A dynamic compartment model for xylem loading and long-distance transport of iron explains the effect of kanamycin on metal uptake in Arabidopsis.

Arabidopsis plants exposed to the antibiotic kanamycin (Kan) display altered metal homeostasis. Further, mutation of the WBC19 gene leads to increased sensitivity to kanamycin and changes in iron (Fe) and zinc (Zn) uptake. Here we propose a model that explain this surprising relationship between metal uptake and exposure to Kan. We first use knowledge about the metal uptake phenomenon to devise a transport and interaction diagram on which we base the construction of a dynamic compartment model. The model has three pathways for loading Fe and its chelators into the xylem. One pathway, involving an unknown transporter, loads Fe as a chelate with citrate (Ci) into the xylem. This transport step can be significantly inhibited by Kan. In parallel, FRD3 transports Ci into the xylem where it can chelate with free Fe. A third critical pathway involves WBC19, which transports metal-nicotianamine (NA), mainly as Fe-NA chelate, and possibly NA itself. To permit quantitative exploration and analysis, we use experimental time series data to parameterize this explanatory and predictive model. Its numerical analysis allows us to predict responses by a double mutant and explain the observed differences between data from wildtype, mutants and Kan inhibition experiments. Importantly, the model provides novel insights into metal homeostasis by permitting the reverse-engineering of mechanistic strategies with which the plant counteracts the effects of mutations and of the inhibition of iron transport by kanamycin.

CRISPR/Cas9-Induced Loss-of-Function Mutation in the Barley Mitogen-Activated Protein Kinase 6 Gene Causes Abnormal Embryo Development Leading to Severely Reduced Grain Germination and Seedling Shootless Phenotype.

The diverse roles of mitogen-activated protein kinases (MAPKs, MPKs) in plant development could be efficiently revealed by reverse genetic studies. In Arabidopsis, mpk6 knockout mutants complete the life cycle; however, ~40% of their embryos show defects in the development leading to abnormal phenotypes of seeds and seedlings' roots. Contrary to the Arabidopsis MPK6, the rice MPK6 (OsMPK6) is an essential gene as transfer DNA (T-DNA) insertion and CRISPR/Cas9 induced loss-of-function mutations in the OsMPK6 cause early embryo arrest. In this study, we successfully developed a viable transgenic barley line with the CRISPR/Cas9-induced heterozygous single base pair cytosine-guanine (CG) deletion [wild type (WT)/-1C] in the third exon of the HvMPK6 gene, a barley ortholog of the Arabidopsis and rice MPK6. There were no obvious macroscopic phenotype differences between the WT/-1C plants and WT plants. All the grains collected from the WT/-1C plants were of similar size and appearance. However, seedling emergence percentage (SEP) from these grains was substantially decreased in the soil in the T2 and T3 generation. The mutation analysis of the 248 emerged T2 and T3 generation plants showed that none of them was a biallelic mutant in the HvMPK6 gene, suggesting lethality of the -1C/-1C homozygous knockout mutation. In the soil, the majority of the -1C/-1C grains did not germinate and the minority of them developed into abnormal seedlings with a shootless phenotype and a reduced root system. Some of the -1C/-1C seedlings also developed one or more small chlorotic leaf blade-like structure/structures. The -1C/-1C grains contained the late-stage developed abnormal embryos with the morphologically obvious scutellum and root part of the embryonic axis but with the missing or substantially reduced shoot part of the embryonic axis. The observed embryonic abnormalities correlated well with the shootless phenotype of the seedlings and suggested that the later-stage defect is predetermined already during the embryo development. In conclusion, our results indicate that barley MPK6 is essential for the embryologically predetermined shoot formation, but not for the most aspects of the embryo and early seedling development.

TALEN-Based HvMPK3 Knock-Out Attenuates Proteome and Root Hair Phenotypic Responses to flg22 in Barley.

Mitogen activated protein kinases (MAPKs) integrate elicitor perception with both early and late responses associated with plant defense and innate immunity. Much of the existing knowledge on the role of plant MAPKs in defense mechanisms against microbes stems from extensive research in the model plant Arabidopsis thaliana. In the present study, we investigated the involvement of barley (Hordeum vulgare) MPK3 in response to flagellin peptide flg22, a well-known bacterial elicitor. Using differential proteomic analysis we show that TALEN-induced MPK3 knock-out lines of barley (HvMPK3 KO) exhibit constitutive downregulation of defense related proteins such as PR proteins belonging to thaumatin family and chitinases. Further analyses showed that the same protein families were less prone to flg22 elicitation in HvMPK3 KO plants compared to wild types. These results were supported and validated by chitinase activity analyses and immunoblotting for HSP70. In addition, differential proteomes correlated with root hair phenotypes and suggested tolerance of HvMPK3 KO lines to flg22. In conclusion, our study points to the specific role of HvMPK3 in molecular and root hair phenotypic responses of barley to flg22.

