Identification of cytochrome P450 heme motif in plants proteome
Akansha Saxena1, Priyanka Singh2, Dharmendra K. Yadav3, Pooja Sharma3, Sarfaraz Alam3, Feroz Khan3*, Sanjog T. Thul4, Rakesh K. Shukla4, Vikrant Gupta4, Neelam S. Sangwan3
1Biomedical Informatics Center, Indian Council of Medical Research, New Delhi, India
2Bioinformatics Infrastructure facility, Department of Biochemistry, University of Lucknow, Lucknow, India
3Metabolic and Structural Biology Department
4Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India
Plant cytochrome (CYP) P450 monooxygenases are heme-containing enzymes that take part in the production of a wide variety of secondary products, a number of which are inhibitory to the survival of insects, pathogens and vertebrate herbivores. Main class of characterized natural plant compounds terpenoids are mostly substrates for plant CYPs. Metabolic engineering of plants using genetic modification of the P450 enzymes has remarkable implications for molecular farming of natural plant chemicals used as pharmaceuticals, disease and insect deterrents. However, molecular information regarding the metabolism of herbicides by plant CYPs is limited and most detoxifying CYPs are expressed at low levels in plants. Therefore, identification of plant CYP enzymes may help in characterization and increase in expression levels followed by design of herbicide-resistant plants. Therefore, in this study we have developed a method to explore the available plant’s proteome for the identification of CYPs on the basis of conserved heme motif. For this we have used position-specific scoring matrix method and correlated the availability of CYPs on the basis of heme motif distribution. We have identified the proteome-wide CYPs on the basis of occurrence and positional arrangements of heme motif in the publicly available nine plant’s proteome viz., Arabidopsis thaliana, Vitis vinifera, Zea mays, Triticum aestivum, Sorghum bicolor, Glycine max, Brassica napus, Brassica oleracea, and Solanum lycopersicum. Results showed that V. vinifera and A. thaliana have high conservation of heme motif in CYP enzymes as well as hypothetical sequences i.e., 88.46%, therefore characterizations of CYP enzymes from these prioritized plants may help to increase the CYPs expression levels followed by design of resistant plants. Among the heme motif sequence, three most variable amino acids i.e., alanine, isoleucine and proline can be targeted to enhance the possibility of functional role in the biosynthesis of secondary metabolites and consequently may increase the production of phytochemicals as a precursor for synthesis of active molecules for drug discovery and phytoremediation.
Pages 1-12 | Full Text PDF | Supplementary data
The effects of glutamate decarboxylase (GAD) RNAi knockout in tissue cultured transgenic tomato (Solanum lycopersicum)
Bee Lynn Chew1*, Graham B. Seymour2
1School of Biological Sciences, Universiti Sains Malaysia, Minden, 11800, Georgetown, Penang, Malaysia
2University of Notttingham, Plant and Crop Science Division, Sutton Bonington, Loughborough, LE12 5RD, United Kingdom
The amino acid glutamate has commonly been associated with the umami or brothy taste which is classified as the fifth sensation other than sweet, sour, salty and bitter. The production of glutamate in plants is linked to the tricarboxylic acid cycle (TCA) and amino acid metabolism. In this study, the effects of down-regulating the tomato glutamate decarboxylase gene (GAD) using an RNAi construct under the control of the 35S promoter are reported. The aim of the experiment was to enhance the levels of glutamate in transgenic tomato plants. The GAD gene sequence was isolated from Micro Tom fruit. Tomato cotyledons were transformed with the GAD gene fragment using Agrobacterium tumefaciens and transformants were observed for the regeneration of transformed plantlets. PCR was performed to confirm the presence of the inserted gene. The infected cotyledons failed to regenerate transformed plantlets using the GAD RNAi transgene. A possible explanation is that the construct resulted in altered levels of gama-aminobutyric acid (GABA) and other amino acids known to be essential for plant survival. This work provides further support for the central role of this GAD gene in tomato.
