[Cloning and characterization of the promoters of the key genes CPT, SRPP and REF involved in Periploca sepium rubber biosynthesis].

Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1, 4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (ß-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.

Cloning and characterization of the promoters of the key genes CPT, SRPP and REF involved in Periploca sepium rubber biosynthesis / 生物工程学报

Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1, 4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (β-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.

ß-cyclodextrin microencapsulation enhanced antioxidant and antihyperlipidemic properties of Tunisian Periploca angustifolia roots condensed tannins in rats.

This study aimed to investigate the microencapsulation of novel condensed tannins isolated from Periploca angustifolia roots, using ß-cyclodextrin macrocyclic oligosaccharides, in order to enhance their antioxidant and antihyperlipidemic potentials. Scanning electron microscopy and Fourier transform infrared spectroscopy results revealed that tannin fraction was successfully included into ß-cyclodextrin cavities proved with an encapsulation efficacy of 70%. Our in vitro findings highlighted that both pure and encapsulated tannins have efficient inhibition capacities of pancreatic lipase activity. However, the inclusion complex has the greatest, in vivo, antioxidant, and antihyperlipidemic effects. In fact, results showed that complexed tannins had markedly restored serum lipid biomarkers, lipid peroxidation, protein carbonyl oxidation, and antioxidant enzyme defense. These findings were additionally confirmed by aortic and myocardial muscle sections of histological examination. Consequently, ß-cyclodextrin microencapsulation may be considered as an effective and promising technique for tannin delivery with improved antioxidant and antihyperlipidemic activities.

Melanogenesis-Inhibitory and Antioxidant Activities of Phenolics from Periploca forrestii.

Two new tetrahydrofuran-type lignans, (-)-gentioluteol 9-O-ß-d-glucopyranoside (1), (-)-berchemol 9-O-ß-d-apiofuranosyl-(1→6)-O-ß-d-glucopyranoside (2), along with sixteen known compounds 3 - 18 were isolated from the 95% EtOH extract of the stems of Periploca forrestii. The structures of the new tetrahydrofuran-type lignans were determined by HR-ESI-MS and various NMR techniques in combination with CD method. Then, their antioxidant abilities were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, and ferric-reducing antioxidant power (FRAP) assays. Meanwhile, a similar trend was obtained in tripartite antioxidant assays, which compounds 7 - 9 and 11 exhibited potent abilities. Subsequently, the evaluation of all compounds against the α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis on the B16F10 cell line, compounds 5 - 11, 15, and 16 exhibited inhibitory effects with no or weak toxicity at low concentration. Of these, compound 8 exhibited the strongest inhibition melanogenesis ability. Furthermore, Western blot analysis suggested that compound 8 could inhibit melanogenesis by suppressing the protein expression of microphthalmia-associated transcription factor (MITF) and tyrosinase.

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.

[Cutting propagation of Periploca forrestii and dynamic analyses of physiological and biochemical characteristitics related to adventitious roots formation].

OBJECTIVE: To study the effects of different auxcin regulators on rooting of Periplocaforrestii cuttings as well as dynamic change rules of endogenous plant hormones and oxidases related to adventitious root formation. METHODS: Cuttings propagation characters of Periploca forrestii were investigated and compared with different concentration treatments of indolebutyric acid (IBA), Rooting Powder No. 1 (ABT1) and naphthylacetic acid (NAA). The dynamic changes of contents of endogenous hormones including indole acetic acid (IAA), abscisic acid (ABA), zeatin ribosides (ZRs) as well as the activities of indoleacetic acid oxidase( IAAO), polyphenol oxidase (PPO), peroxidase (POD) were tested. RESULTS: Rooting percentage of cutting with 150 mg/L IBA, 150 mg/L ABT1 treatment and NAA treatment were 80% ,70% and 68% respectively, rooting percentage of cuttings of the control was 23% only. The adventitious rooting displayed three distinct phases i. e. root-inducing, root-formating and root-elongating phases. During root-inducing phase the contents of IAA, ABA and ZRs decreased,whereas IAAO activity kept at a higher level. The IAA content reached the peak and PPO activity increased obviously during root-formating phase, while activities of IAAO, POD and contents of ABA, ZRs declined to minimum. During root-elongating phase contents of IAA, ABA, ZRs were much steadfast and activities of PPO, IAAO, POD were increased. After that, the activities of the three oxidases decreased slowly. 150 mg/L IBA treatment increased the content of IAA and PPO activity in cuttings during rooting,while the opposite result occurred in contents of ZRs, ABA and the activities of IAAO, POD. CONCLUSION: The dynamic changes of endogenous hormones (IAA, ABA, ZRs) contents and IAAO, PPO, POD activities are tightly related to the rooting process of cuttings in Periploca forrestii.

