Identification of genes associated with biosynthesis of bioactive flavonoids and taxoids in Taxus cuspidata Sieb. et Zucc. plantlets exposed to UV-B radiation.

UV-B radiation is a typical environmental stressor that can promote phytochemical accumulation in plants. Taxus species are highly appreciated due to the existence of bioactive taxoids (especially paclitaxel) and flavonoids. However, the effect of UV-B radiation on taxoid and flavonoid biosynthesis in Taxus cuspidata Sieb. et Zucc. is largely unknown. In the present work, the accumulation of taxoids and flavonoids in T. cuspidata plantlets was significantly induced by 12 and 24 h of UV-B radiation (3 W/m2), and a large number of significantly differentially expressed genes were obtained via transcriptomic analysis. The significant up-regulation of antioxidant enzyme- and flavonoid biosynthesis-related genes (phenylalanine ammonia lyase 1, chalcone synthase 2, flavonol synthase 1, and flavonoid 3', 5'-hydroxylase 2), suggested that UV-B might cause the oxidative stress thus promoting flavonoid accumulation in T. cuspidata. Moreover, the expression of some genes related to jasmonate metabolism and taxoid biosynthesis (taxadiene synthase, baccatin III-3-amino 3-phenylpropanoyltransferase 1, taxadiene-5α-hydroxylase, and ethylene response factors 15) was significantly activated, which indicated that UV-B might initiate jasmonate signaling pathway that contributed to taxoid enhancement in T. cuspidata. Additionally, the identification of some up-regulated genes involved in lignin biosynthesis pathway indicated that the lignification process in T. cuspidata might be stimulated for defense against UV-B radiation. Overall, our findings provided a better understanding of some potential key genes associated with flavonoid and taxoid biosynthesis in T. cuspidata exposed to UV-B radiation.

Effects of light acclimation on shoot morphology, structure, and biomass allocation of two Taxus species in southwestern China.

Acclimation to changing light conditions plays a crucial role in determining the competitive capability of tree species. There is currently limited information about acclimation to natural light gradient and its effect on shoot structure and biomass in Taxus species. We examined the acclimation of the leaf and shoot axis morphology, structure and biomass allocation of Taxus yunnanensis and T. chinensis var. mairei under three different natural light environments, full daylight, 40-60% full daylight and <10% full daylight. The leaf biomass, nitrogen content per unit area, leaf carbon content per dry mass and leaf dry mass to fresh mass ratio increased with light in both species, demonstrating an enhanced investment of photosynthetic biomass and structural investment under high light. The number of leaves per unit shoot axis length and the leaf dry mass per unit shoot axis length increased with light in both species. However, the light increase did not result in the increase of the total shoot mass. T. yunnanensis produced larger leaves under low light and a higher shoot axis length per unit dry mass under high light, whereas the leaf size and biomass yield of T. chinensis var. mairei were not sensitive to light.

Response and Defense Mechanisms of Taxus chinensis Leaves Under UV-A Radiation are Revealed Using Comparative Proteomics and Metabolomics Analyses.

Taxus chinensis var. mairei is a species endemic to south-eastern China and one of the natural sources for the anticancer medicine paclitaxel. To investigate the molecular response and defense mechanisms of T. chinensis leaves to enhanced ultraviolet-A (UV-A) radiation, gel-free/label-free and gel-based proteomics and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The transmission electron microscopy results indicated damage to the chloroplast under UV-A radiation. Proteomics analyses in leaves and chloroplasts showed that photosynthesis-, glycolysis-, secondary metabolism-, stress-, and protein synthesis-, degradation- and activation-related systems were mainly changed under UV-A radiation. Forty-seven PSII proteins and six PSI proteins were identified as being changed in leaves and chloroplasts under UV-A treatment. This indicated that PSII was more sensitive to UV-A than PSI as the target of UV-A light. Enhanced glycolysis, with four glycolysis-related key enzymes increased, provided precursors for secondary metabolism. The 1-deoxy-d-xylulose-5-phosphate reductoisomerase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase were identified as being significantly increased during UV-A radiation, which resulted in paclitaxel enhancement. Additionally, mRNA expression levels of genes involved in the paclitaxel biosynthetic pathway indicated a down-regulation under UV-A irradiation and up-regulation in dark incubation. These results reveal that a short-term high dose of UV-A radiation could stimulate the plant stress defense system and paclitaxel production.

Seasonal-based temporal changes fluctuate expression patterns of TXS, DBAT, BAPT and DBTNBT genes alongside production of associated taxanes in Taxus baccata.

