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Recent Res. Devel. Plant Mol. Biol., 2(2005): 1-29 ISBN: 81-7736-241-0

Molecular physiology and genomics Of of developing barley grains Ulrich Wobus, Nese Sreenivasulu, Ljudmilla Borisjuk, Hardy Rolletschek, Reinhard Panitz, Sabine Gubatz and Winfriede Weschke Institute of Plant Genetics and Crop Plant Research (IPK), Department of Genetics, Corrensstr. 3, D-06466 Gatersleben/Germany

Molecular

Abstract Barley belongs to the most important food and feed plants worldwide. As a diploid cereal Hordeum vulgare has been studied extensively and represents a cereal model plant. This review is focused on specific aspects of the molecular physiology of developing seeds and new insights into seed biology recently gained by genomics approaches. A more general description of the morphology and histology of developing caryopses is followed by an outline of the molecular physiology of growth and storage. Recent EST and array-based analyses revealed insights into metabolic pathways and regulatory networks governing seed development. The data suggest a specific role of seed photosynthesis in the initiation of storage processes in developing grains. New experimental approaches at high spatial resolution demonstrate that photosynthesis in seeds predominantly produces oxygen. This oxygen is mostly used for ATP Correspondance/Reprint request: Prof. Ulrich Wobus, Department of Molecular Genetics, Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Corrensstr. 3, Germany. Email: [email protected]

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production to overcome the energy limitation imposed on intensive storage product biosynthesis in the seed. Sugars, but also nitrogen, play an important role in seed development both as metabolites and as signal molecules. We describe the genes/enzymes and transporters involved in carbohydrate metabolism and their role in the developmental switch from the cell division/pre-storage to the maturation/storage phase. The role of nitrogen (amino acid and peptide) transporters has only recently been studied more intensively. In a final chapter the role of sugars as signal molecules is discussed, and common features in the development of monocot barley and dicot legume seeds are described. The available data underline that seed development follows common general rules in spite of drastic morphological differences.

Introduction Already our ancestors several thousand years ago used the barley grain as staple food. Nowadays the crop still belongs to the most important ones but is predominantly a feed grain in many parts of the world. The popularly best-known use for humans is in the malting industry for brewing beer. Due to its importance barley has been and still is intensively investigated [1, 2, 3, 4]. In the era of genomics it serves as a model plant for temperate cereals mainly due to its diploid genome and the well worked-out genetics including a large number of mutants. With 5 x 109 bp the genome size exceeds that of rice at least ten times and that of Arabidopsis thaliana at least thirty times (http://www.rbgkew.org.uk/cval/homepage.html). Therefore, we can not hope for a total genome sequence soon, but the available rice genome sequence together with the extensive synteny between cereal genomes [5, 6], the accumulating sequences from generich regions of maize [7] and - in the near future - wheat [8] and the rich barley EST collection (see below) offer ample opportunities to make use of genomics technology. Combined with a broad set of other techniques from high-throughput biochemical analyses like metabolite profiling to noninvasive imaging of metabolite gradients within developing plant seeds, a large amount of qualitatively new data will be collected in the near future and hopefully lead to a very detailed but at the same time also more holistic knowledge of barley grain development from pollination to seed germination. The present review does not provide a comprehensive overview on all aspects of seed molecular physiology in barley and related cereals. Whereas chapter 2 conveys a more general overview the following chapters rather focus on specific aspects our laboratory has contributed in the recent past. Cited published work deals mainly with barley and other monocot crop plants but occasionally work in other plants is briefly discussed.

A brief account of barley seed development Morphology and histology of developing caryopses A detailed knowledge of tissue and cell types as well as their differentiation during development is a major prerequisite to understand seed development in general and the measured changes in molecular, biochemical and physiological parameters in particular.

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The mature barley grain consists of the surrounding husks and the caryopsis, a fruit characterized by a fusion between pericarp and seed coat. Enclosed by the seed coat (testa) are embryo and endosperm, the latter differentiated into the outer aleuron/subaleuron layers and the inner starchy endosperm. The embryonic tissues are located in the basal part of the grain and comprise the scutellum, a nursing tissue comparable to a dicot cotyledon, and the embryo proper [for a detailed description see 1]. From anthesis to maturity, caryopsis development progresses through several phases. Generally, seed development is divided into three phases based on general features as ongoing cell division and morphogenesis (cell division or pre-storage phase), storage product accumulation (storage or maturation phase) and water loss (desiccation phase) [9 and Fig. 2A]. For the barley caryopsis, we recently described an intermediate or transition phase (between pre-storage and storage phase) based on dramatic changes at the transcriptional level [10] and numerous physiological parameters (see below). A

B

Figure 1 A general scheme of barley seed development. A, Developmental and biochemical parameters led to the widely accepted definition of three main stages (prestorage, storage and dessication phase) of (barley) seed development. DAF, Days After Flowering. B, Gene expression patterns underlying distinct developmental phases during barley grain development. A hybridized array with about 12,000 cDNA fragments of unique genes expressed in the developing barley grain is over-layed with expression pattern schemes of functional groups of genes. The expression pattern schemes result from a combination of expression analysis results and data mining of more than 40,752 ESTs out of four tissueand seed development-specific cDNA libraries (Sreenivasulu et al., unpubl.).

