Cryptochromes Interact Directly with PIFs to Control Plant Growth in Limiting Blue Light.

Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome wide and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions.

Mapping transcription factor interactome networks using HaloTag protein arrays.

Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.

Spatial Regulation of the Gene Expression Response to Shade in Arabidopsis Seedlings.

The shade avoidance response, which allows plants to escape from nearby competitors, is triggered by a reduction in the PFR form of phytochrome in response to shade. Classic physiological experiments have demonstrated that the shade signal perceived by the leaves is transmitted to the other parts of the plant. Recently, a simple method was developed to analyze the transcriptome in a single microgram tissue sample. In the present study, we adopted this method to conduct organ-specific transcriptomic analysis of the shade avoidance response in Arabidopsis seedlings. The shoot apical samples, which contained the meristem, basal parts of leaf primordia and short fragments of vasculature, were collected from the topmost part of the hypocotyl and subjected to RNA sequencing analysis. Unexpectedly, many more genes were up-regulated in the shoot apical region than in the cotyledons. Spotlight irradiation demonstrated that the apex-responsive genes were mainly controlled by phytochrome in the cotyledons. In accordance with the involvement of many auxin-responsive genes in this category, auxin biosynthesis was genetically shown to be essential for this response. In contrast, organ-autonomous regulation was more important for the genes that were up-regulated preferentially either in the cotyledons or in both the cotyledons and the apical region. Their responses to shade depended variously on auxin and PIFs (phytochrome-interacting factors), indicating the mechanistic diversity of the organ-autonomous response. Finally, we examined the expression of the auxin synthesis genes, the YUC genes, and found that three YUC genes, which were differently spatially regulated, co-ordinately elevated the auxin level within the shoot apical region.

PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis.

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group-A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA-binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1-PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co-immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1-ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase-PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR-mediated ABA signalling.

Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein-protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein-protein interactions simultaneously in plant cells.

Tyrosine phosphorylation regulates the activity of phytochrome photoreceptors.

Phytochromes are red/far-red light receptors that function in photomorphogenesis of plants. Photoisomerization of phytochrome by red light leads to its translocation to the nucleus, where it regulates gene expression. We examined whether phytochrome is phosphorylated in response to light, and we report that phytochrome B (phyB)'s N terminus contains a region with a number of phosphoserines, threonines, and tyrosines. The light-dependent phosphorylation of tyrosine 104 (Y104) appears to play a negative role in phyB's activity, because a phosphomimic mutant, phyBY104E, is unable to complement any phyB-related phenotype, is defective in binding to its signaling partner PIF3, and fails to form stable nuclear bodies even though it retains normal photochemistry in vitro. In contrast, plants stably expressing a nonphosphorylatable mutant, phyBY104F, are hypersensitive to light. The proper response to changes in the light environment is crucial for plant survival, and our study brings tyrosine phosphorylation to the forefront of light-signaling mechanisms.

Functional classification of Arabidopsis peroxisome biogenesis factors proposed from analyses of knockdown mutants.

In higher plants, peroxisomes accomplish a variety of physiological functions such as lipid catabolism, photorespiration and hormone biosynthesis. Recently, many factors regulating peroxisomal biogenesis, so-called PEX genes, have been identified not only in plants but also in yeasts and mammals. In the Arabidopsis genome, the presence of at least 22 PEX genes has been proposed. Here, we clarify the physiological functions of 18 PEX genes for peroxisomal biogenesis by analyzing transgenic Arabidopsis plants that suppressed the PEX gene expression using RNA interference. The results indicated that the function of these PEX genes could be divided into two groups. One group involves PEX1, PEX2, PEX4, PEX6, PEX10, PEX12 and PEX13 together with previously characterized PEX5, PEX7 and PEX14. Defects in these genes caused loss of peroxisomal function due to misdistribution of peroxisomal matrix proteins in the cytosol. Of these, the pex10 mutant showed pleiotropic phenotypes that were not observed in any other pex mutants. In contrast, reduced peroxisomal function of the second group, including PEX3, PEX11, PEX16 and PEX19, was induced by morphological changes of the peroxisomes. Cells of the pex16 mutant in particular possessed reduced numbers of large peroxisome(s) that contained unknown vesicles. These results provide experimental evidence indicating that all of these PEX genes play pivotal roles in regulating peroxisomal biogenesis. We conclude that PEX genes belonging to the former group are involved in regulating peroxisomal protein import, whereas those of the latter group are important in maintaining the structure of peroxisome.

