Effect of Phytase Derived from the E. coli AppA Gene on Weaned Piglet Performance, Apparent Total Tract Digestibility and Bone Mineralization.

The objective of this study was to assess the effect of novel appAT1 and appAT2 phytase inclusion at 250 phytase units (FTU)/kg on weaned piglet performance, the apparent total tract digestibility of P and Ca, and bone mineralization. Piglets (48 males) were randomly divided into four treatment groups: a positive control (PC), with recommended levels of phosphorus (P) and calcium (Ca), a negative control (NC) deficient in P and Ca, and two experimental groups with NC diets supplemented with phytase derived from the appA gene of Escherichia coli. Diets fed in a mashed form were divided into prestarter (0-21 days) and starter (22-42 days) periods. During the whole period of the study, the experimental diets improved (p < 0.05) the body weight gain (BWG) and feed conversion ratio (FCR) compared to the NC diet. The apparent total tract digestibility (ATTD) of the dry matter and crude protein was not significantly different among the diets. Phytase-supplemented diets improved the ATTD of P (p < 0.05) and the ATTD of Ca (p < 0.05). Bone ash content in the third metacarpal and P and Ca content were improved among the phytase supplemented diets compared to the NC diet.

Transcription Factor-Mediated Control of Anthocyanin Biosynthesis in Vegetative Tissues.

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology.

Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves.

Coloration of plant organs such as fruit, leaves and flowers through anthocyanin production is governed by a combination of MYB and bHLH type transcription factors (TFs). In this study we introduced Rosea1 (ROS1, a MYB type) and Delila (DEL, a bHLH type), into Nicotiana benthamiana leaves by agroinfiltration. ROS1 and DEL form a pair of well-characterized TFs from Snapdragon (Antirrhinum majus), which specifically induce anthocyanin accumulation when expressed in tomato fruit. In N. benthamiana, robust induction of a single anthocyanin, delphinidin-3-rutinoside (D3R) was observed after expression of both ROS1 and DEL. Surprisingly in addition to D3R, a range of additional metabolites were also strongly and specifically up-regulated upon expression of ROS1 and DEL. Except for the D3R, these induced compounds were not derived from the flavonoid pathway. Most notable among these are nornicotine conjugates with butanoyl, hexanoyl, and octanoyl hydrophobic moieties, and phenylpropanoid-polyamine conjugates such as caffeoyl putrescine. The defensive properties of the induced molecules were addressed in bioassays using the tobacco specialist lepidopteran insect Manduca sexta. Our study showed that the effect of ROS1 and DEL expression in N. benthamiana leaves extends beyond the flavonoid pathway. Apparently the same transcription factor may regulate different secondary metabolite pathways in different plant species.

An O-methyltransferase modifies accumulation of methylated anthocyanins in seedlings of tomato.

Anthocyanins contribute to the appearance of fruit by conferring to them a red, blue or purple colour. In a food context, they have also been suggested to promote consumer health. In purple tomato tissues, such as hypocotyls, stems and purple fruits, various anthocyanins accumulate. These molecules have characteristic patterns of modification, including hydroxylations, methylations, glycosylations and acylations. The genetic basis for many of these modifications has not been fully elucidated, and nor has their role in the functioning of anthocyanins. In this paper, AnthOMT, an O-methyltransferase (OMT) mediating the methylation of anthocyanins, has been identified and functionally characterized using a combined metabolomics and transcriptomics approach. Gene candidates were selected from the draft tomato genome, and their expression was subsequently monitored in a tomato seedling system comprising three tissues and involving several time points. In addition, we also followed gene expression in wild-type red and purple transgenic tomato fruits expressing Rosea1 and Delila transcription factors. Of the 57 candidates identified, only a single OMT gene showed patterns strongly correlating with both accumulation of anthocyanins and expression of anthocyanin biosynthesis genes. This candidate (AnthOMT) was compared to a closely related caffeoyl CoA OMT by recombinant expression in Escherichia coli, and then tested for substrate specificity. AnthOMT showed a strong affinity for glycosylated anthocyanins, while other flavonoid glycosides and aglycones were much less preferred. Gene silencing experiments with AnthOMT resulted in reduced levels of the predominant methylated anthocyanins. This confirms the role of this enzyme in the diversification of tomato anthocyanins.

