CRISPR/Cas9-mediated mutation of Eil1 transcription factor genes affects exogenous ethylene tolerance and early flower senescence in Campanula portenschlagiana.

Improving tolerance to ethylene-induced early senescence of flowers and fruits is of major economic importance for the ornamental and food industry. Genetic modifications of genes in the ethylene-signalling pathway have frequently resulted in increased tolerance but often with unwanted side effects. Here, we used CRISPR/Cas9 to knockout the function of two CpEil1 genes expressed in flowers of the diploid ornamental plant Campanula portenschlagiana. The ethylene tolerance in flowers of the primary mutants with knockout of only one or all four alleles clearly showed increased tolerance to exogenous ethylene, although lower tolerance was obtained with one compared to four mutated alleles. The allele dosage effect was confirmed in progenies where flowers of plants with zero, one, two, three and four mutated alleles showed increasing ethylene tolerance. Mutation of the Cpeil1 alleles had no significant effect on flower longevity and endogenous flower ethylene level, indicating that CpEil1 is not involved in age-dependent senescence of flowers. The study suggests focus on EIN3/Eils expressed in the organs subjected to early senescence for obtaining tolerance towards exogenous ethylene. Furthermore, the observed allelic dosage effect constitutes a key handle for a gradual regulation of sensitivity towards exogenous ethylene, simultaneously monitoring possibly unwanted side effects.

Structure of a cereal purple acid phytase provides new insights to phytate degradation in plants.

Grain phytate, a mixed metal ion salt of inositol hexakisphosphate, accounts for 60%-80% of stored phosphorus in plants and is a potent antinutrient of non-ruminant animals including humans. Through neofunctionalization of purple acid phytases (PAPhy), some cereals such as wheat and rye have acquired particularly high mature grain phytase activity. As PAPhy activity supplies phosphate, liberates metal ions necessary for seedling emergence, and obviates antinutrient effects of phytate, its manipulation and control are targeted crop traits. Here we show the X-ray crystal structure of the b2 isoform of wheat PAPhy induced during germination. This high-resolution crystal structure suggests a model for phytate recognition that, validated by molecular dynamics simulations, implicates elements of two sequence inserts (termed PAPhy motifs) relative to a canonical metallophosphoesterase (MPE) domain in forming phytate-specific substrate specificity pockets. These motifs are well conserved in PAPhys from monocot cereals, enzymes which are characterized by high specificity for phytate. Tested by mutagenesis, residues His229 in PAPhy motif 4 and Lys410 in the MPE domain, both conserved in PAPhys, are found to strongly influence phytase activity. These results explain the observed phytase activity of cereal PAPhys and open the way to the rational engineering of phytase activity in planta.

Barley Nepenthesin-Like Aspartic Protease HvNEP-1 Degrades Fusarium Phytase, Impairs Toxin Production, and Suppresses the Fungal Growth.

Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs. This study describes the effect of nepenthesin 1 (HvNEP-1) protease from barley (Hordeum vulgare L.) on fungal histidine acid phosphatase (HAP) phytase activity. Signal peptide lacking HvNEP-1 was expressed in Pichia pastoris and biochemically characterized. Recombinant HvNEP-1 (rHvNEP-1) strongly inhibited the activity of Aspergillus and Fusarium phytases, which are enzymes that release inorganic phosphorous from phytic acid. Moreover, rHvNEP-1 suppressed in vitro fungal growth and strongly reduced the production of mycotoxin, 15-acetyldeoxynivalenol (15-ADON), from Fusarium graminearum. The quantitative PCR analysis of trichothecene biosynthesis genes (TRI) confirmed that rHvNEP-1 strongly repressed the expression of TRI4, TRI5, TRI6, and TRI12 in F. graminearum. The co-incubation of rHvNEP-1 with recombinant F. graminearum (rFgPHY1) and Fusarium culmorum (FcPHY1) phytases induced substantial degradation of both Fusarium phytases, indicating that HvNEP-1-mediated proteolysis of the fungal phytases contributes to the HvNEP-1-based suppression of Fusarium.

A Roadmap to Modulated Anthocyanin Compositions in Carrots.

