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ABSTRACT: Plants actively modulate the levels of the various phyto-chrome isoforms during their life cycle to optimize light absorption and perception. For phytochrome A (phyA), one of the most influential methods of control is selective turnover of the photoreceptor upon photoconversion from the red-absorbing form (Pr) to the far-red-absorbing form (Pfr). Whereas the Pr form has a half-life of approximately 1 week, the Pfr form is rapidly degraded with a half-life of 1–2 h. The ubiquitin/26S proteasome pathway has been implicated in phyA breakdown. In this proteolytic pathway, multiple ubiquitins are covalently attached to proteins committed for degradation; these ubiquitin-protein conjugates then serve as intermediates in the breakdown of the target protein by the 26S proteasome, a multi-subunit proteolytic complex. In several plant species, ubiquitin-phyA conjugates have been detected in vivo following Pfr formation that show accumulation and decay kinetics expected for Pfr degradation intermediates. Analyses of phyA mutants and phyA/phyB chimeras expressed in transgenic plants have been particularly useful in mapping domains within the chromoprotein that are necessary for Pfr degradation. Several domains have been identified within both the N- and C-terminal portions of the photoreceptor that presumably serve as recognition and/or acceptor sites for ubiquitination
Plant Cell and Environment 06/2008; 20(6):713 - 721. · 5.22 Impact Factor
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ABSTRACT: The concept that plants exploit polypeptides as post-translational modifiers is rapidly emerging as an important method to manipulate various cellular processes. The best known is Ub (ubiquitin) that serves as reusable tag for selective protein degradation by the 26 S proteasome and for endosomal trafficking. Genomic analyses indicate that Ub pathway alone comprises over 6% of the Arabidopsis proteome with thousands of proteins being targets. Consequently, this pathway influences much of plant biology. Others tags include RUB-1 (related to Ub-1; also known as NEDD8), SUMO (small Ub-like modifier), ATG-8 (autophagy-8) and ATG-12, UFM-1 (Ub-fold modifier-1) and HUB-1 (homology to Ub-1). Preliminary studies indicate that these tags have much more limited sets of targets and provide more specialized functions, including transcriptional regulation, protein localization, autophagic turnover and antagonizing the effects of Ub. On the basis of their widespread distribution and pervasive functions, peptide tags can now be considered as prime players in plant cell regulation.
Biochemical Society Transactions 05/2005; 33(Pt 2):393-9. · 3.71 Impact Factor
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ABSTRACT: Phytochromes comprise a principal family of red/far-red light sensors in plants. Although phytochromes were thought originally to be confined to photosynthetic organisms, we have recently detected phytochrome-like proteins in two heterotrophic eubacteria, Deinococcus radiodurans and Pseudomonas aeruginosa. Here we show that these form part of a widespread family of bacteriophytochromes (BphPs) with homology to two-component sensor histidine kinases. Whereas plant phytochromes use phytochromobilin as the chromophore, BphPs assemble with biliverdin, an immediate breakdown product of haem, to generate photochromic kinases that are modulated by red and far-red light. In some cases, a unique haem oxygenase responsible for the synthesis of biliverdin is part of the BphP operon. Co-expression of this oxygenase with a BphP apoprotein and a haem source is sufficient to assemble holo-BphP in vivo. Both their presence in many diverse bacteria and their simplified assembly with biliverdin suggest that BphPs are the progenitors of phytochrome-type photoreceptors.
Nature 01/2002; 414(6865):776-9. · 36.28 Impact Factor
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ABSTRACT: The 26S proteasome plays a major role in eukaryotic protein breakdown, especially for ubiquitin-tagged proteins. Substrate specificity is conferred by the regulatory particle (RP), which can dissociate into stable lid and base subcomplexes. To help define the molecular organization of the RP, we tested all possible paired interactions among subunits from Saccharomyces cerevisiae by yeast two-hybrid analysis. Within the base, a Rpt4/5/3/6 interaction cluster was evident. Within the lid, a structural cluster formed around Rpn5/11/9/8. Interactions were detected among synonymous subunits (Csn4/5/7/6) from the evolutionarily related COP9 signalosome (CSN) from Arabidopsis, implying a similar quaternary arrangement. No paired interactions were detected between lid, base or core particle subcomplexes, suggesting that stable contacts between them require prior assembly. Mutational analysis defined the ATPase, coiled-coil, PCI and MPN domains as important for RP assembly. A single residue in the vWA domain of Rpn10 is essential for amino acid analog resistance, for degrading a ubiquitin fusion degradation substrate and for stabilizing lid-base association. Comprehensive subunit interaction maps for the 26S proteasome and CSN support the ancestral relationship of these two complexes.
