Two Fatty Acid Desaturases,STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 and FATTY ACID DESATURASE3, Are Involved in Drought and Hypoxia Stress Signaling in Arabidopsis Crown Galls

Agrobacterium tumefaciens-derived crown galls of Arabidopsis (Arabidopsis thaliana) contain elevated levels of unsaturated fatty acids and strongly express two fatty acid desaturase genes, ω3 FATTY ACID DESATURASE3 (FAD3) and STEAROYL-ACYL CARRIER PROTEIN Δ⁹-DESATURASE6 (SAD6). The fad3-2 mutant with impaired α-linolenic acid synthesis developed significantly smaller crown galls under normal, but not under high, relative humidity. This strongly suggests that FAD3 plays a role in increasing drought stress tolerance of crown galls. SAD6 is a member of the SAD family of as yet unknown function. Expression of the SAD6 gene is limited to hypoxia, a physiological condition found in crown galls. As no sad6 mutant exists and to link the function of SAD6 with fatty acid desaturation in crown galls, the lipid pattern was analyzed of plants with constitutive SAD6 overexpression (SAD6-OE). SAD6-OE plants contained lower stearic acid and higher oleic acid levels, which upon reduction of SAD6 overexpression by RNA interference (SAD6-OE-RNAi) regained wild-type-like levels. The development of crown galls was not affected either in SAD6-OE or SAD6-OE-RNAi or by RNA interference in crown galls. Since biochemical analysis of SAD6 in yeast (Saccharomyces cerevisiae) and Escherichia coli failed, SAD6 was ectopically expressed in the background of the well-known suppressor of salicylic acid-insensitive2 (ssi2-2) mutant to confirm the desaturase function of SAD6. All known ssi2-2 phenotypes were rescued, including the high stearic acid level. Thus, our findings suggest that SAD6 functions as a Δ⁹-desaturase, and together with FAD3 it increases the levels of unsaturated fatty acids in crown galls under hypoxia and drought stress conditions.Plant tumors, also referred to as crown galls, develop upon infection of susceptible plants with Agrobacterium tumefaciens. A DNA fragment, the transfer DNA (T-DNA) of the tumor-inducing plasmid of virulent A. tumefaciens strains, is randomly integrated into the genome of a host plant (Thomashow et al., 1980; Kim et al., 2007; Pitzschke and Hirt 2010). Expression of the T-DNA-encoded oncogenes drives increased production of auxin and cytokinin, thereby promoting cell proliferation. Plant tumor growth causes disruption of the epidermal cell layer that is covered by a cuticle and thus is permanently endangered to lose water (Schurr et al., 1996). In order to prevent desiccation and wilting, rescue processes appear to be constitutively activated (Veselov et al., 2003). Thereby, ethylene and abscisic acid trigger the expression of drought stress-responsive genes, the accumulation of osmoprotectants, and suberization of the surface cell layers (Efetova et al., 2007). In addition, cell membrane lipids are the major targets of environmental stresses, and tolerance to drought stress is dependent on high levels of polyunsaturated fatty acids (PUFAs) and the ability to maintain fatty acid (FA) desaturation activity (Berberich et al., 1998; Mikami and Murata, 2003; Torres-Franklin et al., 2009). In Arabidopsis (Arabidopsis thaliana) crown gall tumors, 27% of the genes involved in lipid metabolism are differentially regulated (Deeken et al., 2006). Under drought stress, Arabidopsis increases the ratio of digalactosyl diglyceride (DGDG) to monogalactosyl diglyceride (MGDG) and FA unsaturation (Gigon et al., 2004). An increase in α-linolenic acid levels (18:3, where x:y denotes an FA with x carbons and y double bonds) by overexpression of the ω3 fatty acid desaturases FAD3 and FAD7 has been shown to enhance tolerance to drought stress in Nicotiana tabacum cells (Zhang et al., 2005), whereas nontolerant plants decline their fraction of 18:3 (Monteiro de Paula et al., 1993; Dakhma et al., 1995; Upchurch, 2008).Developing crown gall tumors also face permanent oxidative stress. Reactive oxygen species (ROS) are produced in tumors, and glutathione S-transferases and peroxidases are strongly up-regulated (Jia et al., 1996; Lee et al., 2009). Due to a reduced respiratory and photosynthetic capacity in crown gall tumors, ATP production is predominantly derived from glycolysis and alcoholic fermentation (Deeken et al., 2006). In other words, the hypoxia physiology of the Arabidopsis tumor is governed by heterotrophic metabolism (Deeken et al., 2006). Since dioxygen is a cofactor of desaturases, its depletion limits the de novo synthesis of unsaturated FAs and thus membrane lipids (Brown and Beevers, 1987). In addition, hypoxia appears to be associated with ROS production, peroxidation of PUFAs, and finally, loss of membrane integrity (Blokhina et al., 2003; Upchurch, 2008).The biosynthesis of PUFAs is initiated by introduction of the first double bond into stearic acid (18:0) by STEAROYL-ACYL CARRIER PROTEIN Δ⁹-DESATURASE (SAD). SAD genes exhibit a tissue-specific expression profile, and the encoding enzymes regulate the pools of oleic acid (18:1), a monounsaturated fatty acid (MUFA; Shanklin and Somerville 1991; Thompson et al., 1991; Cahoon et al., 1996, 1998; Whittle et al., 2005). In Arabidopsis, five out of seven members of the SAD gene family (SAD1, SAD3, SAD4, SAD5, and SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2 [SSI2]) are capable of desaturating 18:0 and contribute to the 18:1 pool (Kachroo et al., 2007). A mutation in the Arabidopsis ssi2 locus results in the accumulation of 18:0, whereas the 18:1 content is reduced. Furthermore, the salicylic acid-mediated defense signaling pathway is constitutively active, resulting in lesion formation and increased expression of the PATHOGENESIS-RELATED (PR) genes. The 18:1 MUFAs are incorporated into membrane lipids by two glycerolipid biosynthesis pathways. Phospholipids and galactolipids of photosynthetic membranes in plastids are synthesized by the prokaryotic pathway, while lipids of extraplastidic membranes are produced in the endoplasmic reticulum (ER) by the eukaryotic pathway (Ohlrogge and Browse, 1995). MUFAs are further desaturated to PUFAs by two sets of membrane-bound FADs. These enzymes are either located in plastids or the ER (Ohlrogge and Browse, 1995). In the ER, conversion of the unsaturated phospholipid FAs 18:1 to 18:2 and of 18:2 to 18:3 is carried out by the ω6 desaturase FAD2 and the ω3 desaturase FAD3, respectively (Browse et al., 1993; Okuley et al., 1994; Los and Murata, 1998).This study focuses on the role of desaturases in Arabidopsis crown galls in the context of drought and hypoxia stress. We document that crown galls produce increased levels of α-linolenic acid and strongly express the two FAD genes FAD3 and SAD6. In contrast to the well-known ω3 desaturase FAD3, the function of SAD6, a putative SAD, is unknown, and mutants for this gene are not available. However, the ability of SAD6 to replace the well-characterized SSI2 functionally in the ssi2-2 mutant suggests that SAD6 is a functional SAD. Overexpression of SAD6 decreased stearic acid and increased oleic acid levels. Down-regulation of SAD6 overexpression by RNA interference (RNAi) restored wild-type-like FA levels. The ability of SAD6 to influence the oleic acid levels together with the finding that SAD6 gene expression is restricted to hypoxia suggest that SAD6 catalyzes FA desaturation under hypoxic conditions. Unlike SAD6, the results obtained with the fad3-2 mutant impaired in α-linolenic acid biosynthesis indicate a role of FAD3 in increasing the drought stress tolerance of crown galls. Thus, both desaturases shape the pool of unsaturated FAs in drought stress- and oxidative stress-challenged Arabidopsis crown gall tumors.     

Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA,alpha-tubulin and HSP90 genes: Implications for the evolutionary history of the double karyomastigont of diplomonads

Background  

Fornicata is a relatively recently established group of protists that includes the diplokaryotic diplomonads (which have two similar nuclei per cell), and the monokaryotic enteromonads, retortamonads and Carpediemonas, with the more typical one nucleus per cell. The monophyly of the group was confirmed by molecular phylogenetic studies, but neither the internal phylogeny nor its position on the eukaryotic tree has been clearly resolved.     

Statins but not fibrates improve the atherogenic to anti-atherogenic lipoprotein particle ratio: a randomized crossover study

Background  

Prior studies suggested low density lipoprotein particle (LDLP) size is a predictor of atherosclerosis. Knowledge of effects of lipid lowering drugs on lipoprotein subclasses is useful. We treated subjects with hyperlipidemia sequentially with statins and fibrates, the 2 main classes of lipid lowering therapy and studied changes in NMR lipoprotein subclasses.     

Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs

Background

6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases.

Methodology/Principal Findings

Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome.

Conclusion/Significance

6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.     

A cautionary note on the use of hypervolume kernel density estimators in ecological niche modelling

Blonder et al. ( 2014 , Global Ecology and Biogeography, 23, 595–609) introduced a new multivariate kernel density estimation (KDE) method to infer Hutchinsonian hypervolumes in the modelling of ecological niches. The authors argued that their KDE method matches or outperforms several methods for estimating hypervolume geometries and for conducting species distribution modelling. Further clarification, however, is appropriate with respect to the assumptions and limitations of KDE as a method for species distribution modelling. Using virtual species and controlled environmental scenarios, we show that KDE both under‐ and overestimates niche volumes depending on the dimensionality of the dataset and the number of occurrence records considered. We suggest that KDE may be a viable approach when dealing with large sample sizes, limited sampling bias and only a few environmental dimensions.     

The HET-s prion protein of the filamentous fungus Podospora anserina aggregates in vitro into amyloid-like fibrils.

The HET-s protein of Podospora anserina is a fungal prion. This protein behaves as an infectious cytoplasmic element that is transmitted horizontally from one strain to another. Under the prion form, the HET-s protein forms aggregates in vivo. The specificity of this prion model compared with the yeast prions resides in the fact that under the prion form HET-s causes a growth inhibition and cell death reaction when co-expressed with the HET-S protein from which it differs by 13 residues. Herein we describe the purification and initial characterization of recombinant HET-s protein expressed in Escherichia coli. The HET-s protein self-associates over time into high molecular weight aggregates. These aggregates greatly accelerate precipitation of the soluble form. HET-s aggregates appear as amyloid-like fibrils using electron microscopy. They bind Congo Red and show birefringence under polarized light. In the aggregated form, a HET-s fragment of approximately 7 kDa is resistant to proteinase K digestion. CD and FTIR analyses indicate that upon transition to the aggregated state, the HET-s protein undergoes a structural rearrangement characterized by an increase in antiparallel beta-sheet structure content. These results suggest that the [Het-s] prion element propagates in vivo as an infectious amyloid.     

New insights in Salicornia L. and allied genera (Chenopodiaceae) inferred from nrDNA sequence data

A phylogenetic analysis was performed based on ITS DNA sequences of fourteen samples from different sources of six species of Salicornia, the three allied genera Arthrocnemum, Sarcocornia and Halocnemum of the same tribe Salicornieae, and other genera of the subfamily Salicornioideae used in previous studies. Bassia hirsuta, Camphorosma monspeliaca (subfamily Chenopodioideae) and four species of Suaeda (subf. Suaedoideae) were chosen as outgroups. Results show that the annual genus Salicornia is a sister group to the perennial genera Sarcocornia, Arthrocnemum and Halocnemum. Moreover, the phylogenetic analysis based on ITS results distinguished two groups of Salicornia species which fitted with ploidy level: one group consisted of diploid species, and the second of tetraploid ones. Sarcocornia and Arthrocnemum are shown to be closely related, even though the species investigated here exhibited an evident distance between their ITS sequences. On the basis of our results, these two genera should be united. Bienertia (already separated as Bienertieae) was confirmed as probable outgroup to the subf. Salicornioideae, while Kalidium (subf. Salicornioideae, tribe Halopeplideae) was an outgroup to the rest of the Salicornioideae (tribe Salicornieae). The group Allenrolfea plus Halocnemum was the most basal of the tribe Salicornieae amongst those investigated in this study. The two samples of Halocnemum strobilaceum used in this work displayed numerous changes (transitions and transversions) in their respective sequences, probably related to their morphological and chorological differentiation. On the basis of our analysis, the most probable basal chromosome number for Salicornieae appears to be 2n = 18. The same number would also be the base number for the annual genus Salicornia and the perennial Arthrocnemum ( + Sarcocornia), with polyploidy arising independently in the two groups.