Precipitation and recovery of phosphorus from the wastewater hydrolysis tank.

Phosphorus, a limited resource, is also an environmental pollutant that should be removed from wastewater and ideally reused. A pilot-scale facility was set up and used to precipitate and recover phosphorus from wastewater. The return activated sludge in a hydrolysis tank was flocculated and separated and the solid material returned to the hydrolysis tank; the flocculation process did not harm the microorganisms. Phosphate in the reject water was precipitated with different calcium salts and the phosphorus-containing precipitate recovered. The precipitate consisted mainly of phosphate and calcium, and under 5% of the final product consisted of iron and aluminum. Around 20% of the precipitate was organic material. The pilot-scale test was supplemented with bench-scale tests using calcium salt, magnesium salt, and NaOH/KOH. Without the addition of calcium ions, phosphate could be precipitated by increasing pH to 9.5, resulting in a concentration of phosphorus in the reject water of under 2 mg/L. If calcium salt was added (Ca:P ratio of 2:1), it was possible to remove phosphate at pH 9 (<1 mg/L). In general, the concentration of dissolved phosphate was 8-10 mg/L lower after precipitation when calcium salt was used compared with all other tested salts. This difference increased if additional phosphate was added to the sludge. The bench- and pilot-scale experiments yielded comparable data. At the pilot-scale facility, it was possible to remove 90% of the phosphate by adding calcium salt and regulating the pH to 8.5.

Plant integrity: an important factor in plant-pathogen interactions.

The effect of plant integrity and of aboveground-belowground defense signaling on plant resistance against pathogens and herbivores is emerging as a subject of scientific research. There is increasing evidence that plant defense responses to pathogen infection differ between whole intact plants and detached leaves. Studies have revealed the importance of aboveground-belowground defense signaling for plant defenses against herbivores, while our studies have uncovered that the roots as well as the plant integrity are important for the resistance of the potato cultivar Sarpo Mira against the hemibiotrophic oomycete pathogen Phytophthora infestans. Furthermore, in the Sarpo Mira-P. infestans interactions, the plant's meristems, the stalks or both, seem to be associated with the development of the hypersensitive response and both the plant's roots and shoots contain antimicrobial compounds when the aerial parts of the plants are infected. Here, we present a short overview of the evidence indicating the importance of plant integrity on plant defense responses.

Revealing the importance of meristems and roots for the development of hypersensitive responses and full foliar resistance to Phytophthora infestans in the resistant potato cultivar Sarpo Mira.

The defence responses of potato against Phytophthora infestans were studied using the highly resistant Sarpo Mira cultivar. The effects of plant integrity, meristems, and roots on the hypersensitive response (HR), plant resistance, and the regulation of PR genes were analysed. Sarpo Mira shoots and roots grafted with the susceptible Bintje cultivar as well as non-grafted different parts of Sarpo Mira plants were inoculated with P. infestans. The progress of the infection and the number of HR lesions were monitored, and the regulation of PR genes was compared in detached and attached leaves. Additionally, the antimicrobial activity of plant extracts was assessed. The presented data show that roots are needed to achieve full pathogen resistance, that the removal of meristems in detached leaves inhibits the formation of HR lesions, that PR genes are differentially regulated in detached leaves compared with leaves of whole plants, and that antimicrobial compounds accumulate in leaves and roots of Sarpo Mira plants challenged with P. infestans. While meristems are necessary for the formation of HR lesions, the roots of Sarpo Mira plants participate in the production of defence-associated compounds that increase systemic resistance. Based on the literature and on the presented results, a model is proposed for mechanisms involved in Sarpo Mira resistance that may apply to other resistant potato cultivars.

A set of Lotus japonicus Gifu x Lotus burttii recombinant inbred lines facilitates map-based cloning and QTL mapping.

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype 'Gifu' was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation.

Differential gene induction in resistant and susceptible potato cultivars at early stages of infection by Phytophthora infestans.

Sarpo Mira, a potato variety with high resistance against the late blight pathogen Phytophthora infestans, is being used in breeding programs to increase late blight resistance in commercial varieties. Discovering genes that are important for P. infestans resistance will assist in the development of molecular markers for the selection of new resistant cultivars and the use of resistant varieties will reduce the environmental, health and financial costs associated with the use of pesticides. Using complementary DNA amplified fragment length polymorphism analyses, differentially expressed genes involved in the potato-P. infestans interaction were identified in the susceptible Bintje and in the resistant Sarpo Mira potato cultivars. Forty-eight differentially expressed transcript derived fragments (TDFs) were cloned and sequenced. The expression profiles of some of these genes were analyzed in detail using quantitative RT-PCR at seven time points: 1, 4, 17, 24, 30, 41 and 65 hours after inoculation (hai). We found that five transcripts with homologies to pathogenesis/defense-related genes and two TDFs with homology to transcription factors were significantly induced to higher levels in the resistant cultivar at very early stages of the infection (1 hai). Interestingly, most of these genes showed different expression profiles throughout the whole infection process between both cultivars. Particularly during its biotrophic growth phase, P. infestans triggered the down-regulation of infection responsive genes in the susceptible but not in the resistance cultivar. Our results suggest that these newly identified early-induced transcripts may be good candidates for conferring Sarpo Mira's resistance to late blight and they could be useful molecular markers for the selection of new resistant cultivars.