Balancing of histone H3K4 methylation states by the Kdm5c/SMCX histone demethylase modulates promoter and enhancer function.

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters.

A phospho/methyl switch at histone H3 regulates TFIID association with mitotic chromosomes.

Histone methylation patterns are correlated with eukaryotic gene transcription. High-affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine-4 of histone H3 (H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high-affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3-PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3-mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live-cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho-methyl switch regulates TFIID-mediated transcription during mitotic progression of the cell cycle.

PHF8 targets histone methylation and RNA polymerase II to activate transcription.

Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. PHF8 contains a plant homeodomain (PHD) in its N terminus and is a member of a family of JmjC domain-containing proteins. While PHDs can act as methyl lysine recognition motifs, JmjC domains can catalyze lysine demethylation. Here, we show that PHF8 is a histone demethylase that removes repressive histone H3 dimethyl lysine 9 marks. Our biochemical analysis revealed specific association of the PHF8 PHD with histone H3 trimethylated at lysine 4 (H3K4me3). Chromatin immunoprecipitation followed by high-throughput sequencing indicated that PHF8 is enriched at the transcription start sites of many active or poised genes, mirroring the presence of RNA polymerase II (RNAPII) and of H3K4me3-bearing nucleosomes. We show that PHF8 can act as a transcriptional coactivator and that its activation function largely depends on binding of the PHD to H3K4me3. Furthermore, we present evidence for direct interaction of PHF8 with the C-terminal domain of RNAPII. Importantly, a PHF8 disease mutant was defective in demethylation and in coactivation. This is the first demonstration of a chromatin-modifying enzyme that is globally recruited to promoters through its association with H3K4me3 and RNAPII.

Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice.

Malaria kills >1 million people each year, in particular in sub-Saharan Africa. Although asexual forms are directly responsible for disease and death, sexual stages account for the transmission of Plasmodium parasites from human to the mosquito vector and therefore the spread of the parasite in the population. Development of a malaria vaccine is urgently needed to reduce morbidity and mortality. Vaccines against sexual stages of Plasmodium falciparum are meant to decrease the force of transmission and consequently reduce malaria burden. Pfs48/45 is specifically expressed in sexual stages and is a well established transmission-blocking (TB) vaccine candidate. However, production of correctly folded recombinant Pfs48/45 protein with display of its TB epitopes has been a major challenge. Here, we show the production of a properly folded Pfs48/45 C-terminal fragment by simultaneous coexpression with four periplasmic folding catalysts in Escherichia coli. This C-terminal fragment fused to maltose binding protein was produced at medium scale with >90% purity and a stability over at least a 9-month period. It induces uniform and high antibody titers in mice and elicits functional TB antibodies in standard membrane feeding assays in 90% of the immunized mice. Our data provide a clear perspective on the clinical development of a Pfs48/45-based TB malaria vaccine.

Epitope analysis of the malaria surface antigen pfs48/45 identifies a subdomain that elicits transmission blocking antibodies.

Pfs48/45, a member of a Plasmodium-specific protein family, displays conformation-dependent epitopes and is an important target for malaria transmission-blocking (TB) immunity. To design a recombinant Pfs48/45-based TB vaccine, we analyzed the conformational TB epitopes of Pfs48/45. The Pfs48/45 protein was found to consist of a C-terminal six-cysteine module recognized by anti-epitope I antibodies, a middle four-cysteine module recognized by anti-epitopes IIb and III, and an N-terminal module recognized by anti-epitope V antibodies. Refolding assays identified that a fragment of 10 cysteines (10C), comprising the middle four-cysteine and the C-terminal six-cysteine modules, possesses superior refolding capacity. The refolded and partially purified 10C conformer elicited antibodies in mice that targeted at least two of the TB epitopes (I and III). The induced antibodies could block the fertilization of Plasmodium falciparum gametes in vivo in a concentration-dependent manner. Our results provide important insight into the structural organization of the Pfs48/45 protein and experimental support for a Pfs48/45-based subunit vaccine.

Specific cysteine protease inhibitors act as deterrents of western flower thrips, Frankliniella occidentalis (Pergande), in transgenic potato.

