Transcriptional landscapes of floral meristems in barley.

Organ development in plants predominantly occurs postembryonically through combinatorial activity of meristems; therefore, meristem and organ fate are intimately connected. Inflorescence morphogenesis in grasses (Poaceae) is complex and relies on a specialized floral meristem, called spikelet meristem, that gives rise to all other floral organs and ultimately the grain. The fate of the spikelet determines reproductive success and contributes toward yield-related traits in cereal crops. Here, we examined the transcriptional landscapes of floral meristems in the temperate crop barley (Hordeum vulgare L.) using RNA-seq of laser capture microdissected tissues from immature, developing floral structures. Our unbiased, high-resolution approach revealed fundamental regulatory networks, previously unknown pathways, and key regulators of barley floral fate and will equally be indispensable for comparative transcriptional studies of grass meristems.

Convergent evolution of a metabolic switch between aphid and caterpillar resistance in cereals.

Tailoring defense responses to different attackers is important for plant performance. Plants can use secondary metabolites with dual functions in resistance and defense signaling to mount herbivore-specific responses. To date, the specificity and evolution of this mechanism are unclear. Here, we studied the functional architecture, specificity, and genetic basis of defense regulation by benzoxazinoids in cereals. We document that DIMBOA-Glc induces callose as an aphid resistance factor in wheat. O-methylation of DIMBOA-Glc to HDMBOA-Glc increases plant resistance to caterpillars but reduces callose inducibility and resistance to aphids. DIMBOA-Glc induces callose in wheat and maize, but not in Arabidopsis, while the glucosinolate 4MO-I3M does the opposite. We identify a wheat O-methyltransferase (TaBX10) that is induced by caterpillar feeding and converts DIMBOA-Glc to HDMBOA-Glc in vitro. While the core pathway of benzoxazinoid biosynthesis is conserved between wheat and maize, the wheat genome does not contain close homologs of the maize DIMBOA-Glc O-methyltransferase genes, and TaBx10 is only distantly related. Thus, the functional architecture of herbivore-specific defense regulation is similar in maize and wheat, but the regulating biosynthetic genes likely evolved separately. This study shows how two different cereal species independently achieved herbivore-specific defense activation by regulating secondary metabolite production.

Golden SusPtrit: a genetically well transformable barley line for studies on the resistance to rust fungi.

KEY MESSAGE: We developed 'Golden SusPtrit', i.e., a barley line combining SusPtrit's high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation. Nonhost and partial resistance to Puccinia rust fungi in barley are polygenically inherited. These types of resistance are principally prehaustorial, show high diversity between accessions of the plant species and are genetically associated. To study nonhost and partial resistance, as well as their association, candidate gene(s) for resistance must be cloned and tested in susceptible material where SusPtrit would be the line of choice. Unfortunately, SusPtrit is not amenable to Agrobacterium-mediated transformation. Therefore, a doubled haploid (DH) mapping population (n = 122) was created by crossing SusPtrit with Golden Promise to develop a 'Golden SusPtrit', i.e., a barley line combining SusPtrit's high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation. We identified nine genomic regions occupied by resistance quantitative trait loci (QTLs) against four non-adapted rust fungi and P. hordei isolate 1.2.1 (Ph.1.2.1). Four DHs were selected for an Agrobacterium-mediated transformation efficiency test. They were among the 12 DH lines most susceptible to the tested non-adapted rust fungi. The most efficiently transformed DH line was SG062N (11-17 transformants per 100 immature embryos). The level of non-adapted rust infection on SG062N is either similar to or higher than the level of infection on SusPtrit. Against Ph.1.2.1, the latency period conferred by SG062N is as short as that conferred by SusPtrit. SG062N, designated 'Golden SusPtrit', will be a valuable experimental line that could replace SusPtrit in nonhost and partial resistance studies, especially for stable transformation using candidate genes that may be involved in rust-resistance mechanisms.

Chromatin alterations during pollen development in Hordeum vulgare.

