment resulted in a significant increase of DXR and DXS expression and DXS activity at 6 h, and a sharp decrease at 24 h. Our results suggested that the MVA pathway played a major role in cell growth, while the MEP pathway was the main source of tanshinone biosynthesis. Both cell growth and tanshinone production could partially depend on the crosstalk between the two pathways. The inhibitor-mediated changes of tanshinone production were reflected in transcript and protein levels of genes of the MVA and MEP pathways.