UV responses of Lolium perenne raised along a latitudinal gradient across Europe: a filtration study.

Lolium perenne (cv. AberDart) was grown at 14 locations along a latitudinal gradient across Europe (37-68°N) to study the impact of ultraviolet radiation (UV) and climate on aboveground growth and foliar UV-B absorbing compounds. At each location, plants were grown outdoors for 5 weeks in a replicated UV-B filtration experiment consisting of open, UV-B transparent (cellulose diacetate) and UV-B opaque (polyester) environments. Fourier transform-infrared spectroscopy was used to compare plant metabolite profiles in relation to treatment and location. UV radiation and climatic parameters were determined for each location from online sources and the data were assessed using a combination of anova and multiple regression analyses. Most of the variation in growth between the locations was attributable to the combination of climatic parameters, with minimum temperature identified as an important growth constraint. However, no single environmental parameter could consistently account for the variability in plant growth. Concentrations of foliar UV-B absorbing compounds showed a positive trend with solar UV across the latitudinal gradient; however, this relationship was not consistent in all treatments. The most striking experimental outcome from this study was the effect of presence or absence of filtration frames on UV-absorbing compounds. Overall, the study demonstrates the value of an European approach in studying the impacts of natural UV across a large latitudinal gradient. We have shown the feasibility of coordinated UV filtration at multiple sites but have also highlighted the need for open controls and careful interpretation of plant responses.

Interactome of the plant-specific ESCRT-III component AtVPS2.2 in Arabidopsis thaliana.

The endosomal sorting complexes required for transport (ESCRT) guides transmembrane proteins to domains that bud away from the cytoplasm. The ESCRT machinery consists of four complexes. ESCRT complexes 0-II are important for cargo recognition and concentration via ubiquitin binding. Most of the membrane bending function is mediated by the large multimeric ESCRT-III complex and associated proteins. Here we present the first in vivo proteome analysis of a member of the ESCRT-III complex which is unique to the plant kingdom. We show with LC-MS/MS, yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) that coimmunoprecipitated proteins from Arabidopsis thaliana roots expressing a functional GFP-tagged VACUOLAR PROTEIN SORTING 2.2 (AtVPS2.2) protein are members of the ESCRT-III complex and associated proteins. Therefore we propose that at least in plants the large ESCRT-III membrane scaffolding complex consists of a mixture of SNF7, VPS2 and the associated VPS46 and VPS60 proteins. Apart from transmembrane proteins, numerous membrane-associated but also nuclear and extracellular proteins have been identified, indicating that AtVPS2.2 might be involved in processes beyond the classical ESCRT role. This study is the first in vivo proteome analysis with a tagged ESCRT-III component demonstrating the feasibility of this approach and provides numerous starting points for the investigation of the biological process in which AtVPS2.2 is involved.

UV-B signaling pathways and fluence rate dependent transcriptional regulation of ARIADNE12.

ARI12 belongs to a family of 'RING between RING fingers' (RBR) domain proteins with E3 ligase activity (Eisenhaber et al. 2007). The Arabidopsis genome codes for 14 ARI genes and two pseudogenes (Mladek et al. 2003). Under standard growth conditions ARI12 is predominantly expressed in roots. In addition, ARI12 is strongly induced in leaves following exposure to ultraviolet (UV)-B radiation at dosages similar to those in areas under a reduced ozone layer. With quantitative reverse transcription polymerase chain reaction analyses and promoter:reporter constructs we show that the expression of ARI12 peaks 2-4 h after UV-B radiation exposure. To test if ARI12's transcriptional activation depends on key players of the UV-B signaling pathway, ARI12 expression was quantified in mutants of the ELONGATED HYPOCOTYL5 (HY5), HY5 HOMOLOG (HYH) and the UV RESISTANCE LOCUS8 (UVR8) genes. ARI12 transcription was reduced by 50-70% in hy5, hyh and hy5/hyh double mutants, but not in uvr8 mutants. However, under low fluence rate UV-B conditions ARI12 is not induced in these mutants. Our results show that ARI12 represents a downstream target of the low fluence rate UVR8/HY5/HYH UV-B signaling pathway while under high fluence rates its expression is regulated by the two bZIP transcription factors HY5 and HYH in an UVR8-independent manner.

The Arabidopsis deubiquitinating enzyme AMSH3 interacts with ESCRT-III subunits and regulates their localization.