The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2-dependent protein transport to peroxisomes.

Peroxisome biogenesis requires various complex processes including organelle division, enlargement and protein transport. We have been studying a number of Arabidopsis apm mutants that display aberrant peroxisome morphology. Two of these mutants, apm2 and apm4, showed green fluorescent protein fluorescence in the cytosol as well as in peroxisomes, indicating a decrease of efficiency of peroxisome targeting signal 1 (PTS1)-dependent protein transport to peroxisomes. Interestingly, both mutants were defective in PTS2-dependent protein transport. Plant growth was more inhibited in apm4 than apm2 mutants, apparently because protein transport was more severely decreased in apm4 than in apm2 mutants. APM2 and APM4 were found to encode proteins homologous to the peroxins PEX13 and PEX12, respectively, which are thought to be involved in transporting matrix proteins into peroxisomes in yeasts and mammals. We show that APM2/PEX13 and APM4/PEX12 are localized on peroxisomal membranes, and that APM2/PEX13 interacts with PEX7, a cytosolic PTS2 receptor. Additionally, a PTS1 receptor, PEX5, was found to stall on peroxisomal membranes in both mutants, suggesting that PEX12 and PEX13 are components that are involved in protein transport on peroxisomal membranes in higher plants. Proteins homologous to PEX12 and PEX13 have previously been found in Arabidopsis but it is not known whether they are involved in protein transport to peroxisomes. Our findings reveal that APM2/PEX13 and APM4/PEX12 are responsible for matrix protein import to peroxisomes in planta.

Differential contribution of two peroxisomal protein receptors to the maintenance of peroxisomal functions in Arabidopsis.

Peroxisomes in higher plant cells are known to differentiate in function depending on the cell type. Because of the functional differentiation, plant peroxisomes are subdivided into several classes, such as glyoxysomes and leaf peroxisomes. These peroxisomal functions are maintained by import of newly synthesized proteins containing one of two peroxisomal targeting signals known as PTS1 and PTS2. These targeting signals are known to be recognized by the cytosolic receptors, Pex5p and Pex7p, respectively. To demonstrate the contribution of Pex5p and Pex7p to the maintenance of peroxisomal functions in plants, double-stranded RNA constructs were introduced into the genome of Arabidopsis thaliana. Expression of the PEX5 and PEX7 genes was efficiently reduced by the double-stranded RNA-mediated interference in the transgenic Arabidopsis. The Pex5p-deficient Arabidopsis showed reduced activities for both glyoxysomal and leaf peroxisomal functions. An identical phenotype was observed in a transgenic Arabidopsis overexpressing functionally defective Pex5p. In contrast, the Pex7p-deficient Arabidopsis showed reduced activity for glyoxysomal function but not for leaf peroxisomal function. Analyses of peroxisomal protein import in the transgenic Arabidopsis revealed that Pex5p was involved in import of both PTS1-containing proteins and PTS2-containing proteins, whereas Pex7p contributed to the import of only PTS2-containing proteins. Overall, the results indicated that Pex5p and Pex7p play different roles in the maintenance of glyoxysomal and leaf peroxisomal functions in plants.

Direct interaction and determination of binding domains among peroxisomal import factors in Arabidopsis thaliana.

We analyzed the role of Arabidopsis orthologues of human Pex14p, Pex5p and Pex7p that are central components of peroxisomal protein import machinery. Immunoblot analysis showed that AtPex14p and AtPex5p were present in most organs in Arabidopsis, suggesting that these factors play a role in the main protein import pathways for plant peroxisomes. Two-hybrid analysis showed that AtPex14p interacted with AtPex5p, but not with AtPex7p. In addition, AtPex7p was bound to AtPex5p, indicating that the PTS2 pathway depends on the PTS1 pathway in Arabidopsis. Further analysis showed that the nine WXXXF/Y repeats in the amino acids 231K-450D and 1M-230V of AtPex5p are bound to two N-terminal domains, amino acids 58I-65L and 78R-97R of AtPex14p and the C-terminal amino acids 266Y-317S of AtPex7p, respectively. Since the binding domains of AtPex5p to AtPex14p and AtPex7p do not overlap, AtPex14p, AtPex5p and AtPex7p might form their complex and function cooperatively in peroxisomal protein import.

