The highly divergent Jekyll genes, required for sexual reproduction, are lineage specific for the related grass tribes Triticeae and Bromeae

Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific

Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth

After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions

Jekyll Encodes a Novel Protein Involved in the Sexual Reproduction of Barley

Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues. JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and tobacco (Nicotiana tabacum). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of expression in the nucellar projection, which mediates the maternal–filial interaction during seed filling. Downregulation of jekyll by the RNA interference technique in barley decelerates autolysis and cell differentiation within the nurse tissues. Flower development and seed filling are thereby extended, and the nucellar projection no longer functions as the main transport route for assimilates. A slowing down in the proliferation of endosperm nuclei and a severely impaired ability to accumulate starch in the endosperm leads to the formation of irregular and small-sized seeds at maturity. Overall, JEKYLL plays a decisive role in the differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further suggest that cell autolysis during the differentiation of the nucellar projection allows the optimal provision of basic nutrients for biosynthesis in endosperm and embryo

Caspase-Like Activities Accompany Programmed Cell Death Events in Developing Barley Grains

<div><p>Programmed cell death is essential part of development and cell homeostasis of any multicellular organism. We have analyzed programmed cell death in developing barley caryopsis at histological, biochemical and molecular level. Caspase-1, -3, -4, -6 and -8-like activities increased with aging of pericarp coinciding with abundance of TUNEL positive nuclei and expression of <i>HvVPE4</i> and <i>HvPhS2</i> genes in the tissue. TUNEL-positive nuclei were also detected in nucellus and nucellar projection as well as in embryo surrounding region during early caryopsis development. Quantitative RT-PCR analysis of micro-dissected grain tissues revealed the expression of <i>HvVPE2a</i>, <i>HvVPE2b</i>, <i>HvVPE2d</i>, <i>HvPhS2</i> and <i>HvPhS3</i> genes exclusively in the nucellus/nucellar projection. The first increase in cascade of caspase-1, -3, -4, -6 and -8-like activities in the endosperm fraction may be related to programmed cell death in the nucellus and nucellar projection. The second increase of all above caspase-like activities including of caspase-9-like was detected in the maturating endosperm and coincided with expression of <i>HvVPE1</i> and <i>HvPhS1</i> genes as well as with degeneration of nuclei in starchy endosperm and transfer cells. The distribution of the TUNEL-positive nuclei, tissues-specific expression of genes encoding proteases with potential caspase activities and cascades of caspase-like activities suggest that each seed tissue follows individual pattern of development and disintegration, which however harmonizes with growth of the other tissues in order to achieve proper caryopsis development.</p></div

Expression profiles of the vacuolar processing enzymes VPE2a-VPE2d (A), proteasome subunits PBA and PBB (B) and phytaspase (PhS) genes (C) in the different tissues micro-dissected from the developing barley grains.

<p>Expression profiles of the vacuolar processing enzymes VPE2a-VPE2d (A), proteasome subunits PBA and PBB (B) and phytaspase (PhS) genes (C) in the different tissues micro-dissected from the developing barley grains.</p

Transcript profiling of the proteasome subunits <i>PBA</i> and <i>PBB</i> (A) and phytaspase (<i>PhS</i>) genes (B) in pericarp (left) and endosperm fractions (right) of the developing barley grains determined by real-time quantitative RT-PCR analysis.

<p>Data are means ± SD, <i>n</i> = 3, values followed by the same letter do not differ significantly at <i>p</i>>0.05.</p

Phylogenetic trees of proteasome subunits PBA and PBB (A) and phytaspases (B) drawn with the ClustalW software.

<p>The horizontal scale represents the evolutionary distance expressed as a number of substitutions per amino acid. The putative phytaspases and proteasome subunits PBA1 of barley are shown in bolt. (A) Putative barley (Hv) proteasome subunits PBA and PBB are similar to the corresponding genes from <i>Arabidopsis thaliana</i> (At), <i>Populus tomentosa</i> (Pt) and <i>Oryza sativa</i> (Os). (B) Putative barley (Hv) phytaspases together with phytaspases from <i>Nicotiana tabacum</i> (Nt) and <i>Oryza sativa</i> (Os) belong to the subgroup 1 of subtilase-like proteases. The phytaspases with proven caspase-6 activity are shown in italic. Only one Arabidopsis subtilase-like protease per subgroup is shown (Rautengarten et al., 2008) in order to simplify the figure.</p

Localization of nuclear DNA fragmentation detected by the TUNEL assay at 6 (A–C), 8 (D–F), 10 (G–I), 16 (J, K), and 18 DAF (L–N).

<p>TUNEL-positive nuclei are visualized as green signals and indicated by red arrows. Upper panel demonstrates positions of histological sections used for TUNEL assay at the reconstructed cross and longitudal views of a barley grain. al, aleurone; cl, chlorenchyma, em, embryo; es, endosperm; esr, embryo surrounding region; mvb, main vascular bundle; np, nucellar projection; nu, nucellus; p, pericarp, tc, transfer cells. Bars = 200 µm.</p