Integrative meta-analysis of protein interaction data identified multiple GID/MRCTLH protein complexes in plants
Mercè Miquel, Carlos Vicient*
Centre for Research in Agricultural Genomics (Consejo Superior de Investigaciones Científicas-Institut de Recerca i Tecnologia Agroalimentàries-Universitat Autònoma de Barcelona-Universitat de Barcelona), Campus UAB, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain
Abstract
GID/MRCTLH is a protein complex involved in the regulation of several cellular processes through the polyubiquitination and proteosome degradation. It has been described in yeast and mammals. Genes coding for homologous proteins are also present in plant genomes but have been little studied. BLAST analyses revealed that genes coding for members of the GID/MRCTLH complex are found in multiple copies in plants, compared to mammals and yeast. The potential structure of the Arabidopsis GID/MRCTLH complex was estimated based on the Arabidopsis protein interaction database Interactome 2.0. According to these data, Arabidopsis may contain two GID/MRCTLH complexes instead of the one described in yeast and mammals. The structure of the two Arabidopsis complexes seem to be similar to the yeast GID complex, and seem to interact with several other proteins out of the complex. These data suggest that, similarly to yeast and mammals, the plant GID/MRCTLH complexes are involved in the regulation of several cellular processes through proteosome protein degradation.
Pages 169-175 |Read More| Supplementary Data| doi: 10.21475/poj.10.04.17.pne212…………………………………………………………………………………………………………………………………………………..
A simple protein extraction method for proteomic analysis of mahogany (Swietenia macrophylla) embryos
Noraliza Alias*, Wan Mohd Aizat, Nor Datiakma Mat Amin, Norwati Muhammad, Normah Mohd Noor
Forestry Biotechnology Division, Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor, Malaysia
Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
Natural Product Division, Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor, Malaysia
Abstract
In any proteomic studies, protein extraction and sample preparation are the most crucial steps for obtaining optimal results. This is to ensure extracted proteins are not only high in yield but also clean from contaminants that could affect downstream proteomic applications such as two dimensional gel electrophoresis (2-DE) and mass spectrometry. Tissues from plants and trees such as Swietenia macrophylla are often rich in non-protein contaminating substances, which could interfere in the proteomic applications. S. macrophylla or also known as the mahogany is one of the most valuable tree species in the world. Studies on proteins for this tree as well as its seeds are very limited. We have extracted proteins from S. macrophylla seeds (specifically embryo tissues) using three different methods, each having different lysis buffer recipes. Furthermore, another set of samples were precipitated using trichloroacetic acid/acetone prior to the three extraction methods to further purify the protein samples. The results from 2-DE analysis showed approximately 240 protein spots were detected from the successful protocol using a lysis buffer of 9 M urea, 4% CHAPS, 0.5% triton X-100 and 100 mM DTT without TCA/acetone precipitation. This study highlights the aspects of sample preparation for S. macrophylla embryos, focusing on the total protein extraction and resolution in SDS-PAGE as well as 2-DE. Furthermore, this is the very first report of the proteome 2DE profile from S. macrophylla embryo.
Pages 176-182 |Read More| doi: 10.21475/poj.10.04.17.pne323…………………………………………………………………………………………………………………………………………………..
Transcription Factor-microRNAs regulatory network in response to multiple stresses in Arabidopsis thaliana
Fereshteh Izadi*, Roohollah Nikfekr, Jahad Soorni
Department of Plant Breeding and Genetics, Sari Agricultural sciences and Natural Resources University (SANRU), Mazandaran, IRAN
Department of Plant Breeding and Genetics, Payame Noor University, PO BOX 19395-3697 Tehran, IRAN
Abstract
Transcription factors (TFs) by interacting with the DNA binding sites of stress responsive gene promoters, control a cascade of reactions in plant cells. On the other hand MicroRNAs (miRNAs) act on dozen of genes involving in response to a particular stress condition via many of biological process. In addition less than 6% of the responding transcripts showed antagonistic responses to stress combinations that require delineation of regulatory interactions underlying multiple concurrent stresses instead of a single biotic or abiotic stress. Therefore inferring transcriptional networks including both of TFs and miRNAs as two classes of regulators likely will reveal more aspects of complex interlayers especially in a combination of stresses. Utilizing 207 microarrays, we performed data mining approaches fallowing by network construction. As a result 17 TFs and 12 miRNAs were identified as the most potential regulators in response to a combination of biotic and abiotic stresses. Seven stress responsive TFs targeted 1697 transcripts in ARACNE based constructed network which classified in a cohort of common metabolisms under investigated treatments. The generated genes network was validated via comparison with a reference network and further motif enrichment. The study identified TFs like ERF/AP2 family also miRNAs including miR5658 and miR172 their crosstalk may undertake transcriptome changes in response to stresses.