[Affect of Ag+ and La3+ elicitors on growth and accumulation of adventitious roots of Periploca sepium].

OBJECTIVE: To study the characteristics of the growth and periplocin accumulation of the adventitious roots of Periploca sepium, and on this basis, study the effect of Ag+ and La3+ elicitors on the growth and periplocin accumulation of the adventitious roots. METHOD: The adventitious roots were sampled every four days, and the dry weight and the contents of the periplocin were measured. The curves of the growth and periplocin accumulation of the roots were plotted. The Ag+ and La3+ elicitors with different concentrations were added to the medium when the adventitious roots grew in the stable phase to study the optimal concentration which was good to synthesize the periplocin. Besides, the optimal concentration of Ag+ and La3+ elicitors was added to the different growth phases to study the effect of the elicitors on the growth and periplocin synthesis of adventitious roots. RESULT: The characteristics of the growth of adventitious roots of P. sepium showed a typical growth S-Curve, which displayed a half-coupling relationship with the metabolism of periplocin. The optimal concentrations of Ag+ and La3+ elicitors were both 0.05 mmol L(-1). Besides, it was the best period for the Ag+ and La3+ elicitors to elicit the synthesis of periplocin when in the terminally exponential phase. CONCLUSION: The growth of adventitious roots and the accumulation of periplocin show a half-coupling relationship. Besides, the concentration and additive time of Ag and La3+ elicitors obviously influences the growth of adventitious roots and synthesis of periplocin.

[Induction of Periploca sepium in vitro plantlet and studies on dynamic accumulation of periplocin].

OBJECTIVE: To analyze the content of periplocin in different part of the Periploca sepium in vitro plantlet and study its dynamic variation during the process of differentiation. METHOD: The seeds were generated seedling under aseptic condition, and the cut hypocotyl was induced to form the callus and adventitious buds on the MS culture medium with the hormone of IBA 0.1 mg x L(-1) + BA 1 mg x L(-1). The seedling was cut down when the buds grew up to 3 cm and then the root was cultured in the 1/2 MS culture medium with the hormone of IBA 0.5 mg x L(-1) to form intact plantlet. Different parts of it were collected and the content of periplocin was measured during the process of differentiation. RESULT: The contents of periplocin varied widely in different parts during the process of differentiation, with the highest in the roots and then callus, stem and leaf of intact plantlet, stem and leaf of plantlet without root from high to low. CONCLUSION: The periplocin of the secondary metabolite is more likely to be produced and accumulated in root and callus. Periplocin in stem and leaf is probably transported by conducting tissue.

[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.

Periploca sepium Bunge as a model plant for rubber biosynthesis study.

Periploca sepium Bunge (Chinese silk vine) is a woody climbing vine belonging to the family Asclepiadaceae. It originally comes from Northwest China. Periploca resembles the Para-rubber tree, Hevea brasiliensis, regarding a similar body plan to produce a milky exudate containing rubber latex. The Periploca plant was assessed as a rubber-producing plant by rubber structure elucidation and its molecular weight distribution. A rubber fraction purified from the milky exudate was subjected to 1H NMR analysis, and a characteristic signal derived from cis-polyisoprene was observed. In addition, when the molecular weight distribution of rubber components in the exudate was measured (using size-exclusion chromatography), the number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity (Mw/Mn) were estimated to be Mn = 1.3 x 10(5), Mw = 4.1 x 10(5), and Mw/Mn = 3.1, respectively. Furthermore, the presence of polyisoprene, with Mn = 4.0 x 10(4), Mw = 7.6 x 10(4), and Mw/Mn = 2.5, was also confirmed in plantlets obtained from shoots as a result of tissue culture.