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In the past a number of careful anatomical studies were published [for review see 11]. O-A. Olsen et al. have studied endosperm differentiation in detail [for review see 12, 13], and embryo development has been described by Engell [14]. However, a recent analysis of whole caryopsis development and documentation of histological changes as a reference system for integrating and interpreting molecular data is missing (for wheat grain development see www.wheatbp.net). We have, therefore, carried out a detailed documentation of pre-storage and early-to-mid storage phase by serial sectioning and used these data to construct several 3D computer models [95]. Fig. 2 illustrates all

Figure 2. Representation of barley grain development. A-D, median transverse sections of BMM-embedded grains, stained with toluidene blue; E-H, schematic drawings representing longitudinal sections; I-L, photographs taken of grains without palea and lemma. The following developmental stages are represented: A, E, I, anthesis (0 DAF); B, F, J, caryopsis with syncytial endosperm (3 DAF); C, G, K, cellularized endosperm (5 DAF) and D, H, L, caryopses with starchy endosperm (10 DAF). A, antipodals; CH, chlorenchyma; EA, egg apparatus; EC, endosperm cavity; EM, embryo; ET, endosperm transfer cells; I, integuments; II, inner integument; M, micropyle; N, nucellus; NE, nucellar epidermis; OI, outer integument; P, pericarp; SE, starchy endosperm; sE, syncytial endosperm; VT, vascular tissue. Bars: A – D, 200µm; I - L, 2mm.

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relevant tissues at defined time points during pre-storage, intermediate and early-to-mid storage phase necessary to understand the developmental processes described later. Bethke et al. [15] provide a detailed description illustrated with some black-and white drawings. At anthesis (0 Days After Flowering, DAF) the maternal tissue (Fig. 2A) consists of the pericarp and the embryo sac surrounded by the integuments. The pericarp differentiates into two major tissue types, the inner green, chloroplast-containing chlorenchyma (see Fig. 5) and outer non-green cell layers harboring the (pre-)vascular tissues (see Fig. 2B). Mainly those parts of the chlorenchyma immediately attached to the main vascular bundle as well as pericarp cells surrounding the small lateral veins accumulate starch transiently [see 16]. Endosperm starts its development as a coenocytic tissue surrounding a fluid-filled cavern (Fig. 2B). Rapid cell divisions and cell wall formation result in the cellularized starchy endosperm (Fig. 2C, D). Until the early storage phase the embryo constitutes only a very minor part of the caryopsis (Fig. 2E). During early development, filial tissues are surrounded by the outer and the inner integument, each consisting of two cell-layers. Together with the nucellar epidermis, the persisting inner integuments later develop into the seed coat (testa). Cuticulae in between outer and inner integument and inner integument and nucellar epidermis prevent any nutrient exchange. Nutrients can only reach filial tissues via the the ventral crease region running alongside the seed. This region contains the main vascular tissue releasing assimilates (see [17, 18] for wheat) into the maternal tissues including the nucellar projection (NP, Fig. 2B) which faces the apoplastic gap (endosperm cavity) separating maternal and filial tissues (Fig. 2 C; for detailed description of NP during the storage phase see [19]). The NP contains several cell types undergoing developmental changes with unknown functional consequences. A central cell area at the apoplastic gap undergoes programmed cell death probably involving a cystein-rich protein 140 amino acid residues in length (V.Radchuk et al., unpublished) provisionally described as nucpro-protein (from nucellar projection)[20]). Morphologically, the process leads to the formation of two NP lobes (Fig. 2D). In contrast to endosperm aleurone, starchy endosperm cells undergo programmed cell death during seed maturation and dessication [see 19 for maize].

Molecular physiology of growth and storage Embryo development occurs in typical steps from the first unequal cell division to the formation of a shoot meristem, scutellum and root meristem [for a comparative description see 22]. Since the embryo consists during the pre-storage phase only of a few hundred cells, it has so far not been studied separately and in detail during this developmental phase. Likewise, the molecular physiology of the developing maternal tissues has not been investigated in any detail. Thus, reported data are related either to the whole grain or the endosperm. We started transcript profiling of ~1400 genes in maternal as well as filial tissue preparations from anthesis to early storage phase to obtain data about coordinated transcriptional regulation between the two tissues and to study differences in their metabolism [10 and below]. The results of the study favour a role of the pericarp as a tissue feeding, after an initial growth, the filial seed part. Consistent with this supposed function is the role of an early sink since the irreversible cleavage of sucrose unloaded into the barley pericarp by invertase activities creates a