Anthocyanins extracted from black carrots have received increased interest as natural colorants in recent years. The reason is mainly their high content of acylated anthocyanins that stabilizes the color and thereby increases the shelf-life of products colored with black carrot anthocyanins. Still, the main type of anthocyanins synthesized in all black carrot cultivars is cyanidin limiting their use as colorants due to the narrow color variation. Additionally, in order to be competitive against synthetic colors, a higher percentage of acylated anthocyanins and an increased anthocyanin content in black carrots are needed. However, along with the increased interest in black carrots there has also been an interest in identifying the structural and regulatory genes associated with anthocyanin biosynthesis in black carrots. Thus, huge progress in the identification of genes involved in anthocyanin biosynthesis has recently been achieved. Given this information it is now possible to attempt to modulate anthocyanin compositions in black carrots through genetic modifications. In this review we look into genetic modification opportunities for generating taproots of black carrots with extended color palettes, with a higher percentage of acylated anthocyanins or a higher total content of anthocyanins.

Anthocyanin synthesis in orange carrot cv. Danvers is activated by transgene expression of the transcription factors DcMYB113_NB and DcEGL1_NB from black carrot cv. Nightbird.

Black carrots are potent sources of anthocyanin for the natural food color industry as their anthocyanins contain very high percentages of acylated anthocyanins which are much more stable than non-acylated anthocyanins. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway activated by a triad of R2R3-MYB, bHLH and WD40 transcription factors (TFs). Recent studies in black carrots have elucidated major anthocyanin related structural genes and also regulatory TFs. However, the active TFs responsible for anthocyanin production in black carrots differ between cultivars. We have previously shown by RNAseq that DcMYB113 (LOC108213488), a R2R3-MYB TF, was up-regulated in colored as compared to non-colored tissues of the black carrots 'Superblack' and 'CH05544' and that this upregulation was positively correlated with anthocyanin content. However, this gene showed no upregulation in the black carrot 'Nightbird' also included in that study. In the present study, we present a novel R2R3-MYB DcMYB113_NB (LOC108212072) and a complementary bHLH DcEGL1_NB (LOC108210744) isolated from the RNA of 'Nightbird'. Their functionality as anthocyanin regulators was confirmed by their simultaneous expression under the control of a constitutive promoter in the background of the orange carrot 'Danvers 126'. Transformants showed activation of the structural anthocyanin genes and accumulation of anthocyanins across leaves, stems and taproots. Interestingly, the anthocyanin profile of the transformants showed increases of 20 to 30% in acylated anthocyanins as compared to 'Nightbird' resulting in transformants with almost 100% acylated anthocyanins.

Hydrogen Sulfide and Carnosine: Modulation of Oxidative Stress and Inflammation in Kidney and Brain Axis.

Emerging evidence indicates that the dysregulation of cellular redox homeostasis and chronic inflammatory processes are implicated in the pathogenesis of kidney and brain disorders. In this light, endogenous dipeptide carnosine (ß-alanyl-L-histidine) and hydrogen sulfide (H2S) exert cytoprotective actions through the modulation of redox-dependent resilience pathways during oxidative stress and inflammation. Several recent studies have elucidated a functional crosstalk occurring between kidney and the brain. The pathophysiological link of this crosstalk is represented by oxidative stress and inflammatory processes which contribute to the high prevalence of neuropsychiatric disorders, cognitive impairment, and dementia during the natural history of chronic kidney disease. Herein, we provide an overview of the main pathophysiological mechanisms related to high levels of pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and neurotoxins, which play a critical role in the kidney-brain crosstalk. The present paper also explores the respective role of H2S and carnosine in the modulation of oxidative stress and inflammation in the kidney-brain axis. It suggests that these activities are likely mediated, at least in part, via hormetic processes, involving Nrf2 (Nuclear factor-like 2), Hsp 70 (heat shock protein 70), SIRT-1 (Sirtuin-1), Trx (Thioredoxin), and the glutathione system. Metabolic interactions at the kidney and brain axis level operate in controlling and reducing oxidant-induced inflammatory damage and therefore, can be a promising potential therapeutic target to reduce the severity of renal and brain injuries in humans.

Molecular Properties and New Potentials of Plant Nepenthesins.

Nepenthesins are aspartic proteases (APs) categorized under the A1B subfamily. Due to nepenthesin-specific sequence features, the A1B subfamily is also named nepenthesin-type aspartic proteases (NEPs). Nepenthesins are mostly known from the pitcher fluid of the carnivorous plant Nepenthes, where they are availed for the hydrolyzation of insect protein required for the assimilation of insect nitrogen resources. However, nepenthesins are widely distributed within the plant kingdom and play significant roles in plant species other than Nepenthes. Although they have received limited attention when compared to other members of the subfamily, current data indicates that they have exceptional molecular and biochemical properties and new potentials as fungal-resistance genes. In the current review, we provide insights into the current knowledge on the molecular and biochemical properties of plant nepenthesins and highlights that future focus on them may have strong potentials for industrial applications and crop trait improvement.

