Ecophysiology with barley eceriferum (cer) mutants: the effects of humidity and wax crystal structure on yield and vegetative parameters.

BACKGROUND AND AIMS: In addition to preventing water loss, plant cuticles must also regulate nutrient loss via leaching. The eceriferum mutants in Hordeum vulgare (barley) potentially influence these functions by altering epicuticular wax structure and composition. METHODS: Cultivar 'Bonus' and five of its cer mutants were grown under optimal conditions for vegetative growth and maturation, and nine traits were measured. Nutrient and water amounts going through the soil and the amount of simulated rain as deionized water, affecting phyllosphere humidity, delivered during either the vegetative or maturation phase, were varied. Cer leaf genes and three wilty (wlt) mutations were characterized for reaction to toluidine blue and the rate of non-stomatal water loss. KEY RESULTS: Vegetative phase rain on 'Bonus' significantly decreased kernel weight and numbers by 15-30 %, while in cer.j59 and .c36 decreases of up to 42 % occurred. Maturation phase findings corroborated those from the vegetative phase. Significant pleiotropic effects were identified: cer.j59 decreased culm and spike length and 1000-kernel weight, .c36 decreased kernel number and weight, .i16 decreased spike length and .e8 increased culm height. Excepting Cer.zv and .ym mutations, none of the other 27 Cer leaf genes or wlt mutations played significant roles, if any, in preventing water loss. Cer.zv and .ym mutants lost non-stomatal water 13.5 times faster than those of Cer.j, .yi, .ys and .zp and 18.3 times faster than those of four cultivars and the mutants tested here. CONCLUSIONS: Using yield to measure the net effect of phyllosphere humidity and wax crystal structure revealed that the former is far more important than the latter. The amenable experimental setup described here can be used to delve deeper. Significant pleiotropic effects were identified for mutations in four Cer genes, of which one is known to participate in wax biosynthesis. Twenty-seven Cer leaf genes and three wlt mutations have little if any effect on water loss.

Selection of transformation-efficient barley genotypes based on TFA (transformation amenability) haplotype and higher resolution mapping of the TFA loci.

KEY MESSAGE: The genetic substitution of transformation amenability alleles from 'Golden Promise' can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars. Barley (Hordeum vulgare) cv. 'Golden Promise' is one of the most useful and well-studied cultivars for genetic manipulation. In a previous report, we identified several transformation amenability (TFA) loci responsible for Agrobacterium-mediated transformation using the F2 generation of immature embryos, derived from 'Haruna Nijo' × 'Golden Promise,' as explants. In this report, we describe higher density mapping of these TFA regions with additional SNP markers using the same transgenic plants. To demonstrate the robustness of transformability alleles at the TFA loci, we genotyped 202 doubled haploid progeny from the cross 'Golden Promise' × 'Full Pint.' Based on SNP genotype, we selected lines having 'Golden Promise' alleles at TFA loci and used them for transformation. Of the successfully transformed lines, DH120366 came the closest to achieving a level of transformation efficiency comparable to 'Golden Promise.' The results validate that the genetic substitution of TFA alleles from 'Golden Promise' can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars.

HvZIP7 mediates zinc accumulation in barley (Hordeum vulgare) at moderately high zinc supply.

High expression of zinc (Zn)-regulated, iron-regulated transporter-like protein (ZIP) genes increases root Zn uptake in dicots, leading to high accumulation of Zn in shoots. However, none of the ZIP genes tested previously in monocots could enhance shoot Zn accumulation. In this report, barley (Hordeum vulgare) HvZIP7 was investigated for its functions in Zn transport. The functions of HvZIP7 in planta were studied using in situ hybridization and transient analysis of subcellular localization with a green fluorescent protein (GFP) reporter. Transgenic barley lines overexpressing HvZIP7 were also generated to further understand the functions of HvZIP7 in metal transport. HvZIP7 is strongly induced by Zn deficiency, primarily in vascular tissues of roots and leaves, and its protein was localized in the plasma membrane. These properties are similar to its closely related homologs in dicots. Overexpression of HvZIP7 in barley plants increased Zn uptake when moderately high concentrations of Zn were supplied. Significantly, there was a specific enhancement of shoot Zn accumulation, with no measurable increase in iron (Fe), manganese (Mn), copper (Cu) or cadmium (Cd). HvZIP7 displays characteristics of low-affinity Zn transport. The unique function of HvZIP7 provides new insights into the role of ZIP genes in Zn homeostasis in monocots, and offers opportunities to develop Zn biofortification strategies in cereals.

