Barley remains dated to the dawn of agriculture have been found at several archaeological sites 1,2 . In addition to indications that barley was an important food crop, recent excavations have fuelled speculation that beverages from fermented grains may have motivated early Neolithic hunter-gatherers to erect some of humankind's oldest monuments 3,4 . Moreover, brewing beer may also have played a role in the eastward spread of the crop after its initial domestication in the Fertile Crescent 5,6 . Since 2012, both genetic research and crop improvement in barley have benefited from a partly ordered draft sequence assembly 7 . This community resource has underpinned gene isolation 8,9 and population genomic studies 10 . However, these and other efforts have also revealed limitations of the current draft assembly. The limitations are often direct consequences of two characteristic genomic features: the extreme abundance of repetitive elements, and the severely reduced frequency of meiotic recombination in pericentromeric regions 11 .These factors have limited the contiguity of whole-genome assemblies to kilobase-sized sequences originating from low-copy regions of the genome. Thus, a detailed investigation of the composition of the repetitive fraction of the genome-including expanded gene families-and of the distribution of targets of selection and crop improvement in (genetically defined) pericentromeric regions has been beyond reach.Here we present a map-based reference sequence of the barley genome including the first comprehensively ordered assembly of the pericentromeric regions of a Triticeae genome. The resource highlights a conspicuous distinction between distal and proximal regions of chromosomes that is reflected by the intranuclear chromatin organization. Moreover, chromosomal compartments are differentiated by an exponential gradient of gene density and recombination rate, striking contrasts in the distribution of retrotransposon families, and distinct patterns of genetic diversity.Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlightin...

Legume seed development is characterized by progressive differentiation of organs and tissues resulting in developmental gradients. The whole process is prone to metabolic control, and distinct metabolite profiles specify the differentiation state. Whereas early embryo growth is mainly maternally controlled, the transition into maturation implies a switch to filial control. A signaling network involving sugars, ABA, and SnRK1 kinases governs maturation. Processes of maturation are activated by changing oxygen/energy levels and/or a changing nutrient state, which trigger responses at the level of transcription and protein phosphorylation. This way seed metabolism becomes adapted to altering conditions. In maturing cotyledons photoheterotrophic metabolism improves internal oxygen supply and biosynthetic fluxes and influences assimilate partitioning. Transgenic legumes with changed metabolic pathways and seed composition provide suitable models to study pathway regulation and metabolic control. At the same time, desirable improvements of seed quality and yield may be achieved.

To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUTl and VfSTPl, respectively. Sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUTl and VfSTP 7 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTPl mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUTl transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT7 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability.

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We previously provided evidence that seed coat‐associated invertase is involved in controlling the carbohydrate state of developing seeds and, by this way, triggering developmental processes (Weber et al. (1995) Plant Cell, 7, 1835–1846). To verify our postulate, we compared seed development of two genotypes of Vicia faba differing in seed weight. The seed coat of the large‐seeded genotype formed a higher number of parenchymatous cell layers and matured later. VfCWINV1 encoding a cell wall‐bound invertase is expressed in the unloading zone of the seed coat. mRNA levels peaked later in ‘large’ coats and mRNA was present in more cell layers over a longer time period. Cell wall‐bound invertase activity revealed a similar accumulation pattern, obviously generating the high hexose conditions present in the endospermal cavity bathing the premature cotyledons and thus controlling their carbohydrate state. High hexose conditions were correlated with an extended mitotic activity of the ‘large’ cotyledons. In ‘large’ and ‘small’ cotyledons, sucrose levels rose when hexoses decreased apparently terminating cell divisions and initiating differentiation and storage activities. This developmental switch was delayed in ‘large’ embryos. To prove the outlined relationship, sucrose was added in vitro to mitotically active cotyledons. This treatment favoured nuclear expansion and starch accumulation over cell division. In contrast, a hexose‐based medium maintained cell divisions. We conclude that development of the embryo is coordinately regulated with that of the maternal seed coat which controls, by metabolic signals, the phase of cell division of the embryo and consequently also seed size.

The classic role of SUCROSE NONFERMENTING-1 (Snf1)-like kinases in eukaryotes is to adapt metabolism to environmental conditions such as nutrition, energy, and stress. During pea (Pisum sativum) seed maturation, developmental programs of growing embryos are adjusted to changing physiological and metabolic conditions. To understand regulation of the switch from cell proliferation to differentiation, SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE (SnRK1) was antisense repressed in pea seeds. Transgenic seeds show maturation defects, reduced conversion of sucrose into storage products, lower globulin content, frequently altered cotyledon surface, shape, and symmetry, as well as occasional precocious germination. Gene expression analysis of embryos using macroarrays of 5,548 seed-specific genes revealed 183 differentially expressed genes in two clusters, either delayed down-regulated or delayed up-regulated during transition. Delayed down-regulated genes are related to mitotic activity, gibberellic acid/brassinosteroid synthesis, stress response, and Ca2+ signal transduction. This specifies a developmentally younger status and conditional stress. Higher gene expression related to respiration/gluconeogenesis/fermentation is consistent with a role of SnRK1 in repressing energy-consuming processes in maturing cotyledons under low oxygen/energy availability. Delayed up-regulated genes are mainly related to storage protein synthesis and stress tolerance. Most of the phenotype resembles abscisic acid (ABA) insensitivity and may be explained by reduced Abi-3 expression. This may cause a reduction in ABA functions and/or a disconnection between metabolic and ABA signals, suggesting that SnRK1 is a mediator of ABA functions during pea seed maturation. SnRK1 repression also impairs gene expression associated with differentiation, independent from ABA functions, like regulation and signaling of developmental events, chromatin reorganization, cell wall synthesis, biosynthetic activity of plastids, and regulated proteolysis.

