Phosphate,phytate and phytases in plants: from fundamental knowledge gained in Arabidopsis to potential biotechnological applications in wheat

Phosphorus (P) is an essential macronutrient for all living organisms. In plants, P is taken up from the rhizosphere by the roots mainly as inorganic phosphate (Pi), which is required in large and sufficient quantities to maximize crop yields. In today’s agricultural society, crop yield is mostly ensured by the excessive use of Pi fertilizers, a costly practice neither eco-friendly or sustainable. Therefore, generating plants with improved P use efficiency (PUE) is of major interest. Among the various strategies employed to date, attempts to engineer genetically modified crops with improved capacity to utilize phytate (PA), the largest soil P form and unfortunately not taken up by plants, remains a key challenge. To meet these challenges, we need a better understanding of the mechanisms regulating Pi sensing, signaling, transport and storage in plants. In this review, we summarize the current knowledge on these aspects, which are mainly gained from investigations conducted in Arabidopsis thaliana, and we extended it to those available on an economically important crop, wheat. Strategies to enhance the PA use, through the use of bacterial or fungal phytases and other attempts of reducing seed PA levels, are also discussed. We critically review these data in terms of their potential for use as a technology for genetic manipulation of PUE in wheat, which would be both economically and environmentally beneficial.     

Structure génétique des cécidomyies des céréales en Tunisie

Genetic structure of Hessian flies in Tunisia. The genetic structure of M. destructor and M. hordei was investigated by sampling 21 fields of cereals in 14 localities of central and southern Tunisia. As previously shown, there was no strict association between the cereal species (wheat, barley and oat) and the Mayetiola species. M. destructor males displayed no heterozygosity at the Pgm3 locus, indicating that they were hemizygous as is the PGM locus in North America. In M. hordei, heterozygous males were observed at all loci, but strong heterozygote deficits were found at two loci (Mdh2 et Hk). Since no such deficit was observed in females, the population structure of M. hordei was studied only in females. Although heterozygosity was two fold higher in M. hordei than in M. destructor, the two species were similar for other genetic characteristics, including a low (Fst < 0.05) but significant (P < 0.05) genetic differentiation, no isolation by distance, and similar rates of gene flow (5.7 ≤ Nm ≤ 9.6). These results are discussed in relation to their consequences in the event of controlling Tunisian Hessian flies using wheat cultivars that are resistant to a M. destructor biotype.     

Co-inoculation Effects of Rhizobium sullae and Pseudomonas sp. on Growth,Antioxidant Status,and Expression Pattern of Genes Associated with Heavy Metal Tolerance and Accumulation of Cadmium in Sulla coronaria

Recently, phytoremediation assisted by soil bacteria has emerged as a potential tool to clean up the metal-contaminated/polluted environment. Three plant-growth-promoting bacteria (PGPBs): Rhizobium sullae, Pseudomonas fluorescens, and Pseudomonas sp. were found to tolerate cadmium (Cd) stress. Sulla coronaria inoculated with these PGPBs, and grown under different Cd concentrations (0, 100, and 200 µM), showed increases in dry biomass and proline content. Notable increases in different gas-exchange characteristics such as photosynthesis rate (A), transpiration rate (E), and water-use efficiency (WUE), as well as increases in nitrogen (N) and Cd accumulations were also recorded in inoculated plants compared to non-inoculated Cd stressed plants. The activities of antioxidant enzymes superoxide dismutase (SOD), guaiacol peroxidase (GPOX), catalase (CAT), and ascorbate peroxidase (APX) in S. coronaria roots increased under Cd stress after PGPB co-inoculation, suggesting that these PGPB species could be used for amelioration of stress tolerance in S. coronaria. The expression patterns of ScPCS, ScMT, ScF-box, ScGR, and ScGST in roots of S. coronaria indicated that these genes are differentially expressed under Cd treatments, suggesting their possible roles in Cd and heavy metal stress responses. The results indicate that co-inoculation with R. sullae and Pseudomonas sp. could alleviate Cd toxicity in S. coronaria. In the present study, the obtained data suggest that the application of PGPBs could be a promising strategy for enhancing the phytostabilization efficiency of Cd-contaminated soils.