Pages 183-189 |Read More| Supplementary Data| doi: 10.21475/poj.10.04.17.pne516…………………………………………………………………………………………………………………………………………………..
Identification of microRNA elements from genomic data of European hazelnut (Corylus avellana L.) and its close relatives
Bihter Avsar*, Danial Esmaeili Aliabadi
Sabanci University, Faculty of Engineering and Natural Sciences, Istanbul, TURKEY
Abstract
Plant microRNAs (miRNAs) are small and non-coding endogenous RNAs which have numerous regulatory roles in cells. These critical players regulate pathways either by inducing translational repression or messenger RNA (mRNA) decay. Newly developed bioinformatics tools and computational methods have been increased to identify miRNAs with their targets inside the genome. In this study, we predicted and identified 57 putative miRNAs through Corylus avellana (C. avellana) genomic data in silico. We also predicted some other putative miRNAs from Arabidopsis thaliana (A. thaliana), Ricinus communis (R. communis), Populus trichocarpa (P. trichocarpa) and Vitis vinifera (V. vinifera) to compare with the C. avellana organism since previous studies have indicated high similarities between these genomes and proteome atlases. The miRBase 21 was used as a reference dataset and the putative miRNAs were identified for the genome of each organism. We used homology conserved method to identify putative miRNAs. Based on our findings, C. avellana miRNA content was found to be highly similar to V. vinifera, R. communis and P. trichocarpa. Also, we found the targets of these hazelnut putative miRNAs and their possible functions inside the cell. One of our major discoveries is that miR171 families are highly represented (the copy number of miRNA) in the hazelnut genome to provide clues for microRNA domestication. The miR396, miR482, and miR2118 families were found as in silico expressed putative miRNAs by using computational methods. All these findings may help us better understanding the miRNA repertoire of the hazelnut organism and provide valuable insight about the regulatory roles of predicted putative miRNAs which are shared with other organisms (A. thaliana, R. communis, P. trichocarpa, V. vinifera) for further studies.
Pages 190-196 |Read More| doi: 10.21475/poj.10.04.17.pne707…………………………………………………………………………………………………………………………………………………..
Proteomic analysis of long term salt responsive proteins in the halophyte Suaeda maritima
Shrikanth Saraswathi Krishnamurthi*, Suja George, Sankaramasubramanian Meenakshisundram, Ajay Parida
Department of Plant Molecular Biology, M.S. Swaminathan Research Foundation, Taramani Institutional Area, Chennai 600 113, India
Institute of Life Science, Bhubaneswar 751023, India
Abstract
A proteomic approach was carried out to identify proteins responsive to long term salt treatment in the halophyte Suaeda maritima. A 3-month-old S. maritima seedlings were hydroponically exposed to Modified Hoagland’s treated with salt solution of 200mM were grown for a period of 14 days in a growth chamber maintained at 24 ± 3°C, 70-75% relative humidity with 14 h light (200 μmol m-2 s-1)/10 h dark cycle. The untreated set maintained in Modified Hoagland’s solution was considered as control with similar conditions. Proteins extracted from the leaves of S. maritima control and salt treated seedlings were separated by Two-Dimensional gel electrophoresis (2DE). Using PDquest software analysis, we observed ~ 50 protein spots were reproducibly detected on gels, out of which 18 differentially expressed protein spots showed at least two-fold differences on 2DE maps some of them were up-regulated and few others were down-regulated in treated compared with the control. From that, we identified 6 up-regulated protein spots shows the maximum level of fold differences and these protein spots were performed trypsin digestion and Matrix Assisted Laser Desorption Ionization -Time of Flight (MALDI-TOF) analysis. The results showed that S. maritima could withstand up to 200mM NaCl for long term period of 14 days by up regulating proteins that are mainly involved in protein transport, vesicle trafficking, heme/iron binding, protein folding and assembly, chromosome segregation, cell maintenance. Our study has identified salt responsive proteins such as RAB2B, CYP71A8, SCC3 that are not previously identified by expressed sequence tag analyses or transcriptome analyses in this species. This is the first report of proteome analysis and provides insights into the molecular mechanisms of long term salt tolerance in S. maritima.