Pages 13-17 | Full Text PDF
The Date Palm (Phoenix dactylifera L.) leaf proteome: identification of a gender biomarker to screen male parents
Sonia Dakhlaoui-Dkhil S.1, Laurent Coquet L.2, Pascal Cosette2, Salem Elkahoui3, Philippe Chan Tchi Song4, David Vaudry4, Amel Salhi Hannachi1, Neila Trifi1,
Férid Limam3 and Thierry Jouenne2*
1Laboratoire de Génétique Moléculaire, immunologie et biotechnologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092 El Manar Tunis, Tunisie
2Laboratoire Polymères, Biopolymères, Surfaces, UMR CNRS 6270, Plate-Forme protéomique PISSARO, Université de Rouen, Institute for Research and Innovation in Biomedicine, 76821 Mont-Saint-Aignan, cedex, France
3Laboratoire des Substances Biologiquement Actives, Centre de Biotechnologie de Borj Cedria, Hammam Lif. , Tunisie
4Laboratoire DC2N, Laboratoire International Associé Samuel de Champlain, U INSERM 982, Plate-Forme protéomique PISSARO, Université de Rouen, 76821 Mont-Saint-Aignan, cedex, France
To establish a proteomic reference map of date palm leaves (Deglet Nour cultivar), we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, respectively. In total, 284 spots were excised from gel and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among them, 158 were successfully identified (i.e, a success rate of 55.6%) conducting to the identification of 126 unique proteins. These proteins were then clustered according to their functional annotations. Identified proteins were involved in metabolism, electron transport, photosynthesis, protein synthesis, cell structure or defence. However, 29.4 % of the identifications gave unknown function. We then compared the proteome map of female and male trees. Only one discriminated spot was found to be specific of the gender. We identified the corresponding protein as an ABC superfamily ATP binding cassette transporter, ABC protein, a protein whose an ortholog in Arabidopsis thaliana was already reported as required for male fertility and pollen formation. The relevance of this protein as gender biomarker was then confirmed in four other cultivars, i.e., Aligue, Khouet Aligue, Kentichi and Kenta. Such biomarker should be helpful in rapidly distinguishing date palm gender of immature trees.
Pages 18-23 | Full Text PDF | Supplementary data
Proteomic analysis of early responsive resistance proteins of wheat (Triticum aestivum) to yellow rust (Puccinia striiformis f. sp. tritici) using ProteomeLab PF2D
Dilara Maytalman1, Zafer Mert2, A. Tarik Baykal3, Cihan Inan4, Aslihan Günel5 and Semra Hasançebi3*
1Istanbul Universty, Faculty of Science, Department of Biology, 34118, Vezneciler, Istanbul, Turkey
2Field Crop Research Institute, P.O Box: 226, Lodumlu, Ankara, Turkey
3The Scientific and Technological Research Council Of Turkey (TUBITAK), Marmara Research Center (MRC), Genetic Engineering and Biotechnology Institute (GEBI), P.O Box: 21, 41470, Gebze, Kocaeli, Turkey
4Istanbul Universty, Faculty of Science, Department of Molecular Biology and Genetic, 34118, Vezneciler, Istanbul, Turkey
5Ahi Evran Universty, Faculty of Science and Arts, Department of Chemistry, Kirsehir, Turkey
Wheat (Triticum aestivum L.) yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. To clarify the molecular details and components of the resistance response in wheat offers further possibilities to combat yellow rust. In this study, differentially regulated early response proteins in wheat leaves infected by Pst isolates were investigated by proteomic approaches. Total proteins extracts from leaves harvested at 24 hour post inoculation (hpi) were separated by two dimensional liquid chromatography system, ProteomeLab PF2D. Following PF2D analysis, six hundred and thirty-seven protein peaks were compared one by one between protein patterns obtained from pathogen- and mock-inoculated leaf tissue. Among those differentially expressed 33 proteins were identified in Pst-infected plants as compared with mock-inoculated controls by nanoLC-ESI-MS/MS. Six proteins were exhibited homology to fungal proteins. Two fungal proteins, including E3 ubiquitin protein ligase and Ubiquitin-like protein, are important members of ubiquitin-proteasome system which the importance of the its proteolytic function in regulating the virulence of pathogenic fungi has just been realized recently. Other identified 27 proteins were host proteins in response to Pst and classified in five groups based on their roles in diverse biological processes. The results indicated that identified defence related proteins such as pathogene related protein 1 and 4 (PR1, PR4), Glutathione S transferase (GST) are major component for systemic acquired resistance (SAR) which is one of the strong disease resistance form in plants and appears within several days following the initial pathogen attack.
Pages 24-35 | Full Text PDF
Identification of a co-segregative protein associated with the tillering trait in rice (Oryza sativa L.)
