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The pan-eukaryotic protein kinase GCN2 (General Control Nonderepressible2) regulates the translation of mRNAs in response to external and metabolic conditions. Although GCN2 and its substrate, translation initiation factor 2 (eIF2) α, and several partner proteins are substantially conserved in plants, this kinase has assumed novel functions in plants, including in innate immunity and retrograde signaling between the chloroplast and cytosol. How exactly some of the biochemical paradigms of the GCN2 system have diverged in the green plant lineage is only partially resolved. Specifically, conflicting data underscore and cast doubt on whether GCN2 regulates amino acid biosynthesis; also whether phosphorylation of eIF2α can in fact repress global translation or activate mRNA specific translation via upstream open reading frames; and whether GCN2 is controlled in vivo by the level of uncharged tRNA. This review examines the status of research on the eIF2α kinase, GCN2, its function in the response to xenobiotics, pathogens, and abiotic stress conditions, and its rather tenuous role in the translational control of mRNAs. • When GCN2 kinase is active, translation is often repressed, but how the former leads to the latter remains to be established. • GCN2 operates together with one obligatory cofactor, GCN1, and one nonessential cofactor, an ATP-binding cassette protein. • GCN2 kinase alone integrates a wide variety of stresses, qualifying it as the regulator of the 'integrated stress response' in plants. • The role of plant GCN2 in direct amino acid sensing remains controversial. [ABSTRACT FROM AUTHOR]

Highlights: [•] uORFs are common control elements for posttranscriptional gene regulation. [•] Certain uORFs function as sensors for metabolites. [•] uORFs regulate development. [•] uORFs challenge us to solve the biochemistry of translation reinitiation. [Copyright &y& Elsevier]

Light is one of the most important environmental factors that regulate plant development. Here we report that a mutation in the Arabidopsis FIERY1 gene ( FRY1) caused a shortened hypocotyl and shorter petioles, most dramatically under low-intensity red light and less pronounced under far-red and blue-light conditions. Furthermore, the fry1 mutant flowered late, probably due to a reduced level of FLOWERING LOCUS T ( FT) transcript. However, although the transcript level of FRY1 was light-regulated, the chlorophyll level and the expression of typical light-regulated genes were not affected in the fry1 mutant. FRY1 is known as a regulator of abiotic stress responses, and its protein product has dual enzymatic activity comprising inositol polyphosphate-1-phosphatase and 3′(2′),5′-bisphosphate nucleotidase activity. Genetic complementation data obtained using cDNA of the FRY1 paralog AHL (Arabidopsis HAL2-like) and the similar phenotype of an xrn2/xrn3 double mutant suggest that FRY1 attenuates light responses via its 3′(2′),5′-bisphosphate nucleotidase activity rather than its inositol polyphosphate-1-phosphatase activity. We discuss the relationship between the FRY1-associated nucleotidase activity, a step in the pathway for sulfur metabolism and utilization, and the Arabidopsis light response. [ABSTRACT FROM AUTHOR]

Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved kcat, as well as elevated luminescence in Arabidopsis. This advances the utility of RLUC as a reporter protein, biosensor, or resonance energy donor. [ABSTRACT FROM AUTHOR]

Three structurally related protein complexes, the COP9 signalosome, the proteasome lid, and the eukaryotic translation initiation factor 3, are revealing new insights into developmental processes and into cell cycle control in healthy cells and cells exposed to genotoxic stress. Newly discovered cullin-RING E3 ubiquitin ligases assembled on the CUL4 platform may provide links between DNA replication, chromatin, and proteolysis. [Copyright &y& Elsevier]

The COP9 signalosome is an eight-subunit protein complex that regulates protein ubiquitination and protein turnover in a variety of plant developmental and physiological contexts, including light-regulated development, hormone signaling, and defense against pathogens. In all eukaryotes tested, the COP9 signalosome is able to posttranslationally modify the cullin subunit of E3-ubiquitin-ligase complexes by cleaving off the covalently coupled peptide, Nedd8. Two contrasting models ascribe stimulatory or inhibitory roles to the modification of cullin/E3 that is mediated by the COP9 signalosome. There is considerable disagreement as to whether Nedd8 cleavage underlies all of the COP9 signalosome’s numerous cellular and phenotypic effects. This is because macroscopic phenotypes do not always correlate with biochemical defects in COP9 signalosome mutants. Additional biochemical activities, including protein interactions with the cellular machineries for protein phosphorylation, protein turnover, and protein translation, have been proposed to account for the role of the COP9 signalosome in development and disease. [Copyright &y& Elsevier]