Homozygous Transgenic Barley (Hordeum vulgare L.) Plants by Anther Culture.

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

Allelic Variants of CRISPR/Cas9 Induced Mutation in an Inositol Trisphosphate 5/6 Kinase Gene Manifest Different Phenotypes in Barley.

Inositol trisphosphate 5/6 kinases (ITPK) constitute a small group of enzymes participating in the sequential phosphorylation of inositol phosphate to inositol hexakisphosphate (IP6), which is a major storage form of phosphate in cereal grains. The development of lines with reduced IP6 content could enhance phosphate and mineral bioavailability. Moreover, plant ITPKs participate in abiotic stress signaling. To elucidate the role of HvITPK1 in IP6 synthesis and stress signaling, a barley itpk1 mutant was created using programmable nuclease Cas9. Homozygous single bp insertion and deletion mutant lines were obtained. The mutants contained altered levels of phosphate in the mature grains, ranging from 65% to 174% of the wild type (WT) content. Homozygous mutant lines were tested for their response to salinity during germination. Interestingly, insertion mutant lines revealed a higher tolerance to salinity stress than deletion mutants. Mature embryos of an insertion mutant itpk1-2 and deletion mutant itpk1-33 were cultivated in vitro on MS medium supplemented with NaCl at 50, 100, and 200 mM. While both mutants grew less well than WT on no or low salt concentrations, the itpk1-2 mutant was affected less than the WT and itpk33 when grown on the highest NaCl concentration. The expression of all ITPKs was induced in roots in response to salt stress. In shoots, the differential effect of high salt on IPTK expression in the two iptk1 mutants was consistent with their different sensitivities to salt stress. The results extend the evidence for the involvement of ITPK genes in phosphate storage and abiotic stress signaling.

Recombinant expression of osmotin in barley improves stress resistance and food safety during adverse growing conditions.

BACKGROUND: Although many genetic manipulations of crops providing biofortified or safer food have been done, the acceptance of biotechnology crops still remains limited. We report on a transgenic barley expressing the multi-functional protein osmotin that improves plant defense under stress conditions. METHODS: An Agrobacterium-mediated technique was used to transform immature embryos of the spring barley cultivar Golden Promise. Transgenic barley plants of the T0 and T1 generation were evaluated by molecular methods. Transgenic barley tolerance to stress was determined by chlorophyll, total protein, malondialdehyde and ascorbate peroxidase content. Methanol extracts of i) Fusarium oxysporum infected or ii) salt-stressed plants, were characterized by their acute toxicity effect on human dermal fibroblasts (HDF), genotoxicity and affection of biodiversity interactions, which was tested through monitoring barley natural virus pathogen-host interactions-the BYDV and WDV viruses transmitted to the plants by aphids and leafhoppers. RESULTS: Transgenic plants maintained the same level of chlorophyll and protein, which significantly declined in wild-type barley under the same stressful conditions. Salt stress evoked higher ascorbate peroxidase level and correspondingly less malondialdehyde. Osmotin expressing barley extracts exhibited a lower cytotoxicity effect of statistical significance than that of wild-type plants under both types of stress tested on human dermal fibroblasts. Extract of Fusarium oxysporum infected transgenic barley was not able to damage DNA in the Comet assay, which is in opposite to control plants. Moreover, this particular barley did not affect the local biodiversity. CONCLUSION: Our findings provide a new perspective that could help to evaluate the safety of products from genetically modified crops.

Mutation Breeding in Barley: Historical Overview.

The discovery of radioactivity at the end of the nineteenth century played a key role in a series of historical landmarks that would lead to contemporary mutation breeding in agricultural crops. The aim of the earliest experiments was to test the effects of radiation on living organisms beginning with fruit flies. Exposure of plants to X-rays provided the first incontrovertible proof that phenotypic changes could be induced. Chemicals were a second type of mutagen tested from the 1940s and both forms are used today. This chapter is an overview of some of the historical developments that led to the use of mutagenesis in plants, with a focus on barley, a model species for mutation genetics and breeding as well as a major cereal crop. Perhaps the most well-known examples of mutant barley cultivars are Diamant, Golden Promise, and their hybrids.