Cyanidin based anthocyanin biosynthesis in orange carrot is restored by expression of AmRosea1 and AmDelila, MYB and bHLH transcription factors.

KEY MESSAGE: The simultaneous expression of AmRosea1 and AmDelila transcription factors from snapdragon can activate the anthocyanin pathway in orange carrots, leading to the synthesis and accumulation of anthocyanins in the taproots. Anthocyanins are phenolic compounds produced in various parts of plants. They are used as natural food dyes and are reported as beneficial antioxidants for humans. Black carrot is an important source for anthocyanins; however, the reason for the lack of anthocyanin production in the orange carrot is unknown. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway that has previously been reported to be activated by a triad of R2R3-MYB, basic helix-loop helix (bHLH) and WD40 transcription factors (TFs). In the current study, orange carrots were turned purple by simultaneous expression of R2R3-MYB and bHLH TFs, i.e. AmRosea1 and AmDelila from snapdragon (Antirrhinum majus). Simultaneous transgenic expression of the TFs under a constitutive promoter in the orange carrot cultivar 'Danvers 126' lead to consistent upregulation of anthocyanin-related biosynthetic genes and significant accumulation of anthocyanins in leaves, stems and taproots. Highest overall content of soluble anthocyanins in the taproot among the transformants amounted to 44.38 mg g-1 dry weight. The anthocyanin profile of the transformants were significantly different from the profile in the reference black carrot 'Deep Purple'. The main anthocyanins present in the transformed taproots were cyanidin 3-xylosyl(sinapoylglucosyl)galactoside, whereas the main anthocyanin present in Deep Purple was cyanidin 3-xylosyl(feruloylglucosyl)galactoside. This study confirms the presence of the necessary biosynthetic genes in orange carrots for production of anthocyanins and demonstrates the absence of suitable R2R3-MYB and bHLH TFs for stimulating anthocyanin biosynthesis in the orange carrot.

Identification of Azoxystrobin Glutathione Conjugate Metabolites in Maize Roots by LC-MS.

Xenobiotic detoxification in plant as well as in animals has mostly been studied in relationship to the deactivation of the toxic residues of the compound that, surely for azoxystrobin, is represented by its ß-methoxyacrylate portion. In maize roots treated for 96 h with azoxystrobin, the fungicide accumulated over time and detoxification compounds or conjugates appeared timewise. The main detoxified compound was the methyl ester hydrolysis product (azoxystrobin free acid, 390.14 m/z) thought to be inactive followed by the glutathione conjugated compounds identified as glutathione conjugate (711.21 m/z) and its derivative lacking the glycine residue from the GSH (654.19 m/z). The glycosylated form of azoxystrobin was also found (552.19 m/z) in a minor amount. The identification of these analytes was done by differential untargeted metabolomics analysis using Progenesis QI for label free spectral counting quantification and MS/MS confirmation of the compounds was carried out by either Data Independent Acquisition (DIA) and Data Dependent Acquisition (DDA) using high resolution LC-MS methods. Neutral loss scanning and comparison with MS/MS spectra of azoxystrobin by DDA and MSe confirmed the structures of these new azoxystrobin GSH conjugates.

Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS.

Background: Zinc accumulates in the embryo, aleurone, and subaleurone layers at different amounts in cereal grains. Our hypothesis is that zinc could be stored bound, not only to low MW metabolites/proteins, but also to high MW proteins as well. Methods: In order to identify the most abundant zinc binding proteins in different grain tissues, we microdissected barley grains into (1) seed coats; (2) aleurone/subaleurone; (3) embryo; and (4) endosperm. Initial screening for putative zinc binding proteins from the different tissue types was performed by fractionating proteins according to solubility (Osborne fractionation), and resolving those via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by polyvinylidene fluoride (PVDF) membrane blotting and dithizone staining. Selected protein fractions were subjected to Zn2+-immobilized metal ion affinity chromatography, and the captured proteins were identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Results: In the endosperm, the most abundant zinc binding proteins were the storage protein B-hordeins, gamma-, and D-hordeins, while in the embryo, 7S globulins storage proteins exhibited zinc binding. In the aleurone/subaleurone, zinc affinity captured proteins were late abundant embryogenesis proteins, dehydrins, many isoforms of non-specific lipid transfer proteins, and alpha amylase trypsin inhibitor. Conclusions: We have shown evidence that abundant barley grain proteins have been captured by Zn-IMAC, and their zinc binding properties in relationship to the possibility of zinc storage is discussed.

Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum.

Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5-9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5-8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum.

Aspergillus ficuum phytase activity is inhibited by cereal grain components.

In the current study, we report for the first time that grain components of barley, rice, wheat and maize can inhibit the activity of Aspergillus ficuum phytase. The phytase inhibition is dose dependent and varies significantly between cereal species, between cultivars of barley and cultivars of wheat and between Fusarium graminearum infected and non-infected wheat grains. The highest endpoint level of phytase activity inhibition was 90%, observed with grain protein extracts (GPE) from F. graminearum infected wheat. Wheat GPE from grains infected with F. graminearum inhibits phytase activity significantly more than GPE from non-infected grains. For four barley cultivars studied, the IC50 value ranged from 0.978 ± 0.271 to 3.616 ± 0.087 mg×ml-1. For two non-infected wheat cultivars investigated, the IC50 values were varying from 2.478 ± 0.114 to 3.038 ± 0.097 mg×ml-1. The maize and rice cultivars tested gaveIC50 values on 0.983 ± 0.205 and 1.972 ± 0.019 mg×ml-1, respectively. After purifying the inhibitor from barley grains via Superdex G200, an approximately 30-35 kDa protein was identified. No clear trend for the mechanism of inhibition could be identified via Michaelis-Menten kinetics and Lineweaver-Burk plots. However, testing of the purified phytase inhibitor together with the A. ficuum phytase and the specific protease inhibitors pepstatin A, E64, EDTA and PMSF revealed that pepstatin A repealed the phytase inhibition. This indicates that the observed inhibition of A. ficuum phytase by cereal grain extracts is caused by protease activity of the aspartic proteinase type.

Biosynthesis and chemical transformation of benzoxazinoids in rye during seed germination and the identification of a rye Bx6-like gene.

Benzoxazinoids are secondary metabolites with plant defense properties and possible health-promoting effects in humans. In this study, the transcriptional activity of ScBx genes (ScBx1-ScBx5; ScBx6-like), involved in benzoxazinoid biosynthesis, was analyzed during germination and early seedling development in rye. Our results showed that ScBx genes had highest levels of expression at 24-30 h after germination, followed by a decrease at later stages. For ScBx1-ScBx5 genes expression was higher in shoots compared with root tissues and vice versa for ScBx6-like gene transcripts. Moreover, methylated forms of benzoxazinoids accumulated in roots rather than in shoots during seedling development, in particular reaching high levels of HMBOA-glc in roots. Chemical profiles of benzoxazinoid accumulation in the developing seedling reflected the combined effects of de novo biosynthesis of the compounds as well as the turnover of compounds either pre-stored in the embryo or de novo biosynthesized. Bioinformatic analysis, together with the differential distribution of ScBx6-like transcripts in root and shoot tissues, suggested the presence of a ZmBx6 homolog encoding a 2-oxoglutarate dependent dehydrogenase in rye. The ScBx6-like cDNA was expressed in E. coli for functional characterization in vitro. LC-MS/MS analysis showed that the purified enzyme was responsible for the oxidation of DIBOA-glc into TRIBOA-glc, strongly suggesting the ScBX6-like enzyme in rye to be a functional ortholog of maize ZmBX6.

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

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.

Label-Free Differential Proteomics and Quantification of Exoenzymes from Isolates of the Entomopathogenic Fungus Beauveria bassiana.

Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression (quality/quantity) of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w/v of dead intact American cockroach (Periplaneta americana) or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion.

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.

Effect of pH and recombinant barley (Hordeum vulgare L.) endoprotease B2 on degradation of proteins in soaked barley.

Nonfermented soaking of barley feedstuff has been established as an in vitro procedure prior to the feeding of pigs as it can increase protein digestibility. In the current study, two feed cultivars of barley (Finlissa and Zephyr) were soaked in vitro either nonbuffered or buffered at pH 3.6 and 4.3. Solubilized and degraded proteins evaluated by biuret, SDS-PAGE, and differential proteomics revealed that pH 4.3 had the greatest impact on both solubilization and degradation. In order to boost proteolysis, the recombinant barley endoprotease B2 (rec-HvEP-B2) was included after 8 h using the pH 4.3 regime. Proteolysis evaluated by SDS-PAGE and differential proteomics confirmed a powerful effect of adding rec-HvEP-B2 to the soaked barley, regardless of the genotype. Our study addresses the use of rec-HvEP-B2 as an effective feed enzyme protease. HvEP-B2 has the potential to increase the digestibility of protein in the pig, either supplied as recombinant additive or as possible new selection criterion in barley breeding.

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

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.