Identification and characterisation of thiamine pyrophosphate (TPP) riboswitch in Elaeis guineensis.

The oil palm (Elaeis guineensis) is an important crop in Malaysia but its productivity is hampered by various biotic and abiotic stresses. Recent studies suggest the importance of signalling molecules in plants in coping against stresses, which includes thiamine (vitamin B1). Thiamine is an essential microelement that is synthesized de novo by plants and microorganisms. The active form of thiamine, thiamine pyrophosphate (TPP), plays a prominent role in metabolic activities particularly as an enzymatic cofactor. Recently, thiamine biosynthesis pathways in oil palm have been characterised but the search of novel regulatory element known as riboswitch is yet to be done. Previous studies showed that thiamine biosynthesis pathway is regulated by an RNA element known as riboswitch. Riboswitch binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA. TPP binds specifically to TPP riboswitch to regulate thiamine biosynthesis through a variety of mechanisms found in archaea, bacteria and eukaryotes. This study was carried out to hunt for TPP riboswitch in oil palm thiamine biosynthesis gene. Riboswitch detection software like RiboSW, RibEx, Riboswitch Scanner and Denison Riboswitch Detector were utilised in order to locate putative TPP riboswitch in oil palm ThiC gene sequence that encodes for the first enzyme in the pyrimidine branch of the pathway. The analysis revealed a 192 bp putative TPP riboswitch located at the 3' untranslated region (UTR) of the mRNA. Further comparative gene analysis showed that the 92-nucleotide aptamer region, where the metabolite binds was conserved inter-species. The secondary structure analysis was also carried out using Mfold Web server and it showed a stem-loop structure manifested with stems (P1-P5) with minimum free energy of -12.26 kcal/mol. Besides that, the interaction of riboswitch and its ligand was determined using isothermal titration calorimetry (ITC) and it yielded an exothermic reaction with 1:1 stoichiometry interaction with binding affinities of 0.178 nM, at 30°C. To further evaluate the ability of riboswitch to control the pathway, exogenous thiamine was applied to four months old of oil palm seedlings and sampling of spear leaves tissue was carried out at days 0, 1, 2 and 3 post-treatment for expression analysis of ThiC gene fragment via quantitative polymerase chain reaction (qPCR). Results showed an approximately 5-fold decrease in ThiC gene expression upon application of exogenous thiamine. Quantification of thiamine and its derivatives was carried out via HPLC and the results showed that it was correlated to the down regulation of ThiC gene expression. The application of exogenous thiamine to oil palm affected ThiC gene expression, which supported the prediction of the presence of TPP riboswitch in the gene. Overall, this study provides the first evidence on the presence, binding and the functionality of TPP riboswitch in oil palm. This study is hoped to pave a way for better understanding on the regulation of thiamine biosynthesis pathway in oil palm, which can later be exploited for various purposes especially in manipulation of thiamine biosynthesis pathways in combating stresses in oil palm.

Pressurized hot water extraction of hydrosable tannins from Phyllanthus tenellus Roxb.

BACKGROUND: Safety, environmental and economic setbacks are driving industries to find greener approaches to extract bioactive compounds from natural resources. Pressurized hot water extraction (PHWE) is among the solvent free and efficient methods for extracting bioactive compounds. EXPERIMENTAL: In this study, the suitability of PHWE for extracting bioactive compounds such as phenolics, hydrolysable tannins and flavonoids from Phyllanthus tenellus was investigated by UPLC-qTOF-MS. RESULTS: Solvent properties of water are significantly increased through imposing temperature at 121 °C and pressure at 15 p.s.i. Pressurized hot water extraction obtained 991-folds higher hydrolysable tannins than methanol extraction. CONCLUSION: The extraction yields of hydrolysable tannins with PHWE was almost double of absolute methanol extraction.

The Effect of Oxidative Stress Towards The Expression of Thiamine Biosynthesis Genes (THIC and THI1/THI4) in Oil Palm (Elaeis guineensis).

Thiamine is known to be an important compound in human diet and it is a cofactor required for vital metabolic processes such as acetyl-CoA biosynthesis, amino acid biosynthesis, Krebs and Calvin cycle. Besides that, thiamine has been shown to be involved in plant protection against stress. In this study, the level of expression of THIC and THI1/THI4, the genes for the first two enzymes in the thiamine biosynthesis pathway were observed when oil palm (Elaeis guineensis) was subjected to oxidative stress. Primers were designed based on the consensus sequence of thiamine biosynthesis genes obtained from Arabidopsis thaliana, Zea mays, Oryza sativa, and Alnus glutinosa. Oxidative stress were induced with various concentrations of paraquat and samplings were done at various time points post-stress induction. The expression of THIC and THI1/THI4 genes were observed via RT-PCR and qPCR analysis. The expression of THIC was increased 2-fold, while THI1/THI4 gene transcript was increased 4-fold upon induction of oxidative stress. These findings showed that oil palm responded to oxidative stress by over-expressing the genes involved in thiamine biosynthesis. These findings support the suggestion that thiamine may play an important role in plant protection against stress.

Artificial symbiotic association and growth induction of embryogenic calli (Elaeis guineensis Jacq.) inoculated with Herbaspirillum seropedicae (Z78)

Aims: The purpose of this experiment was to determine the artificial symbiosis interaction of Herbaspirillum seropedicae (Z78) on oil palm embryogenic calli. Methodology and results: For this purpose, symbiotic associations were established between Z78 and embryogenic calli of oil palm tissue cultured. A total of five treatments involved, in particular: i) + 3.0 mg/L 2,4-D + 100% N MS medium (control), ii) + Z78 pellet cells (1 mL) + 25% N MS medium, iii) + Z78 supernatant (1 mL) + 25% N MS medium, iv) + Z78 broth culture (1 mL) + 25% N MS medium, and v) + Z78 sonicated cells (1 mL) + 25% N MS medium. All treatments were supplied with minimal N sources (25% N), ammonium nitrate and potassium nitrate, while the control was treated with 100% N sources. Treated samples were harvested on D80 and observed for biomass and diameter increment (%), formation of embryoids, and Z78 colonization. The results showed embryogenic calli in the inoculated treatments that contained depleted N produced similar result to the control treatment which contained 100% N nutrients. Positive interactions occurred between the diazotroph and host plant tissues as viewed under FESEM and EFTEM. Among the treatments, Z78 sonicated cell showed better growth of embryogenic calli compared to others. Conclusion, significance and impact study: The in vitro nitrogen-depleted artificial symbiosis environment allowed the diazotroph (Z78) to be expressed and provide the nitrogen sources and indole-3-acetic acid for cell growth. This study represents beneficial co-culture interaction effects of different inocula of diazotrophic bacterial cells with in vitro embryogenic calli of oil palm.