Enhancement of artemisinin content and biomass in <Emphasis Type="Italic">Artemisia annua</Emphasis> by exogenous GA<Subscript>3</Subscript> treatment

Artemisinin is a promising and potent antimalarial drug naturally produced by the plant Artemisia annua L. but in very low yield. Its artemisinin content is known to be greatly affected by both genotype and environmental factors. In this study, the production of artemisinin and leaf biomass in Artemisia annua L. was significantly increased by exogenous GA₃ treatment. The effect of GA₃ application on expression of proposed key enzymes involved in artemisinin yield was examined in both wild type (007) and FPS-overexpression (253-2) lines of A. annua. In the wild type (007) at 6 h post GA₃ application there was an abrupt rise in FPS, ADS and CYP71AV1 expression and at 24 h a temporary and significant peak in artemisinin (1.45-fold higher than the control). After GA₃ application in line 253-2, there was a dramatic rise in expression of FPS at 3 h, CYP71AV1 at 9 h and ADS at 72 h and accumulation of artemisinin after 7 days, which was a delay when compared with the wild type plant. Thus, increased artemisinin content from exogenous GA₃ treatment was associated with increased expression of key enzymes in the artemisinin biosynthesis pathway. Interestingly, exogenous GA₃ continuously enhanced artemisinin content from the vegetative stage to flower initiation in both plant lines and gave significantly higher leaf biomass than in control plants. Consequently, the artemisinin yield in GA₃-treated plants was much higher than in control plants. Although the maximum artemisinin content was found at the full blooming stage [2.1% dry weight (DW) in 007 and 2.4% DW in 253-2], the highest artemisinin yield in GA₃-treated plants was obtained during the flower initiation stage (2.4 mg/plant in 007 and 2.3 mg/plant in 235-2). This was 26.3 and 27.8% higher, respectively, than in non-treated plants 007 and 253-2. This study showed that exogenous GA₃ treatment enhanced artemisinin production in pot experiments and should be suitable for field application.     

Effect of down-regulating 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate reductoisomerase by RNAi on growth and artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), an important enzyme in the 2-c-methyl-d-erythritol-4-phosphate (MEP) pathway in plant plastids, provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the roles of the MEP pathway in regulating growth, development and artemisinin biosynthesis of Artemisia annua L., we used RNA interference technology to generate transgenic plants with suppressed expression of DXR in A. annua (AaDXR). Suppression of AaDXR resulted in shorter stems, decreased branch numbers and leaf area, lower density of leaf trichomes. Although AaDXR-RNAi plants had no significant changes on the stomatal conductance, the net photosynthesis rate was decreased by 20.0–31.4% due to the marked decline in the contents of chlorophyll. Decreased levels of endogenous gibberellic acid (GA₃) and abscisic acid were also detected in the transgenic lines. The artemisinin contents in leaves of all tested transgenic lines declined by 41.8–73.4% at the vegetative stage and 61.5–63.6% at the stages of flowering. The enhancement of artemisinin contents by methyl jasmonate at 300 µM has been abolished at seedling and vegetative stages in AaDXR-RNAi plants. These results demonstrate that AaDXR play import roles in the control of plan vegetative growth and artemisinin biosynthesis in A. annua.     

The response of terpenoids to exogenous gibberellic acid in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. at vegetative stage

In this study the influence of gibberellic acid (GA₃) on plastidic and cytosolic terpenoids and on two key enzymes, 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), for terpenoid biosynthesis was compared in vegetative cannabis plants. Treatment with GA₃ resulted in a decrease of DXS activity in comparison with the control plants. The amount of chlorophylls a, b and total carotenoids declined when plants treated by GA₃ in a concentration dependent manner. The α-tocopherol content of cannabis plants decreased in 50 μM GA₃ treatment and increased in 100 μM GA₃ treatment. Exogenous GA₃ caused an increase in HMGR activity. Concomitant with this result, the amount of squalene and phytosterols increased with GA₃ treatment. The amount of THC and CBD did not change at 50 μM GA₃ treatment, but applying of 100 μM GA₃ increased THC and CBD content in leaf plant in comparison with control plants. GA₃ treatment declined number and percentage of monoterpenes in treated plants. Also the number of sesquiterpenes decreased in response to GA₃ treatment but among the remainder of them, the amount of some sesquiterpenes decreased and some sesquiterpenes increased with GA₃ treatment. Our results showed that GA₃ treatment had opposite effect on primary terpenoid biosynthesis by the plastidic 2C-methyl-d-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways. But secondary terpenoids showed different response to GA₃ treatment probably due to interference of two biosynthetic pathways in their formation.     