The EMBO Journal 01/2002; 20(24):7096-107. · 9.20 Impact Factor
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ABSTRACT: The ubiquitin/26S proteasome pathway is a major route for selectively degrading cytoplasmic and nuclear proteins in eukaryotes. In this pathway, chains of ubiquitins become attached to short-lived proteins, signalling recognition and breakdown of the modified protein by the 26S proteasome. During or following target degradation, the attached multi-ubiquitin chains are released and subsequently disassembled by ubiquitin-specific proteases (UBPs) to regenerate free ubiquitin monomers for re-use. Here, we describe Arabidopsis thaliana UBP14 that may participate in this recycling process. Its amino acid sequence is most similar to yeast UBP14 and its orthologues, human IsoT1-3 and Dictyostelium UbpA, and it can functionally replace yeast UBP14 in a ubp14Delta mutant. Like its orthologues, AtUBP14 can disassemble multi-ubiquitin chains linked internally via epsilon-amino isopeptide bonds using Lys48 and can process some, but not all, translational fusions of ubiquitin linked via alpha-amino peptide bonds. However, unlike its yeast and Dictyostelium orthologues, AtUBP14 is essential in Arabidopsis. T-DNA insertion mutations in the single gene that encodes AtUBP14 cause an embryonic lethal phenotype, with the homozygous embryos arresting at the globular stage. The arrested seeds have substantially increased levels of multi-ubiquitin chains, indicative of a defect in ubiquitin recycling. Taken together, the data demonstrate an essential role for the ubiquitin/26S proteasome pathway in general and for AtUBP14 in particular during early plant development.
The Plant Journal 10/2001; 27(5):393-405. · 6.16 Impact Factor
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ABSTRACT: The committed step in the biosynthesis of the phytochrome chromophore phytochromobilin involves the oxidative cleavage of heme by a heme oxygenase (HO) to form biliverdin IXalpha. Through positional cloning of the photomorphogenic mutant hy1, the Arabidopsis HO (designated AtHO1) responsible for much of phytochromobilin synthesis recently was identified. Using the AtHO1 sequence, we identified families of HO genes in a number of plants that cluster into two subfamilies (HO1- and HO2-like). The tomato (Lycopersicon esculentum) yg-2 and Nicotiana plumbaginifolia pew1 photomorphogenic mutants are defective in specific HO genes. Phenotypic analysis of a T-DNA insertion mutant of Arabidopsis HO2 revealed that the second HO subfamily also contributes to phytochromobilin synthesis. Homozygous ho2-1 plants show decreased chlorophyll accumulation, reduced growth rate, accelerated flowering time, and reduced de-etiolation. A mixture of apo- and holo-phyA was detected in etiolated ho2-1 seedlings, suggesting that phytochromobilin is limiting in this mutant, even in the presence of functional AtHO1. The patterns of Arabidopsis HO1 and HO2 expression suggest that the products of both genes overlap temporally and spatially. Taken together, the family of HOs is important for phytochrome-mediated development in a number of plants and that each family member may uniquely contribute to the phytochromobilin pool needed to assemble holo-phytochromes.
Plant physiology 07/2001; 126(2):656-69. · 6.53 Impact Factor
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ABSTRACT: The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.
Journal of Biological Chemistry 04/2001; 276(10):6959-66. · 4.77 Impact Factor
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ABSTRACT: Ubiquitin-specific proteases (UBPs) are a family of unique hydrolases that specifically remove polypeptides covalently linked via peptide or isopeptide bonds to the C-terminal glycine of ubiquitin. UBPs help regulate the ubiquitin/26S proteolytic pathway by generating free ubiquitin monomers from their initial translational products, recycling ubiquitins during the breakdown of ubiquitin-protein conjugates, and/or by removing ubiquitin from specific targets and thus presumably preventing target degradation. Here, we describe a family of 27 UBP genes from Arabidopsis that contain both the conserved cysteine (Cys) and histidine boxes essential for catalysis. They can be clustered into 14 subfamilies based on sequence similarity, genomic organization, and alignments with their closest relatives from other organisms, with seven subfamilies having two or more members. Recombinant AtUBP2 functions as a bona fide UBP: It can release polypeptides attached to ubiquitins via either alpha- or epsilon-amino linkages by an activity that requires the predicted active-site Cys within the Cys box. From the analysis of T-DNA insertion mutants, we demonstrate that the AtUBP1 and 2 subfamily helps confer resistance to the arginine analog canavanine. This phenotype suggests that the AtUBP1 and 2 enzymes are needed for abnormal protein turnover in Arabidopsis.