In this study, the effects of the accumulation of cysteine protease inhibitors on the food preferences of adult female western flower thrips, Frankliniella occidentalis (Pergande), were investigated. Representative members of the cystatin and thyropin gene families (stefin A, cystatin C, kininogen domain 3 and equistatin) were expressed in potato (Solanum tuberosum) cv. Impala, Kondor and Line V plants. In choice assays, a strong time- and concentration-dependent deterrence from plants expressing stefin A and equistatin was observed. Cystatin C and kininogen domain 3 were not found to be active. All tested inhibitors were equally or more active than stefin A at inhibiting the proteolytic activity of thrips, but, in contrast with stefin A, they were all expressed in potato as partially degraded proteins. The resistance of cysteine protease inhibitors against degradation in planta by endogenous plant proteases may therefore be relevant in explaining the observed differences in the deterrence of thrips. The results demonstrate that, when given a choice, western flower thrips will select plants with low levels of certain cysteine protease inhibitors. The novel implications of the defensive role of plant cysteine protease inhibitors as both deterrents and antimetabolic proteins are discussed.

Engineered multidomain cysteine protease inhibitors yield resistance against western flower thrips (Frankliniella occidentalis) in greenhouse trials.

Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), cause very large economic damage on a variety of field and greenhouse crops. In this study, plant resistance against thrips was introduced into transgenic potato plants through the expression of novel, custom-made, multidomain protease inhibitors. Representative classes of inhibitors of cysteine and aspartic proteases [kininogen domain 3 (K), stefin A (A), cystatin C (C), potato cystatin (P) and equistatin (EIM)] were fused into reading frames consisting of four (K-A-C-P) to five (EIM-K-A-C-P) proteins, and were shown to fold into functional inhibitors in the yeast Pichia pastoris. The multidomain proteins were expressed in potato and found to be more resistant to degradation by plant proteases than the individual domains. In a time span of 14-16 days, transgenic potato plants expressing EIMKACP and KACP at a similar concentration reduced the number of larvae and adults to less than 20% of the control. Leaf damage on protected plants was minimal. Engineered multidomain cysteine protease inhibitors thus provide a novel way of controlling western flower thrips in greenhouse and field crops, and open up possibilities for novel insect resistance applications in transgenic crops.

Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production.

Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desirée) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P(1)-positions (asparagine [Asn]-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed.

Cloning, functional expression in Pichia pastoris, and purification of potato cystatin and multicystatin.

In the tubers and leaves of potato, Solanum tuberosum, cysteine protease inhibitors are thought to play roles in the defence against herbivores and in regulating physiological processes like senescence and cell death. The cDNAs for two such inhibitors, potato multicystatin (PMC) with 8 cystatin domains and potato cystatin (PC) with a single domain, were cloned and expressed in the yeast Pichia pastoris. PC yielded on average 100 mg of purified active protein from 1l of culture supernatant. Purification to homogeneity was done in one step by cation exchange. The apparent equilibrium dissociation constant (K(i)) for papain was 0.1 nM. Cloning of the PMC cDNA was successful despite apparent toxicity for Escherichia coli and a high frequency of recombination events in RecA- strains of E. coli. In yeast, the expression of the cloned full length PMC gene was poor compared to that of the single domain.

Optimization of the expression of equistatin in Pichia pastoris.

To improve the expression of equistatin, a proteinase inhibitor from the sea anemone Actinia equina, in the yeast Pichia pastoris, we prepared gene variants with yeast-preferred codon usage and lower repetitive AT and GC content. The full gene optimization approximately doubled the level of steady-state mRNA and protein accumulated in the culture medium. The removal of a short stretch of 12 additional nucleotides from the multiple cloning site (MCS) sequence in the vector pPIC9 had an enhancement effect similar to full gene optimization (factor 1.5) at the mRNA level. However, at the protein level, this increase was 4- to 10-fold. The optimized gene without the MCS sequence yielded 1.66 g/L active protein in a bioreactor and was purified by a new two-step procedure with a recovery of activity that was >95%. This production level constitutes an overall improvement of about 20-fold relative to our previously published results. The characteristics of the MCS sequence element are discussed in the light of its apparent ability to act as negative expression regulator.