The Interaction of Plant Growth Regulators and Vernalization on the Growth and Flowering of Cauliflower (Brassica oleracea var. botrytis)

The growth and flowering response of a cold-requiring cauliflower (Brassica oleracea var. botrytis cv. 60 day) to a range of temperatures under 10 h photoperiod and to growth regulator application were investigated. Endogenous gibberellin A₁(GA₁) concentrations were also assessed under these treatments. Flowering and growth of the inflorescence stalk were correlated with plant developmental stage at the time of a vernalizing cold treatment. Temperature and its duration also affected flowering and inflorescence development. The most effective temperature for inflorescence induction was 10 °C. Flowering did not occur in non-vernalized plants (25 °C) even though they had been treated with GA₃. Application of GA₃ promoted inflorescence stalk elongation greatly in vernalized plants (10 °C), but less so in partially vernalized plants (15 °C or 20 °C). Paclobutrazol (PP₃₃₃) sprayed at the 8–9 leaf stage significantly suppressed inflorescence stalk length and slightly delayed flower bud formation and anthesis. Vernalization at 10 °C increased endogenous GA₁ content in both leaves and the inflorescence stalk irrespective of GA₃ or PP₃₃₃ treatment. Application of GA₃ tended to increase GA₁ levels, while PP₃₃₃ significantly reduce GA₁, both irrespective of vernalization. Vernalization is an important factor for flowering, but not curd formation in this cauliflower cv. 60 day and GA₁ is likely a causal factor in inflorescence stalk elongation.     

Exogenous salicylic acid-mediated modulation of arsenic stress tolerance with enhanced accumulation of secondary metabolites and improved size of glandular trichomes in <Emphasis Type="Italic">Artemisia annua</Emphasis> L<Emphasis Type="Italic">.</Emphasis>

The present study was undertaken to find out individual and interactive effects of arsenic (As) and salicylic acid (SA) on an important medicinal plant, Artemisia annua. As uptake and its accumulation was detected and found to be maximum in roots at higher As concentration (150 μM). Under As treatments, H₂O₂ and MDA content were induced. Biomass and chlorophyll content were negatively affected under As treatments. Furthermore, enzymatic (SOD, CAT, APX, and GR) and non-enzymatic antioxidants were also enhanced under As treatments. Exogenous application of SA reduced the extent of H₂O₂ and O₂ ^? generation and lipid peroxidation, while reverted biomass and chlorophyll content to overcome oxidative stress. Simultaneous application of SA with As increased endogenous SA level, artemisinin, and dihydroartemisinic acid as compared with individual As treatment and pre-application of SA with As treatments. The expression of four key artemisinin biosynthetic pathway genes, i.e., ADS, CYP71AV1, DBR2, and ALDH1 were upregulated at a maximum in plants simultaneously treated with SA and As. Similar pattern of artemisinin accumulation and glandular trichome size was observed which attest that SA has a stimulatory impact on artemisinin biosynthesis under As stress. Our study suggests that exogenous application of SA and As together induced more tolerance in A. annua than a comparable dose of SA pre-treatment. The study may provide a platform with dual benefits by developing As-tolerant plants to be used for phytoremediation of arsenic from As-contaminated soil and obtaining high artemisinin-producing A. annua plants.     