Plant physiology 01/2001; 124(4):1828-43. · 6.53 Impact Factor
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ABSTRACT: The recent discovery of phytochrome-like photoreceptors, collectively called bacteriophytochromes, in a number of bacteria has greatly expanded our understanding of the origins and modes of action of phytochromes in higher plants. These primitive receptors contain an N-terminal domain homologous to the chromophore-binding pocket of phytochromes, and like phytochromes, they bind a variety of bilins to generate photochromic holoproteins. Following the chromophore pocket is a domain similar to two-component histidine kinases, suggesting that these bacterial photoreceptors function in phosphorelay cascades that respond to the light environment. Their organization and distribution support the views that higher-plant phytochromes evolved from a cyanobacterial precursor and that they act as light-regulated kinases. With the ability to exploit bacterial genetics, these bacteriophytochromes now offer simple models to help unravel the biochemical and biophysical events that initiate phytochrome signal transmission.
Seminars in Cell and Developmental Biology 01/2001; 11(6):511-21. · 6.65 Impact Factor
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ABSTRACT: Phytochromes are a family of photoreceptors used by green plants to entrain their development to the light environment. The distribution of these chromoproteins has been expanded beyond photoautotrophs with the discovery of phytochrome-like proteins in the nonphotosynthetic eubacteria Deinococcus radiodurans and Pseudomonas aeruginosa. Like plant phytochromes, the D. radiodurans receptor covalently binds linear tetrapyrroles autocatalytically to generate a photochromic holoprotein. However, the attachment site is distinct, using a histidine to potentially form a Schiff base linkage. Sequence homology and mutational analysis suggest that D. radiodurans bacteriophytochrome functions as a light-regulated histidine kinase, which helps protect the bacterium from visible light.
Science 12/1999; 286(5449):2517-20. · 31.20 Impact Factor
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ABSTRACT: The ubiquitin/26S proteasome pathway is a major route for degrading abnormal and important short-lived regulatory proteins in eukaryotes. Covalent attachment of ubiquitin, which triggers entry of target proteins into the pathway, is accomplished by an ATP-dependent reaction cascade involving the sequential action of three enzymes, E1s, E2s and E3s. Although much of the substrate specificity of the pathway is determined by E3s (or ubiquitin-protein ligases, UPLs), little is known about these enzymes in plants and how they choose appropriate targets for ubiquitination. Here, we describe two 405 kDa E3s (UPL1 and 2) from Arabidopsis thaliana related to the HECT-E3 family that is essential in yeast and animals. UPL1 and 2 are encoded by 13 kbp genes 26 cM apart on chromosome I, that are over 95% identical within both the introns and exons, suggesting that the two loci arose from a recent gene duplication. The C-terminal HECT domain of UPL1 is necessary and sufficient to conjugate ubiquitin in vitro in a reaction that requires the positionally conserved cysteine within the HECT domain, E1, and an E2 of the UBC8 family. Given that HECT E3s help define target specificity of the ubiquitin conjugation, a continued characterization of UPL1 and 2 should be instrumental in understanding the functions of ubiquitin-dependent protein turnover in plants and for identifying pathway substrates.
The Plant Journal 11/1999; 20(2):183-95. · 6.16 Impact Factor
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ABSTRACT: The 26S proteasome, a multisubunit complex, is the primary protease of the ubiquitin-mediated proteolytic system in eukaryotes. We have recently characterized MCB1 (RPN10), a subunit of the 26S complex that has affinity for multiubiquitin chains in vitro and as a result may function as a receptor for ubiquitinated substrates. To define the role of MCB1 further, we analyzed its function in Physcomitrella patens by generating MCB1 gene disruptions using homologous recombination. PpMCB1, which is 50 to 75% similar to orthologs from other eukaryotes, is present in the 26S proteasome complex and has a similar affinity for multiubiquitin chains, using a conserved hydrophobic domain within the C-terminal half of the polypeptide. Unlike yeast Deltamcb1 strains, which grow normally, P. patens Deltamcb1 strains are viable but are under developmental arrest, generating abnormal caulonema that are unable to form buds and gametophores. Treatment with auxin and cytokinin restored bud formation and subsequent partial development of gametophores. Complementation of a Deltamcb1 strain with mutated versions of PpMCB1 revealed that the multiubiquitin chain binding site is not essential for the wild-type phenotype. These results show that MCB1 has an important function in the 26S proteasome of higher order eukaryotes in addition to its ability to bind multiubiquitin chains, and they provide further support for a role of the ubiquitin/26S proteasome proteolytic pathway in plant developmental processes triggered by hormones.
The Plant Cell 09/1999; 11(8):1457-72. · 8.99 Impact Factor
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ABSTRACT: The 26S proteasome is a multi-subunit ATP-dependent protease responsible for degrading most short-lived intracellular proteins targeted for breakdown by ubiquitin conjugation. The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylindrical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP-dependence and the specificity for ubiquitinated proteins. Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, designated RPT1-6 for regulatory particle triple-A ATPase, that form a distinct family within the AAA superfamily. Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation. Here, we describe the RPT gene family from Arabidopsis thaliana. From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts. Five of the six RPT sub- units are encoded by two genes; the exception being RPT3 which is encoded by a single gene. mRNA for each of the six proteins is present in all tissue types examined. Five of the subunits (RPT1 and 3-6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent. Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non-redundant functions.
