Molecular characterisation of the day neutral flowering (dnf) mutant in Arabidopsis thaliana

For many plants day length is critical for the control of flowering time, as the ability to respond to environmental signals is fundamental for the induction of flowering in optimal conditions. Arabidopsis is a facultative long day plant and as such flowers under both long and short days but sooner under long day conditions. To identify novel flowering mutants of the photoperiodic pathway a T-DNA tagged population of Arabidopsis was obtained from NASC (INRA Versailles lines) and screened for altered flowering times in long and short days. A novel flowering time mutant, termed day neutral flowering (dnf) was isolated as a result of this screen which displayed no short-day inhibition of flowering. The dnf mutant, therefore, flowers at the same time under both long and short day conditions. Complementation of the dnf mutant was carried out by re-introducing the wild-type DNF gene under the control of its endogenous promoter. Further complementation was also achieved using N-terminal TAP-tag fusion proteins. This complementation confirmed that the dnf mutation is responsible for the observed phenotype. The ability to perceive daylength in Arabidopsis is achieved by the coincidence of light with (CONSTANS) CO expression. This promotes the expression of FLOWERING LOCUS T (FT) which in turn leads to the promotion of flowering. Quantitative real-time PCR (qRT-PCR) has shown that the expression of both CO and FT is altered in the dnf mutant, which is indicative of its early flowering phenotype. Furthermore the expression of GIGANTEA (GI) was unaltered in dnf. These results position DNF upstream of CO and downstream of GI within this photoperiodic flowering pathway. Crosses between dnf and the co-2 mutant flowered at the same time as the co-2 mutant and as such confirms that DNF lies on the photoperiodic flowering pathway. The fact that dnf flowers early in SD combined with the qRT-PCR analysis of GI, CO and FT suggest that DNF plays a role in the repression of CO in such photoperiods. The DNF protein is 141 amino acids in length and contains a predicted membrane spanning domain between residues 13 and 33 and a putative RING finger domain between residue 79 and 121. This putative ring finger domain within DNF shows sequence similarity to both an E3 ligase and a PHD domain. Many E3 ubiquitin ligases have been shown to target specific proteins for degradation by the 26S proteosome whereas PHD domains have been implicated in the regulation of gene transcription through chromatin remodelling. The biochemical function of DNF is unknown, however one method to elucidate its role is to look at possible protein interactors. With this aim in mind we have decided to use an in vivo pull-down approach and have produced plants expressing a TAP tagged DNF protein for this purpose. However, due to time constraints pull down and MASS SPEC analysis was unable to be performed. The results of EGFP expression assays revealed that DNF protein is localised in the cytoplasm when driven by its native promoter and in the cytoplasm and nucleus of plant cells when over-expressed from the strong P35S promoter. The hypothesised role of DNF in the control of flowering will be discussed

A specific group of genes respond to cold dehydration stress in cut Alstroemeria flowers whereas ambient dehydration stress accelerates developmental senescence expression patterns

Petal development and senescence entails a normally irreversible process. It starts with petal expansion and pigment production, and ends with nutrient remobilization and ultimately cell death. In many species this is accompanied by petal abscission. Post-harvest stress is an important factor in limiting petal longevity in cut flowers and accelerates some of the processes of senescence such as petal wilting and abscission. However, some of the effects of moderate stress in young flowers are reversible with appropriate treatments. Transcriptomic studies have shown that distinct gene sets are expressed during petal development and senescence. Despite this, the overlap in gene expression between developmental and stress-induced senescence in petals has not been fully investigated in any species. Here a custom-made cDNA microarray from Alstroemeria petals was used to investigate the overlap in gene expression between developmental changes (bud to first sign of senescence) and typical post-harvest stress treatments. Young flowers were stressed by cold or ambient temperatures without water followed by a recovery and rehydration period. Stressed flowers were still at the bud stage after stress treatments. Microarray analysis showed that ambient dehydration stress accelerates many of the changes in gene expression patterns that would normally occur during developmental senescence. However, a higher proportion of gene expression changes in response to cold stress were specific to this stimulus and not senescence related. The expression of 21 transcription factors was characterized, showing that overlapping sets of regulatory genes are activated during developmental senescence and by different stresses

Deubiquitinating enzymes AtUBP12 and AtUBP13 and their tobacco homologue NtUBP12 are negativeregulators of plant immunity

Signalling by ubiquitination is implicated in diverse aspects of the plant lifecycle, and enzymes of ubiquitin metabolism are overrepresented in the Arabidopsis genome compared with other model eukaryotes. Despite the importance of ubiquitination in the regulation of signalling, little is known about deubiquitinating enzymes, which reverse the process of ubiquitination. Transgenic RNA interference-based cosuppression and the isolation of Atubp12/13 double mutants collectively provides the first report that AtUBP12 and AtUBP13 are functionally redundant and are required for immunity against virulent Pseudomonas syringae pv tomato in Arabidopsis. The Solanaceous AtUBP12 orthologue NtUBP12 was identified. Viral-induced gene silencing and transient gain-of-function assays were employed to establish that the NtUBP12 protein functions as a negative regulator of the Cf-9-triggered hypersensitive response. Here, we demonstrate that NtUBP12 and AtUBP12 are bona fide deubiquitinating enzymes capable of cleaving lysine-48-linked ubiquitin chains. AtUBP12 and NtUBP12 are functionally interchangeable and their deubiquitinating activity is required to suppress plant cell death. Overall, our data implicate AtUBP12- and NtUBP12-dependent deubiquitination in the stabilization of common substrates across Solanaceae and Brassicaceae which regulate disease resistance

Cooperative effects of Th2 cytokines and allergen on normal and asthmatic bronchial epithelial cells

In sensitized individuals, exposure to allergens such as Dermatophagoides pteronyssinus (Der p) causes Th2 polarization and release of cytokines, including IL-4 and IL-13. Because Der p extracts also have direct effects on epithelial cells, we hypothesized that allergen augments the effects of Th2 cytokines by promoting mediator release from the bronchial epithelium in allergic asthma. To test our hypothesis, primary bronchial epithelial cultures were grown from bronchial brushings of normal and atopic asthmatic subjects. RT-PCR showed that each culture expressed IL-4R?, common ?-chain, and IL-13R?1, as well as IL-13R?2, which negatively regulates IL-13 signaling; FACS analysis confirmed IL-13R?2 protein expression. Exposure of epithelial cultures to either Der p extracts, TNF-?, IL-4, or IL-13 enhanced GM-CSF and IL-8 release, and this was partially suppressible by corticosteroids. Simultaneous exposure of the epithelial cultures to IL-4 or IL-13 together with Der p resulted in a further increase in cytokine release, which was at least additive. Release of TGF-? was also increased by TNF-? and combinations of IL-4, IL-13, and Der p; however, this stimulation was only significant in the asthma-derived cultures. These data suggest that, in an allergic environment, Th2 cytokines and allergen have the potential to sustain airway inflammation through a cooperative effect on cytokine release by the bronchial epithelium. Our novel finding that IL-4, IL-13, and allergen enhance release of TGF-?, a ligand for the epidermal growth factor receptor that stimulates fibroblast proliferation and goblet cell differentiation, provides a potential link between allergen exposure, Th2 cytokines, and airway remodelling in asthma