COP1 destabilizes DELLA proteins in Arabidopsis.

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis thaliana Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed in Nicotiana benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.

DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis.

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

Tandem Affinity Purification of Protein Complexes from Arabidopsis Cell Cultures.

Tandem affinity purification (TAP) coupled to mass spectrometry has become a powerful approach to identify protein-protein interactions from different biological systems, including plants, in a proteome-wide manner. By using two sequential affinity purification steps, TAP allows for isolation of high-purity TAP-tagged proteins of interest and their associated proteins. Here we describe optimized procedures to use the GSRhino TAP technology for protein complex isolation from Arabidopsis cell suspension cultures.

Epigenetic switch from repressive to permissive chromatin in response to cold stress.

Switching from repressed to active status in chromatin regulation is part of the critical responses that plants deploy to survive in an ever-changing environment. We previously reported that HOS15, a WD40-repeat protein, is involved in histone deacetylation and cold tolerance in Arabidopsis However, it remained unknown how HOS15 regulates cold responsive genes to affect cold tolerance. Here, we show that HOS15 interacts with histone deacetylase 2C (HD2C) and both proteins together associate with the promoters of cold-responsive COR genes, COR15A and COR47 Cold induced HD2C degradation is mediated by the CULLIN4 (CUL4)-based E3 ubiquitin ligase complex in which HOS15 acts as a substrate receptor. Interference with the association of HD2C and the COR gene promoters by HOS15 correlates with increased acetylation levels of histone H3. HOS15 also interacts with CBF transcription factors to modulate cold-induced binding to the COR gene promoters. Our results here demonstrate that cold induces HOS15-mediated chromatin modifications by degrading HD2C. This switches the chromatin structure status and facilitates recruitment of CBFs to the COR gene promoters. This is an apparent requirement to acquire cold tolerance.

Prefoldins Negatively Regulate Cold Acclimation in Arabidopsis thaliana by Promoting Nuclear Proteasome-Mediated HY5 Degradation.

The process of cold acclimation is an important adaptive response whereby many plants from temperate regions increase their freezing tolerance after being exposed to low non-freezing temperatures. The correct development of this response relies on proper accumulation of a number of transcription factors that regulate expression patterns of cold-responsive genes. Multiple studies have revealed a variety of molecular mechanisms involved in promoting the accumulation of these transcription factors. Interestingly, however, the mechanisms implicated in controlling such accumulation to ensure their adequate levels remain largely unknown. In this work, we demonstrate that prefoldins (PFDs) control the levels of HY5, an Arabidopsis transcription factor with a key role in cold acclimation by activating anthocyanin biosynthesis, in response to low temperature. Our results show that, under cold conditions, PFDs accumulate into the nucleus through a DELLA-dependent mechanism, where they interact with HY5, triggering its ubiquitination and subsequent degradation. The degradation of HY5 would result, in turn, in anthocyanin biosynthesis attenuation, ensuring the accurate development of cold acclimation. These findings uncover an unanticipated nuclear function for PFDs in plant responses to abiotic stresses.

The Arabidopsis Iron-Sulfur Protein GRXS17 is a Target of the Ubiquitin E3 Ligases RGLG3 and RGLG4.

The stability of signaling proteins in eukaryotes is often controlled by post-translational modifiers. For polyubiquitination, specificity is assured by E3 ubiquitin ligases. Although plant genomes encode hundreds of E3 ligases, only few targets are known, even in the model Arabidopsis thaliana. Here, we identified the monothiol glutaredoxin GRXS17 as a substrate of the Arabidopsis E3 ubiquitin ligases RING DOMAIN LIGASE 3 (RGLG3) and RGLG4 using a substrate trapping approach involving tandem affinity purification of RING-dead versions. Simultaneously, we used a ubiquitin-conjugating enzym (UBC) panel screen to pinpoint UBC30 as a cognate E2 UBC capable of interacting with RGLG3 and RGLG4 and mediating auto-ubiquitination of RGLG3 and ubiquitination of GRXS17 in vitro. Accordingly, GRXS17 is ubiquitinated and degraded in an RGLG3- and RGLG4-dependent manner in planta. The truncated hemoglobin GLB3 also interacted with RGLG3 and RGLG4 but appeared to obstruct RGLG3 ubiquitination activity rather than being its substrate. Our results suggest that the RGLG family is intimately linked to the essential element iron.