Transcriptome analysis of Bipolaris sorokiniana - Hordeum vulgare provides insights into mechanisms of host-pathogen interaction.

Spot blotch disease incited by Bipolaris sorokiniana severely affects the cultivation of barley. The resistance to B. sorokiniana is quantitative in nature and its interaction with the host is highly complex which necessitates in-depth molecular analysis. Thus, the study aimed to conduct the transcriptome analysis to decipher the mechanisms and pathways involved in interactions between barley and B. sorokiniana in both the resistant (EC0328964) and susceptible (EC0578292) genotypes using the RNA Seq approach. In the resistant genotype, 6,283 genes of Hordeum vulgare were differentially expressed out of which 5,567 genes were upregulated and 716 genes were downregulated. 1,158 genes of Hordeum vulgare were differentially expressed in the susceptible genotype, out of which 654 genes were upregulated and 504 genes were downregulated. Several defense-related genes like resistant gene analogs (RGAs), disease resistance protein RPM1, pathogenesis-related protein PRB1-2-like, pathogenesis-related protein 1, thaumatin-like protein PWIR2 and defensin Tm-AMP-D1.2 were highly expressed exclusively in resistant genotype only. The pathways involved in the metabolism and biosynthesis of secondary metabolites were the most prominently represented pathways in both the resistant and susceptible genotypes. However, pathways involved in MAPK signaling, plant-pathogen interaction, and plant hormone signal transduction were highly enriched in resistant genotype. Further, a higher number of pathogenicity genes of B. sorokiniana was found in response to the susceptible genotype. The pathways encoding for metabolism, biosynthesis of secondary metabolites, ABC transporters, and ubiquitin-mediated proteolysis were highly expressed in susceptible genotype in response to the pathogen. 14 and 11 genes of B. sorokiniana were identified as candidate effectors from susceptible and resistant host backgrounds, respectively. This investigation will offer valuable insights in unraveling the complex mechanisms involved in barley- B. sorokiniana interaction.

Retrotransposon BARE displays strong tissue-specific differences in expression.

The BARE retrotransposon comprises c. 10% of the barley (Hordeum vulgare) genome. It is actively transcribed, translated and forms virus-like particles (VLPs). For retrotransposons, the inheritance of new copies depends critically on where in the plant replication occurs. In order to shed light on the replication strategy of BARE in the plant, we have used immunolocalization and in situ hybridization to examine expression of the BARE capsid protein, Gag, at a tissue-specific level. Gag is expressed in provascular tissues and highly localized in companion cells surrounding the phloem sieve tubes in mature vascular tissues. BARE Gag and RNA was not seen in the shoot apical meristem of young seedlings, but appeared, following transition to flowering, in the developing floral spike. Moreover, Gag has a highly specific localization in pre-fertilization ovaries. The strong presence of Gag in the floral meristems suggests that newly replicated copies there will be passed to the next generation. BARE expression patterns are consistent with transcriptional regulation by predicted response elements in the BARE promoter, and in the ovary with release from epigenetic transcriptional silencing. To our knowledge, this is the first analysis of the expression of native retrotransposon proteins within a plant to be reported.

Asymmetric growth of root epidermal cells is related to the differentiation of root hair cells in Hordeum vulgare (L.).

The root epidermis of most vascular plants harbours two cell types, namely trichoblasts (capable of producing a root hair) and atrichoblasts. Here, in vivo analysis, confocal laser-scanning microscopy, transmission electron microscopy, histological analysis, and three-dimensional reconstruction were used to characterize the cell types present in the barley root epidermis and their distribution in the tissue. Both trichoblasts and atrichoblasts were present in the wild-type cultivars and could be distinguished from one another at an early stage. Trichoblast/atrichoblast differentiation depended on asymmetric cell expansion after a period of symmetrical cell division. After asymmetric growth, only the shorter epidermal cells could produce root hairs, whereas the longer cells became atrichoblasts. Moreover, the root epidermis did not develop root hairs at all if the epidermal cells did not differentiate into two asymmetric cell types. The root hairless phenotype of bald root barley (brb) and root hairless 1.b (rhl1.b) mutants was caused by a mutation in a gene related to the asymmetric expansion of the root epidermal cells. Additionally, the results showed that the mechanism of trichoblast/atrichoblast differentiation is not evolutionally conserved across the subfamilies of the Poaceae; in the Pooideae subfamily, both asymmetric division and asymmetric cell expansion have been observed.