Interactive effects of gibberellin A<Subscript>3</Subscript> and ascorbic acid on lipid peroxidation and antioxidant enzyme activities in <Emphasis Type="Italic">Glycine max</Emphasis> seedlings under nickel stress

The effects of nickel in combination with ascorbic acid (AsA) and gibberellin on 7-day-old soybean seedlings were examined. Exposure to 0.25 mM NiCl₂ × 6H₂O for 5 days resulted in toxicity symptoms, such as formation of reddish-brown mottled spots on the leaf blade. Addition of 0.05 mM GA₃ or 1 mM AsA reduced toxic effects of nickel. After their simultaneous application, these symptoms did not appear. Ni decreased dry weights of roots and shoots and reduced chlorophyll content in leaves. An enhanced level of lipoxygenase activity and malondialdehyde, and changes in the activities of the antioxidant enzymes, catalase, guaiacol peroxidase and ascorbate peroxidase, in both roots and leaves indicated that Ni caused an oxidative stress in soybean plants. The Ni-stressed seedlings exposed to AsA or GA₃, especially to GA₃ plus AsA, exhibited an improved growth as compared to Ni-treated plants. GA₃ decreased Ni uptake by roots, while ascorbic acid considerably reduced root-to-shoot translocation of Ni. Interaction of AsA plus GA₃ prevented the decrease in chlorophyll content and lipid peroxidation as well as increased the activities of the antioxidant enzymes. These results suggest that GA₃ plus AsA treatment counteracts the negative effects of Ni on soybean seedlings. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 85–91. The text was submitted by the authors in English.     

Influence of gibberellin A<Subscript>3</Subscript> application,pH of the medium,photoperiod and temperature on the enhancement of in vitro flowering in <Emphasis Type="Italic">Vitex negundo</Emphasis> L.

Vitex negundo L. is an economically important small medicinal tree and an essential oil is extracted from its flowers. In nature, flowering is season specific and irregular, appearing only after a long vegetative phase. A very much improved protocol has been developed for the enhancement of flowering in vitro, one which increases both the number of flowers and the percentage (98.6%) of micro-plants flowering. The inclusion of gibberellin A₃ (GA₃) in the culture medium played the key role in controlling the in vitro flowering in V. negundo. The promotive effect of GA₃ was further improved by optimising pH, photoperiod and temperature. Our in vitro flower induction procedures provide an extremely effective method for further research on flowering regulation mechanisms in this important medicinal tree.     

Plant water status,H<Subscript>2</Subscript>O<Subscript>2</Subscript> scavenging enzymes,ethylene evolution and membrane integrity of <Emphasis Type="Italic">Cicer arietinum</Emphasis> roots as affected by salinity

The chickpea genotype, CSG-8962 was raised in screenhouse to study salinity induced changes in ethylene evolution, antioxidative defence system and membrane integrity in relation to changes in plant water and mineral content. At vegetative stage (60 d after sowing), the plants were exposed to single saline irrigation (0, 2.5, 5.0 and 10.0 dS m^–1). Sampling was done 3 d after saline treatments. The other sets of treated plants were re-irrigated with water and sampled after further 3 d. The _w of leaf and _s of leaf and roots decreased from –0.47 to –0.61 MPa, –0.67 to –1.23 MPa and from –0.57 to –0.95 MPa, respectively, with increasing salinity. Similarly, RWC of leaf and roots reduced from 87.5 to 72.3 % and 96.7 to 84.35 %, respectively. The decline in s of roots was mainly due to accumulation of proline and total soluble sugar. With salinity, increase in ethylene evolution, 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC oxidase activity was reported. Similarly, marked increase in H₂O₂ content (20 – 182 %) and lipid peroxidation (43 – 170 %) was observed. The defense mechanism activated in roots was confirmed by the increased activities of superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX), glutathione transferase (GTase), glutathione reductase (GR) and catalase (CAT) but ascorbic acid (AA) content was decreased. About 3-fold increase in Na⁺/K⁺ ratio and 2.5 fold increase in Cl^– content was observed. Upon desalinization, a partial recovery was observed in most of the parameters studied.     