Fangyu Chen1, Liangrong Jiang1, Jingsheng Zheng1, Rongyu Huang1,2, Houcong Wang1, Zonglie Hong2*, Yumin Huang1*
1School of Life Sciences, Xiamen University, Xiamen 361005, China
2Department of Plant, Soil, and Entomological Sciences, and Program of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Idaho 83844, USA
Tillering is an important trait for grain production in rice. To understand the mechanism underlying tillering regulation in rice, a combined proteomic and genetic approach was taken to analyze potential protein markers associated with the high-tillering dwarf phenotype. Total proteins were extracted from basal internodes of rice plants and subjected to 2-dimensional gel electrophoresis. Proteomes were compared between control rice cultivars and tillering mutant lines that are known to have a reduced number of culms or contain the d10 allele. A total of 33 polymorphic protein markers were identified between the wild-type cultivar GLA4 and the mutant line JHCA with dwarf and high tillering phenotype. A protein spot was found to behave as a qualitative trait, cosegregating with the normal-tillering phenotype in the F2 population. This protein was present in the wild-type plants, but was undetectable in the mutant line JHCA and the high-tillering dwarf F2 plants. Mass spectrometry analysis identified the protein as putative carboxyvinyl-carboxyphosphonate phosphorylmutase (CPPM; EC 188.8.131.52), which may catalyze the formation of a unique C-P bond from phosphoenolpyruvate and may participate in the biosynthesis of unidentified compounds with an inhibitory effect on tillering in rice. Real-time PCR analysis revealed that the mRNA level of CPPM was down-regulated in the high-tillering d10 dwarf plants, suggesting that the expression of the CPPM gene requires the function of D10. We propose that CPPM may play a role that is related to the tillering regulation in rice.
Pages 36-45| Full Text PDF | Supplementary data
In search for new players of the oxidative stress network by phenotyping an Arabidopsis T-DNA mutant collection on reactive oxygen species-eliciting chemicals
Veselin Petrov1,2,*, Jos Schippers3,4, Maria Benina1,2, Ivan Minkov1,2, Bernd Mueller-Roeber3,4, Tsanko Gechev1,2
1Department of Plant Physiology and Molecular Biology, University of Plovdiv, 24 Tsar Assen str., Plovdiv 4000, Bulgaria
2Institute of Molecular Biology and Biotechnologies, 105 Ruski Blvd., Plovdiv 4000, Bulgaria
3Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam, Germany
4Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany
The ability of some chemical compounds to cause oxidative stress offers a fast and convenient way to study the responses of plants to reactive oxygen species (ROS). In order to unveil potential novel genetic players of the ROS-regulatory network, a population of ~2,000 randomly selected Arabidopsis thaliana T-DNA insertion mutants was screened for ROS sensitivity/resistance by growing seedlings on agar medium supplemented with stress-inducing concentrations of the superoxide-eliciting herbicide methyl viologen or the catalase inhibitor 3-amino-triazole. A semi-robotic setup was used to capture and analyze images of the chemically treated seedlings which helped interpret the screening results by providing quantitative information on seedling area and healthy-to-chlorotic tissue ratios for data verification. A ROS-related phenotype was confirmed for three of the initially selected 33 mutant candidates, which carry T-DNA insertions in genes encoding a Ring/Ubox superfamily protein, ABI5 binding protein 1 (AFP1), previously reported to be involved in ABA signaling, and a protein of unknown function, respectively. In addition, we identified six mutants, most of which have not been described yet, that are related to growth or chloroplast development and show defects in a ROS-independent manner. Thus, semi-automated image capturing and phenotyping applied on publically available T-DNA insertion collections adds a simple means for discovering novel mutants in complex physiological processes and identifying the genes involved.
