APRF1 Interactome Reveals HSP90 as a New Player in the Complex That Epigenetically Regulates Flowering Time in Arabidopsis thaliana.

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana, such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 (aprf1) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

The SAH7 Homologue of the Allergen Ole e 1 Interacts with the Putative Stress Sensor SBP1 (Selenium-Binding Protein 1) in Arabidopsis thaliana.

In this study, we focused on a member of the Ole e 1 domain-containing family, AtSAH7, in Arabidopsis thaliana. Our lab reports for the first time on this protein, AtSAH7, that was found to interact with Selenium-binding protein 1 (AtSBP1). We studied by GUS assisted promoter deletion analysis the expression pattern of AtSAH7 and determined that the sequence 1420 bp upstream of the transcription start can act as a minimal promoter inducing expression in vasculature tissues. Moreover, mRNA levels of AtSAH7 were acutely increased under selenite treatment in response to oxidative stress. We confirmed the aforementioned interaction in vivo, in silico and in planta. Following a bimolecular fluorescent complementation approach, we determined that the subcellular localization of the AtSAH7 and the AtSAH7/AtSBP1 interaction occur in the ER. Our results indicate the participation of AtSAH7 in a biochemical network regulated by selenite, possibly associated with responses to ROS production.

GUS Reporter-Aided Promoter Deletion Analysis of A. thaliana POLYAMINE OXIDASE 3.

Polyamine oxidases (PAOs) have been correlated with numerous physiological and developmental processes, as well as responses to biotic and abiotic stress conditions. Their transcriptional regulation is driven by signals generated by various developmental and environmental cues, including phytohormones. However, the inductive mechanism(s) of the corresponding genes remains elusive. Out of the five previously characterized Arabidopsis PAO genes, none of their regulatory sequences have been analyzed to date. In this study, a GUS reporter-aided promoter deletion approach was used to investigate the transcriptional regulation of AtPAO3 during normal growth and development as well as under various inductive environments. AtPAO3 contains an upstream open reading frame (uORF) and a short inter-cistronic sequence, while the integrity of both appears to be crucial for the proper regulation of gene expression. The full-length promoter contains several cis-acting elements that regulate the tissue-specific expression of AtPAO3 during normal growth and development. Furthermore, a number of TFBS that are involved in gene induction under various abiotic stress conditions display an additive effect on gene expression. Taken together, our data indicate that the transcription of AtPAO3 is regulated by multiple environmental factors, which probably work alongside hormonal signals and shed light on the fine-tuning mechanisms of PAO regulation.

RNAi silencing of the Arabidopsis thaliana ULCS1 gene results in pleiotropic phenotypes during plant growth and development.

WD40-repeat-containing proteins (WDRs) are highly abundant in all eukaryotes. Several have been implicated as subunits of multi-protein CRL E3 ligase complexes that regulate ubiquitination mediated protein degradation and thus various cellular and developmental processes. Impairment of the WDR protein ULCS1 from Arabidopsis causes pleiotropic phenotypes during plant development, including reduced lignification, anther indehiscence, and sterility. Here we show that RNAi-mediated downregulation of ULCS1 results in a fast-growing phenotype during vegetative development. Due to accelerated growth, ulcs1i mutants reach their vegetative to reproductive transition point earlier than WT plants. However, their comparable germination rate and their similar number of secondary branches and rosette leaves at bolting indicate that ulcs1i is not an early flowering time mutant. GUS staining of progeny, obtained from crosses between ulcs1i and CYCB1::GUS plants, revealed an increased number of mitotic cell divisions in the root meristems of ulcs1i compared to WT. Immunolabeling of homogalacturonans (HGAs) epitopes showed significant fluorescent signal differences at the cell walls and the mucilage of the seeds between ulcs1i and WT. Furthermore, we demonstrate that ULCS1 interacts with the UBA-like protein in a yeast two-hybrid assay, suggesting a direct or indirect physical coupling of these proteins in Arabidopsis.

Investigation of the interaction of DAD1-LIKE LIPASE 3 (DALL3) with Selenium Binding Protein 1 (SBP1) in Arabidopsis thaliana.

Phospholipase PLA1-Iγ2 or otherwise DAD1-LIKE LIPASE 3 (DALL3) is a member of class I phospholipases and has a role in JA biosynthesis. AtDALL3 was previously identified in a yeast two-hybrid screening as an interacting protein of the Arabidopsis Selenium Binding Protein 1 (SBP1). In this work, we have studied AtDALL3 as an interacting partner of the Arabidopsis Selenium Binding Protein 1 (SBP1). Phylogenetic analysis showed that DALL3 appears in the PLA1-Igamma1, 2 group, paired with PLA1-Igammma1. The highest level of expression of AtDALL3 was observed in 10-day-old roots and in flowers, while constitutive levels were maintained in seedlings, cotyledons, shoots and leaves. In response to abiotic stress, DALL3 was shown to participate in the network of genes regulated by cadmium, selenite and selenate compounds. DALL3 promoter driven GUS assays revealed that the expression patterns defined were overlapping with the patterns reported for AtSBP1 gene, indicating that DALL3 and SBP1 transcripts co-localize. Furthermore, quantitative GUS assays showed that these compounds elicited changes in activity in specific cells files, indicating the differential response of DALL3 promoter. GFP::DALL3 studies by confocal microscopy demonstrated the localization of DALL3 in the plastids of the root apex, the plastids of the central root and the apex of emerging lateral root primordia. Additionally, we confirmed by yeast two hybrid assays the physical interaction of DALL3 with SBP1 and defined a minimal SBP1 fragment that DALL3 binds to. Finally, by employing bimolecular fluorescent complementation we demonstrated the in planta interaction of the two proteins.

