November 2012 issue | Plant Omics Journal

Enhancement of artemisinin biosynthesis by overexpressing dxr, cyp71av1 and cpr in the plants of Artemisia annua L.

Lien Xiang, Lixia Zeng, Yuan Yuan, Min Chen, Fang wang, Xiaoqiang Liu, Lingjiang Zeng, Xiaozhong Lan, Zhihua Liao

Abstract

Artemisinin is extracted from a traditional Chinese medicinal herb Artemisia annua L., which is regarded as the most efficient drug against malaria in the world. In recent years, attention has been paid to increase the artemisinin content through transgenic methods because of the low content of artemisinin in wild plants. In this article, three functional artemisinin-related genes namely dxr, cyp71av1 and cpr, were used to genetically modify the artemisinin biosynthesis pathway in A. annua. Four independent transgenic lines of A. annua plants with overexpression of dxr and five independent transgenic A. annua lines with overexpression of both cyp71av1 and cpr were obtained and confirmed through genomic PCR. All the transgenic A. annua plants with overexpression of the dxr gene showed higher levels of artemisinin than the wild type. The content of artemisinin in the Line D24 with overexpression of dxr (1.21±0.01 mg•g-1 DW) was more than two times compared with that in the wild type (0.52±0.01 mg•g-1 DW). All the five lines of co-overexpressing cyp71av1 and cpr had an increase of artemisinin production compared with the wild-type A. annua. Line No. 16 with overexpression of cyp71av1 and cpr had the highest content of artemisinin (2.44±0.13 mg•g-1 DW) at nearly three times of the wild-type A. annua (0.91±0.02 mg•g-1 DW). Thus, the present study demonstrated that genetic modification of the upstream 2-C-methyl-D-erythritol 4-phosphate pathway or metabolic engineering of the artemisinin-specific pathway could, respectively, enhance artemisinin biosynthesis. These strategies could be applied to develop transgenic A. annua with higher levels of artemisinin.

Page 503-507 | Full Text PDF

Application of Fourier Transform Infrared Microspectroscopy (FTIR) and Thermogravimetric Analysis (TGA) for quick identification of Chinese herb
Solanum lyratum

Yiling Wang
Abstract

Solanum lyratum is a medicinal plant used to treat cancers and tumors. The objective of this study was to apply Fourier transform infrared microspectroscopy (FTIR) and thermogravimetric analysis (TGA) to quick identification of S. lyratum fourteen samples from its main geographic origins in China. The hierarchical dendrogram based on principal component analysis (PCA) of FTIR and TGA data showed that these samples could be divided into three ecotypes according to their geographic distances to the ocean: inland type, middle type and inshore type. Differences in cell compositions and structures by FTIR and TGA indicated that higher content of cell wall polysaccharides, hermo-oxidative degradation of hemicellulose (HODH) and degradation of lignin (DL) are appointed for inshore type compared to inland type and middle type. These indices (cell wall polysaccharides, HODH and DL) can be characteristic values for identifying S. lyratum from different region in China. The FTIR-TGA method, providing compositional and structural differences in their macromolecules in cell for the herbs rapidly and accurately, would be capable of further identification of other Chinese medicinal plant.

Page 508-513 | Full Text PDF

Histological analyses of PLBs of Dendrobium sonia-28 in the recognition of cell competence for regeneration and Agrobacterium infection

Advina Lizah Julkifle, Ranjetta Poobathy, Razip Samian and Sreeramanan Subramaniam

Abstract

Protocorm-like bodies (PLBs), resembling the orchid embryo, are excellent target explants for many orchid experiments. Histology and scanning electron microscopy (SEM) were applied on PLBs of Dendrobium sonia-28. Histological observations revealed that globular masses arose from the epidermal cell layers of the PLBs, giving rise to new PLBs. The primordial and young leaves differentiated from the shoot apical meristem, transforming PLBs into plantlets. The SEM analysis revealed an abundance of stomata and a scarcity of trichomes on the PLB surface, favourable for Agrobacterium infection.

