Giuseppe Dionisio

Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat, Barley, Maize, and Rice

Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.

Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat ( Triticum aestivum L.), Barley ( Hordeum vulgare L.), Maize ( Zea maize L.) and Rice ( Oryza sativa L.)

Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.

Cisgenic barley with improved phytase activity

The cisgenesis concept implies that plants are transformed only with their own genetic materials or genetic materials from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. We used a barley phytase gene (HvPAPhy_a) expressed during grain filling to evaluate the cisgenesis concept in barley. The marker gene elimination method was used to obtain marker-free plant lines. Here, the gene of interest and the selection gene are flanked by their own T-DNA borders to allow unlinked integration of the two genes. We analysed the transformants for co-transformation efficiency, increased phytase activities in the grain, integration of the kanamycin resistance gene of the vector-backbone and segregation between the HvPAPhy_a insert and the hygromycin resistance gene. The frequencies of the four parameters imply that it should be possible to select 11 potentially cisgenic T(1) -lines out of the 72 T(0) -lines obtained, indicating that the generation of cisgenic barley is possible at reasonable frequencies with present methods. We selected two potential cisgenic lines with a single extra copy of the HvPAPhy_a insert for further analysis. Seeds from plants homozygous for the insert showed 2.6- and 2.8-fold increases in phytase activities and the activity levels were stable over the three generations analysed. In one of the selected lines, the flanking sequences from both the left and right T-DNA borders were analysed. These sequences confirmed the absence of truncated vector-backbone sequences linked to the borders. The described line should therefore be classified as cisgenic.

Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling

BackgroundThe potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, Scmv1 and Scmv2, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions.ResultsBy employing time course microarray experiments we identified 68 significantly differentially expressed sequences within the different time points. The majority of differentially expressed genes differed between the near-isogenic line carrying Scmv1 resistance locus at chromosome 6 and the other isogenic lines. Most differentially expressed genes in the SCMV experiment (75%) were identified one hour after virus inoculation, and about one quarter at multiple time points. Furthermore, most of the identified mapped genes were localised outside the Scmv QTL regions. Annotation revealed differential expression of promising pathogenesis-related candidate genes, validated by qRT-PCR, coding for metallothionein-like protein, S-adenosylmethionine synthetase, germin-like protein or 26S ribosomal RNA.ConclusionOur study identified putative candidate genes and gene expression patterns related to resistance to SCMV. Moreover, our findings support the effectiveness and reliability of the combination of different expression profiling approaches for the identification and validation of candidate genes. Genes identified in this study represent possible future targets for manipulation of SCMV resistance in maize.

The degradation of phytate by microbial and wheat phytases is dependent on the phytate matrix and the phytase origin

BACKGROUND Phytases increase utilization of phytate phosphorus in feed. Since wheat is rich in endogenous phytase activity it was examined whether wheat phytases could improve phytate degradation compared to microbial phytases. Moreover, it was investigated whether enzymatic degradation of phytate is influenced by the matrix surrounding it. Phytate degradation was defined as the decrease in the sum of InsP₆ + InsP₅. RESULTS Endogenous wheat phytase effectively degraded wheat Ins₆ + InsP₅ at pH 4 and pH 5, while this was not true for a recombinant wheat phytase or phytase extracted from wheat bran. Only microbial phytases were able to degrade InsP₆ + InsP₅ in the entire pH range from 3 to 5, which is relevant for feed applications. A microbial phytase was efficient towards InsP₆ + InsP₅ in different phytate samples, whereas the ability to degrade InsP₆ + InsP₅ in the different phytate samples ranged from 12% to 70% for the recombinant wheat phytase. CONCLUSION Wheat phytase appeared to have an interesting potential. However, the wheat phytases studied could not improve phytate degradation compared to microbial phytases. The ability to degrade phytate in different phytate samples varied greatly for some phytases, indicating that phytase efficacy may be affected by the phytate matrix.

Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis

Hemoglobins modulate embryogenesis by regulating programmed cell death. Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somatic embryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somatic embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somatic embryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species.

Drought Stress Responses in Soybean Roots and Nodules

Drought is considered to be a major threat to soybean production worldwide and yet our current understanding of the effects of drought on soybean productively is largely based on studies on above-ground traits. Although the roots and root nodules are important sensors of drought, the responses of these crucial organs and their drought tolerance features remain poorly characterized. The symbiotic interaction between soybean and rhizobia facilitates atmospheric nitrogen fixation, a process that provides essential nitrogen to support plant growth and development. Symbiotic nitrogen fixation is important for sustainable agriculture, as it sustains plant growth on nitrogen-poor soils and limits fertilizer use for crop nitrogen nutrition. Recent developments have been made in our understanding of the drought impact on soybean root architecture and nodule traits, as well as underpinning transcriptome, proteome and also emerging metabolome information, with a view to improve the selection of more drought-tolerant soybean cultivars and rhizobia in the future. We conclude that the direct screening of root and nodule traits in the field as well as identification of genes, proteins and also metabolites involved in such traits will be essential in order to gain a better understanding of the regulation of root architecture, bacteroid development and lifespan in relation to drought tolerance in soybean.

High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene

The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a.

Fructan accumulation and transcription of candidate genes during cold acclimation in three varieties of Poa pratensis.

Poa pratensis, a type species for the grass family (Poaceae), is an important cool season grass that accumulates fructans as a polysaccharide reserve. We studied fructan contents and expression of candidate fructan metabolism genes during cold acclimation in three varieties of P. pratensis adapted to different environments: Northern Norway, Denmark, and the Netherlands. Fructan content increased significantly during cold acclimation and varieties showed significant differences in the level of fructan accumulation. cDNA sequences of putative fructosyltransferase (FT), fructan exohydrolase (FEH), and cold acclimation protein (CAP) genes were identified and cloned. In agreement with a function in fructan biosynthesis, transcription of a putative sucrose:fructan 6-fructosyltransferase (Pp6-SFT) gene was induced during cold acclimation and fructan accumulation in all three P. pratensis varieties. Transcription of putative PpFEH and PpCAP genes was also induced by cold acclimation; however, transcription of these two genes was several-fold higher in the variety from Norway compared to the other two varieties. The results presented here suggest that Pp6-SFT is involved in fructan biosynthesis in P. pratensis. FEHs have previously been suggested to be involved in fructan biosynthesis and freezing tolerance, and induced expression of PpFEH during fructan accumulation could also suggest a role in fructan biosynthesis. However, based on the different PpFEH transcription rates among varieties and similar expression of PpFEH and PpCAP, we suggest that PpFEH is more likely to be involved in mediating freezing tolerance, e.g., by regulating the cell osmotic potential through fructan degradation.

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains

Summary The phytase purple acid phosphatase (HvPAPhy_a) expressed during barley seed development was evaluated as transgene for overexpression in barley. The phytase was expressed constitutively driven by the cauliflower mosaic virus 35S‐promoter, and the phytase activity was measured in the mature grains, the green leaves and in the dry mature vegetative plant parts left after harvest of the grains. The T2‐generation of HvPAPhy_a transformed barley showed phytase activity increases up to 19‐fold (29 000 phytase units (FTU) per kg in mature grains). Moreover, also in green leaves and mature dry straw, phytase activities were increased significantly by 110‐fold (52 000 FTU/kg) and 57‐fold (51 000 FTU/kg), respectively. The HvPAPhy_a‐transformed barley plants with high phytase activities possess triple potential utilities for the improvement of phosphate bioavailability. First of all, the utilization of the mature grains as feed to increase the release of bio‐available phosphate and minerals bound to the phytate of the grains; secondly, the utilization of the powdered straw either directly or phytase extracted hereof as a supplement to high phytate feed or food; and finally, the use of the stubble to be ploughed into the soil for mobilizing phytate‐bound phosphate for plant growth.