KEY MESSAGE: Environmental cues have synergistic or antagonistic regulatory roles on transcription activity and taxanes accumulation in yew, though DBAT activity is less influenced, could be accordingly a rate-limiting enzyme. The current work was undertaken to elucidate the consequences of some environmental cues (i.e., day length, temperature, sunlight and relative humidity) on the expression patterns of TXS, DBAT, BAPT and DBTNBT genes contributed to the taxol biosynthetic pathway along with the accumulation of some taxanes in needles and stems of Taxus baccata over year 2013-2014. In both tissues, light intensity and temperature correlated with the production of 10-DAB III and total taxanes, and TXS activity, while a lack of significant association was deduced for day length and relative humidity. Furthermore, in both tissues, a weak correlation was observed between BAC III and light intensity, temperature, day length and relative humidity, and the corresponding gene, DBAT. Surprisingly, DBAT activity was not co-induced with TXS in both tissues, and remained expressed at basal levels over year, supporting that the conversion of 10-DAB III into BAC III could presumably be a rate limiting step in the taxol biosynthetic pathway. Similar to BAC III, no strong correlation was detected between production of taxol in both tissues and all the meteorological data, while the corresponding genes BAPT and DBTNBT, in some cases, exhibited significant correlated results. Notably, despite higher activities of BAPT and DBTNBT in both tissues over year, taxol production was still in small quantities, probably owing to the low amounts of its precursors rather than low volumes of BAPT and DBTNBT transcripts. The results, altogether, could provide us new insights towards the potential regulatory roles of environmental cues on the production of taxanes in yew trees.

Responses in the morphology, physiology and biochemistry of Taxus chinensis var. mairei grown under supplementary UV-B radiation.

The effects of supplemental UV-B radiation on Taxus chinensis var. mairei were studied. Leaf traits, gas exchange parameters and the concentrations of photosynthetic pigments, cellular defense system products, secondary metabolites and ultrastructure were determined. UV-B radiation significantly decreased leaf area (p<0.05). Leaf number, secondary branch number, leaf weight per plant and leaf moisture all increased dramatically (p<0.05). Neither the leaf weight nor the specific leaf weight (SLW) exhibited significant differences between ambient and enhanced UV-B radiation. Gas exchange parameters were all dramatically reduced by enhanced UV-B radiation (p<0.05). The contents of chlorophyll and the chlorophyll a/b ratio were not distinctly affected by UV-B radiation, while carotenoids content significantly decreased (p<0.05). Supplemental UV-B treatment induced significant flavonoid accumulation (p<0.05), which was able to protect plant from radiation damage. Meanwhile, the appendage content, abaxial stomatal density, papilla density and particulate matter content in substomatic chambers increased noticeably by supplemental UV-B radiation, whereas the aperture size of single stomata was diminished. The number and area of plastoglobuli were apparently reduced by UV-B radiation, but stroma and grana lamellae were not destroyed. Our results demonstrated that T. chinensis var. mairei can activate several defense mechanisms against oxidative stress injury caused by supplemental UV-B radiation.

Leaf age as a factor in anatomical and physiological acclimative responses of Taxus baccata L. needles to contrasting irradiance environments.

To evaluate the acclimative ability of current-year and previous-year needles of a shade tolerant conifer Taxus baccata L. to contrasting irradiance conditions, seedlings were raised under 27% solar irradiance and at 3 years of age they were transferred to an experimental garden and grown for one season under full irradiance (HL), 18% irradiance (ML) or 5% irradiance (LL). Whereas previous year needles did not change anatomically, current year needles in HL were thicker and had a thicker palisade and spongy mesophyll, and greater leaf mass per area than ML or LL needles. LL needles had greater nitrogen concentration than HL needles irrespective of age but only previous year LL needles also had an increased N per area content, thanks to their lack of reduction in LMA. Adjustment of chlorophyll and carotenoid content occurred in both needle age classes with LL and ML needles having much higher concentrations but, in current year needles, only slightly higher per area content than HL needles. Chlorophyll a/b ratio was not affected by age or irradiance. These modifications had no significant effect on photosynthetic capacities, which did not significantly differ between the age classes in HL or LL treatment and between treatments. On the other hand, high growth irradiance resulted in a greater photochemical yield, photochemical quenching, apparent electron transport rate and inducible non-photochemical quenching in needles formed in the current season. In previous year needles, however, only inducible NPQ was enhanced by high irradiance with other parameters remaining identical among treatments. To test sensitivity to photoinhibition, at the end of the summer plants from the three irradiance levels were transferred to a HL situation and F (v)/F (M) was determined over the following 18 days. Sensitivity to photoinhibition was negatively related to growth irradiance and previous year needles were less photoinhibited than current year needles. Thus, differences in acclimation ability between needle age classes were most pronounced at the level of anatomy and light reactions of photosynthesis, both of which showed almost no plasticity in previous year needles but were considerably modified by irradiance in current year needles.

Photosynthetic activity in winter needles of the evergreen tree Taxus cuspidata at low temperatures.

