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.

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.

APRF1 promotes flowering under long days in Arabidopsis thaliana.

Arabidopsis thaliana flowering time mutants revealed the function of numerous genes that regulate the transition from vegetative to reproductive growth. Analyses of their loci have shown that many of them act as chromatin modifiers. In this study, a combination of molecular and genetic approaches have been implemented, to characterize the function of APRF1 (ANTHESIS POMOTING FACTOR 1) gene in A. thaliana and to investigate its role in plant development. APRF1 encodes for a low molecular weight nuclear WDR protein which displays functional homology to the Swd2 protein, an essential subunit of the yeast histone methylation COMPASS complex. Compared to WT plants, total loss-of-function aprf1 mutants exhibited shoot apical meristem (SAM) alterations and increased growth rates. However, the vegetative phase of aprf1 plants was prolonged and bolting was delayed, indicating an impairment in flowering under long days (LD). On the contrary, overexpression of APRF1 accelerates flowering. Consistent with the late flowering phenotype, the molecular data confirmed that FLC and SOC1 expression were significantly altered in the aprf1 mutants. Our data suggest that APRF1 acts upstream of FLC and promotes flowering under LD.

RNAi-mediated silencing of the Arabidopsis thaliana ULCS1 gene, encoding a WDR protein, results in cell wall modification impairment and plant infertility.

Ubiquitin mediated protein degradation constitutes one of the most complex post translational gene regulation mechanisms in eukaryotes. This fine-tuned proteolytic machinery is based on a vast number of E3 ubiquitin ligase complexes that mark target proteins with ubiquitin. The specificity is accomplished by a number of adaptor proteins that contain functional binding domains, including the WD40 repeat motif (WDRs). To date, only few of these proteins have been identified in plants. An RNAi mediated silencing approach was used here to functionally characterize the Arabidopsis thaliana ULCS1 gene, which encodes for a small molecular weight WDR protein. AtULCS1 interacts with the E3Cullin Ring Ligase subunit DDB1a, regulating most likely the degradation of specific proteins involved in the manifestation of diverse developmental events. Silencing of AtULCS1 results in sterile plants with pleiotropic phenotypes. Detailed analysis revealed that infertility is the outcome of anther indehiscence, which in turn is due to the impairment of the plants to accomplish secondary wall modifications. Furthermore, IREGULAR XYLEM gene expression and lignification is diminished in anther endothecium and the stem vascular tissue of the silenced plants. These data underline the importance of AtULCS1 in plant development and reproduction.