Ubiquitination and deubiquitination regulate various cellular processes. We have recently shown that the deubiquitinating enzyme Associated Molecule with the SH3 domain of STAM3 (AMSH3) is involved in vacuole biogenesis and intracellular trafficking in Arabidopsis thaliana. However, little is known about the identity of its interaction partners and deubiquitination substrates. Here, we provide evidence that AMSH3 interacts with ESCRT-III subunits VPS2.1 and VPS24.1. The interaction of ESCRT-III subunits with AMSH3 is mediated by the MIM1 domain and depends on the MIT domain of AMSH3. We further show that AMSH3, VPS2.1, and VPS24.1 localize to class E compartments when ESCRT-III disassembly is inhibited by coexpression of inactive Suppressor of K+ transport Defect 1 (SKD1), an AAA-ATPase involved in the disassembly of ESCRT-III. We also provide evidence that AMSH3 and SKD1 compete for binding to VPS2.1. Furthermore, we show that the loss of AMSH3 enzymatic activity leads to the formation of cellular compartments that contain AMSH3, VPS2.1, and VPS24.1. Taken together, our study presents evidence that AMSH3 interacts with classical core ESCRT-III components and thereby provides a molecular framework for the function of AMSH3 in plants.

Nuclear expression of apurinic/apyrimidinic endonuclease increases with progression of ovarian carcinomas.

OBJECTIVE: Apurinic/apyrimidinic endonuclease (APE alias Ref-1) is a key enzyme in the base excision repair pathway. Besides its function in DNA repair, APE serves to maintain several transcription factors in an active reduced state such as c-Fos, c-Jun, NF-kappaB, p53 and HIF-1alpha, all of which have been shown to play a role in tumorigenesis. Because of the importance of APE in maintaining genomic stability and gene regulation, we examined whether APE expression is associated with survival and histopathological parameters of patients with ovarian cancer. METHODS: Tissue sections of primary epithelial ovarian carcinomas from 141 patients were immunostained using a monoclonal antibody directed against APE. RESULTS: Nuclear expression of APE was clearly associated with progression of ovarian carcinomas. Patients with Federation of Gynecology and Obstetrics (FIGO) stages III and IV showed a higher nuclear APE expression level than patients with FIGO stages I and II (P < 0.0001). Similarly, nuclear APE expression was associated with histological grading (grade 1 vs. 2 vs. 3; P = 0.025). In contrast, cytoplasmic and stromal APE expression were not associated with progression. The fraction of APE-positive nuclei (P = 0.0185), the intensity of nuclear staining (P = 0.0496) and a combination of both (P = 0.0070) were associated with survival of ovarian cancer patients, as evidenced by a univariable proportional hazards model. CONCLUSIONS: Multivariable analysis, adjusted to FIGO stage, histological grade and type as well as residual tumor after surgery showed that APE is not independent from "classical" prognostic factors of ovarian cancer. An unexpected observation was the inverse correlation between nuclear and cytoplasmic expression of APE. Tumors with strong cytoplasmic APE reactivity showed a higher fraction of APE-negative nuclei than tumors with weak or negative cytoplasmic APE expression (P = 0.045). This suggests that nuclear translocation of APE is impaired during ovarian carcinogenesis. In conclusion, we have shown that nuclear APE expression increases during tumor progression. This suggests that increased base excision repair capacity and/or APE-mediated activation of transcription factors may contribute to more aggressive proliferation of ovarian carcinomas.

Blood flow velocity waveforms of the fetal middle cerebral artery in a normal population: reference values from 18 weeks to 42 weeks of gestation.

The aim of this prospective cross-sectional study was to establish new Doppler reference curves for peak blood flow velocities (Vmax, Vmean, Vmin) and impedance indices (PI, RI) of the middle cerebral artery at 18-42 weeks of gestation by an automatic wave form analysis integrated into the ultrasound device. In 962 low-risk pregnancies, blood flow velocities were derived from the middle cerebral artery with pulsed color Doppler ultrasonography. Reference curves were constructed for the individual parameters based on a growth function from a four-parameter class of monotonic continuous functions according to the smallest square principle for maximum blood flow velocities, as well as on a polynomial function for resistance indices. Further assessed was intra-observer reliability. The results obtained for maximum blood flow velocities demonstrate a continuous increase (Vmax = 26.8 cm/s to 59.1 cm/s, Vmean = 11.2 cm/s to 29.7 cm/s and Vmin = 4.9 to 15.8 cm/s) over the period from 18 to 42 weeks of gestation. In contrast, reference curves for the pulsatility and the resistance index are characterized by a parabolic pattern (18 weeks: 1.5; 28 weeks: 1.9; 42 weeks: 1.1 and 18 weeks: 0.68; 28 weeks 0.8; 42 weeks: 0.61). Intraobserver reliability for PI, RI, Vmax, Vmean, and Vmax were 87%, 83%, 80%, 83% and 93%, respectively. At constant measurement conditions, the reference ranges for blood flow velocities and impedance indices in the middle cerebral artery established by this study were found to be a useful non-invasive tool in risk assessment for fetal anemia, as well as in the Doppler ultrasound observation of the course in fetuses with intrauterine growth restriction.