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sucrose gradient and guarantees high sucrose influx [23]. Before and shortly after fertilization the maternal nucellus has a role either as a nutritional source or even as a regulating tissue for megagametophyte/early filial tissue development [24]. Within the embryo sac, the two synergids and antipodial cells degenerate and may also contribute to the developmental growth of the embryo and early endosperm [25, 26]. However, molecular-physiological studies of the tissues embedded within the ovary have not been reported. Since early seed development is in several ways decisive for final seed number and seed size [27] a detailed study using the much-refined molecular, biochemical and physiological methods now available would be highly timely. Early in development, starch is transiently synthesized and accumulated only in the pericarp. Later (5-6 DAF) considerable amounts of starch are visible in that part of the maternal tissue flanking the main vascular bundle (see [16] and below). In parallel, starch accumulation starts in the wings and the median cell rows of the starchy endosperm. At the end of the pre-storage phase, endosperm cell division stops in those regions accumulating starch, and the grain is reprogrammed within a few days during the intermediate or transition phase to massive storage product synthesis in both monocot (see following chapter) and dicot seeds. This phase is characterized in the filial seed part by a switch from invertase-controlled to a sucrose-synthase controlled sucrose cleavage, by a switch from a high hexoses/sucrose to a high sucrose/hexoses ratio, by strongly increasing expression of sucrose transporter genes and active function of endosperm transfer cells [see 16, 23], by a strong increase of transcriptional activity of photosynthesis-associated as well as ATP-producing and transporting genes and by a switch between the pathways of energy production related to oxygen availability within the endosperm [see 10, 28]. Some of these processes will be described in detail below. During the following storage phase barley grains accumulate mainly starch [~5565% of grain dry weight [29], composed of 20-25% un-branched amylose and 75-80% branched amylopectin [1] and storage proteins (~12%) [30]. Most of the carbon skeletons used to synthesize seed storage products are derived from currently synthesized photoassimilates transported into the seed mainly from flag leaf, stem and green parts of the ear. Therefore, early flag leaf emergence and delay in leaf senescence results in a longer grain filling period and higher yields as shown in wheat [31]. However, it is likely that half or more of the photosynthate in mature grains is temporarily deposited in one or more reserve pools before being transferred to the grain [32]. The composition of starch accumulated in barley grains has been reviewed [33], and a more recent paper [34] reviews seed carbohydrates. Storage starch accumulation starts earlier than storage protein biosynthesis and marks the end of the cell-division phase [16, 35]. Despite of the fact that storage protein synthesis also relies on carbon skeletons derived from sucrose there seems to be no common control of starch and storage protein synthesis and distribution in barley grains as discussed in [10] (see also [36] for wheat as well as [37, 38] for dicots), possibly because of the different intracellular localization of the two processes. Endosperm starch localization is highly controlled during development and dependent on both sucrose and energy supply (see p. 15). For some time highly disputed, it is now clear that in cereals most of the ADP-glucose for plastid starch synthesis is produced in the endosperm cell cytoplasm by a cytoplasmic isoform of ADPglucose-pyrophosphorylase (AGPase) and transported into plastids by a respective transporter [39]). The small subunit of this isoform is missing in the low-

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starch barley mutant Riso16 [40]. In maize seed weight is dependent on the amount of endosperm AGPase [41], and manipulation of AGPase can result in higher seed yield in wheat and rice [42, 43]. Barley storage protein is a complex mixture of different components, which have been classified for nearly a century according to the Osborne fractionation scheme based on protein solubility in different solvents. The major endosperm storage proteins of barley are the alcohol/water soluble prolamins, called hordeins. Based on sequence relationships cereal prolamins are further classified into high molecular weight (HMW) prolamins (D hordein), sulfur-poor prolamins (C-hordein) and sulfur-rich prolamins (γ- and B-hordein) [30, 44]. They are exclusively synthesized in the starchy endosperm whereas storage globulins (related to the 7S vicilins of legumes) soluble in dilute salt solutions are mainly found in the aleurone layer and the embryo (see [45] for a recent review). Grass seed 7S globulins have been best studied in maize embryos [reviewed in 46]. In spite of intensive research in the past, genomics approaches discovered hitherto unknown storage proteins in both maize [47] and wheat [48] demonstrating the power of the new technology. Also restricted to the endosperm is a family of trypsin/α-amylase inhibitors, which make up a substantial portion of water-soluble smaller proteins. Their synthesis precedes that of the main storage proteins [10]. Their actual function in vivo is unknown. Besides of their suggested function in plant defense [reviewed in 49] they may protect accumulated starch or proteins against degradation. An embryo-specific group of barley seed proteins are the lectins but their concentration is low (<0.5% of soluble embryo protein) [reviewed in 50]. Protein studies during the last few decades were either focused on storage proteins or specific enzymes relevant for seed development. With the advent of new technologies more global approaches to protein analysis during seed development are possible. A proteomics study of barley grain filling and maturation [51] focused on low salt-soluble proteins, i.e. the major storage proteins were excluded. Protein spots in 2d gels were grouped according to intensity changes in time and 36 were identified, for instance trypsin/α-amylase inhibitors, serpins (serine proteinase inhibitors) and rubisco. However, mainly due to the lack of extensive barley sequence data, spot identification is still difficult. Nevertheless, proteomics technologies will be of increasing importance as most recently shown by a study of the starch granule proteome [52]. Lipids make up a minor percentage (~3%) and are mainly found in aleurone and embryo [53, 54]. Transcript profiling [10] can help to understand how seed lipid synthesis, mobilization and composition are controlled and thus assist molecular breeding for grains with a higher lipid content. Such grains are favored as feed [15]. Much attention has been devoted to the molecular biology and cell biology of the aleurone layer especially with respect to gibberellin action (for a recent review see [55]).