     

Structure of the M2 transmembrane segment of GLIC, a prokaryotic Cys loop receptor homologue from Gloeobacter violaceus, probed by substituted cysteine accessibility

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.     

Alleviation of drought stress in faba bean (Vicia faba L.) by exogenous application of β-aminobutyric acid (BABA)

Physiology and Molecular Biology of Plants - Drought stress is one of the most prevalent environmental factors limiting faba bean (Vicia faba L.) crop productivity. β-aminobutyric acid (BABA)...     

Functional anatomy of the Arabidopsis cytokinesis-specific syntaxin KNOLLE

In plant cytokinesis, Golgi/trans-Golgi network-derived vesicles are targeted to the plane of cell division where they fuse with one another to form the partitioning membrane (cell plate). This membrane fusion requires a specialised syntaxin (Qa-SNARE), named KNOLLE in Arabidopsis. KNOLLE is only made during the M-phase of the cell cycle, targeted to the plane of cell division and degraded in the vacuole at the end of cytokinesis. To identify the parts of KNOLLE required for proper targeting and function in membrane fusion, we generated chimeric syntaxins comprising complementary fragments from KNOLLE and MVB-localized PEP12 (SYP21). Surprisingly, targeting of the chimeric protein was not specified by the C-terminal membrane anchor. Rather the N-terminal region including helix Ha and the adjacent linker to helix Hb appeared to played a critical role. However, deletion of this N-terminal fragment from KNOLLE (KN(Δ1-82) ) had the same effect as its presence in the chimeric protein (KN(1-82) -PEP12(64-279) ), suggesting that targeting to the plane of cell division occurs by default, i.e. when no sorting signal would target the syntaxin to a specific endomembrane compartment. Once the full-length syntaxin accumulated at the plane of division, phenotypic rescue of the knolle mutant only required the SNARE domain plus the adjacent linker connecting helix Hc to the SNARE domain from KNOLLE. Our results suggest that targeting of syntaxin to the plane of cell division occurs without active sorting, whereas syntaxin-mediated membrane fusion requires sequence-specific features.     

Structural requirements for nucleocapsid protein-mediated dimerization of avian leukosis virus RNA

The avian leukosis virus (ALV) belongs to the alpha group of retroviruses that are widespread in nature. The 5'-untranslated region of ALV genome contains the L3 element that is important for virus infectivity and the formation of an unstable RNA dimer in vitro. The L3 sequence is predicted to fold into a long stem-loop structure with two internal loops and an apical one. Phylogenetic analysis predicts that the L3 stem-loop is conserved in alpharetroviruses. Furthermore, a significant selection mechanism maintains a palindrome in the apical loop. The nucleocapsid protein of the alpharetroviruses (NCp12) is required for RNA dimer formation and replication in vivo. It is not known whether L3 can be an NCp12-mediated RNA dimerization site able to bind NCp12 with high affinity. Here, we report that NCp12 chaperones formation of a stable ALV RNA dimer through L3. To investigate the NCp12-mediated L3 dimerization reaction, we performed site-directed mutagenesis, gel retardation and heterodimerization assays and analysis of thermostability of dimeric RNAs. We show that the affinity of NCp12 for L3 is lower than its affinity for the microPsi RNA packaging signal. Results show that conservation of a long stem-loop structure and a loop-loop interaction are not required for NCp12-mediated L3 dimerization. We show that the L3 apical stem-loop is sufficient to form an extended duplex and the whole stem-loop L3 cannot be converted by NCp12 into a duplex extending throughout L3. Three-dimensional modelling of the stable L3 dimer supports the notion that the extended duplex may represent the minimal dimer linkage structure found in the genomic RNA.     

