Albrecht G. von Arnim

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

BackgroundPlant 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.ResultsWe 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.ConclusionThe 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.

Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues

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: (i) 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.

Discrete Domains Mediate the Light-Responsive Nuclear and Cytoplasmic Localization of Arabidopsis COP1

The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) protein plays a critical role in the repression of photomorphogenesis during Arabidopsis seedling development. We investigated the control of COP1 partitioning between nucleus and cytoplasm, which has been implicated in the regulation of COP1 activity, by using fusion proteins between COP1 and β-glucuronidase or the green fluorescent protein. Transient expression assays using onion epidermal cells and data from hypocotyl cells of stably transformed Arabidopsis demonstrated that COP1 carries a single, bipartite nuclear localization signal that functions independently of light. Nuclear exclusion was mediated by a novel and distinct signal, bordering the zinc-finger and coiled-coil motifs, that was able to redirect a heterologous nuclear protein to the cytoplasm. The cytoplasmic localization signal functioned in a light-independent manner. Light regulation of nuclear localization was reconstituted by combining the individual domains containing the nuclear localization signal and the cytoplasmic localization signal; the WD-40 repeat domain of COP1 was not required. However, phenotypic analysis of transgenic seedlings suggested that the constitutively nuclear-localized WD-40 repeat domain was able to mimic aspects of COP1 function, as indicated by exaggerated hypocotyl elongation under light conditions.

YABBYs and the Transcriptional Corepressors LEUNIG and LEUNIG_HOMOLOG Maintain Leaf Polarity and Meristem Activity in Arabidopsis

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.

PCI complexes: pretty complex interactions in diverse signaling pathways

Three protein complexes (the proteasome regulatory lid, the COP9 signalosome and eukaryotic translation initiation factor 3) contain protein subunits with a well defined protein domain, the PCI domain. At least two (the COP9 signalosome and the lid) appear to share a common evolutionary origin. Recent advances in our understanding of the structure and function of the three complexes point to intriguing and unanticipated connections between the cellular functions performed by these three protein assemblies, especially between translation initiation and proteolytic protein degradation.

Translational Regulation via 5′ mRNA Leader Sequences Revealed by Mutational Analysis of the Arabidopsis Translation Initiation Factor Subunit eIF3h

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.

Arabidopsis eIF3e (INT-6) Associates with Both eIF3c and the COP9 Signalosome Subunit CSN7

The Arabidopsis COP9 signalosome is a multisubunit repressor of photomorphogenesis that is conserved among eukaryotes. This complex may have a general role in development. As a step in dissecting the biochemical mode of action of the COP9 signalosome, we determined the sequence of proteins that copurify with this complex. Here we describe the association between components of the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known also as INT-6) and eIF3c (p105). To obtain a biochemical marker forArabidopsis eIF3, we cloned the Arabidopsisortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and eIF3b cofractionated by gel filtration chromatography in a complex that was larger than the COP9 signalosome. Whereas eIF3, as detected through eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also found in the nucleus. This suggests that eIF3e and eIF3c are probably components of multiple complexes and that eIF3e and eIF3c associate with subunits of the COP9 signalosome, even though they are not components of the COP9 signalosome core complex. This interaction may allow for translational control by the COP9 signalosome.

Regulation of plant translation by upstream open reading frames.

We review the evidence that upstream open reading frames (uORFs) function as RNA sequence elements for post-transcriptional control of gene expression, specifically translation. uORFs are highly abundant in the genomes of angiosperms. Their negative effect on translation is often attenuated by ribosomal translation reinitiation, a process whose molecular biochemistry is still being investigated. Certain uORFs render translation responsive to small molecules, thus offering a path for metabolic control of gene expression in evolution and synthetic biology. In some cases, uORFs form modular logic gates in signal transduction. uORFs thus provide eukaryotes with a functionality analogous to, or comparable to, riboswitches and attenuators in prokaryotes. uORFs exist in many genes regulating development and point toward translational control of development. While many uORFs appear to be poorly conserved, and the number of genes with conserved-peptide uORFs is modest, many mRNAs have a conserved pattern of uORFs. Evolutionarily, the gain and loss of uORFs may be a widespread mechanism that diversifies gene expression patterns. Last but not least, this review includes a dedicated uORF database for Arabidopsis.

Bioluminescence resonance energy transfer: Monitoring protein-protein interactions in living cells

Publisher Summary This chapter describes a resonance energy transfer method based on bioluminescence. Fluorescence resonance energy transfer (FRET) is a well-established phenomenon that is useful in cellular microscopy. When two fluorophores (the “donor” and the “acceptor”) with overlapping emission/absorption spectra are within ∼50Ǻ of one another and their transition dipoles are appropriately oriented, the donor fluorophore is able to transfer its excited state energy to the acceptor fluorophore. Protein–protein interactions are known to play an important role in a variety of biochemical systems. To date, thousands of protein–protein interactions are identified by using the conventional two-hybrid system, but this method is limited in that the interaction must occur in the yeast nucleus. This means interactions that strictly depend on cell type-specific processing and/or compartmentalization cannot be detected. Therefore, a number of new methods are developed that rely on reconstitution of biochemical function in vivo , such as fluorescence resonance energy transfer (FRET), protein mass spectrometry, or evanescent wave spectroscopy.

Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24

BackgroundWithin 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).ResultsHere 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.ConclusionWe 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 uORF-containing mRNAs and, in turn, to orchestrate plant development.