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Comprehensive computational analysis of different classes of Glutathione S-transferases in Triticum aestivum L.

B. Pandey_,P. Sharma,D.M Pandey, J. Varshney, S. Sheoran, M. Singh, R. Singh, I. Sharma and R. Chatrath

Abstract

Glutathione S-transferases (GSTs) appear to be ubiquitous in plants and have defined roles in herbicide detoxification of a wide variety of xenobiotic compounds. A newly discovered plant GST subclass has been intensively studied in numerous stress responses, including those arising from oxidative stress, pathogen attack and heavy metal toxicity. In the current study, we investigated a comprehensive role of the GSTs in Triticum aestivum L. using in silico analytical approaches. The motifs predicted in GST classes were aligned to generate the position weight matrix and information content by using Perl script. cis-acting regulatory elements present within the 5’ regulatory region of the wheat GSTs, were identified using Plant CARE and PLACE databases. Prediction of folding state of wheat GSTs indicated that only PhiGST have disordered amino acid residues. Considerable degree of homology was seen in alignment of all available GST sequences in different cereal crops by ClustalW2 and Neighbour-joining method. In addition, we discovered expression profile pattern of Tau and Phi classes using the available EST information of wheat GST genes. We described the structure model for both Phi and Zeta classes using homology model, essential for defining the active site and also for designing, improving docking of small ligands to the complex target protein. This study revealed the possible role of cis-acting regulatory elements in the expression and regulation of GST gene families in T. aestivum during cellular development or environmental stress conditions.

Page 518-531 | Full Text PDF | Supplementary data

Expression of aquaporin gene (Os PIP1-3) in salt-stressed rice (Oryzasativa L.) plants pre-treated with the neurotransmitter (dopamine)

Amal F. Abdelkader, Sahar El-khawas, Nahla Amin Safaa El-Din El-Sherif, Raifa A. Hassanein, Manal Asem Emam, Rasha El-Sherif Hassan

Abstract

Rice (Oryza sativa L.) plants are influenced by salinity, which is a global soil problem. Plants utilise different mechanisms to regulate solute homeostasis and to balance cellular water transport. The objective of the current study was to investigate the presence of any potential role of dopamine (D, a natural product synthesised in the catecholamine pathway from tyrosine) in enhancing salinity tolerance in two-month-old rice (Oryza sativa L.) plants through modulating the plasma membrane intrinsic proteins (PIPs). Using RT-PCR technique, the level of OsPIP 1-3 expression was up-regulated in response to mild salt treatment (0.15 M NaCl), but gene expression was considerably down regulated in response to dopamine, indicating the possible regulatory role of dopamine in water permeation. Relative water content in stressed rice plants was highly retrieved in response to 0.2 µgml^-1dopamine. The content of pigment and proline was regulated significantly when dopamine was administered to plants before their exposure to salt stress. Fluorescence emission spectra showed non-significant changes in 0.2 µgml^-1D plants and slight intensity increase in 0.4 µgml^-1D plants, whereas no change was detected in 0.6 µgml^-1D plants. Decreased sodium uptake in response to 0.4 µgml^-1 D is explained by the low expression of PIP1-3 and, hence related to the possible block of aquaporins. A high detection of membrane stability index in stressed-rice plants pre-treated with dopamine was associated with low membrane leakage. We concluded two facts; (1) the participation of OsPIP1-3gene in water permeation in salinity-stressed rice and, (2) the role of exogenous dopamine in OsPIP1-3 gene expression regulation, which was shown to be concentration-dependent. Subsequently, dopamine applications in a few doses are recommended as cheap and potential material which ameliorates salt stress in rice plants through its effect on plasma membrane aquaporins.

