Salicylic Acid Alters Antioxidant and Phenolics Metabolism in Catharanthus Roseus Grown Under Salinity Stress

Background

Salicylic acid (SA) acts as a potential non-enzymatic antioxidant and a plant growth regulator, which plays a major role in regulating various plant physiological mechanisms. The effects of salicylic acid (SA; 0.05 mM) on physiological parameters, antioxidative capacity and phenolic metabolism, lignin, alkaloid accumulation in salt stressed Catharanthus roseus were investigated.

Materials and Methods

Catharanthus roseus seeds were grown for two months in a glass house at 27–30°C in sunlight, and then divided into four different groups and transplanted with each group with the following solutions for one month: group I (non-saline control), group II, 100 mM NaCl, group III, 0.05 mM SA, group IV, 100 mM NaCl+0.05 mM SA and to determine the physiological parameters (DW, FW, WC), chlorophyll contents, carotenoid contents, lipid peroxidation, phenolics, lignin, alkaloid and enzymatic assays in each leaf pairs and roots.

Results

SA exhibited growth-promoting property, which correlated with the increase of dry weight, water content, photosynthetic pigments and soluble proteins. SA has additive effect on the significant increase in phenylalanine ammonia-lyase (PAL) activity, which is followed by an increase in total soluble phenolics and lignin contents in all leaf pairs and root of C. roseus. SA enhances malondialdehyde content in all leaf pairs and root. The antioxidant enzymes (catalase, glutathione reductase, glutathione-S-tranferase, superoxide dismutase, peroxidase) as well as alkaloid accumulation increased in all treatments over that of non-saline control but the magnitude of increase was found more in root. Further, the magnitude of increase of alkaloid accumulation was significantly higher in 100 mM NaCl, but highly significant was found in presence of 0.05 mM SA and intermediate in presence of both 0.05 mM SA+100 mM NaCl.

Conclusion

We concluded that applied SA to salt stress, antioxidant and phenolic metabolism, and alkaloid accumulation were significantly altered and the extent of alteration varied between the SA and salt stress.     

From the Cover: Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them.     

长春花脱乙酰氧基文多灵-4-羟化酶基因启动子的克隆与生物信息学分析

通过基因组步行技术克隆到长春花脱乙酰氧基文多灵-4-羟化酶基因上游905bp启动子序列,生物信息学分析发现该序列中存在多个与光照诱导有关的顺式元件,同时也发现一个与长春花特异的顺式作用元件JERE类似的短序列,推测该序列是茉莉酸诱导途径中转录因子ORCA3的特异结合位点。     

营养及环境因子对农杆菌诱导的长春花发根生长和生物碱生成的影响

将A4发根农杆菌感染长春花(Catharanthus roseus(L.)G.Don)后所长出的发根(hairy-root)进行培养，比较不同营养成分、碳源及初始糖浓度、外源激素以及温度和摇床转速对发根生长及产碱的影响。结果表明，当培养基为1/2B5培养基时发根生长良好；一定范围内初始糖浓度增加，有利于发根生长和生物碱生成；加入500mg/L的L-色氨酸利于发根的生长和产碱；将发根在28℃、75r／min的条件下悬浮培养10d可获得较高产量的生物碱。     

Catharanthus roseus cell cultures: Growth,alkaloid synthesis and antidiabetic activity

Suspension cultures of Catharanthus roseus were established from three different cell lines namely, CWS, CWS-A and CWS-G on MS medium supplemented with 2,4-D (0.4 ppm). The three cell lines were cultured in growth and production media. Cell line CWS grown in production medium showed a PCV (packed cell volume) of 70% in 21 days. The cells synthesized 0.10% ajmalicine in the production medium and the cell extract caused a 71% decrease in blood sugar in diabetes induced rats. In growth medium the cells showed a PCV of 97% in 21 days and produced trace amounts of alkaloids. The cell extracts did not show any antidiabetic activity. The CWS-A cell line showed a PCV of 98% in 21 days and synthesized 0.036% ajmalicine in production medium. The extracts had no hypoglycaemic effect. In growth medium the cells showed a 98% PCV in 21 days and produced trace amounts of alkaloids. The cell extract caused an 86% decrease in blood sugar. The CWS-G cell line grown in flasks failed to synthesize significant levels of alkaloids both in growth and production media. The cell extracts did not show any antidiabetic activity.     

长春花突变细胞生理特征的研究

目的研究以秋水仙碱处理的长春花突变细胞在继代培养过程中的生长规律、吲哚生物碱积累的规律及营养成分的适宜浓度等方面的特征,期望得到适于工业化细胞培养的理想材料。方法将突变细胞培养于MS固体培养基上,定期称取其鲜质量,用有机溶剂萃取吲哚生物碱,应用RP-HPLC法检测药用成分阿玛碱和长春质碱。结果突变细胞培养至第30代时,生长速度和吲哚总碱积累得最快,培养至第45代时,两者均明显下降。而药用成分的量于第20代达到最高;在培养基中添加适量的色氨酸能提高药用成分的量;培养基中Ca2 和Zn2 的质量浓度分别为1760和12.6mg/L时,明显促进了药用成分的积累。结论长春花突变细胞可能是一种适于工业化细胞培养的理想材料。     

长春花突变细胞生理特征的研究

目的研究以秋水仙碱处理的长春花突变细胞在继代培养过程中的生长规律、吲哚生物碱积累的规律及营养成分的适宜浓度等方面的特征,期望得到适于工业化细胞培养的理想材料。方法将突变细胞培养于MS固体培养基上,定期称取其鲜质量,用有机溶剂萃取吲哚生物碱,应用RP-HPLC法检测药用成分阿玛碱和长春质碱。结果突变细胞培养至第30代时,生长速度和吲哚总碱积累得最快,培养至第45代时,两者均明显下降。而药用成分的量于第20代达到最高;在培养基中添加适量的色氨酸能提高药用成分的量;培养基中Ca2 和Zn2 的质量浓度分别为1760和12.6mg/L时,明显促进了药用成分的积累。结论长春花突变细胞可能是一种适于工业化细胞培养的理想材料。     

生物技术在长春花研究中的应用

长春花为重要的抗癌药用植物,对细胞组织培养,Ti和Ri质粒遗传化等生物技术在长春花研究中的应用作一综述,并对长春花的开发利用和长春花生物磁的工业化生产存在问题和前景作了分析.     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.