Barley Anther Culture.

The production of doubled haploid (DH) barley plants through anther culture is a very useful yet simple in vitro technique. DH plants derive from divisions of haploid microspores that have undergone a developmental switch under the appropriate conditions. The successive divisions lead to the formation of an embryo or callus rather than the formation of mature pollen grains. Plants that regenerate from these embryos are often either haploid, in which case their chromosome set can be doubled by treatment with colchicine, or spontaneous double haploids. The efficiency of DH plant production is highly variable depending on the genotype of the source material. Despite this limitation, DH plants have been widely used in breeding and research programs. Compared to conventional approaches, breeding strategies that makes use of DH plants achieve a homozygous state, allowing transgene or mutation stabilization in the genome, within a considerably shorter time, thus accelerating workflow or reducing work volume.

Pre-caecal digestible phosphorus in maize and wheat for broiler chickens.

1. The objective of this study was to determine the coefficient of pre-caecal digestion of P in maize (3.9 g/kg of total P, 0.83 g/kg of phytate P, 138 FTU [phytase units]/kg) and wheat (3.17 g/kg of total P, 1.94 g/kg of phytate P, 666 FTU/kg) in broilers according to the WPSA protocol. 2. For the diets, monosodium phosphate was used as an additional P supplement. Two sets of diets containing 200, 460 and 740 g/kg of wheat or 200, 500 and 740 g/kg of maize were formulated. A total of 288 21-d-old male broilers (Ross 308) were assigned to 24 cages (8 birds per cage) and the 6 test diets were assigned to cages. The coefficient of pre-caecal digestion of P was determined by the indicator method and linear regression. 3. In both ingredients, pre-caecal digestible P increased linearly with increasing inclusion levels of maize or wheat (P < 0.05). The coefficients of digestion of pre-caecal P were estimated to be 0.18 for wheat and 0.33 for maize.

Overexpression of cytokinin dehydrogenase genes in barley (Hordeum vulgare cv. Golden Promise) fundamentally affects morphology and fertility.

Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.

Electrophoretic and chromatographic evaluation of transgenic barley expressing a bacterial dihydrodipicolinate synthase.

Nutritional quality of human and animal foodstuffs is determined by the content of essential amino acids. Barley is the fourth most important cereal of the world and the second most important cereal grown in the Czech Republic. Cereal grains such as barley contain insufficient levels of some essential amino acids, especially lysine. Dihydrodipicolinate synthase is the key enzyme involved in the regulatory step for lysine biosynthesis. Two constructs pBract214::sTPdapA and pBract214::mdapA containing the dapA gene from Escherichia coli coding for the bacterial dihydrodipicolinate synthase were used for transformation of barley. An Agrobacterium-mediated technique was used for transformation of immature embryos of spring barley cv. Golden Promise. Transgenic barley plants of the T0 and T1 generations were evaluated by PCR, real-time PCR, gel electrophoresis, and Western blot. Amino acid content was analyzed by HPLC after HCl hydrolysis. The lysine content in leaves of the T1 generation plant no. 5/5 was 50% higher than in wild-type plants; the lysine content in seeds of T2 generation plant no. 5/16 was 30% higher than in wild-type seeds of spring barley cv. Golden Promise.

Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels.

Plant hormones, cytokinins (CKs), have been for a long time considered to be involved in plant responses to stress. However, their exact roles in processes linked to stress signalization and acclimatization to adverse environmental conditions are unknown. In this study, expression profiles of the entire gene families of CK biosynthetic and degradation genes in maize (Zea mays) during development and stress responses are described. Transcript abundance of particular genes is discussed in relation to the levels of different CK metabolites. Salt and osmotic stresses induce expression of some CK biosynthetic genes in seedlings of maize, leading to a moderate increase of active forms of CKs lasting several days during acclimatization to stress. A direct effect of CKs to mediate activation of stress responses does not seem to be possible due to the slow changes in metabolite levels. However, expression of genes involved in cytokinin signal transduction is uniformly down-regulated within 0.5 h of stress induction by an unknown mechanism. cis-Zeatin and its derivatives were found to be the most abundant CKs in young maize seedlings. We demonstrate that levels of this zeatin isomer are significantly enhanced during early stress response and that it originates independently from de novo biosynthesis in stressed tissues, possibly by elevated specific RNA degradation. By enhancing their CK levels, plants could perhaps undergo a reduction of growth rates maintained by abscisic acid accumulation in stressed tissues. A second role for cytokinin receptors in sensing turgor response is hypothesized besides their documented function in CK signaling.