Page 532-541 | Full Text PDF

Characterization of differentially expressed genes induced by virulent and avirulent Magnaporthe grisea strains in rice

Hu Haiyan, Zhuang Jieyun, Zheng Kangle, Liu Mingjiu

Abstract

To identify differentially expressed genes of rice induced by virulent and avirulent of pathogen Magnaporthe grisea, an indica rice Zhong156 with race-specific resistance to leaf blast, was used to establish two subtractive cDNA libraries. For this purpose a Suppression Subtractive Hybridization was used after inoculation with an avirulent strain ZC15 and a virulent strain ZB1 of Magnaporthe grise. Then differential screening, sequencing, function annotation and RT-PCR were carried out and 25 cDNA clones showing differential expression (during 72 h of infection) between the avirulent and virulent strains were identified. The expression profile showed that (1) these differentially expressed genes were induced or suppressed after inoculation with the two strains, but the changing trends, degree and time of expression varied in different interactions. These differences were due to the different strains of the pathogen infection; (2) the genes induced or repressed by both strains were considered participating in the fundamental defense responses and (3) genes induced by the avirulent strain which repressed by the virulent strain (or opposite trends) might play a pivotal role in regulating the race-special resistance.

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Transcription of mitochondrial and chloroplast related genes in rice plants under anoxic stress

Camila Pegoraro, Mariana Madruga Krüger, Railson Schreinert dos Santos, Liliane Marcia Mertz, Luciano Carlos da Maia, Cesar Valmor Rombaldi, Antônio Costa de Oliveira

Abstract

Most of mitochondrial and chloroplast proteins are encoded by nuclear genes and their import into organelles of destiny involve protein complexes located in the mitochondria (TIM and TOM) and chloroplast (TIC and TOC) membranes. Any damage in mitochondrial and chloroplast membranes impair the movement of important nuclear genome encoded proteins by these complexes, leading to plants death. The aim of this study was to evaluate the gene expression of TIM/TOM and TIC/TOC complexes in rice (Oryza sativa L.) plants under anoxic as well as other abiotic stresses, seeking to associate the expression of these genes with membrane integrity and transport of proteins into mitochondria and chloroplast under stress conditions. Expression data obtained from Genevestigator for 16 genes showed that the transcript levels of most TIM/TOM and TIC/TOC genes are increased when rice is under abiotic stress. The results from qRT-PCR analyses for the genes TIM17/22-Os03g0305600, TIM17/22-Os04g0376100 and TIM17/22-Os10g0519700 presented high levels of expression 24 h after anoxic conditions, suggesting a role in the primary response to anoxic stress adaptation. After 72 h under anoxia, most genes were inhibited, suggesting that an interruption in protein transport into mitochondria and chloroplast does occur after this period of stress.

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Regulatory TGACG-motif may elicit the secondary metabolite production through inhibition of active Cyclin-dependent kinase/Cyclin complex

Ehsan Sadegh Nejad, Hossein Askari, Sattar Soltani

Abstract

Cell division and expansion have a pivotal importance for enhancement of efficient metabolite production through cell cycle regulation in transcriptional levels. The Gap1/Synthesis (G1/S) transition of the cell cycle could predict the products for synthesis as well as the distribution of the secondary metabolite between the cell and the medium with cessation of cell cycle in this transition. In this study, 1000 bp upstream sequences of G1/S transition genes as promoter regions from Arabidopsis thaliana were analyzed to introduce new generation of pharmaceutical metabolite stimuli. Identification and analysis of potential cis-regulatory motifs with using Botany Array Resource database (BAR) detected ten cis-regulatory elements that had significant differences than other cis-regulatory elements. Then, we evaluated the function of ten Cis-regulatory elements with PlantCARE database. TGACG-motif as Methyl jasmonate responsive element was found among ten putative cis-regulatory elements. The presence of this element in promoter region of cell cycle inhibitors such as Kip-related proteins (KRPs) suggests inhibiting active Cyclin-dependent kinase/Cyclin complex in G1/S checkpoint. Cell cycle arrest in G1/S checkpoint will also increase in the production of plant secondary metabolites in non-cycling cells.