Functional differentiation of peroxisomes revealed by expression profiles of peroxisomal genes in Arabidopsis thaliana.

It is well known that peroxisomal matrix proteins contain one of two targeting signals, PTS1 and PTS2. We comprehensively surveyed genes related to peroxisomal function and biogenesis in the entire Arabidopsis genome sequence. Here, we identified 256 gene candidates of PTS1- and PTS2-containing proteins and another 30 genes of non-PTS-containing proteins. Of these, only 29 proteins have been reported to be functionally characterized as peroxisomal proteins in higher plants. We extensively investigated expression profiles of genes described above in various organs of Arabidopsis: Statistical analyses of these expression profiles revealed that peroxisomal genes could be divided into five groups. One group showed ubiquitous expression in all organs examined, while the other four were classified as showing organ-specific expression in seedlings, cotyledons, roots and in both cotyledons and leaves. These data proposed more detailed description of differentiation of plant peroxisomes.

Molecular characterization of an Arabidopsis acyl-coenzyme a synthetase localized on glyoxysomal membranes.

In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the beta-oxidation pathway, fatty acids need to be activated to acyl-coenzyme As (CoAs) by the enzyme acyl-CoA synthetase (ACS; EC 6.2.1.3). Here, we report the characterization of an Arabidopsis cDNA clone encoding for a glyoxysomal acyl-CoA synthetase designated AtLACS6. The cDNA sequence is 2,106 bp long and it encodes a polypeptide of 701 amino acids with a calculated molecular mass of 76,617 D. Analysis of the amino-terminal sequence indicates that acyl-CoA synthetase is synthesized as a larger precursor containing a cleavable amino-terminal presequence so that the mature polypeptide size is 663 amino acids. The presequence shows high similarity to the typical PTS2 (peroxisomal targeting signal 2). The AtLACS6 also shows high amino acid identity to prokaryotic and eukaryotic fatty acyl-CoA synthetases. Immunocytochemical and cell fractionation analyses indicated that the AtLACS6 is localized on glyoxysomal membranes. AtLACS6 was overexpressed in insect cells and purified to near homogeneity. The purified enzyme is particularly active on long-chain fatty acids (C16:0). Results from immunoblot analysis revealed that the expression of both AtLACS6 and beta-oxidation enzymes coincide with fatty acid degradation. These data suggested that AtLACS6 might play a regulatory role both in fatty acid import into glyoxysomes by making a complex with other factors, e.g. PMP70, and in fatty acid beta-oxidation activating the fatty acids.

Ped3p is a peroxisomal ATP-binding cassette transporter that might supply substrates for fatty acid beta-oxidation.

Glyoxysomes, a group of specialized peroxisomes, are organelles that degrade fatty acids by the combination of fatty acid beta-oxidation and glyoxylate cycle. However, the mechanism underlying the transport of the fatty acids across the peroxisomal membrane is still obscure in higher plant cells. We identified and analyzed the PED3 gene and its gene product, Ped3p. The phenotype of the Arabidopsis ped3 mutant indicated that the mutation in the PED3 gene inhibits the activity of fatty acid beta-oxidation. Ped3p is a 149-kDa protein that exists in peroxisomal membranes. The amino acid sequence of Ped3p had a typical characteristic for "full-size" ATP-binding cassette (ABC) transporter consisting of two transmembrane regions and two ATP-binding regions. This protein was divided into two parts, that had 32% identical amino acid sequences. Each part showed a significant sequence similarity with peroxisomal "half" ABC transporters so far identified in mammals and yeast. Ped3p may contribute to the transport of fatty acids and their derivatives across the peroxisomal membrane.

Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor.

Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.