Genomics of barley seed development Although substantial progress has been made in the past years in elucidating the biochemical pathways of seed metabolism, the current knowledge about the interplay of the many cellular and metabolic events coordinated by a complex regulatory network during seed development is rather scarce. With the advent of Expressed Sequence Tag (EST) generation from a wide range of cDNA libraries of various stages of cereal grain

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development, gene discovery and elucidation of metabolic pathways are increasingly pursued. cDNAs and the respective single path sequences are used to generate oligo-, micro- and macro arrays for transcriptome analysis. From such analyses, we are able to gain insights into the global developmental and metabolic regulatory network and to identify candidate key regulatory genes. In order to understand the possible functions of such genes, a number of other multi-parallel techniques such as random and targeted mutagenesis [56, 57], complementation, microarray-based expression screening and promoter-trapping strategies [58] are needed together with approaches like genetical genomics [59]. Recently published studies on cereal seeds focussed on transcriptome analysis by EST inquiry (in silico expression analyses), oligonucleotide- and cDNA-based arrays as well as profiling techniques (SAGE = 'Serial Analysis of Gene Expression' and MPSS = 'Massively Parallel Signature Sequencing') (see [10, 20, 60] for barley, [48, 61] for wheat, [62, 63, 64] for maize and [65, 66] for rice). Milligan et al. [67] published a broader review on functional genomics of seed development in cereals, and van der Geest [68] discussed in a general way how genomics technologies will push seed science. Generation and analysis of expressed sequence tags (ESTs) In cereals, large numbers of ESTs were generated which have great potential to provide functional genomics information (refer to web pages http://www.ncbi.nlm.nih.gov/dbEST/ dbEST_summary.html; http://harvest.ucr.edu/; http//www.tigr.org/tdb/tgi.plant.shtml). Based on the gene index report of the TIGR database, dated 05.01.04, the highest number of 494,195 ESTs was generated from Triticum aestivum, followed by 364,267 from Zea mays, 343,206 from Hordeum vulgare, 228,475 from Oryza sativa, 130,897 from Sorghum bicolour and 8,971 from Secale cereale. Barley EST projects were initiated by several research institutes using different cultivars such as ‘Barke’ (~110,000 ESTs; Institute of Plant Genetics and Crop Plant Research; http://pgrc.ipk-gatersleben.de/databases.php), ‘Morex’ (61,439 ESTs; Clemson University Genomic Institute; http://www.genome.clemson.edu/projects/ barley/), ‘Optic’ (40,000 ESTs; Scottish Crop Research Institute; http://wwwexternal.scri.sari.ac.uk/SCRI/web/site/home/home.asp); ‘Saana’ (50,000 ESTs; University of Helsinki; http://www.biocenter.helsinki.fi/bi/bare-1_html/est.htm); ‘Haruna’ ‘Nijo’, ‘Akashinriki’ and ‘H602’ (92,675 ESTs; Okayama university; http://www.shigen.nig.ac.jp/barley/) and small numbers from other laboratories. The number of ESTs generated so far solely from cDNA libraries of developing seeds amounts to 96,125 as compared to a total of 343,206 barley ESTs. Table 1 lists the EST collections generated from developing barley seeds. As listed in the table, our laboratory produced about 40,000 ESTs from four different cDNA libraries representing maternal and filial tissues of the barley grain during the pre-storage phase (0-7 DAF) and whole caryopses of the early to mid storage phase (8 - 15 DAF) and late storage/dessication phase (16 - 25 DAF). Such large EST collections produced from different tissues at different developmental stages of non-normalized libraries can be used to gain in silico expression data by analyzing the relative numbers of sequences within specific clusters representing putative genes (unigenes) assigned to specific cDNA libraries. This digital in silico method to quantify expression data has first been implemented to assess genes preferentially expressed in seeds by using the available

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Table 1. EST collections derived from cDNA libraries of developing barley seeds Cultivar

Deposited source

Tissue Source

Barke Barke Barke Barke

IPK IPK IPK IPK

maternal, 0-7DAF filial, 0-7 DAF whole caryopsis, 8-15DAF whole caryopsis, 16-25DAF