Detection by real time PCR of walnut allergen coding sequences in processed foods.

A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB-phenol-chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays.

Detection of almond allergen coding sequences in processed foods by real time PCR.

The aim of this work was to develop and analytically validate a quantitative RT-PCR method, using novel primer sets designed on Pru du 1, Pru du 3, Pru du 4, and Pru du 6 allergen-coding sequences, and contrast the sensitivity and specificity of these probes. The temperature and/or pressure processing influence on the ability to detect these almond allergen targets was also analyzed. All primers allowed a specific and accurate amplification of these sequences. The specificity was assessed by amplifying DNA from almond, different Prunus species and other common plant food ingredients. The detection limit was 1 ppm in unprocessed almond kernels. The method's robustness and sensitivity were confirmed using spiked samples. Thermal treatment under pressure (autoclave) reduced yield and amplificability of almond DNA; however, high-hydrostatic pressure treatments did not produced such effects. Compared with ELISA assay outcomes, this RT-PCR showed higher sensitivity to detect almond traces in commercial foodstuffs.

Targeted degradation of abscisic acid receptors is mediated by the ubiquitin ligase substrate adaptor DDA1 in Arabidopsis.

CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)-related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDB1-ASSOCIATED1 (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABA-mediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability.

A Novel Proteomic Analysis of the Modifications Induced by High Hydrostatic Pressure on Hazelnut Water-Soluble Proteins.

Food allergies to hazelnut represent an important health problem in industrialized countries because of their high prevalence and severity. Food allergenicity can be changed by several processing procedures since food proteins may undergo modifications which could alter immunoreactivity. High-hydrostatic pressure (HHP) is an emerging processing technology used to develop novel and high-quality foods. The effect of HHP on allergenicity is currently being investigated through changes in protein structure. Our aim is to evaluate the effect of HHP on the protein profile of hazelnut immunoreactive extracts by comparative proteomic analysis with ProteomeLab PF-2D liquid chromatography and mass spectrometry. This protein fractionation method resolves proteins by isoelectric point and hydrophobicity in the first and second dimension, respectively. Second dimension chromatogram analyses show that some protein peaks present in unpressurized hazelnut must be unsolubilized and are not present in HHP-treated hazelnut extracts. Our results show that HHP treatment at low temperature induced marked changes on hazelnut water-soluble protein profile.

Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis.

Anthocyanins are natural pigments that accumulate only in light-grown and not in dark-grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark-grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post-translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants.

Real Time PCR to detect hazelnut allergen coding sequences in processed foods.

A quantitative RT-PCR method, employing novel primer sets designed on Cor a 9, Cor a 11 and Cor a 13 allergen-coding sequences has been setup and validated. Its specificity, sensitivity and applicability have been compared. The effect of processing on detectability of these hazelnut targets in complex food matrices was also studied. The DNA extraction method based on CTAB-phenol-chloroform was the best for hazelnut. RT-PCR using primers for Cor a 9, 11 and 13 allowed a specific and accurate amplification of these sequences. The limit of detection was 1 ppm of raw hazelnut. The method sensitivity and robustness were confirmed with spiked samples. Thermal treatments (roasting and autoclaving) reduced yield and amplificability of hazelnut DNA, however, high-hydrostatic pressure did not affect. Compared with an ELISA assay, this RT-PCR showed higher sensitivity to detected hazelnut traces in commercial foodstuffs. The RT-PCR method described is the most sensitive of those reported for the detection of hazelnut traces in processed foods.