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Mathur J Radhamony R Sinclair AM Donoso A Dunn N Roach E Radford D Mohaghegh PS Logan DC Kokolic K Mathur N 《Plant physiology》2010,154(4):1573-1587
Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs. 相似文献
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Dhiraj R. Thakare Radhamony Resminath N. J. Chikhale S. R. Bhat R. Srinivasan 《Journal of plant biochemistry and biotechnology.》2006,15(1):7-13
An Arabidopsis thaliana mutant, exhibiting anther specific GUS expression, identified from a mutant population of Arabidopsis tagged with a promoterless β-glucuronidase (GUS), carries the T-DNA insertions at two distinct loci. We have been able to segregate the two inserts from each other by backcrossing with wild type plants. The insertion responsible for anther specific GUS expression in segregating population has been identified and confirmed to be in the upstream region of a putative peroxidase gene, AT2G24800. Here we report detailed histochemical and molecular characterization of the mutant Anth85, carrying a single insertion of T-DNA in the peroxidase gene. In Anth85, the GUS expression was observed in the anthers and rosette of the young seedlings. The expression of GUS in the anthers was restricted to the tapetum and microspores. The mutant has no developmental defects and the gene appears to be redundant for normal plant growth. Cloning of upstream region and detailed deletion study of upstream region in transgenic plants is likely to lead to the identification of anther specific promoter elements. 相似文献
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Sivanandan C Sujatha TP Prasad AM Resminath R Thakare DR Bhat SR Srinivasan 《Biochimica et biophysica acta》2005,1731(3):202-208
From a T-DNA tagged Arabidopsis population, a line, M-57 showing GUS (beta-glucuronidase) expression in the vascular regions of young roots was identified. Southern analysis revealed presence of a single T-DNA insert. Using inverse PCR, the plant sequence flanking the T-DNA insertion was cloned. The insertion was identified to be in the intergenic area between loci At4G13940 and At4G13930, coding for SAHH (S-Adenosyl-l-Homocysteine Hydrolase) and SHMT (Serine Hydroxy Methyl Transferase) genes, respectively. A 452-bp fragment immediately upstream of the T-DNA insertion when cloned and mobilized as a GUS fusion was capable of driving a similar root-specific expression of reporter gene in transgenic Arabidopsis plants and their progenies. This cryptic promoter element does not show the presence of any known root-specific promoter element. 相似文献
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Radhamony Resminath Anand Mohan Prasad Dhiraj R. Thakare C. Sivanandan S. R. Bhat Srinivasan 《Journal of plant biochemistry and biotechnology.》2005,14(1):1-8
A new promoter trap vector was constructed based on the juxtaposition of T-DNA right border to coding sequence of GUS. The new vector pRN-1 carried an intron in the GUS coding region. Promoter trap vectors pGKB5 and pRN-1 vectors were used to transform Arabidopsis ecotype Columbia using the floral dip transformation system. The transformants were selected on appropriate selection media and the primary transformants were confirmed by PCR using gene specific primers. Approximately 50 % of the T2 lines segregated for a 3:1 ratio indicating presence of T-DNA at single locus. Approximately 15% of the transformed lines showed expression of GUS. Morphological mutants for male sterility and dwarfism were also identified in the T2 population. A T-DNA tagged line was identified in T2 with GUS expression specifically in the floral parts. The number of T-DNA loci in this line was confirmed by Southern blot hybridization. T-DNA flanking region isolated from this line suggested insertions into chromosome 2 at two closely linked loci. The results demonstrate that the population generated can be used effectively to identify and characterize gene regulatory elements. 相似文献
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Chloroplast protein import is generally believed to occur posttranslationally through the interaction of a precursor protein with the Toc and Tic transport apparatus in the plastid envelope membranes. The cleavable N-terminal transit peptide present on translocated proteins has been considered to be essential and sufficient for targeting. This idea was recently challenged when an analysis of the chloroplast proteome revealed many proteins without a predicted transit peptide. A recent study demonstrates the existence of a novel chloroplast targeting pathway, starting with protein entry into the endoplasmic reticulum and involving the Golgi apparatus. 相似文献
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