Exogenous GA<Subscript>3</Subscript> overcomes bud deterioration in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) bulbs during dry storage by promoting endogenous IAA activity in the internodes

Endogenous free IAA was examined with an immunohistochemical method for its involvement in the reduction of bud deterioration after GA₃ was injected into the bulbs. We found that tulip bulbs stored at 20°C constantly developed severe bud deterioration, whereas the symptoms of deterioration was lighter in the bulbs with GA₃ injection and not observed in the bulbs with 4°C treatment. 73% success in overcoming bud deterioration was achieved in 20°C with GA₃ treatment after 8 weeks of bulb storage, and the success rate was 7% after 12 weeks of storage. IAA was detected in the parenchyma cells in the internodes of the shoot after the bulbs were stored at 4°C or at 20°C with GA₃ injection for 4 weeks, but little was detected in the bulbs stored at 20°C constantly. Moreover, a weak IAA signal was present in between the cells of the internodes irrespective of bulb treatment. After planting, the bulbs that had been treated differently exhibited different flowering ability. The bulbs stored at 4°C for 4, 8 and 12 weeks attained high flowering percentage, which was lower in the 20°C with GA₃ treatment and lowest in the 20°C treatment. It may be concluded that GA₃ injection decreases bud deterioration of tulip bulbs during dry storage at 20°C by promoting the endogenous IAA in the internodes.     

Effect of gibberellic acid on the cyanobacterium <Emphasis Type="Italic">Nostoc linckia</Emphasis>

We investigated the influence of gibberellic acid (GA₃; 0, 1, 10, and 100 μM) on Nostoc linckia culture at 7, 14, and 21 days. The fresh and dry weight of N. linckia was increased considerably by the 10 and 100 μM GA₃ treatments. A reduction in heterocyst frequency was observed in cultures treated with 1 and 10 μM GA₃. Adding GA₃ to N. linckia culture had a little effect on cell size. The amount of chlorophyll a and carotenoids decreased at all concentrations of GA₃. The amount of phycocyanin increased up to twofold in 7-day-old culture treated with 1 μM GA₃, and similar changes were observed for allophycocyanin and phycoerythrin content after 7 days. The effect of GA₃ on reducing sugar content was different and was dependent on the growth period. A reduction in soluble sugar content was detected after GA₃ application in 7- and 14-day-old cyanobacteria. Cultures treated with GA₃ had a higher protein content after 14 days and a lower protein content after 7 and 21 days, and reduced nitrogenase activity after 7, 14, and 21 days. Our data show that GA₃ application can be a suitable and inexpensive way to increase N. linckia biomass and phycobiliprotein production.     

Improvement of pistillate flowers yield with GA3 in heavy metals treated plants

GA₃ can be beneficial to improve the flowering yield in Lagenaria siceraria (Mol.) Standl. and Luffa cylindrica L. (Roem) treated with HgCl₂ and . This paper reviews the literature on the possible involvement of gibberellins in flower development and suggests that plants under the stress of heavy metals can be treated with GA₃ to improve growth parameters, to avoid delay in flowering and likewise quality of fruit can be improved. Applied GA₃ (400 mg/l) caused precocious flowering, increasing the number of flowers in the treated plants. All data was compared with control plants grown under similar conditions. Both heavy metals caused significant delay in flowering, consequently leading to reduction in the number of flowers. However, when GA₃ was applied with HgCl₂ and , there was less decrease in staminate and pistillate flowers, revealing the dominant effect of GA₃. Current work reveals that inhibitory effects of heavy metals on flowering can be partially restored with GA₃ application.     

Root proteomics reveals cucumber 24-epibrassinolide responses under Ca(NO<Subscript>3</Subscript>)<Subscript>2</Subscript> stress

Key message

The application of exogenous 24-epibrassinolide promotes Brassinosteroids intracellular signalling in cucumber, which leads to differentially expressed proteins that participate in different life process to relieve Ca(NO ₃ ) ₂ damage.