Novel interactions of Selenium Binding Protein family with the PICOT containing proteins AtGRXS14 and AtGRXS16 in Arabidopsis thaliana.

During abiotic stress the primary symptom of phytotoxicity can be ROS production which is strictly regulated by ROS scavenging pathways involving enzymatic and non-enzymatic antioxidants. Furthermore, ROS are well-described secondary messengers of cellular processes, while during the course of evolution, plants have accomplished high degree of control over ROS and used them as signalling molecules. Glutaredoxins (GRXs) are small and ubiquitous glutathione (GSH) -or thioredoxin reductase (TR)-dependent oxidoreductases belonging to the thioredoxin (TRX) superfamily which are conserved in most eukaryotes and prokaryotes. In Arabidopsis thaliana GRXs are subdivided into four classes playing a central role in oxidative stress responses and physiological functions. In this work, we describe a novel interaction of AtGRXS14 with the Selenium Binding Protein 1 (AtSBP1), a protein proposed to be integrated in a regulatory network that senses alterations in cellular redox state and acts towards its restoration. We further show that SBP protein family interacts with AtGRXS16 that also contains a PICOT domain, like AtGRXS14.

Molecular and functional characterization of Arabidopsis thaliana VPNB1 gene involved in plant vascular development.

Armadillo (ARM) repeat containing proteins constitute a large family in plants and are involved in diverse cellular functions, like signal transduction, proliferation and differentiation. In animals, ARM repeat proteins have been implicated in cancer development. In this study, we aimed in characterizing the VPNB1 gene from Arabidopsis thaliana and its role in plant development, by implementing a number of genetic and molecular approaches. AtVPNB1 encodes for an ARM repeat protein of unknown function, exclusively expressed in the cambium as well as in the differentiating xylem and phloem cells of the vascular system. Subcellular localization experiments showed that VPNB is confined in nucleoplasmic speckle-like structures unrelated to cajal bodies. Transgenic VPNB-impaired plants exhibit a slower growing phenotype and a non-canonical pattern of xylem tissue. On the contrary, VPNB overexpression lines display an inverted phenotype of increased growth, accompanied by an increased deposition of phloem and xylem cell layers. In line with the above data, qPCR analysis revealed a deregulation of several key master genes of secondary wall biosynthesis, underlining the involvement of VPNB1 in the regulation and differentiation of the root and shoot vascular tissue.

Transcriptional regulation and functional involvement of the Arabidopsis pescadillo ortholog AtPES in root development.

The Pescadillo gene is highly conserved from yeasts to human and has been shown to impact on both the cell cycle and on ribosome biogenesis. However, the biological function and transcriptional regulation of the plant orthologs remain unclear. In the present study, we have implemented a combination of molecular and genetic approaches, in order to characterize the Arabidopsis thaliana pescadillo ortholog (AtPES) and its role in root development. The RNAi transgenic lines displayed severely compromised meristem structures and a reduction of the primary root length of up to 70%. The correct pattern of the cell files is distorted, whereas in the root elongation and differentiation zone the epidermal and cortex cells appear abnormally enlarged. Yeast two hybrid and BiFC experiments confirmed that AtPES interacts physically with AtPEIP1 and AtPEIP2, the orthologs of the murine Bop1 and WDR12. Promoter deletion analysis revealed that AtPES expression depends on a number of transcription factor binding sites, with the TELO-box being a crucial site for regulating its accurate tissue-specific manifestation. Our results indicate that AtPES is firmly regulated at the transcriptional level and that the corresponding protein plays a role in root developmental processes.

Comparison of two gamma interferon release assays and tuberculin skin testing for tuberculosis screening in a cohort of patients with rheumatic diseases starting anti-tumor necrosis factor therapy.

Gamma interferon release assays (IGRAs) are increasingly used for latent Mycobacterium tuberculosis infection (LTBI) screening in patients with rheumatic diseases starting anti-tumor necrosis factor (anti-TNF) therapies. We compared the performances of two IGRAs, an enzyme-linked immunospot release assay (T-SPOT.TB) and an enzyme-linked immunosorbent assay (QuantiFERON-TB Gold In Tube [QFT-GIT]), to that of tuberculin skin testing (TST) for LTBI screening of 157 consecutive rheumatic patients starting anti-TNF therapies. Among 155 patients with valid results, 58 (37%) were positive by TST, 39 (25%) by T-SPOT.TB assay, and 32 (21%) by QFT-GIT assay. IGRAs were associated more strongly with at least one risk factor for tuberculosis (TB) than TST. Risk factors for a positive assay included chest X-ray findings of old TB (TST), advanced age (both IGRAs), origin from a country with a high TB prevalence, and a positive TST (T-SPOT.TB assay). Steroid use was negatively associated with a positive QFT-GIT assay. The agreement rate between IGRAs was 81% (kappa rate = 0.47), which was much higher than that observed between an IGRA and TST. If positivity by either TST or an IGRA was required for LTBI diagnosis, then the rate of LTBI would have been 46 to 47%, while if an IGRA was performed only for TST-positive patients, the respective rate would have been 11 to 17%. In conclusion, IGRAs appear to correlate better with TB risk than TST and should be included in TB screening of patients starting anti-TNF therapies. In view of the high risk of TB in these patients, a combination of one IGRA and TST is probably more appropriate for LTBI diagnosis.