Page 553-558 | Full Text PDF

Epigenetic inheritance of spine formation in sago palm (Metroxylon sagu Roettb)

Annabelle U. Novero, Ma. Brigida Mabras, Hannah Jean Esteban

Abstract

The formation of spine is a primitive evolutionary event in plants. In sago palm, studies reporting the results of RAPD and AFLP analyses showed lack of significant genetic variation among spiny and non-spiny plants. This observation led us to the hypothesis that spine formation may be an epigenetic event. The inheritance of spine formation is of high interest to plant breeders because spineless plants are preferred due to their more cost efficient handling. In this study, we analyzed the DNA methylation patterns of sago palm DNA by distinguishing methylated (5dmC) and non-methylated cytosine (dC) in HPLC analysis. We optimized HPLC conditions (nucleoside digestion, retention time of mobile phase and stop time) and identified the factors (growth environment and age of the palms) which may have affected DNA methylation levels. Our data show that a flow rate of 0.2 mL/min using C18 columns was most suitable in distinguishing between dC and 5mdC peaks. There was significant difference in methylation percentage between spiny (21.5%) and non-spiny (11.5%) palms at P = 0.05, indicating that the formation of spine was an epigenetic event. Further, there was an indication that the wet environment may have caused the epigenetic event and that spine formation was also age dependent. The influence of these factors will be confirmed using a more sensitive technique such as the methylation-sensitive amplified polymorphism (MSAP) analysis.

Page 559-566 | Full Text PDF

Comparative secretome analysis of differentially-induced proteins in rice lesion mimic mutant spotted leaf 11 (spl11)

Yiming Wang, Jingni Wu, Dong Yeol Lee, Yu Ji Kim, Yong Chul Kim, In Soo Choi, Beom-Gi Kim, Sang-Ryeol Park, Sang Gon Kim, Kyu Young Kang, Sun Tae Kim
Abstract

Rice spotted leaf 11 (spl11) mutant produces lesions caused by spontaneous cell death without environmental stresses at the three- to four-leaf stages. However, the differential regulation of secreted proteins during cell death process has not yet been explored. Proteins secreted from spotted leaves of the spl11 mutant plants and normal leaves of the wild type were extracted using a calcium chloride extraction method, followed by phenol extraction. Comparative secretome analysis using 2-DGE coupled with MALDI-TOF-MS was then applied to these secreted proteins and 28 protein spots were found to be differentially regulated in spl11 compared to wild type rice. Two of them were highly accumulated in the wild type, three were highly expressed in spl11, and 22 were only detected in spl11 mutant. MALDI-TOF MS analysi of 19 spots revealed that they were related to multiple molecular functions, such as photosynthesis (oxygen-evolving enhancer protein, ribulose bisphosphate carboxylase), plant defense (thaumatin-like protein, beta-1,3-glucanase), ROS detoxifying (Cu/Zn-superoxide dismutase, peroxidase), and glycolysis (glyceraldehyde-3-phosphate dehydrogenase). Overall, the results presented here represent the first report of a secretome analysis of spl11 mutants as a model system and demonstrate that spontaneous cell death progress was tightly associated with host defense related protein expression and secretion, which was similar to auto-activation of the host defense process.

