Dataset Information

Visualization of Multicolored in vivo Organelle Markers for Co-Localization Studies in Oryza sativa.

ABSTRACT: Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in monocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on well-established targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our generated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the sub-cellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.

SUBMITTER: Dangol S

PROVIDER: S-EPMC5712512 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Visualization of Multicolored in vivo Organelle Markers for Co-Localization Studies in Oryza sativa.

Dangol Sarmina S Singh Raksha R Chen Yafei Y Jwa Nam-Soo NS

Molecules and cells 20171106 11

Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into ...[more]

PMID: 29113428

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Project description:BACKGROUND: Histone acetyltransferases (HATs) play an important role in eukaryotic transcription. Eight HATs identified in rice (OsHATs) can be organized into four families, namely the CBP (OsHAC701, OsHAC703, and OsHAC704), TAFII250 (OsHAF701), GNAT (OsHAG702, OsHAG703, and OsHAG704), and MYST (OsHAM701) families. The biological functions of HATs in rice remain unknown, so a comprehensive protein sequence analysis of the HAT families was conducted to investigate their potential functions. In addition, the subcellular localization and expression patterns of the eight OsHATs were analyzed. RESULTS: On the basis of a phylogenetic and domain analysis, monocotyledonous CBP family proteins can be subdivided into two groups, namely Group I and Group II. Similarly, dicotyledonous CBP family proteins can be divided into two groups, namely Group A and Group B. High similarities of protein sequences, conserved domains and three-dimensional models were identified among OsHATs and their homologs in Arabidopsis thaliana and maize. Subcellular localization predictions indicated that all OsHATs might localize in both the nucleus and cytosol. Transient expression in Arabidopsis protoplasts confirmed the nuclear and cytosolic localization of OsHAC701, OsHAG702, and OsHAG704. Real-time quantitative polymerase chain reaction analysis demonstrated that the eight OsHATs were expressed in all tissues examined with significant differences in transcript abundance, and their expression was modulated by abscisic acid and salicylic acid as well as abiotic factors such as salt, cold, and heat stresses. CONCLUSIONS: Both monocotyledonous and dicotyledonous CBP family proteins can be divided into two distinct groups, which suggest the possibility of functional diversification. The high similarities of protein sequences, conserved domains and three-dimensional models among OsHATs and their homologs in Arabidopsis and maize suggested that OsHATs have multiple functions. OsHAC701, OsHAG702, and OsHAG704 were localized in both the nucleus and cytosol in transient expression analyses with Arabidopsis protoplasts. OsHATs were expressed constitutively in rice, and their expression was regulated by exogenous hormones and abiotic stresses, which suggested that OsHATs may play important roles in plant defense responses.

Project description:Microarray-based markers such as Diversity Arrays Technology (DArT) have become the genetic markers of choice for construction of high-density maps, quantitative trait loci (QTL) mapping and genetic diversity analysis based on their efficiency and low cost. More recently, the DArT technology was further developed in combination with high-throughput next-generation sequencing (NGS) technologies to generate the DArTseq platform representing a new sequencing tool of complexity-reduced representations. In this study, we used DArTseq markers to investigate genetic diversity and genome-wide association studies (GWAS) of grain quality traits in rice (Oryza sativa L.). The study was performed using 59 rice genotypes with 525 SNPs derived from DArTseq platform. Population structure analysis revealed only two distinct genetic clusters where genotypes were grouped based on environmental adaptation and pedigree information. Analysis of molecular variance indicated a low degree of differentiation among populations suggesting the need for broadening the genetic base of the current germplasm collection. GWAS revealed 22 significant associations between DArTseq-derived SNP markers and rice grain quality traits in the test genotypes. In general, 2 of the 22 significant associations were in chromosomal regions where the QTLs associated with the given traits had previously been reported, the other 20 significant SNP marker loci were indicative of the likelihood discovery of novel alleles associated with rice grain quality traits. DArTseq-derived SNP markers that include SNP12_100006178, SNP13_3052560 and SNP14_3057360 individually co-localised with two functional gene groups that were associated with QTLs for grain width and grain length to width ratio on chromosome 3, indicating trait dependency or pleiotropic-effect loci. This study demonstrated that DArTseq markers were useful genomic resources for genome-wide association studies of rice grain quality traits to accelerate varietal development and release.

Dataset Information

Visualization of Multicolored in vivo Organelle Markers for Co-Localization Studies in Oryza sativa.

Publications

Visualization of Multicolored in vivo Organelle Markers for Co-Localization Studies in Oryza sativa.

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