Symbiotic effectiveness and response to mannitol-mediated osmotic stress of various chickpea–rhizobia associations

Thirty-six symbiotic associations involving six chickpea cultivars against six rhizobial strains were evaluated for symbiotic performance and responses to osmotic stress applied by mannitol (50 mM) in aerated hydroponic cultures. Analyses in different symbioses were focused on biomass production, nodulation, nitrogen fixation, and their modulation under osmotic stress conditions, as well as expression of nodular antioxidant enzymes. Mesorhizobium ciceri reference (835) and local (CMG6) strains, as well as the local (C₁₁) M. mediterraneum allowed the best symbiotic efficiency for all chickpea cultivars. The osmotic stress induces severe decrease ranging 30–50% in aerial biomass and 50–70% for nitrogen fixation. Nevertheless, plants inoculated with M. ciceri (835) and M. mediterraneum (C₁₁) preserve a relatively high growth (4 g plant⁻¹) with nitrogen-fixing activity (25 μmols h⁻¹ plant⁻¹). The bacterial partner was the most important factor of variance of the analysed parameters in osmotic stress or physiological conditions where it gets to 60–85%. The strains allowing the best competent symbioses were proposed for field assays. Under osmotic stress, nodular peroxidase (POX) and ascorbate peroxidase (APX) activities were significantly enhanced. The increase of POX and APX was inversely correlated with the inhibition of aerial biomass production (p = 0.05) and nitrogen-fixing capacity (p = 0.01), suggesting a protective role of these enzymes in nodules. Superoxide dismutase (SOD) was also activated in stressed nodules. However, the spectacular decrease in catalase (CAT) activity discounts its involvement in osmotic stress response.     

Sensitivity of Zymoseptoria tritici Isolates from Tunisia to Pyraclostrobin,Fluxapyroxad, Epoxiconazole,Metconazole, Prochloraz and Tebuconazole

Sensitivity of 159 isolates of Zymoseptoria tritici collected from durum wheat fields in Tunisia in 2012 was analysed towards pyraclostrobin, fluxapyroxad, epoxiconazole, metconazole, prochloraz and tebuconazole using microtiter tests. All isolates were found to be highly sensitive to pyraclostrobin with EC₅₀ <0.01 mg/l with the exception of three isolates from the same field with higher EC₅₀ values (>0.5 mg/l). These three isolates carried a mutation in the cytochrome b gene encoding the G143A substitution. This is the first report of quinone outside inhibitors (QoI) resistance in Z. tritici in Tunisia. Sensitivity towards r fluxapyroxad was in a narrow range with EC₅₀ values ranging between 0.013 and 0.125 mg/l, which can serve as baseline sensitivity data for the future. Demethylation inhibitors sensitivity varied across a broad range with the data indicating a slight shift in sensitivity when compared to a previous study on the 2010 population. No highly sensitive strains were isolated from samples from fields, which had received three or four DMI applications.     

Characterization of rhizosphere prokaryotic diversity in a horizontal subsurface flow constructed wetland using a PCR cloning-sequencing based approach

Performance of biological wastewater treatment systems may be related to the composition and activity of microbial populations they contain. However, little information is known regarding microbial community inhabiting these ecosystems. The purpose of this study was to investigate archaeal and bacterial diversity, using cultivation-independent molecular techniques, in a constructed wetland receiving domestic wastewater. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by PCR using primers specific for archaeal and bacterial domains. A high microbial diversity was detected. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 31.3 % of the OTUs, followed by the Bacteroidetes (14.8 %), Planctomycetales (13.8 %), Actinobacteria (12 %), and Chloroflexi (8.2 %). Sequences affiliated with minor phylogenetic divisions such as the TM7, Nitrospira, OP10, and BRC1 are represented by <6 % of total OTUs. The Archaea domain was represented by the Thaumarchaeota phylum dominated by the Candidatus Nitrososphaera genus.