Page 567-575 | Full Text PDF | Supplementary data

VvTMT2 encodes a putative tonoplast monosaccharide transporter expressed during grape berry (Vitis vinifera cv. Sultanine) ripening
Birsen Çakir, Refika RA Giachino

Abstract

Sugars produced by photosynthesis are transported to the berries as sucrose via the phloem and imported into the fleshy cells through the plasma membrane and they accumulate in the vacuoles as glucose and fructose during ripening. Therefore, this process requires sugar transporter activity of plasma membrane as well as tonoplast. To examine sugar transport processes in Vitis vinifera, a full-length cDNA clone encoding a monosaccharide transporter designated as VvTMT2 (Vitis vinifera Tonoplast Monosaccharide Transporter 2) was isolated by RT-PCR from the ripening stage of V. vinifera ‘Sultanine’ berries (GeneBank accession number: JX233818). VvTMT2 cDNA contains a 2220-bp ORF encoding a predicted protein of 739 amino acids with a molecular mass of 79.2 kDa. According to the hydropathy pattern, VvTMT2 possesses 11 transmembrane-spanning domains with an extended cytoplasmic loop between transmembrane helices 6 and 7. This loop spans 371 amino acids, which is approximately 4-5 times longer than the corresponding structures in all other known monosaccharide transporters from prokaryotes and eukaryotes. VvTMT2 exhibited high similarities with other tonoplast monosaccharide transporters from different plant species. A search for cis-regulatory elements in the VvTMT2 promoter region showed that this gene is probably regulated by phytohormones, sugars and abiotic stresses. A semiquantitative RT-PCR was conducted to determine the VvTMT2 expression pattern using mRNAs isolated from various organs such as berries, young and mature leaves, tendrils, roots and pollens. VvTMT2 was highly expressed during the initiation of ripening and over-ripening of berries. However, transcript levels of VvTMT2 were undetectable in some vegetative organs, suggesting that expression of VvTMT2 gene is tissue and organ specific. Thus, VvTMT2 gene may be involved in hexose transport from the cytoplasm to the vacuoles during berry ripening and over-ripening.

Page 576-583 | Full Text PDF

Efficient isolation of high quality RNA from tropical palms for RNA-seq analysis

Yong Xiao, Yaodong Yang, Hongxing Cao, Haikuo Fan, Zilong Ma, Xintao Lei, Annaliese S. Mason, Zhihui Xia, Xi Huang

Abstract

Currently, RNA-seq as a high throughput technology is being widely applied to various species to elucidate the complexity of the transcriptome and to discover large number of novel genes. However, the technology has had poor success in elucidating the transcriptome of tropical palms, as it is difficult to isolate high quality RNA from tropical palm tissues due to their high polysaccharide and polyphenol content. Here, we developed an RNA-isolation protocol for tropical palms, the MRIP method (Methods for RNA Isolation from Palms). The integrity of the RNA molecules extracted using this protocol was determined to be of high quality by means of gel electrophoresis and Agilent 2100 Bioanalyzer microfluidic electrophoresis chip examination with a RIN (RNA Integrity Number) value of more than 9, indicating that the mRNAs were of good integrity. Subsequently the isolated RNA was used for transcription analysis without further purification. With Illumina sequencing, we obtained 54.9 million short reads and then conducted de novo assembly to gain 57,304 unigenes with an average length of 752 base pairs. Moreover, the RNA isolated with this protocol was also successfully used for real-time RT-PCR. These results suggested that the RNA isolated was suitable for Illumina RNA sequencing and quantitative real-time RT-PCR. Furthermore, this method was also successful in isolating total RNA from the leaves of various Palmaceae species.

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Rhizobiales-like protein phosphatases (Rhilphs): A role in plant defence responses?

Mikhail A. Kutuzov, Alexandra V. Andreeva

Abstract

We previously identified a novel group of protein phosphatases (“Rhilphs”), shared by plants and some a-Proteobacteria, including purple photosynthetic bacteria. In this work, we (1) identified genes that show expression correlation with Rhilphs; (2) examined the physiological stimuli affecting Rhilph expression, and (3) examined characterised Arabidopsis thaliana (L.) Heynh. mutants with altered Rhilph expression. We found that Rhilph expression correlated best with the genes associated with defence responses, carbohydrate metabolism, membrane trafficking and cell wall modification. Using available expression profiling data, we found that Rhilph-1 (but not Rhilph-2) is induced by flg22 treatment (but not by its inactive analogue from Agrobacterium tumefaciens). Both isoforms are induced in response to Pseudomonas syringae infection. This induction is impaired in A. thaliana mutants deficient in salicylic acid production. Examination of available data for characterised A. thaliana mutants showed that Rhilph-1 expression is elevated in plants lacking MPK4 or both MKK1 and MKK2, components of MAP kinase signalling that regulates innate immune responses. Taken together, these data suggest that Rhilph functions are likely associated with defence responses / innate immunity and cell wall-related processes, possibly cell wall remodelling during pathogen attack.