Iron and ferritin accumulate in separate cellular locations in Phaseolus seeds.

BACKGROUND: Iron is an important micronutrient for all living organisms. Almost 25% of the world population is affected by iron deficiency, a leading cause of anemia. In plants, iron deficiency leads to chlorosis and reduced yield. Both animals and plants may suffer from iron deficiency when their diet or environment lacks bioavailable iron. A sustainable way to reduce iron malnutrition in humans is to develop staple crops with increased content of bioavailable iron. Knowledge of where and how iron accumulates in seeds of crop plants will increase the understanding of plant iron metabolism and will assist in the production of staples with increased bioavailable iron. RESULTS: Here we reveal the distribution of iron in seeds of three Phaseolus species including thirteen genotypes of P. vulgaris, P. coccineus, and P. lunatus. We showed that high concentrations of iron accumulate in cells surrounding the provascular tissue of P. vulgaris and P. coccineus seeds. Using the Perls' Prussian blue method, we were able to detect iron in the cytoplasm of epidermal cells, cells near the epidermis, and cells surrounding the provascular tissue. In contrast, the protein ferritin that has been suggested as the major iron storage protein in legumes was only detected in the amyloplasts of the seed embryo. Using the non-destructive micro-PIXE (Particle Induced X-ray Emission) technique we show that the tissue in the proximity of the provascular bundles holds up to 500 microg g(-1) of iron, depending on the genotype. In contrast to P. vulgaris and P. coccineus, we did not observe iron accumulation in the cells surrounding the provascular tissues of P. lunatus cotyledons. A novel iron-rich genotype, NUA35, with a high concentration of iron both in the seed coat and cotyledons was bred from a cross between an Andean and a Mesoamerican genotype. CONCLUSIONS: The presented results emphasize the importance of complementing research in model organisms with analysis in crop plants and they suggest that iron distribution criteria should be integrated into selection strategies for bean biofortification.

The conserved cysteine-rich domain of a tesmin/TSO1-like protein binds zinc in vitro and TSO1 is required for both male and female fertility in Arabidopsis thaliana.

Development of reproductive tissue and control of cell division are common challenges to all sexually reproducing eukaryotes. The Arabidopsis thaliana TSO1 gene is involved in both these processes. Mild tso1 mutant alleles influence only ovule development, whereas strong alleles have an effect on all floral tissues and cause cell division defects. The tso1 mutants described so far carry point mutations in a conserved cysteine-rich domain, the CRC domain, but the reason for the range of phenotypes observed is poorly understood. In the present study, the tesmin/TSO1-like CXC (TCX) proteins are characterized at the biochemical, genomic, transcriptomic, and functional level to address this question. It is shown that the CRC domain binds zinc, offering an explanation for the severity of tso1 alleles where cysteine residues are affected. In addition, the phylogenetic and expression analysis of the TCX genes suggested an overlap in function between AtTSO1 and the related gene AtTCX2. Their expression ratios indicated that pollen, in addition to ovules, would be sensitive to loss of TSO1 function. This was confirmed by analysis of novel tso1 T-DNA insertion alleles where the development of both pollen and ovules was affected.

Concerted evolution of a tandemly arrayed family of mating-specific genes in Phytophthora analyzed through inter- and intraspecific comparisons.

Multigene families are features of most eukaryotic genomes, which evolve through a variety of mechanisms. This study describes the structure, expression, and evolution of a novel family in the oomycete Phytophthora. In the heterothallic species P. infestans, M96 is expressed specifically during sexual sporogenesis, and encodes a low-complexity extracellular protein that may be a component of oospore walls. Intriguingly, M96 exists in P. infestans as 22 relatively homogeneous loci tandemly repeated at a single site, which is partitioned by inversions and retroelements into subclusters exhibiting semi-independent evolution. M96 relatives were detected in other heterothallic and homothallic oomycetes including species closely (P. mirabilis, P. phaseoli) or distantly (P. ramorum, P. sojae) related to P. infestans. Those M96 relatives also exhibit oosporogenesis-specific expression and are arrayed multigene families. Nucleotide changes and repeat expansion diversify M96 in each species, however, paralogues are more related than orthologues. Concerted evolution through gene conversion and not strong purifying selection appears to be the major contributor to intraspecific homogenization. Divergence and concerted evolution was also detected between isolates of P. infestans. The divergence of M96 proteins between P. infestans, P. ramorum, and P. sojae exceeds that of typical proteins, reflecting trends in reproductive proteins from other kingdoms.