Activation tagging is a powerful method of insertional mutagenesis for generating gain-of-function mutations in plants. Current activation-tagging cassettes, based on the 35S enhancer of the cauliflower mosaic virus, have limited utility in genetic backgrounds containing 35S promoter sequences because they may cause homology-dependent gene silencing. We constructed series of novel activation-tagging vectors that do not contain the CaMV 35S enhancer but instead contain multiple tandem copies of an alternative enhancer from cassava vein mosaic virus. For selection, the T DNAs confer Basta herbicide resistance. Resulting activation-tagging cassettes were introduced into Arabidopsis thaliana to demonstrate stable integration of the TDNA. Vectors described here may be suitable for, but not limited to, activation-tagging projects in genetic backgrounds harboring transgenes driven by the CaMV 35S promoter. [ABSTRACT FROM AUTHOR]

Background: Meiotically heritable epimutations affecting transgene expression are not well understood, even and in particular in the plant model species, Arabidopsis thaliana. The Arabidopsis trans-silencer locus, C73, which encodes a fusion protein between the repressor of photomorphogenesis, COP1, and green fluorescent protein (GFP-COP1), heritably modifies the expression pattern and cop1-like cosuppression phenotypes of multiple GFP-COP1 target loci by transcriptional gene silencing. Results: Here we describe three additional features of trans-silencing by the C73 locus. First, the silencing phenotype of C73 and of similar complex loci was acquired epigenetically over the course of no more than two plant generations via a stage resembling posttranscriptional silencing. Second, imprints imposed by the C73 locus were maintained heritably for at least five generations in the absence of the silencer with only sporadic spontaneous reversion. Third, the pairing of two other GFP-COP1 transgene loci, L91 and E82, showed an increased tendency for epigenetic modification when L91 carried an epigenetic imprint from C73, but not when E82 bore the imprint. Conclusions: The latter data suggest a transfer of trans-silencing activity from one transgene locus, C73, to another, namely L91. These results extend our operational understanding of interactions among transgenes in Arabidopsis. [ABSTRACT FROM AUTHOR]

Demonstrates that light inactivation of Arabidopsis photomorphogenic repressor COP1 involves a cell-specific regulation of its nucleocytoplasmic partitioning. Role of light-regulated COP1 nucleocytoplasmic partitioning during morphogenesis; Optimal development regulation.

Substantial guidance is available on undergraduate quantitative training for biologists, including reports focused on biomedical science. Far less attention has been paid to the graduate curriculum and the particular challenges of the diversity of specialization within the life sciences. We propose an innovative approach to quantitative education that goes beyond recommendations of a course or set of courses or activities, derived from analysis of the expectations for students in particular programs. Due to the plethora of quantitative methods, it is infeasible to expect that biomedical PhD students can be exposed to more than a minority of the quantitative concepts and techniques employed in modern biology. We collected key recent papers suggested by the faculty in biomedical science programs, chosen to include important scientific contributions that the faculty consider appropriate for all students in the program to be able to read with confidence. The quantitative concepts and methods inherent in these papers were then analyzed and categorized to provide a rational basis for prioritization of those concepts to be emphasized in the education program. This novel approach to prioritization of quantitative skills and concepts provides an effective method to drive curricular focus based upon program-specific faculty input for science programs of all types. The results of our particular application to biomedical science training highlight the disconnect between typical undergraduate quantitative education for life science students, focused on continuous mathematics, and the concepts and skills in graphics, statistics, and discrete mathematics that arise from priorities established by biomedical science faculty. There was little reference in the key recent papers chosen by faculty to classic mathematical areas such as calculus which make up a large component of the formal undergraduate mathematics training of graduate students in biomedical areas. [ABSTRACT FROM AUTHOR]

