Over-expression of Escherichia coli transaldolase in the cytosol of Arabidopsis thaliana

Transaldolase (TAL) is an enzyme of the oxidative pentose phosphate pathway (OPPP) which catalyzes the reversible reaction of sedoheptulose-7-phophate into fructose-6-phosphate and erythrose-4-phosphate. In some micro-organisms, fungi and plants, erythrose-4-phosphate condenses with phosphoenolpyruvate (PEP) from glycolysis to form chorismate which is a precursor for many secondary metabolic pathways such as aromatic amino acids, flavonoids, lignin, indole acetate and UV light protectants. An analysis of plant genome databases reveals that the OPPP is incomplete in the cytosol of plants as no genes encoding for a cytosolic transaldolase (TAL) and transketolase (TK) have been identified so far. Thus, this study attempted to complete the compartmentation of TAL in the cytosol and plastid of plants by over-expressing it in the cytosol of A. thaliana. For this purpose, homozygous transgenic plants were obtained in these studies; it was found that the transaldolase activity of transgenic lines increased as compared to wild type plants. The findings of the current study also demonstrated that transgenic plants did not show any distinct phenotypes and there was no difference in a range of growth parameters compared with A. thaliana Col-0 (wild type)

Development of salt-tolerant lines of Malaysian indica rice (Oryza sativa L. cv. MR219) using tissue culture approach

Rice is one of the most important staple foods for human. However, millions hectares of rice land in the South and Southeast Asia were left uncultivated and/or grown with very low yields due to salinity. Due to this, development and production of salt-tolerant rice seed as apart of integrated management practices is needed. By using tissue culture approach, salt-tolerant lines of Malaysia indica rice cv. MR219 were produced. This work started with production and propagation of MR219 callus. Then, callus was sub-cultured separately on MS media supplemented with 2 mg/L 2,4-D and different concentration of NaCl (0, 50, 100, 200, and 300 mM NaCl) to produce salt-tolerant MR219 callus. Screening and selection of salt-tolerant MR219 callus were conducted using morphological and biochemical markers which are total proline content, total soluble sugar, lipid peroxidase and the activity of ascorbate peroxidase and catalase. At regeneration of salt-tolerant plantlets, selected salt-tolerant callus was sub-cultured on MS media supplemented with 2 mg/L kinetin and 1 mg/L BAP for shoot induction, followed by sub-cultured in MS media supplemented with 0.5 mg/L BAP, 1 mg/L kinetin, 1 mg/L IBA and 0.5 mg/L NAA for root formation. At acclimatization stage, MR219 plantlets from 50 mM NaCl found survived and transferred to pots containing paddy soil. These plantlets is called First generation (F1) salt-tolerant MR219. After 70 days, seeds of F1-salt-tolerant MR219 lines was successfully obtained. The grain characteristics of mother plant and F1-salt-tolerant MR219 lines were compared. Germination capability of F1-salt-tolerant MR219 seed in saline showed that seeds of F1-salt-tolerant MR219 able to germinate and growth in 50 -100 mM NaCl. As conclusion, salt-tolerant MR219 rice was produced in vitro and have potential to be commercialized. The protocol to produce salt-tolerant rice can be used to produce other salt-tolerant plant

Isolation, similarity and subcellular localisation of transaldolase from sugarcane (Saccharum officinarum)

This study focused on isolation, cloning of TAL from sugarcane. Transaldolase is one of the enzymes of the Pentose Phosphate Pathway (PPP). Transaldolase in non-oxidative phase of OPPP transfer a three carbon dihydroxyacetone moiety from sedoheptulose-7-phosphate and glyceraldehyde-3-phophate to produce Erythrose-4-Phophate (E4P) and fructose-6-phophate. E4P is the precursor for many secondary metabolic pathways including aromatic amino acids, lignin and flavonoid synthesis. Earlier studies revealed that OPPP is incomplete in the cytosol of plants as no genes encoding for a cytosolic TAL. Moreover, there is no study about the TAL genes from sugarcane until to date. Thus, the objective of this study is to isolate TAL gene from sugarcane, to compare its similarity with other plants, to determine its subcellular localization. A total of 1601 bp of TAL has been isolated by PCR. Similarity, studies by ClustalW revealed that TAL show highest similarity (75%) with Zea mays. Analysis of subcellular localization by using Target 1.1 revealed that of TAL from sugarcane was not located in the plastidic