Morex Morex Morex Morex

Clemson University Clemson University Clemson University Clemson University

spike 5-45 DAF spike 20 DAF spike 0-8 DAF, infected with Fusarium Testa/pericarp

Saana Saana

University of Helsinki University of Helsinki

whole caryopsis, 0-9 DAF whole caryopsis, 12-18 DAF

Optic Optic Optic Optic Optic Optic Optic Optic Optic Optic Optic Optic Optic Optic Optic

IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI IGF; SCRI

embryo 12 DAF 14 DAF embryo 21 DAF embryo 28 DAF embryo 40 DAF embryo 4-6 DAF embryosac 4 DAF maternal 8 DAF maternal 12 DAF maternal 21 DAF maternal 28 DAF maternal 4 DAF pistil 8 DAF pistil 12 DAF pistil 24 DAF pistil

no. of ESTs 10,041 12,763 10,261 7,689 4,876 4,937 4,867 4,487 10,841 10,693 1,163 1,186 1,174 1,183 1,131 1,225 1,035 949 836 252 1,198 1,029 924 603 782

Stack PACK clustering data of all 110,000 IPK barley EST-sequences. All information regarding the consensus sequences resulting from StackPACK are stored in our CR-EST database (http://pgrc.ipk-gatersleben.de/est/). Fig. 3 provides a few examples and focusses on genes with expression patterns largely restricted to specific tissues and/or developmental stages. Among the abundantly expressed seed-specific genes, three main clusters were found with expression predominantly confined for maternal (HZ; cluster 1) and filial (HA; cluster 2) caryopsis tissues during the pre-storage and storage phase of seed development (HB and HF, respectively; cluster 3 in Fig. 3). Such genes with very specific expression patterns provide a valuable source for the isolation of tissue specific promoters for further research and genetic engineering. The same EST data set has also been used to unravel functional categories by calculating the abundance of ESTs resulting from genes associated to specific metabolic pathways. The semi-quantitative data obtained, refined by expression data from a 1400 unigene array [10], were compiled into a general scheme of barley seed development with respect to major metabolic and cellular processes (Fig. 1B). The following points

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Figure 3. Genes abundantly expressed in seeds identified by digital expression analysis. StackPack clustering of 110,981 barley ESTs from cDNA libraries of developing and germinating seeds and reproductive and vegetative tissues [124] resulted in identification of abundantly expressed clusters [Tentative Unigene Consensi (TUCs)] with more than 50 ESTs. By scoring the EST count of every abundant TUC within each of the 16 cDNA libraries, digital expression analysis was performed. By doing so, we identified genes highly expressed in maternal (HZ) and filial (HA) seed tissues during early development (0-7 DAF) and whole caryopsis during storage phase (HB, HF). The genes highly expressed in specific tissues identified by both data mining and cDNA array expression analysis are marked by *. Library specific abbreviations: HA-filial tissues of developing seeds (0-7 DAF); HZ-maternal tissues of developing seeds (0-7 DAF); HB- whole developing seeds (8-15 DAF; storage phase I); HF-whole developing seed (16-25 DAF; storage phase II); HT- aleuron/endosperm of germinating seeds 2HAI (hours after imbibition); HS-embryo+scutellum of germinating seeds 2 HAI; HU and HV- germinating seeds 16-48 and 48-96 HAI, resp.; HM-male inflorescence; HI- female inflorescence; HCcoleoptile; HG-green leaves; HR-root; HX-apex; HD-callus; HO-epidermis infected by Bgh/Bgt (Blumeria graminis f. sp. Hordei/ Blumeria graminis f. sp. Tritici, resp.) are of special interest: (1) Expression of starch biosynthesis genes precedes that of genes involved in storage protein synthesis. (2) Nearly all genes known to be involved in the lipid metabolism of Arabidopsis [38, 69] are also found in barley seeds despite of the fact that seeds of Arabidopsis store mainly oil, but those of barley mainly starch. (3) There is a temporally defined peak in the activity of photosynthesis-related genes, which will be discussed later. To identify key regulators of starch, storage protein and lipid metabolism, we surveyed and identified the abundantly expressed putative transcription factors as well as kinases during pre-storage, storage and desiccation phase. Approximately 439 putative kinase genes and 388 putative unique transcription factor genes were predicted from about 40,000 ESTs derived from developing barley seeds. These data provide a valuable resource to identify and characterize key regulatory factors controlling storage product accumulation during seed development.