Similarities between the three related ‘PCI’ complexes – eIF3, the COP9 signalosome and the proteasome lid – have hinted at novel pathways controlling protein homeostasis. Recent experiments with fission yeast have begun to weigh in with genetic evidence. [Copyright &y& Elsevier]

Background: Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis. Methodology/Principal Findings: Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation. Conclusion/Significance: eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis. [ABSTRACT FROM AUTHOR]

Background: Within the scanning model of translation initiation, reinitiation is a non-canonical mechanism that operates on mRNAs harboring upstream open reading frames. The h subunit of eukaryotic initiation factor 3 (eIF3) boosts translation reinitiation on the uORF-containing mRNA coding for the Arabidopsis bZip transcription factor, AtbZip11, among others. The RPL24B protein of the large ribosomal subunit, which is encoded by SHORT VALVE1, likewise fosters translation of uORF-containing mRNAs, for example mRNAs for auxin response transcription factors (ARFs). Results: Here we tested the hypothesis that RPL24B and eIF3h affect translation reinitiation in a similar fashion. First, like eif3h mutants, rpl24b mutants under-translate the AtbZip11 mRNA, and the detailed spectrum of translational defects in rpl24b is remarkably similar to that of eif3h. Second, eif3h mutants display defects in auxin mediated organogenesis and gene expression, similar to rpl24b. Like AtbZip11, the uORF-containing ARF mRNAs are indeed undertranslated in eif3h mutant seedlings. Conclusion: We conclude that, similar to eIF3h, RPL24B bolsters the reinitiation competence of uORF-translating ribosomes. Coordination between eIF3 and the large ribosomal subunit helps to fine-tune translation of uORFcontaining mRNAs and, in turn, to orchestrate plant development. [ABSTRACT FROM AUTHOR]

Background: Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results: We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion: The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis. [ABSTRACT FROM AUTHOR]

Essentially all aboveground plant tissues develop from the stem cells in the primary shoot apical meristem. Proliferation of the stem cell population in the Arabidopsis shoot apical meristem is tightly controlled by a feedback loop formed primarily by the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA ligand-receptor system. In this study, it is shown that mutation of a translation initiation factor, eIF3h, causes a tendency to develop a strikingly enlarged shoot apical meristem with elevated and ectopic expression of WUS and CLAVATA3 (CLV3). Many of the mRNAs that function in apical meristem maintenance possess upstream open reading frames (uORFs), translational attenuators that render translation partially dependent on eIF3h. Specifically, the mRNA for the receptor kinase, CLV1, is undertranslated in the eif3h mutant as shown by transient and transgenic expression assays. Concordant phenotypic observations include defects in organ polarity and in translation of another uORF-containing mRNA, ASYMMETRIC LEAVES 1 (AS1), in eif3h. In summary, the expression of developmental regulatory mRNAs is attenuated by uORFs, and this attenuation is balanced in part by the translation initiation factor, eIF3h. Thus, translational control plays a key role in Arabidopsis stem cell regulation and organogenesis. [ABSTRACT FROM AUTHOR]

Background Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as nonpolysomal mRNAs to examine the defects in translation in a poly(A)binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1. Results The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected twelve biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs. Conclusions These data represent the first genome-wide analysis of translation in a eukaryote defective in the large ribosomal subunit. RPL24 and eIF3h play similar but nonidentical roles in eukaryotic translation. The data also shed light on the fine structure of the regulon of ribosomal protein mRNAs. [ABSTRACT FROM AUTHOR]

In Arabidopsis thaliana, FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3) encode YABBY domain proteins that regulate abaxial patterning, growth of lateral organs, and inflorescence phyllotaxy. In this study, we show that YABs physically interact with components of a transcriptional repressor complex that include LEUNIG (LUG), LEUNIG_HOMOLOG (LUH), the LUG-associated coregulator SEUSS, and related SEUSS-LIKE proteins. Consistent with the formation of a LUG-YAB complex, we find that lug mutants enhance the polarity and growth defects of fil yab3 mutant leaves and that this enhancement is due to a loss of LUG activity from the abaxial domain. We performed a more extensive genetic analysis, which included the characterization of yab triple and quadruple mutants, lug luh/+ (heterozygous only for luh) mutants, and plants expressing artificial microRNAs targeting LUG or LUH. These analyses showed that the LUG-YAB complex also promotes adaxial cell identity in leaves as well as embryonic shoot apical meristem (SAM) initiation and postembryonic SAM maintenance. Based on the likely formation of the LUG-YAB complex in the abaxial domain of cotyledons and leaves, we propose that this complex has numerous non-cell-autonomous functions during plant development. [ABSTRACT FROM AUTHOR]

The roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits are largely unclear, although some are essential, while others are thought to have regulatory roles. The ‘e’ subunit, also known as Int-6/Int6, is a candidate for a regulatory subunit as it is not essential for translation initiation in yeasts. eIF3e associates with the COP9 signalosome, and localizes to the nucleus in certain tissues. To further elucidate the roles of eIF3e, we have taken a genetic approach using Arabidopsis as a model system. Overexpression of eIF3e results in defects similar to mutations in the COP9 signalosome. eIF3e protein, but not transcript, over accumulates in csn mutants, and eIF3e is degraded in a proteasome-dependent fashion. In vitro and in vivo assays suggest that excess eIF3e inhibits translation. We conclude that the COP9 signalosome maintains a precise regulation of eIF3e levels, which is necessary for normal development in Arabidopsis. [ABSTRACT FROM AUTHOR]

The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP–rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families. [ABSTRACT FROM PUBLISHER]

FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (11 dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein α (C/EBPα) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations. [ABSTRACT FROM AUTHOR]

Bioluminescence resonance energy transfer (BRET) is a natural biophysical phenomenon that underlies an emerging technique to monitor protein–protein interactions in living cells in real time. Here, we present a series of technical advances to enhance the utility of the BRET assay in plants. A series of recombination cloning vectors was generated to accelerate the expression of proteins tagged with Renilla luciferase or yellow fluorescent protein under transient assay conditions and in stable transgenic plants. Working in stably transformed Arabidopsis or tobacco, we then detected BRET between three pairs of candidate interaction partners: dimerization of the E3 ubiquitin ligase COP1, interaction between COP1 and the B-box protein STH, and interaction between the light regulatory bZip transcription factors HY5 and HYH. A codon-optimized version of the Renilla luciferase gene resulted in improved expression in Arabidopsis. Renilla luciferase was active in a variety of subcellular organelles, including plastids, mitochondria, peroxisomes and Golgi stacks. In a survey of the Arabidopsis light signaling machinery as a model system, we estimated the likelihood that a known protein–protein interaction can be documented using BRET. Finally, we show that Renilla luciferase may serve as a reporter of protein stability in a cycloheximide chase assay. [ABSTRACT FROM AUTHOR]

Eukaryotic translation initiation factor 3 (eIF3) consists of core subunits that are conserved from yeast to man as well as less conserved, noncore, subunits with potential regulatory roles. Whereas core subunits tend to be indispensable for cell growth, the roles of the noncore subunits remain poorly understood. We addressed the hypothesis that eIF3 noncore subunits have accessory functions that help to regulate translation initiation, by focusing on the Arabidopsis thaliana eIF3h subunit. Indeed, eIF3h was not essential for general protein translation. However, results from transient expression assays and polysome fractionation indicated that the translation efficiency of specific 5′ mRNA leader sequences was compromised in an eif3h mutant, including the mRNA for the basic domain leucine zipper (bZip) transcription factor ATB2/AtbZip11, translation of which is regulated by sucrose. Among other pleiotropic developmental defects, the eif3h mutant required exogenous sugar to transit from seedling to vegetative development, but it was hypersensitive to elevated levels of exogenous sugars. The ATB2 mRNA was rendered sensitive to the eIF3h level by a series of upstream open reading frames. Moreover, eIF3h could physically interact with subunits of the COP9 signalosome, a protein complex implicated primarily in the regulation of protein ubiquitination, supporting a direct biochemical connection between translation initiation and protein turnover. Together, these data implicate eIF3 in mRNA-associated translation initiation events, such as scanning, start codon recognition, or reinitiation and suggest that poor translation initiation of specific mRNAs contributes to the pleiotropic spectrum of phenotypic defects in the eif3h mutant. [ABSTRACT FROM AUTHOR]