Cloning, expression and characterization of sugarcane (Saccharum officinarum L.) transketolase

Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants

The effects of herbal plant extracts on the growth and sporulation of Colletotrhichum gloesporioides

Objectives: The antifungal activities of the leaf extracts of fifteen selected medicinal plants; Alpinia galanga L., Alstonia spatulata Blume., Annona muricata L., Blechnum orientale L., Blumea balsamifera L., Centella asiatica L., Dicranopteris linearis, Dillenia suffruticosa, Litsea garciae Vidal., Melastoma malabathricum L.,Momordica charantia L., Nephrolepis biserrata (Sw.)., Pangium edule Reinw., Piper betle L., and Polygonum minus Huds., were evaluated on the plant pathogenic fungus; C.loeosporioides isolated from mango. Methodology and results: Different antifungal assays were employed, i.e. Agar-Disc Dilution assay as primary screening assay, followed by determination of Minimum Inhibition Concentration (MIC), and the rate of sporulation assay. The antifungal assay was carried out in Potato Dextrose Media in five different treatments, i.e.; distilled water as negative control, crude extract of leaves in methanol, chloroform, acetone and Benomyl as positive control. A. galanga extracts were most effective and exhibited highest antifungal activities against C. gloeosporioides. Methanol crude extract reduced radial growth of C. gloeosporioides by 66.39%, followed by chloroform crude extract 63.26%, and 61.56% for acetone crude extracts. The exact concentrations that have definite potential to fully restrict the growth of C. gloeosporioides (MICs) for A.galanga is 15.00 mg/mL in methanol, 17.50 mg/mL in chloroform, and 17.50 mg/mL in acetone. The sporulation assay also revealed that A. galanga leaves crude extracts showed highest inhibition of spore germination of C. gloeosporioides overall at concentration of 10 mg/mL; with 68.89% inhibition by methanol extracts, 64.13% by chloroform extracts, and 62.86% by acetone extracts. Conclusion and application of findings: Numerous natural products of plant origin are pesticidal and have the potential to control fungal diseases of crops. Thus, considerable effort should be devoted to screening plants in order to develop new natural fungicides as alternative to existing. In this study, the leaf crude extracts of A. galanga exhibited effectiveness against C. gloeosporioides and should be considered for further evaluation

Nutritional and biochemical properties of Malaysian okra variety

The nutritional and biochemical contents of Malaysian okra fruits and leaves were studied by analysing the nutritional contents in the fruits and leaves such as protein, carbohydrate, moisture, oil, ash and fibre, while the biochemical contents in the fruits and leaves such as chlorophyll, phenolics and flavonoids were also analysed. The result of the proximate analysis revealed a significant difference (p < 0.05) among the fruits and leaves with highest percentage of crude protein (4.81%) and ash (2.44%) were present in the leaf. Mature fruits contain highest percentage of fiber (2.44%), oil (0.4%) and carbohydrate (11.7%) respectively, while the young fruits showed highest moisture contents of 88.47%. The results of the biochemical analysis showed significant differences (p < 0.05) among the fruits and leaves with the highest total chlorophyll content in mature leaves (32.99 mg/1 g). The total highest phenolics content was found in young leaves (0.99 mgTNE/1 g) and the total flavonoid was highest in mature leaves (0.79 mgQE/1 g). This paper showed that nutritional and the biochemical contents of okra were higher in the leaves than in the fruits

Isolation, subcellular localization and phylogenetic of transaldolase genes from Zea mays cv sweet corn bi-color

The Pentose Phosphate Pathway (PPP) also is known as 6-phosphogluconate pathway which occurs in the cytosol of the plant cell. There are two main arms in PPP; the oxidative and non-oxidative arms. The main functions of this pathway is to generate reducing equivalents in the form of NADPH, for reductive biosynthesis reactions within cells in the biosynthesis of fatty acids and steroid. This pathway also provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids. Transaldolase (TAL) is an enzyme which plays an important role in the non-oxidative portion of the pentose phosphate pathway. The actual reaction is between glyceraldehydes 3-phosphate and sedoheptulose 7-phosphate, resulting the formation of C4 product erythrose 4-phosphate and fructose 6-phosphate. In this study, transaldolase (TAL) has been successfully isolated and identified from Zea mays cv Sweet corn bi-color. The objectives of the study were achieved where the specific primer designed has function effectively in isolated TAL with 773 bp of nucleotide sequences. Analysis of homology through multiple sequence alignment revealed that TAL from Z. mays cv Sweet corn bi-color is highly similar with the plant species compared to animal and bacteria. ClustalW analysis found that TAL from Z. mays hit the score of 85.0 as they are close related in terms of taxonomy level which they are from the family of Poaceae. Other plants such as Solanum lycopersicum and Solanum tuberosum are from family Solanaceae, Dimorcarpus longan is from family Sapindaceae whereas Hyacinthus orientalis is from Hyacinthus family. However, it is discovered that TAL from Z. mays cv Sweet corn bi-color with TAL in Oryza sativa subsp. Indica only possess score of 64.0

