Transgenic plants of Artemisia annua L., a medicinal plant that produces the compound artemisinin which has an anti-malarial activity, were developed following Agrobacterium tumefaciens-mediated transformation of leaf explants. A. tumefaciens strain EHA105 carrying either pCAMBIA1301 or pCAMBIAFPS was used. Both plasmids harbored the hygromycin phosphotransferase II (hptII) gene as a selectable gene, but the latter plasmid also harbored the gene encoding for farnesyl pyrophosphate synthase (FPS), a key enzyme for artemisinin biosynthesis. Shoot regeneration was observed either directly from leaf sections or via intervening callus when explants were incubated on solidified Murashige and Skoog (MS) (1962) medium containing 0.1 mg l -1 a-naphthaleneacetic acid (NAA), 1 mg l -1 N 6 -benzyladenine (BA), 30 mg l -1 meropenem and 10 mg l -1 hygromycin. Applying vacuum infiltration dramatically increased transformation efficiency up to 7.3 and 19.7% when plasmids with and without FPS gene were used, respectively. All putative transgenic regenerants showed positive bands of hptII gene following Southern blot analysis. Expression of FPS was observed in all transgenic lines, and FPS over-expressed lines exhibited higher artemisinin content and yield, of 2.5-and 3.6-fold, respectively, than that detected in wild-type plants. A relatively high correlation (R 2 = 0.78) was observed between level of expression of FPS and artemisinin content. However, gene silencing was detected in some transgenic lines, especially for those lines containing two copies of the FPS transgene, and with some lines exhibiting reduced growth.
Water deficit limits plant growth and yield. Arbuscular mycorrhizal (AM) symbiosis is viewed as one of the several methods to improve growth under water deficit. The present study investigated the growth performance in relation to water deficit in two cultivars (''H2'' and ''660'') of AM treated macadamia (Macadamia tetraphylla L.) plants. AM treatment significantly improved the growth in macadamia plants that have been subjected to water deficit (7 % soil water content) for 14 days. Leaf water content (LWC) and maximum quantum yield of PSII (F v /F m ) in AM-associated plants were maintained better than those in the control (well-watered) plants. A positive correlation was observed between LWC and F v /F m in ''H2'' cultivar. AM treatment enhanced proline and soluble sugar content in ''H2'' cultivar under water deficit stress. In contrast, only soluble sugars were accumulated in the AMassociated plants of ''660'' cultivar under water deficit stress. The study concludes that soluble sugars and proline are involved as key signals of osmoregulation defense response, improve water relation in plant tissues, and thereby resulting in improved growth in AM-associated macadamia plants.
Soluble carbohydrates play a key role as osmolytes and significantly contribute in salt defence mechanism, especially in halophyte species. The objective of this study is to investigate the transcriptional expression of starch-related genes, sugar profile and physiological performances of two contrasting rice genotypes, Pokkali (salt tolerant) and IR29 (salt sensitive), in response to salt stress. Total soluble sugars, glucose and fructose levels in the flag leaf of salt-stressed Pokkali rice were enhanced relative to soluble starch accumulation in plants exposed to EC = 13.25 dS m(-1) (salt stress) for 3 days. In Pokkali, the net photosynthetic rate and starch metabolism may play a key role as energy resources under salt stress. In contrast, photosynthetic performance, indicated by photosynthetic pigment levels and chlorophyll fluorescence parameters, in salt-stressed IR29 was significantly reduced, leading to delayed starch biosynthesis. The reduction in photosynthetic ability and lack of defence mechanisms in IR29 caused growth inhibition and yield loss. Soluble starch and soluble sugar enrichment in Pokkali rice may function alternatively as osmotic adjustment in salt defence mechanism and strengthen carbon energy reserves, greater survival prospects under salt stress and enhanced productivity.
The aim of this research was to investigate betaine aldehyde dehydrogenase (BADH) and glycinebetaine (Glybet) biosynthesis in photoautotrophic rice seedlings. The role of Glybet on physiological and growth responses to salt stress in both salt-tolerant and salt-sensitive lines is to be investigated. The BADH activity in salt-tolerant seedlings cultured under extreme salt stress (342 mm NaCl) progressively increased during the first few hours until it peaked after 72 h. This was about 2.5 times greater than in salt-sensitive plants. Similarly, the amount of Glybet detected in salt-tolerant lines was 1.3 times more than in salt-sensitive lines at 96 h salt exposure. The BADH activities were positively related to Glybet accumulation in both salt-tolerant and salt-sensitive lines. The accumulation of Glybet in salt-tolerant lines was directly correlated with pigment stabilization. Relative water content in the salt-tolerant lines was closely related to water oxidation in photosystem II (PSII), defined by maximum quantum yield of PSII (F v /F m ). In addition, a high concentration of total chlorophyll is more efficient in capturing light energy, defined by photochemical quenching. The concentrations of chlorophyll a and total carotenoid were positively related to the quantum efficiency of PSII (U PSII ) and nonphotochemical quenching, respectively, resulting in a high net-photosynthetic rate (NPR) and the promotion of growth. The high level of Glybet in salt-tolerant lines plays a role as a salt defensive response mechanism in terms of pigment stabilization and water oxidation in PSII, resulting in high NPR and growth efficiency.
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 GA 3 treatment. The effect of GA 3 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 GA 3 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 GA 3 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 GA 3 treatment was associated with increased expression of key enzymes in the artemisinin biosynthesis pathway. Interestingly, exogenous GA 3 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 GA 3 -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 GA 3 -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 GA 3 treatment enhanced artemisinin production in pot experiments and should be suitable for field application.
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