The glucocorticoid-inducible GVG system causes severe growth defects in both root and shoot of the model legume Lotus japonicus.

During the past decade, the legume Lotus japonicus has emerged as an important model system for study of symbiotic nitrogen fixation. Controlled expression of genes involved in symbiosis from an inducible promoter at specific time points would be a valuable tool for investigating gene function in L. japonicus. We have attempted to study the function of the putative transcription factors LjNDX and LjCPP1 by expression from the GVG inducible system. This study showed that the GVG system itself causes growth disturbances in L. japonicus. Shoot internode elongation and root pericycle cell division are affected when the chimeric GVG transcription factor is activated. We suggest that deficient auxin signaling could cause the phenotype observed and conclude that the GVG inducible system is not well suited for use in the model legume L. japonicus.

A gene expressed during sexual and asexual sporulation in Phytophthora infestans is a member of the Puf family of translational regulators.

A gene from Phytophthora infestans that was previously identified as being induced during the development of sexual spores was also found to be active during asexual sporulation. The gene, M90, was expressed as a 3.1-kb primary transcript containing two introns and was predicted to encode a member of the Puf family of translational regulators. The protein showed up to 51% amino acid identity to other Puf proteins within its 353-amino-acid RNA-binding domain. Little similarity extended beyond this region, as noted for other members of the family. Expression of M90 was measured by using RNA blots and transformants of P. infestans expressing a fusion between the M90 promoter and the beta-glucuronidase (GUS) gene. A 1.3-kb promoter fragment conferred the normal M90 pattern of expression to the GUS reporter in transformants. In matings, expression was first detected in male and female gametangial initials and persisted in mature oospores. Expression was also observed in hyphal tips just prior to asexual sporulation, in sporangiophores, in mature sporangia, and in zoospores. The signal quickly disappeared once spores made the transition to hyphae after germination. Nutrient limitation did not induce the gene. Potential roles for a translational regulator during both sexual development and asexual sporulation are discussed.

Stable transformation of the oomycete, Phytophthora infestans, using microprojectile bombardment.

Germinated asexual sporangia, zoospores, and mycelia of Phytophthora infestans were transformed to G418-resistance by microprojectile bombardment. After optimization, an average of 14 transformants/shot were obtained, using 10(6) germinated sporangia and gold particles coated with 1 microg of vector. Transformants displayed tandem or simple insertions of vector sequences within chromosomes. Most primary transformants were heterokaryons of transformed and wild-type nuclei, a state which generally persisted for generations, even with G418 selection. Transgenic homokaryons were easily obtained from primary transformants through G418 selection of zoospores. To facilitate the optimization of transformation, experiments were performed using a vector containing neomycin phosphotransferase (npt) and beta-glucuronidase (GUS) genes fused to oomycete transcriptional regulatory sequences. To indicate which orientations of transgenes would maximize their expression, head-to-head, head-to-tail, or tail-to-tail orientations of npt and GUS were compared. Each yielded similar rates of transformation and levels of GUS activity, indicating little transcriptional interference.

Stage-specific gene expression during sexual development in Phytophthora infestans.

Eight genes that are upregulated during sexual development in the heterothallic oomycete, Phytophthora infestans, were identified by suppression subtractive hybridization. Two genes showed very low but detectable expression in vegetative hyphae and became induced about 40- to >100-fold early in mating, before gametangial initials appeared. The remaining six loci were not induced until later in mating, coincident with the formation of gametangia and oospores, with induction levels ranging from 60- to >100-fold. Five genes were single copy, and three were members of families. Sequence analysis revealed that the predicted products of three of the genes had similarity to proteins that influence RNA stability, namely a ribonuclease activator, the pumilio family of RNA-binding proteins and RNase H. The products of two other mating-induced genes resembled two types of Phytophthora proteins previously shown to elicit plant defence responses. Each mating-induced gene was also expressed in a self-fertile strain, which was shown to be a heterokaryon. However, quantitative and qualitative differences existed in their expression in normal matings and in the self-fertile heterokaryon. Besides the mating-induced genes, two extrachromosomal RNA elements were identified.