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Macro-array analysis of early and mid barley seed development To study gene expression in the maternal and filial seed part during the pre-storage and initial storage phase of barley grain development, two types of macro arrays were used: a 711 cDNA array with at least 620 unigenes [20] and a 1400 cDNA array [10]; see also http://pgrc.ipk-gatersleben.de/seeds/). Presently, a high-density array containing 11,787 tentative unique genes from cDNA libraries specific for developing seeds is used for more detailed analyses of seed development in 2-day steps from anthesis to early desiccation. Tissue separations (maternal and filial tissues during early development; endosperm and embryo during seed maturation) increase the resolution of the analysis. Identification of functional classes of genes expressed specifically in maternal and filial tissues during caryopses development. During caryopsis development an intimate interaction between maternal (mostly pericarp) and filial (mostly endosperm and embryo) tissues can be expected. For instance, during early development the pericarp functions as the major sink of the seed whereas later, beginning with the transition phase, filial tissues become the predominant sink. Which genes are responsible for the tissue-specific functions and how their activities relate to the functional changes during development is largely unknown. In an initial analysis we identified, by using 1184 unigenes, 79 genes up-regulated more than 2x in the maternal tissue and 258 genes up-regulated to the same extent in the filial part of the caryopsis between anthesis and mid-maturation [10]. By applying more stringent criteria (i.e. an approximately 4 fold difference with respect to maternal versus filial tissue expression values [R = 6]), 36 'maternal' genes depicted in Fig. 4A. and 58 'filial' genes depicted in Fig. 4B were selected. The 'maternal' gene cluster is characterized by a peak of expression of specific genes especially during pericarp degeneration (8 to 12 DAF). Non-specific lipid transfer protein and lipoxygenase 2 genes pointing to processes of lipid degradation were detected exclusively in the maternal seed part as shown in Fig. 3/column HZ. Functional categorization of the ESTs found exclusively in the maternal tissues revealed mainly sets of genes related to transient storage, reserve mobilization and degradation processes, but most of the genes highly expressed in the maternal part show no significant homology to known sequences [10]. Their function has to be elucidated to explain the exact role of maternal tissues for seed development. The 'filial' gene set exhibits two distinct patterns (Fig. 4, lower panel), characterized by higher expression either during early seed development or during early storage phase. To the latter class belong a number of genes involved in carbohydrate metabolism, storage protein accumulation and protease inhibition. Carbohydrate metabolism will be discussed in more detail later. Transcriptional reprogramming during transition from the pre-storage to the storage phase. Principle component analysis (PCA) of expression data (39,788 data points) of maternal and filial tissues during 0-12 DAF (obtained at 2-day intervals) revealed differences between maternal and filial tissue samples and grouped expression profiles to defined developmental stages [see Fig. 1 in 10]. From the PCA result we conclude that gene expression regulating barley grain development is not a continuous process but proceeds in distinct steps. This finding is true for both, the maternal and the filial seed part, but is much more evident in the filial tissues. The distances between the data points allude to large transcriptional changes, i. e. regulatory re-programming

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Figure 4. Genes predominantely expressed in the maternal (a) and filial fraction (b) of developing barley seeds 0-12 DAF. The bright-red, red, green and bright-green color (for scale see bottom of the figure) visualizes differences in expression level, calculated by using the log2 value of the normalized expression intensity of every gene being member of the respective cluster at one of the defined time points (between 0 and 12 DAF). Each time point is represented by two independent experiments /experiment 1 and 2). NSH in the clone description on the right represents clones showing no significant homology to sequences available from the net, i. e. their BLAST scores are fairly low.

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during the intermediate or transition phase (5-9 DAF) (see Fig. 1B). The transition stage of barley grain development is correlated with a bell-shaped expression profile of all those genes on the macro array filter involved in photosynthesis and ATP production and transport ([10] and Fig. 5E). In the following chapter, the role of photosynthesis during seed development is discussed with special emphasis on barley.

Role of pericarp photosynthesis during seed development Photosynthetic tissues and photosynthesis related genes During the pre-storage and accumulation phase, the barley caryopsis shows more or less extended areas of green tissue (Fig. 2I-L). Microscopy revealed that two layers of chlorophyll-containing cells envelop the underlying integuments/nucellar epidermis and the endosperm. (Fig. 5). Those cells flanking the symplasmic way of assimilates out of the main vascular bundle into the endospermal cavity contain masses of chloroplasts, make up the main part of the chlorenchyma (Fig. 5C) and transiently accumulate masses of starch [16 and Fig. 10D].

Figure 5. Photosynthesis in developing barley caryopses: chlorophyll localisation (A-C), chlorophyll accumulation (D), and profiles of photosynthesis-related genes (E). Chlorophyll (red colour; analysed by laser-scanning microscopy) is located in the chlorenchyma in close vicinity to the endosperm. VT, vascular tissue; NP, nucellar projection; P, pericarp; TC, endosperm transfer cells; E, starchy endosperm. Bars: 200 µm (A), 500 µm (B).