The dynamics of posttranslational histone modifications in relation to nuclear architecture has been analyzed during pollen development in Hordeum vulgare L. cv. Igri. Notwithstanding the asymmetry of cytokinesis associated with pollen mitosis I, immunolabeling revealed that the vegetative and generative nuclei initially display identical chromatin modification patterns. Yet, differential chromatin modification patterns between vegetative and generative nuclei emerge with the development of conspicuous differences in nuclear morphology as visualized by 4',6-diamidino-2-phenylindole staining. The temporal and spatial distribution of most histone modifications observed is in agreement with reduced gene activity in the generative nucleus and increased expression in the vegetative nucleus as indicated by immunolabeling of active RNA polymerase II. Signals of trimethylation of histone H3 lysine 27 proved to be particularly enriched in euchromatic domains of subtelomeric regions. In the context of nuclear differentiation in bicellular pollen, this modification became restricted to the vegetative nucleus, indicating a role in activating rather than suppressing gene expression. The presence of acetylated histone H3 at lysine 9 in the cytoplasm of the generative cell is indicative of a more complex, still unknown function of this particular modification.

Time-lapse imaging of the initiation of pollen embryogenesis in barley (Hordeum vulgare L.).

Pollen embryogenesis provides exciting opportunities in the areas of breeding and biotechnology as well as representing a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular. A cell culture system was devised in which immature barley pollen could be cultured as a monolayer trapped between the bottom glass-cover slip of a live-cell chamber and a diaphanous PTFE membrane within a liquid medium over a period of up to 28 d, allowing the process of embryogenesis to be tracked in individual pollen. Z-stacks of images were automatically captured every 3min, starting from the unicellular pollen stage up to the development of multicellular, embryogenic structures. The method should prove useful for the elucidation of ultrastructural features and molecular processes associated with pollen embryogenesis.

The use of cyanobacteria as filler in nitrocellulose capillaries improves ultrastructural preservation of immature barley pollen upon high pressure freezing.

The high pressure freezing (HPF) followed by freeze substitution technique has advantages over chemical fixation in the context of preserving sample ultrastructure. However, when HPF is applied to cultured pollen grains, the large intercellular spaces present lead to a poor level of ultrastructure preservation. We report here that the mixing of cyanobacteria with immature barley pollen grains succeeded in greatly reducing the volume of liquid present between the large pollen grains, and so improved the loading of the sample into a nitrocellulose capillary. The use of yeast or cyanobacteria paste to surround the filled capillaries was beneficial in speeding the transfer of heat during the freezing process. This modification of the HPF method resulted in a greatly improved level of ultrastructure preservation.

Parthenogenetic egg cells of wheat: cellular and molecular studies.

The 'Salmon' system of wheat comprises three isogenic alloplasmic lines with either zygotic (aS) or autonomous, fertilisation-independent (cS kS) embryo development. While the initiation of embryogenesis from the isolated sexual egg cell depends on in vitro fertilisation, the corresponding parthenogenetic egg cell develops into an early embryo without fertilisation. This demonstrates that parthenogenesis is an inherent feature of the isolated egg cell. Based on this observation, we have constructed egg-cell-specific cDNA libraries and report first results of a sequencing project aimed at the isolation of putative egg-cell-specific and parthenogenesis-related genes.

In vitro development of wheat (Triticum aestivum L.) from zygote to plant via ovule culture.

Ovules of the wheat breeding line Veery #5 were excised and transferred to culture within 24 h after pollination. When ovules were cultured on Phytagel-solidified medium, and the pericarp removed exclusively at the micropylar tip and the abaxial side, zygotes from up to 79.2% of the ovules underwent embryogenesis with the same developmental pattern as found in planta. Embryos from more than 50% of the cultured ovules germinated when transferred to regeneration medium. More than 100 plantlets were randomly chosen for transfer to soil, all of which developed to phenotypically normal and fertile plants. With this system, the entire process of zygotic embryogenesis can be studied using living material. Furthermore, the method could be used as an embryo rescue technique for plant breeding purposes.