Page 590-596 | Full Text PDF | Supplementary data 1 | Supplementary data 2

Proline related genes expression and physiological changes in indica rice response to water-deficit stress

Suravoot Yooyongwech, Suriyan Cha-um, Kanyaratt Supaibulwatana
Abstract

Water deficit stress is a major abiotic stress causing to reduce crop productivity especially rice. Rice has been reported as drought susceptible, which decline plant growth and development in both seedling and reproductive developmental stages. Induced mutant in rice crop against water deficit stress is a fruitful topic. Proline osmolyte is a candidate metabolite to maintain the osmotic pressure in cellular level of water deficit stressed plants. The key enzymes i.e. P5CS, P5CR and ProDH in proline biosynthesis and degradation are well established. In present study, P5CS, P5CR and ProDH and the final product, proline in rice genotypes at booting stage was investigated when subjected to water-deficit and recovery processes. The expression levels of the P5CS gene in PT1 rice (drought susceptible) and the EE12 mutant line were up-regulated when rice genotypes were exposed to severe water-deficit (7% SWC), whereas P5CR genes in NSG19, IR20 and PT1 were up-regulated by the recovery process to a significant degree (p£0.01). A positive relationship between P5CS expression level and proline content in rice genotypes subjected to water-deficit stress was evidently stated (R² = 0.60). In addition, the expression level of ProDH in rice genotypes was exhibited in the recovery process. Moreover, physiological changes, including maximum quantum yield of PSII (F_v/F_m), water use efficiency (WUE) and net photosynthetic rate (P_n) were significantly reduced when plants were subjected to severe water-deficit stress (7% SWC), leading to retard plant height.

Page 597-603 | Full Text PDF

Comparative proteomic analysis of the effects of nitric oxide on alleviating Cd-induced toxicity in rice (Oryza sativa L.)

Xiufeng Zhao, Chengqiang Ding, Lin Chen, Shaohua Wang, Qiangsheng Wang and Yanfeng Ding
Abstract

Nitric oxide (NO) is a signaling molecule that mediates many physiological processes. To help understand the effect of NO on cadmium (Cd)-induced toxicity in rice (Oryza sativa L.), a hydroponic experiment was conducted to investigate the effects of exogenous sodium nitroprusside (SNP), an NO donor, on the physiology and proteomics of rice seedlings. Rice seedlings were treated with 0.1 mM Cd and the same Cd concentration with a range of SNP concentrations (0.005, 0.05, 0.1 and 0.2 mM) for 8 days. A concentration of 0.005 mM SNP significantly ameliorated the Cd-induced decrease in dry weight and length of both the shoots and roots, whereas the hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content in the rice seedlings decreased with the addition of NO. The differential expression of proteins in the rice leaves and roots was analyzed using a two-dimensional electrophoresis and MALDI-TOF/MS approach. Compared with the plants under Cd stress, 92 protein spots from the rice leaves and roots were differentially expressed after NO application, and 41 of those 92 proteins were successfully identified. A total of 16 proteins were expressed in the rice leaves, and 25 proteins were expressed in the rice roots. As expected, the identified proteins were involved in photosynthesis, carbohydrate metabolism, nitrogen metabolism, oxidative phosphorylation, oxidative stress responses, signal transductions and cell division.

Page 604-614 | Full Text PDF | Supplementary data