Solute content and energy status of roots of barley plants cultivated at different pH on nitrate- or ammonium-nitrogen.

Barley seedlings (Hordeum vulgare cv. 'Gaulois') were precultured hydroponically for 4d on nutrient solutions with nitrate (3 mM) as N-source at pH 4.7, and were then transferred to solutions with ammonium or nitrate as N-source (3 mM), buffered and adjusted to different pH (4.0, 5.5, 6.8). Variations in shoot and root growth and solute contents were examined and grouped into pH-effects, ammonium effects and interactions. Shoot biomass was not affected under all conditions. Root fresh weight was insensitive to the external pH when nitrate was the N-source, but was drastically affected by a combination of ammonium and low pH. In contrast, root length was negatively affected by low pH per se. In nitrate-grown plants, ammonium levels in roots and shoots were low (0.5 to 1 mM). After transfer of plants to ammonium solution, roots accumulated ammonium within 24 h about sixfold (18 mM) above the external concentration. At pH 5.5 or 6.8, but not at pH 4, root ammonium contents decreased afterwards to a lower steady state value (10 mM). Leaves also accumulated ammonium, especially at the most acidic pH. Concentrations of major inorganic cations in roots were markedly but differentially affected by acidic pH and ammonium. The magnesium content of roots was drastically decreased (from 18 to 2 mM) by ammonium nutrition, and this was independent of the external pH. In contrast, calcium levels in roots were decreased by low external pH, independent of the N-source. Potassium levels in roots were rather insensitive to both low pH and ammonium. The pH of crude root homogenates was measured in order to obtain at least crude information about trends in possible cellular pH changes. At an external pH of 4.0 and 5.5, the pH of the homogenates was 5.8, and it increased to 6.3 when the external pH was 6.8. The pH of the homogenates was not affected by the N-source. In spite of the drastic effects of the N-source on the concentration of ammonium and magnesium in root tissues, ATP/ADP-ratios were not affected. Also, sugar levels were unchanged or even increased. Thus, growth impairment could not be traced back to impaired carbohydrate or ATP-supply, which might occur as a consequence of ammonium accumulation or Mg2+ -deficiency.

Competition between micro-organisms and roots of barley and sorghum for iron accumulated in the root apoplasm.

Graminaceous plant species respond to iron (Fe)-deficiency stress by enhancing the release of phytosiderophores from the roots and the uptake of Fe-phytosiderophores. For studying the mobilization and uptake of apoplasmic root Fe by barley (inherently high phytosiderophore release) and sorghum (inherently low phytosiderophore release) in axenic and nonaxenic (inoculated) nutrient solution, Fe pools in the root apoplasm were loaded during plant preculture with 10-4 M Fe(III)-EDTA. After 27 d growth in Fe-deficient nutrient solution, inoculated barley plants developed moderate Fe-deficiency chlorosis compared with the less chlorotic axenic plants. In inoculated plants, recovery of phytosiderophores and mobilization of apoplasmic root Fe tended to be slightly lower than in axenic plants, and in both treatments apoplasmic root Fe was completely depleted at harvest. As determined by the nonsoluble Fe fraction (> 0·2 µm) in the nutrient solution and at the rhizoplane, the microbial uptake and immobilization of apoplasmic root Fe was estimated at about 3% of the total amount of apoplasmic root Fe after preculture and at less than 10% of plant Fe uptake. Under axenic conditions, Fe-deficient sorghum also depleted apoplasmic root Fe and developed moderate Fe-deficiency chlorosis, although phytosiderophore recovery was 5-10-fold lower than in barley. By contrast, in inoculated sorghum plants, phytosiderophore recovery and Fe mobilization were extremely low. At harvest, in inoculated sorghum plants apoplasmic Fe pools were still considerably loaded and plant Fe uptake was c. 60% lower than that of axenic plants, resulting in severe Fe-deficiency chlorosis. Thus, in Fe-deficient sorghum plants, the lower rate of phytosiderophore release and its degradation restricted an efficient mobilization of apoplasmic root Fe in the presence of micro-organisms. In barley, however, the higher rate of phytosiderophore release allowed a complete mobilization of apoplasmic root Fe even in inoculated nutrient solution. Furthermore, the results show that the dominating effect of micro-organisms in their competition with barley and sorghum for apoplasmic root Fe is the degradation of phytosiderophores rather than the immobilization or uptake of Fe.