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The described pattern of chlorophyll distribution is maintained during the entire development (Fig. 5A and B), and is very similar to that in wheat [70]. According to biochemical analyses, the amount of pigment increases strongly during the intermediate growth phase (between 6 to 10 days after pollination, see Fig. 5D). The chlorophyll A/B ratio increases slightly during the entire growth period. A transient burst in the expression of photosynthesis related genes (Rubisco small and large subunit, chlorophyll a/b binding protein and others, see Fig. 5E) precedes the rapid accumulation of pigments, and represents the molecular basis for the assembly of the complete photosynthetic apparatus. The induction of photosynthesis-related genes is an integral part of the transcriptional reprogramming during the intermediate growth phase following endosperm cellularization [10]. Photosynthetic activity of cereal seeds There is much experimental evidence confirming photosynthetic activity of caryopses. For instance, it was demonstrated that the green pericarp layer is able to fix carbon dioxide in the light [71, 72]. Furthermore it was shown, that developmental changes in the photosynthetic activity coincide with chlorophyll content [71, 72]. Pericarp chloroplasts were shown to catalyse a Hill reaction with rates similar to that of leaves. During the main storage phase, photosynthetic oxygen evolution in the light even exceeded the oxygen demand of the respiring endosperm, leading to a net oxygen evolution of the whole caryopsis [73, 74]. Despite of all these findings a significant role of seed photosynthesis in product accumulation was mostly neglected mainly due to two sets of experiments: (1) measurements of enzyme activities associated with photosynthesis (e.g. Rubisco) have shown very low activities [75], and even more important, (2) estimates on carbon balances revealed that only 2% of the weight of starch found in the mature caryopsis is fixed via seed photosynthesis [72]. However, this does not exclude that, for instance, starch accumulating in the crease-chlorenchyma (see above) may mainly result from photosynthesis. Generally, measurements of carbon budgets are technically difficult due to refixation of internally supplied CO2 generated by endosperm respiration [72, 76]. Thus, there is some doubt whether especially older estimates of the contribution of seed photosynthesis to product accumulation are reliable. Role of photosynthetic oxygen production for internal O2 levels In recent years, it became evident that aspects other than the CO2-fixation potential of seed photosynthesis may play an important role. For example, legume embryos inside seeds were found to develop in a hypoxic environment, but seed photosynthesis contributes significantly to a relief from this hypoxic stress [77]. In cereal grains, the induction of genes related to fermentative metabolism during aleurone development (i. e. during the ongoing storage product accumulation process in the starchy endosperm) led also to the hypothesis that developing caryopses are subject to hypoxia. One reason is that the transparent outer pericarp layer functions as a barrier to O2 uptake, thus restricting gas exchange and external oxygen supply, respectively [73]. Very recently, the use of O2sensitive microsensors enabled us to test if photosynthetic oxygen production might be of relevance for seed internal oxygen levels. The experiments provided first direct evidence for hypoxic conditions in regions of the endosperm filled with starch granules [79]. Within specific tissues (nucellar projection cells/endospermal

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cavity/endospermal transfer cells) oxygen levels fall below 0.1 % saturation (approx. 2.5 µM). These tissues represent an essential part of the main transport route for photoassimilates [80]. Furthermore, oxygen levels are remarkably lower during night (Fig. 6A). Both, nutrient transport to the endosperm and storage activity within the endosperm might be dependent on photo-synthetically derived oxygen. The data on internal oxygen levels can be related to earlier findings on the incorporation of 14C-labelled sucrose into starch and its stimulation by light [81]. All together these data clearly show the importance of photosynthetic oxygen production for the internal oxygen balance of grains, and thereby for overall metabolism.

Figure 6. Oxygen and ATP levels in barley caryopses. A, O2 concentration measured by micro-sensors in light and darkness at the storage phase, for abbreviations see Fig. 2. B, C, Developmental changes in the level of ATP and adenylate energy charge. D, spatial distribution of ATP (coloured) measured within cryosections (E); F, gradient in size of starch grains in the peripheral endosperm region. Grains are large in fully differentiated starchy endosperm (SE), middle-sized in aleuron-attached cells (AA) and barel -detectable in aleurone (AL). Bars: 1 mm (D, E), 200 µm (F). Energy state of caryopses is related to oxygen supply and storage pattern There is growing evidence that biosynthesis in seeds is energy-limited [82, 83]. To analyse the energy state of developing barley grains and a possible rate-limiting role for energy supply, the concentration of adenine nucleotides has to be considered. In barley caryopses, the ATP level is more or less stable during early development, but peaks at the onset of the storage phase, and declines significantly thereafter (Fig. 6B). The amount of available energy, represented by the adenylate energy charge [84], decreases