Pages 197-204 |Read More| Supplementary Data| doi: 10.21475/poj.10.04.17.pne734…………………………………………………………………………………………………………………………………………………..
Research Note
Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses
Edgardo H. Avico, Raúl M. Acevedo, Pablo I. Calzadilla, Oscar A. Ruiz, Pedro A. Sansberro*
Laboratorio de Biotecnología Aplicada y Genómica Funcional. Instituto de Botánica del Nordeste (CONICET), Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Sargento Cabral 2131, CC: 209, W3402BKG, Corrientes, Argentina
Unidad de Biotecnología 1. Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (UNSAM-CONICET). Av. Intendente Marino Km 8,200 CC: 164. B7130IWA, Chascomús, Buenos Aires, Argentina
Abstract
Extraction of high quality RNA is a prerequisite for downstream application in functional genomics analyses. However, the extraction and purification of pure nucleic acids from root tissues is generally difficult due to the high concentration of carbohydrates and secondary metabolites. Furthermore, the presence of enzymatic inhibitors such as fulvic and humic acids can also negatively affect extraction quality, when extracting from clay soil-grown roots. In this work, total RNA was extracted from soil-grown roots of Ilex paraguariensis using four commercially available kits: SpectrumTM, RNeasy®, TRI Reagent®, and SV Total RNA Isolation System. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The SpectrumTM and RNeasy® protocols provided the highest quantity and quality of RNA; however, the former revealed superior extraction performance. Consequently, total RNA was extracted from the roots of non-stressed and drought-stressed plants using the SpectrumTM method and six RNA-seq libraries were prepared from polyA + mRNAs by means of TruSeq mRNA library construction protocol to convert RNA to complementary DNA (cDNA). More than 80 million raw read sequences were obtained from each condition with an average read length of 150 bp. The yield and quality of the total RNA were consistently high and the RNA could be used for further analyses as demonstrated by cDNA library construction, RT-PCR, and transcriptome sequencing. Thus, SpectrumTM method can be used to isolate high quality RNA from roots of normal and drought stressed I. paraguariensis plants.
Pages 205-209 |Read More| doi: 10.21475/poj.10.04.17.pne745…………………………………………………………………………………………………………………………………………………..
Identification of suitable reference genes by quantitative real-time PCR for gene expression normalization in sunflower
Ana Claudia Ochogavía*, María Angelina Novello, Liliana Amelia Picardi, Graciela María Nestares
IICAR, UNR, CONICET (Instituto de Investigaciones en Ciencias Agrarias de Rosario, Universidad Nacional de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas), Zavalla, Santa Fe, Argentina
CIUNR (Consejo de Investigaciones de la Universidad Nacional de Rosario), Argentina
Abstract
Quantitative real-time PCR (qPCR) is currently the most accurate method for detecting differential gene expression, but depends greatly on normalization to stably expressed housekeeping genes. Transcriptomics analyses and experimental validation in different plant species have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. Thus, reliable validation of reference genes is required to ensure proper normalization. This paper presents a systematic comparison of ten potential reference genes in sunflower: five commonly used genes (Actin, Elongation Factor1, Plastid-encode RNA polymerase, Tubulin, and Ubiquitin, as ACT, EF1, PEP, TUB, and UBQ respectively), as well as five new candidates (Translation initiation factor, MicroRNA precursors 171 and 156, Ask-interacting protein, and Protein of unknown function, as ETIF5, MIR171, MIR156, SKIP, and UNK2 respectively). Reference gene expression stability was examined by qPCR across 20 biological samples, representing different tissues at various developmental stages. Expression of all 10 genes was variable to some extent, but that of ACT, UNK2, and EF1 was overall the most stable. A combination of ETIF5/UNK2/EF1 would be appropriate to use as a reference panel for normalizing gene expression data among vegetative tissues, whereas the combination of ACT/MIR156/UNK2 is most suitable for reproductive tissues. Reference genes selected in this study were further validated by examining relative expression of ahas1, one of three acetohydroxyacid synthase genes of sunflower. Our identification and validation of suitable normalizer genes will be of use to ensure accurate results in future transcriptomics studies in this crop.
Pages 210-218 |Read More | Supplementary Data| doi: 10.21475/poj.10.04.17.pne831RSS