Proximate analyses and anti-nutritional factors in local and improved cowpea varieties

Cowpea (Vigna unguiculata) seeds from local and improved varieties obtained from Abuja market, International Institute of Tropical Agriculture (IITA), and National Centre for Genetic Resources and Biotechnology (NACGRAB) were analyzed for the proximate determination (protein, moisture and ash) and anti-nutritional composition (Phytate, Alkaloid and Tannin). The seed protein content in the local and improved cowpea varieties ranged from 22.61% to 27.92%. The highest crude protein was found in NG/SA/066-1 (27.92%) and lowest was in Sampea 10 (23.76%). There was no significant difference in the moisture and ash content among the local and improved cowpea varieties. The result of the anti-nutritional composition showed that the highest phytate content (1.94 mg/g) was found in Big white variety while the lowest phytate content was found in Butter beans (0.84 mg/g). White cowpea variety recorded the highest alkaloid content at (2.54 mg/g) while Butter beans recorded the lowest alkaloid content at (0.24 mg/g). The highest tannin content was found in Big white at (5.72±0.15 mg/g) while the lowest was found in NG/AO/035 at (1.92±0.03 mg/g). The results herein can aid in cowpea breeding and conservation

Morphological and molecular systematic for selected Blechnum from Peninsular Malaysia

Systematic of Blechnum spp. using both morphological and molecullar studies were resucued in a monophyletic tree. The use of coding rbcL and non-coding tmH-psbA genes amplified from chloroplast were used to support the morphological differences. From the study, five species of Blechnum, Blechnum fraseri, Blechnum vestitum, Blechnum indicum, Blechnum orientale and Blechnum finlaysonianum were clustered independently according to morphological characters of scales, rhizome, stipe, rachis, laminae and their fertile and sterile pinnae. On the basis of morphological similarity, both B. fraseri and B. vestitum were lomariod species; their fertile pinnae much reduced in size compared to their sterile pinnae. This result was supported by molecular analyses by having 0.034 genetic distances and the phylogenetic tree represented here shown both of them more related. However, some of the relationships have been previously suspected on the basis of morphological similarity and supported as well by the DNA analyses were shown in this study for B. oriental and B. finlaysomanium. They were similar in rhizome, scales and laminae characters. Most importantly, both of them are eublecdnoid species; the fertile and sterile pinnae are similar in size and the genetic distance for B. orientale and B. finlaysonianum is 0.017. However. B. indicum was independently because of morphological distinct and no obvious relatives to other species. Morphological and molecular variations were consistently complemented to each other, and may be useful for further phylogenetic and taxonomical studies

In vitro regeneration of Cucumis sativus cv. MTi2 by using shoot apical meristem as explant

Cucumber variety MTi2 is one of the famous cucumber variety in Malaysia that consumed by local people. Therefore it is important to maintain the good supply of this favorable variety and to conserve the species to ensure its continuance in the future. Besides, the conventional means of propagation commonly yield non uniform plants that may reduce the crop quality. This might be a huge problem for large scale multiplication industries and for conservation purpose of the species. As an alternative, in vitro regeneration was carried out to optimize the protocol in obtaining uniform plantlet through culturing shoot apical meristem (SAM) of cucumber. Therefore the SAM culture was optimized in this study to generate identical plantlet. The effect of plant growth regulators (PGR) in in vitro regeneration was also studied through application of 10 treatments of full strength MS media supplemented with different concentration and combination of PGR, with MS media served as control treatment. The result showed that meristem tissue of cucumber can be regenerated into whole plant through in vitro micropropagation. The combination treatment of 0.01 mgL-1 IAA and 0.1 mgL-1 KIN produced highest percentage survival of plantlets which was 88%