SummaryRecent applications of oxygen-sensitive microsensors have demonstrated steep oxygen gradients in developing seeds of various crops. Here, we present an overview on oxygen distribution, major determinants of the oxygen status in the developing seed and implications for seed physiology. The steady-state oxygen concentration in different seed tissues depends on developmental parameters, and is determined to a large extent by environmental factors. Photosynthetic activity of the seed significantly diminishes hypoxic constraints, and can even cause transient, local hyperoxia. Changes in oxygen availability cause rapid adjustments in mitochondrial respiration and global metabolism. We argue that nitric oxide (NO) is a key player in the oxygen balancing process in seeds, avoiding fermentation and anoxia in vivo. Molecular approaches aiming to increase oxygen availability within the seed are discussed.Abbreviations: At, Arabidopsis thaliana; AOX, alternative oxidase; COX, cytochrome C oxidase; HIF1, hypoxia-inducible factor; NO, nitric oxide; ROS, reactive oxygen species. I. IntroductionThe modern atmosphere contains approx. 21 kPa oxygen. However, over the course of the past 550 million yr (Phanerozoic time), during which time the vascular plants invaded the land surface, plants have adapted to levels of atmospheric oxygen ranging from 13 to 51 kPa (Raven, 1991). This variation has been a major driver of plant evolution, and has led to the tuning New Phytologist (2009) 182: 17-30 doi: 10.1111/j. 1469-8137.2008.02752.x Key words: hypoxia, nitric oxide (NO), oxygen diffusion, oxygen sensing, seed development, seed photosynthesis, storage metabolism. Tansley review Review 18New Phytologist ( of plant architecture/ultrastructure and metabolism to tolerate both low and high oxygen supply (Berner, 1999). Although over a shorter time-scale the atmospheric oxygen level may appear stable, plants must be able to adapt to variation in oxygen provision imposed by the local environment. For example, little oxygen is available to the plant root in a temporarily waterlogged soil, so plants have developed a number of strategies for acclimatization, avoidance and escape (Armstrong et al., 1994; Crawford & Brändle, 1996; Drew, 1997;Vartapetian et al., 2008). The diffusion of gas is 10 000 fold slower through a liquid medium than through air, so waterlogging rapidly leads to hypoxic and eventually even to anoxic conditions. Under hypoxia, the concentration of oxygen limits mitochondrial ATP production (oxidative phosphorylation), whereas under anoxia there is essentially no oxygen available for mitochondrial respiration. A restricted capacity for oxygen diffusion, in conjunction with a high rate of cellular metabolism, can generate hypoxia even in aerial organs such as the fruit (Ke et al., 1995), certain vascular tissues (Kimmers & Stringer, 1988), the pollen grain (Leprince & Hoekstra, 1998) and the seed (Rolletschek et al., 2002). From a historical perspective, seed germination and subsequent seedling growth has been one of...

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14 C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14 C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15 N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.Legume seeds are a major source of plant-derived proteins and economically important for worldwide feed and food. Vicia and pea (Pisum sativum) seeds contain globulin storage proteins, hexameric legumins, and trimeric vicilins/convicilins, which together account for the majority of seed protein. The remainder consists of albumins, including lectins, lipoxygenases, proteinase inhibitors, late embryogenesis abundant proteins, and many other soluble proteins (Casey et al., 1993). Storage protein accumulation in legumes occurs in the embryo during maturation. Gln and/or Asn are translocated through the phloem (Miflin and Lea, 1977) and are symplastically unloaded into the seed coat where they are metabolized and reconstructed (Rochat and Boutin, 1991;Lanfermeijer et al., 1992). Mainly Gln, Ala, and Thr are released from the pea seed coat (Lanfermeijer et al., 1992) and, at maturation, Asn is also unloaded (Rochat and Boutin, 1991). Efflux of amino acids (and Suc) from pea seed coats is passive with linear kinetics, probably mediated by nonselective pores (DeJong et al., 1996(DeJong et al., , 1997. Amino acid uptake into soybean (Glycine max) and pea embryos is partially passive, especially during the early stage...