Photosystems harvest light energy, yet this energy cannot be efficiently employed for CO(2) assimilation at the below-freezing temperatures to which plants are typically exposed during winter in the temperate and boreal zones. To elucidate the mechanisms whereby this energy is dissipated, I evaluated performance of photosystems in winter needles of the evergreen tree Taxus cuspidata Sieb. et Zucc. Chloroplasts were localized adjacent to plasma membranes in needle cells in summer, whereas they congregated together in the centers of the cells during winter. When winter needles were acclimated to a temperature of 20 degrees C, their chloroplasts gradually dispersed to the edges of the cells, as in the summer. Acclimation-dependent relocalization coincided with changes in CO(2) uptake. Examination of photosystem II fluorescence kinetics in winter needles indicated that the quinone electron acceptor (Q(A)) reduction rate exceeded the Q(A) oxidation rate at low temperatures. The majority of Q(A) remained reduced even when winter needles were subjected to a temperature of -5 degrees C at low irradiance.

Pulsed electric field stimulates plant secondary metabolism in suspension cultures of Taxus chinensis.

The effects of pulsed electric field (PEF) on growth and secondary metabolite production by plant cell culture were investigated by using suspension cultures of Taxus chinensis as a model system. Cultured cells in different growth phases were exposed to a PEF (50 Hz, 10 V/m) for various periods of time. A significant increase in intracellular accumulation of taxuyunnanine C (Tc), a bioactive secondary metabolite, was observed by exposing the cells in the early exponential growth phase to a 30-min PEF. The Tc content (i.e., the specific production based on dry cell weight) was increased by 30% after exposure to PEF, without loss of biomass, compared with the control. The combination of PEF treatment and sucrose feeding proved useful for improving secondary metabolite formation. Production levels of reactive oxygen species, extracellular Tc, and phenolics were all increased, whereas cell capacitance was decreased with PEF treatment. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolite biosynthesis in plant cell cultures.

Improved cell growth and Taxol production of suspension-cultured Taxus chinensis var. mairei in alternating and direct current magnetic fields.

The growth of suspension cultures of Taxus chinensis var. mairei and Taxol production were promoted both by a sinusoidal alternating current magnetic field (50 Hz, 3.5 mT) and by a direct current magnetic field (3.5 mT). Taxol production increased rapidly from the 4th d with the direct current magnetic field but most slowly with the alternating current magnetic field. The maximal yield of Taxol was 490 microg l(-1) with the direct current magnetic field and 425 microg l(-1) with the alternating current magnetic field after 8 d of culture, which were, respectively, 1.4-fold and 1.2-fold of that without exposure to a magnetic field.

Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures.

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US.

[Effects of ultraviolet-C irradiation on membrane lipid peroxidation and activity of PS II electron transport in chloroplasts of Taxus cuspidata needles].

The isolated chloroplasts of Taxus cuspidata needles treated with 12 W.m-2 of ultraviolet-C (UV-C, 254 nm) irradiation were studied under laboratory conditions. The results showed that with the increase of UV-C irradiation, the carotenoids (Car) and glutathione (GSH) contents and superoxide dismutase (SOD) activity were decreased, while the malondialdehyde content (MDA) and the relative permeability of chloroplasts membrane were increased to various extents. The PS II electron transport activity was conspicuous decreased, which had a significant inverse ratio with photosynthetically active radiation (PAR). UV-C irradiation was insensitive on chlorophyll. The results indicated that membrane lipid peroxidation of chloroplasts caused by UV-C radiation was the reason of the decrease of PS II electron transport activity.

The influence of ultraviolet radiation on the content of pharmacologically active taxoids in yew tissues.

The aim of the work was the investigation of the influence of UV radiation on the taxoids contents in yew tissues. The experiment was performed using Taxus baccata var. Aurea Corr. twigs irradiated with UV C (lambda = 254 nm) and UV A (lambda = 366 nm) in comparison to control samples. Multistep procedure of sample preparation was applied before the analysis of paclitaxel and 10-DAB III: SPE using alumina - for purification from the chlorophylles, waxes and polar balasts as well as zonal micropreparative TLC on silica - for isolation of partially separated fractions. The quantitation of some taxoids in the isolated fractions was performed using RP-HPLC procedure in system C18/acetonitrile + water. The experiments with UV-A and especially UV-C radiation showed changes in concentrations of paclitaxel and its precursor-10-deacetylbaccatin III. The results can be utilized to increase the yield of the taxoids isolated for medicinal and practical purposes.

[Effect of far-ultraviolet radiation on lipid peroxidation and inherent protection system in seedlings of Taxus cuspidata].

The effect of far-ultraviolet radiation on lipid peroxidation and inherent protection system in seedlings of Taxus cuspidata was examined with simulated UV-BC irradiation treatments. The electrolyte leakage, lipid peroxidation, tissue autoxidation and H2O2 content increased, while the contents of soluble protein, ascorbic acid, carotenoids, and chlorophyll and the photosynthetic electron transport rate of Photosystem II decreased with the increasing time of treatment. The lipid peroxidation of the needles was inhibited by exogenous scavengers sodium benzoate and ascorbate, but enhanced to some extent by methyl viologen and DDC (an inhibitor of SOD). The mechanisms of the injury of the seedlings induced by UV-BC radiation might be similar to that of photooxidation injuries of conifers in winter. However, Taxus cuspidata was much more resistant to UV-BC radiation than crops.