Abstract

NO₃ ^? and Ca²⁺ are the main anion and cation of soil secondary salinization during greenhouse cultivation. Brassinosteroids (BRs), steroidal phytohormones, regulate various important physiological and developmental processes and are used against abiotic stress. A two-dimensional electrophoresis gel coupled with MALDI-TOF/TOF MS was performed to investigate the effects of exogenous 24-epibrassinolide (EBL) on proteomic changes in cucumber seedling roots under Ca(NO₃)₂ stress. A total of 80 differentially accumulated protein spots in response to stress and/or exogenous EBL were identified and grouped into different categories of biological processes according to Gene Ontology. Under Ca(NO₃)₂ stress, proteins related to nitrogen metabolism and lignin biosynthesis were induced, while those related to cytoskeleton organization and cell-wall neutral sugar metabolism were inhibited. However, the accumulation of abundant proteins involved in protein modification and degradation, defence mechanisms against antioxidation and detoxification and lignin biosynthesis by exogenous EBL might play important roles in salt tolerance. Real-time quantitative PCR was performed to investigate BR signalling. BR signalling was induced intracellularly under Ca(NO₃)₂ stress. Exogenous EBL can alleviate the root indices, effectively reduce the Ca²⁺ content and increase the K⁺ content in cucumber roots under Ca(NO₃)₂ stress. This study revealed the differentially expressed proteins and BR signalling-associated mRNAs induced by EBL in cucumber seedling roots under Ca(NO₃)₂ stress, providing a better understanding of EBL-induced salt resistance in cucumber seedlings. The mechanism for alleviation provides valuable insight into improving Ca(NO₃)₂ stress tolerance of other horticultural plants.

     

Changes in carbohydrate contents of <Emphasis Type="Italic">Zantedeschia</Emphasis> leaves under gibberellin-stimulated flowering

The effect of gibberellic acid (GA₃) on the carbohydrate accumulation in relation to vegetative growth of Zantedeschia ‘Black Magic’ plants undergoing transition to flowering was investigated. In response to GA treatment the carbohydrate level increased independently of earlier stimulation of shoot emergence. Under vegetative growth stage the content of reducing sugars of leaf blades was 2.5-fold higher than in control plants, and suggests the stimulation of photosynthetic activity. The changes observed during the flowering, in principle noted in petiole tissues, support the GA-effect on assimilate transport to the sink organs. Moreover, the high level of non-structural carbohydrates in petiole tissues, in particular reducing sugars, can be an effect of photosynthetic activity of these organs and/or essential for osmoregulation and high turgor pressure. The results indicate that apart from the influence on the shoot emergence, the GAs may stimulate the photosynthetic activity from the beginning of shoot growth and are thus responsible for the enhancement of callas flower yield.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Salicylic acid activates artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

This paper provides evidence that salicylic acid (SA) can activate artemisinin biosynthesis in Artemisia annua L. Exogenous application of SA to A. annua leaves was followed by a burst of reactive oxygen species (ROS) and the conversion of dihydroartemisinic acid into artemisinin. In the 24 h after application, SA application led to a gradual increase in the expression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene and a temporary peak in the expression of the amorpha-4,11-diene synthase (ADS) gene. However, the expression of the farnesyl diphosphate synthase (FDS) gene and the cytochrome P450 monooxygenase (CYP71AV1) gene showed little change. At 96 h after SA (1.0 mM) treatment, the concentration of artemisinin, artemisinic acid and dihydroartemisinic acid were 54, 127 and 72% higher than that of the control, respectively. Taken together, these results suggest that SA induces artemisinin biosynthesis in at least two ways: by increasing the conversion of dihydroartemisinic acid into artemisinin caused by the burst of ROS, and by up-regulating the expression of genes involved in artemisinin biosynthesis.     

Physical,chemical, and regulatory properties of glycolate oxidase in C<Subscript>3</Subscript> and C<Subscript>4</Subscript> plants

Physical, chemical, and regulatory properties of glycolate oxidase (GO) isolated from the leaves of C₄ and C₃ plants (Zea mays L., cv. Voronezhskaya 76 and Glycine max (L.) Merr., cv. Pripyat’, respectively) were studied. The homogenous preparations were obtained by multistage enzyme purification from soybean leaves and maize mesophyll and bundle sheath. The glycolate oxidase (GO) preparations obtained consisted of two types of subunits, 37 and 44 kD. The GO isolated from C₃ plant leaves had many in common with that extracted from C₄ plant bundle sheath as regards physical, chemical, and catalytic properties. The primary function of GO in both plant types is metabolism of glycolate, which is a product of ribulosebisphosphate oxalacetic acid oxidation and is used by plants for biosynthesis of hydrocarbons and amino acids.