The Plant Journal 07/1999; 18(5):529-39. · 6.16 Impact Factor
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ABSTRACT: The hy1 mutants of Arabidopsis thaliana fail to make the phytochrome-chromophore phytochromobilin and therefore are deficient in a wide range of phytochrome-mediated responses. Because this defect can be rescued by feeding seedlings biliverdin IXalpha, it is likely that the mutations affect an enzyme that converts heme to this phytochromobilin intermediate. By a combination of positional cloning and candidate-gene isolation, we have identified the HY1 gene and found it to be related to cyanobacterial, algal, and animal heme oxygenases. Three independent alleles of hy1 contain DNA lesions within the HY1 coding region, and a genomic sequence spanning the HY1 locus complements the hy1-1 mutation. HY1 is a member of a gene family and is expressed in a variety of A. thaliana tissues. Based on its homology, we propose that HY1 encodes a higher-plant heme oxygenase, designated AtHO1, responsible for catalyzing the reaction that opens the tetrapyrrole ring of heme to generate biliverdin IXalpha.
Proceedings of the National Academy of Sciences 06/1999; 96(11):6541-6. · 9.68 Impact Factor
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ABSTRACT: As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN 10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10delta strains which grow normally, Physcomitrella rpn10delta strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.
Molecular Biology Reports 05/1999; 26(1-2):137-46. · 2.93 Impact Factor
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ABSTRACT: A major goal of plant biotechnology is the production of genetically engineered crops that express natural or foreign proteins at high levels. To enhance protein accumulation in transgenic plants, we developed a set of vectors that express proteins and peptides as C-terminal translational fusions with ubiquitin (UBQ). Studies of several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins can be readily expressed in plants as UBQ fusions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused protein moieties in free forms; (c) the synthesis of a protein as a UBQ fusion can significantly augment its accumulation; (d) proper processing and localization of a protein targeted to either the apoplast or the chloroplast is not affected by the N-terminal UBQ sequence; and (e) single amino acid substitutions surrounding the cleavage site can inhibit in vivo processing of the fusion by Ubps. Noncleavable UBQ fusions of beta-glucuronidase became extensively modified, with additional UBQs in planta. Because multiubiquitinated proteins are the preferred substrates of the 26S proteasome, noncleavable fusions may be useful for decreasing protein half-life. Based on their ability to augment protein accumulation and the sequence specificity of Ubps, UBQ fusions offer a versatile way to express plant proteins.
Plant physiology 03/1999; 119(2):713-24. · 6.53 Impact Factor
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ABSTRACT: Photoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr from initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal domain is essential for phyA degradation, the N-terminal domain is responsible for the selective recognition and ubiquitination of Pfr.
The Plant Journal 02/1999; 17(2):155-67. · 6.16 Impact Factor
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BioTechniques 10/1998; 25(3):374-6, 378. · 2.67 Impact Factor
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ABSTRACT: The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived and abnormal intracellular proteins, especially those targeted by ubiquitin conjugation. The 700-kD complex exists as a hollow cylinder comprising four stacked rings with the catalytic sites located in the lumen. The two outer rings and the two inner rings are composed of seven different alpha and beta polypeptides, respectively, giving an alpha7/beta7/beta7/alpha7 symmetric organization. Here we describe the molecular organization of the 20S proteasome from the plant Arabidopsis thaliana. From an analysis of a collection of cDNA and genomic clones, we identified a superfamily of 23 genes encoding all 14 of the Arabidopsis proteasome subunits, designated PAA-PAG and PBA-PBG for Proteasome Alpha and Beta subunits A-G, respectively. Four of the subunits likely are encoded by single genes, and the remaining subunits are encoded by families of at least 2 genes. Expression of the alpha and beta subunit genes appears to be coordinately regulated. Three of the nine Arabidopsis proteasome subunit genes tested, PAC1 (alpha3), PAE1 (alpha5) and PBC2 (beta3), could functionally replace their yeast orthologs, providing the first evidence for cross-species complementation of 20S subunit genes. Taken together, these results demonstrate that the 20S proteasome is structurally and functionally conserved among eukaryotes and suggest that the subunit arrangement of the Arabidopsis 20S proteasome is similar if not identical to that recently determined for the yeast complex.
Genetics 07/1998; 149(2):677-92. · 4.01 Impact Factor
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ABSTRACT: Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is 'brighter' because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.
Plant Molecular Biology 04/1998; 36(4):521-8. · 4.15 Impact Factor