Colonization of plant roots by egg-parasitic and nematode-trapping fungi.

• The ability of the nematode-trapping fungus Arthrobotrys oligospora and the nematode egg parasite Verticillium chlamydosporium to colonize barley (Hordeum vulgare) and tomato (Lycopersicum esculentum) roots was examined, together with capability of the fungi to induce cell wall modifications in root cells. • Chemotropism was studied using an agar plate technique. Root colonization was investigated with light microscopy and scanning electron microscopy, while compounds involved in fungus-plant interactions were studied histochemically. • Only A. oligospora responded chemotropically to roots. Colonization of barley and tomato by both fungi involved appressoria to facilitate epidermis penetration. V. chlamydosporium colonized tomato root epidermis and produced chlamydospores. Papillae, appositions and lignitubers ensheathing hyphae on tomato were also found. Phenolics (including lignin), protein deposits and callose were present in papillae in both hosts. Both fungi were still present in epidermal cells 3 months after inoculation. • Nematophagous fungi colonized endophytically monocotyledon and dicotyledon plant roots. Arthrobotrys oligospora seemed to be more aggressive than V. chlamydosporium on barley roots. Both fungi induced cell wall modifications, but these did not prevent growth. The response of root cells to colonization by nematophagous fungi may have profound implications in the performance of these organisms as biocontrol agents of plant parasitic nematodes.

Carbohydrate-ethanol transition in cereal grains under anoxia.

• Cereal grains differ greatly in their reponses to anaerobiosis. Here, the in vivo conversion of carbohydrates to ethanol and CO2 under anoxia is reported for three cereal grains. • The conversion of glucose, fructose or sucrose to ethanol under anaerobic conditions was investigated in rice (Oryza sativa), barley (Hordeum vulgare) and wheat (Triticum aestivum) grains; alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.1) activities were also analysed under aerobic and anaerobic incubation. • Our data suggest that rice grains are able to produce ethanol under anoxia for the whole period of anoxic treatment, whereas barley and wheat grains can produce this terminal product of fermentation only during the first days of anaerobiosis. The level of enzymes involved in the fermentation pathway increases strongly under anoxic conditions in all three cereals. • Conversion of hexose to CO2 is nearly unaffected by anoxia in wheat, barley and rice, whereas only rice grains are able to degrade and utilize sucrose efficiently under anoxia. By contrast, wheat and barley do not utilize sucrose efficiently under anaerobic conditions.

Location of (1 → 3)- and (1 → 3),(1 → 4)-ß-D-glucans in vegetative cell walls of barley (Hordeum vulgare) using immunogold labelling.

• (1 â†’ 3),(1 â†’ 4)-ß-Glucans occur only in the cell walls of the family Poaceae (grasses and cereals) and related families, but little is known about their distribution among walls of different cell types or within walls. • The locations of (1 â†’ 3)- and (1 â†’ 3),(1 â†’ 4)-ß-glucans in the walls of the coleoptile, first leaf and root tip of barley (Hordeum vulgare) seedlings were determined using immunogold labelling. • All the walls were labelled with the (1 â†’ 3),(1 â†’ 4)-ß-glucan antibody, except those of the outer root cap cells. Labelling of the primary walls was heavy in the coleoptile and leaf, but light or very light in the root tip. Two types of primary wall labelling occurred: in the coleoptiles (except the walls of the epidermis and two layers of parenchyma under this) and in the leaf, labelling was throughout the walls; in the root tips, labelling was only adjacent to the plasma membrane. Small amounts of labelling occurred with the (1 â†’ 3)-ß-glucan antibody, mostly over plasmodesmata. Both antibodies labelled cell plates. • (1 â†’ 3),(1 â†’ 4)-ß-Glucans occur widely in the cell walls of vegetative organs of the barley seedlings, including the walls of meristematic tissues.