Pages 46-54 | Full Text PDF | Supplementary data
Molecular cloning and in silico analysis of novel oleate desaturase gene homologues from Brassica juncea through sub-genomic library approach
Suresha G.S1, 2* and Santha I.M1
1Division of Biochemistry, Indian Agricultural Research Institute (IARI), New Delhi, 110012, India
2Present address: Scientist (Biochemistry), Sugarcane Breeding Institute (ICAR), Coimbatore, Tamil Nadu-641007, India
The oleate desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Using the sequence information of Bjfad2 (EF639848) gene that was isolated and characterized earlier from Brassica juncea, three additional novel fad2 gene homologues designated as Bjfad2a (FJ696650), Bjfad2b (FJ696651) and Bjfad2c (FJ696652) were isolated from sub-genomic library of B. juncea. Sequence analysis of these genes revealed the sequence size of 2526 bp (Bjfad2a), 2326 bp (Bjfad2b) & 2325 bp (Bjfad2c), an open reading frame of 1155 bp that encodes a single polypeptide of 384 amino acids. Sequence comparison of isolated Bjfad2 gene sequences shared 99% (Bjfad2a), 96% (Bjfad2b) and 95% (Bjfad2c) sequence similarity to reported B. juncea fad2 gene (EF639848). The deduced amino acids of the these gene homologues displayed the typical three histidine boxes, a characteristic of all membrane bound desaturases along with five membrane spanning domains that are different from known plant desaturases. The signature amino acid sequence ‘YNNKL’ was found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the genomic structures alignment grouped them within the cluster of fad2 gene(s) of Brassicacea and are distinct from fad2 gene sequences from sesame (42%), soybean (38%) and sunflower (19%). Comparison of the Bjfad2 genomic structures with other plant fad2 genes revealed the presence of single large intron in their 5’ UTR region which is evolutionarily conserved. However, the length of the intron varies across the plant species. Variation in the restriction sites of all three Bjfad2 genes and its further confirmation through restriction analysis showed that the isolated genes are different homologues but code for the same enzyme, oleate desaturase. These observations confirm that atleast four copies of fad2 gene exist which is consistent with the tetraploid nature of B. juncea genome.
Pages 55-64 | Full Text PDF
Expression analysis of kenaf cinnamate 4-hydroxylase (C4H) ortholog during developmental and stress responses
Jonggeun Kim1, Bosung Choi1, Savithiry Natarajan2, Hanhong Bae1,*
1School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of Korea
2US Department of Agriculture, Agricultural Research Service, Soybean Genomics and Improvement Laboratory, 10300, Baltimore Avenue, Beltsville, MD 20705, USA
This study was conducted to clone and analyze the expression pattern of a C4H gene encoding cinnamate 4-hydroxylase from kenaf (Hibiscus cannabinus L.). The full-length of the C4H ortholog was cloned using degenerate primers and the RACE (rapid amplification of cDNA ends) method. The full-length C4H ortholog contained a 1,518-bp open reading frame (ORF) encoding 505 amino acids. The deduced amino acid sequence showed that kenaf C4H had the highest similarity (95%) with that of Gossypium arboretum. We examined the expression patterns of the C4H ortholog in diverse tissues and at different developmental stages, as well as in response to abiotic stress conditions such as wounding, NaCl, cold, H2O2, ABA (abscisic acid) and SA (salicylic acid). Three-week-old stem tissues were used to examine the effects of abiotic stresses on C4H ortholog expression. While the highest transcript level of C4H ortholog was observed at an early stage in both stems and leaves, the transcripts were most abundant in the late stage flowers. In cases of wounding and SA, early induction of the C4H ortholog was observed. Conversely, H2O2 led to intermediate induction, while cold and ABA led to late induction. NaCl treatment showed different expression patterns such as complex biphasic expression. In summary, the C4H ortholog was expressed in all tissues and organs, as well as in response to various treatments.
Pages 65-72 | Full Text PDF
Metabolic profiling of millet (Panicum miliaceum) using gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) for quality assessment
Jae Kwang Kim,1 Soo-Yun Park,1 Yunsoo Yeo,1 Hyun Suk Cho,1 Yeon Bok Kim,2 Hanhong Bae,3 Cheol Ho Park,4 Jai-Heon Lee 5,*, Sang Un Park2,*
1National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Korea
2Department of Crop Science, Chungnam National University, 99 Daehak-Ro, Yuseong-Gu, Daejeon 305-764, Korea
3School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Korea
4Department of Bio-Health Technology, College of Biomedical Science, Kangwon National University, 192-1 Hyoja 2-Dong, Chuncheon 200-701, Korea
5Department of Genetic Engineering, Dong-A University, Busan 604-714, Korea
Gas-chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS) was used to determine the diversity among primary metabolites and phenolic acids in three varieties of millet (Panicum miliaceum L.). Three cultivars of millet seeds were germinated in a greenhouse, and the seedlings were transferred to the field and allowed them to grow for a period of 4 months. A total of 48 metabolites were identified from millet, including 43 primary metabolites and five phenolic acids. The metabolite profiles were subjected to principal component analysis (PCA) and partial least-squares discriminate analysis (PLS-DA) to evaluate the differences among varieties. PCA and PLS-DA fully distinguished the three varieties tested. Joongjuk millet was separated from the other varieties based on the high levels of metabolites, and appears to be a good candidate for future breeding programs because of its high phenolic acids levels. This GC-TOFMS-based metabolic profiling approach is a viable alternative method for evaluating food quality.
Pages 73-78 | Full Text PDF
Water relation and aquaporin genes (PIP1;2 and PIP2;1) expression at the reproductive stage of rice (Oryza sativa L. spp. indica) mutant subjected to water deficit stress
Suravoot Yooyongwech1, Suriyan Cha-um2*, Kanyaratt Supaibulwatana3
1Division of Agricultural Science, Mahidol University, Kanchanaburi campus, Kanchanaburi, Thailand
2National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Paholyothin Road, Klong Nuang, Klong Luang, Pathumthani, Thailand
3Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand
Rice (Oryza sativa L. spp. indica) is a carbohydrate crop that grows well in the aquatic habitat. In the drought prone areas, adaptive growth ability and plant defence mechanisms coping with less water are challenging tasks for rice breeder to maintain rice productivity. Rice plants, including Thai jasmine rice (cv. KDML105; wild type), M401 mutant line derived from KDML105 (upon treatment with EMS and gamma-irradiation), and Pathumthani 1 (PT1, a negative check -drought susceptible), were grown in the pot culture until booting stage (inflorescence formation) and then subjected to water withholding for 14 days (water deficit stress) represented by 5.28% soil water content (SWC) and well watering (control; 31.9% SWC). Water use efficiency (WUE), net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (gs) and expression of PIP1;2 and PIP2;1 were examined in flag leaf tissues of rice grown under control and water deficit stress. WUE in MT401 mutant plant subjected to water deficit stress was increased, whereas Pn in both MT401 and KDML105 was maintained. Transcriptional levels of OsPIP1;2 and OsPIP2;1 in the MT401 grown under water deficit stress were up-regulated by 2.0-2.5 folds higher than those in KDML105 and PT1 genotype. The expression of OsPIP2;1 in MT401 mutant plant was maintained when plants were exposed to water deficit condition, resulting in stabilization of WUE at the cellular levels. In addition, panicle length and number of spikelets per panicle in MT401 mutant were retained well under water deficit, suggesting MT401 as water deficit tolerant type.
Pages 79-85 | Full Text PDF
Molecular cloning, characterization and transcriptional variability study of resistance gene candidates from wild Curcuma spp. for resistance against Pythium aphanidermatum
Basudeba Kar, Sanghamitra Nayak, Raj Kumar Joshi*
Centre of Biotechnology, Siksha O Anusandhan University, Bhubaneswar, India-751003
The genetic base of preferred turmeric (Curcuma longa L.) genotypes has eroded due to their continuous domestication through exclusive vegetative routes thus making them highly susceptible to various pests and pathogens. Molecular cloning of resistance related sequences from wild genotypes can result in efficient turmeric improvement by evolving more effective resistance specificities compared to cultigens. In this study, a PCR mediated approach has been made using degenerate primers designed on conserved region (P-loop and GLPL) of the NBS domain from R-genes that provides the source for cloning analogous sequences called resistance gene candidates (RGCs) from three wild turmeric- Curcuma aromatica, Curcuma angustifolia and Curcuma zedoaria. Twenty-one wild turmeric RGCs were isolated and grouped into four phenetic classes. A strong amino acid identity ranging from 41 to 53% together with presence of internal conserved motifs provided evidence that the isolated RGCs belong to the non-toll interleukin receptor (non-TIR) NBS-LRR R-gene sub-family. Southern hybridization showed a high copy representation of turmeric RGCs. Expression variability of wild turmeric RGCs was analyzed through reverse transcription PCR in root tissues of the three wild turmeric plants resistant or susceptible to Pythium aphanidermatum. Cap12 and Can12 showed a constitutive expression in both resistant and susceptible plants of Curcuma aromatica and Curcuma angustifolia respectively while Czp11 expression was realized only in Pythium aphanidermatum resistant lines of Curcuma zedoaria as well as Curcuma longa L. This result can pave way towards the identification and characterization of a potential Pythium aphanidermatum resistance gene in turmeric.
Pages 86-94 | Full Text PDF | Supplementary data