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during the storage phase (Fig. 6C), mainly due to the decrease in ATP and a concomitant increase in AMP. Obviously, the storage phase becomes increasingly energy-limited [80]. This limitation is a result of two aspects. First, the elevated metabolic activity leads to an increased energy demand. The activity of starch biosynthesis is directly correlated to the respiratory flux [85]. Second, the respiratory ATP supply is limited by low O2concentrations (as discussed above). The decrease in energy charge during storage seems to contradict the correlation of high-energy supply and storage activity. However, the spatial arrangement of both storage activity and energy levels has to be considered [86]. The method of quantitative bioluminescence can be used to image ATP. According to such analysis, it could recently be shown that remarkable ATP gradients occur during barley seed development [79]. During the storage phase, high levels of ATP exist in lateral and peripheral regions of the endosperm, which are well supplied with oxygen via pericarp photosynthesis (Fig. 6D-F). Concomitantly, these regions correspond to metabolic activities involved in storage biosynthesis. Based on these topographical correlations we suggest that endospermal storage activity (coupled with high energy demand) occurs in regions favourably supplied with O2 resulting from pericarp photosynthesis, and containing elevated concentrations of ATP. It is reasonable to assume that a high ATP level/energy state might even be necessary to fuel the metabolic fluxes increased during storage. Photosynthesis may affect the energy state and overall metabolic activity, respectively, either directly by providing both ATP and reducing equivalents, or indirectly by providing oxygen which is instantly used for mitochondrial respiration/ATP production. The latter seems to be of particular importance [81, 86, 87].

Sucrose and amino acid uptake, transport and metabolization Sucrose metabolization Sugars are of utmost importance in plants both as metabolites and as signal molecules (for a recent review see [88]). Sink organs as seeds receive photo-assimilates mainly in the form of sucrose, which has to be cleaved and re-synthesized if necessary. To understand the role of sugars in seed development and seed function, we need a detailed knowledge of the enzymes involved in sucrose metabolism, their gene expression patterns and their regulation. The most important enzymes involved are invertases, sucrose synthases (SUS) and sucrose-phosphate synthases (SPS). Invertases irreversibly cleave sucrose into the hexoses glucose and fructose. Depending on conditions sucrose synthases can either cleave sucrose into fructose and UDP-glucose or synthesize sucrose from the just mentioned monosaccharides. In sink tissues, the predominant role of the enzyme is believed to cleave sucrose. According to recent work, the enzyme may even catalyze the de novo production of ADP-glucose linked to starch biosynthesis [89]. Sucrose-phosphate synthases catalyze the reaction of UDP-glucose and fructose-6-phosphate to sucrose phosphate. The remaining phosphate group is immediately removed by sucrose phosphate phosphatase (SPP). This enzyme has only recently been purified from rice leaves and a cDNA clone isolated and characterized [91]. Related sequences were found in maize, wheat and barley EST databases [90]. In our own barley seed EST database 26 SPP sequences derived mainly from developing and storing tissues were detected. Sucrose metabolism has been reviewed recently [91, 92].

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Expression patterns of invertases, sugar transporters and sucrose synthases Invertases. Analysis of a large collection of barley ESTs derived from seed cDNA libraries revealed three cell wall-bound invertase isoforms and no isoforms of vacuolar invertase, which are known from rice and maize. Instead, four sequences homologous to fructosyl-transferase genes were found [23]. One of them, HvSF6FT1, had already been described earlier and the enzyme shown to be able to work as vacuolar invertase in the presence of sucrose as the only substrate [93]. A fructosyl transferase with vacuolar invertase activity may be typical for northern hemisphere grasses with an intensified fructan metabolism as a climatic adaptation. HvSF6FT1-mRNA was found around 2 DAF nearly exclusively within the pericarp (see Fig. 7A) and appears 1-2 days later in the ventral pericarp and the endospermal transfer cells (Fig. 7B) where it becomes

Figure 7. Tissue-specific accumulation of HvSF6FT-and HvSTP2-mRNA in transverse sections of young developing caryopses (dark field images) (A, B, C, D) and of HvCWINV2-mRNA (DIG-labelling) in a longitudinal section (E) after in situ hybridization. A, 2 DAF, HvSF6FT-mRNA is localized in the pericarp. Most intense label is found over the inner cell rows on the dorsal side and ventral cells flanking the main vascular bundle. B, 4 DAF, HvSF6FT-mRNA is mainly localized in the ventral part of the pericarp and in the endosperm transfer cells. Note that those cells immediately flanking the main vascular bundle are completely free of label. C, 6 DAF, intense labeling of HvSF6FT-mRNA is now exclusively found in the endosperm transfer cells. D, 2 DAF, localization of HvSTP2-mRNA in the pericarp. Labeling is found mainly in the region of the dorsal vascular bundle, weaker labeling in some cell rows surrounding the dorsal part of the embryo sac and in the region of the lateral vascular bundle. E, HvCWINV2-mRNA is mainly localized in pericarp cells immediately below the style. A gradient of mRNA expression in the maternal tissues is visible with strong label on top.

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concentrated around 6 DAF (Fig. 7C). This mRNA distribution roughly parallels the levels of vacuolar/acid soluble invertase activity measured in isolated maternal and filial tissue fractions (Fig. 8). We interpreted this pattern as follows [23].

Figure 8. HvSF6SFT-mRNA level and activity of vacuolar invertase in maternal and filial tissues of developing caryopses. Results are presented from one out of two independent sets of phytochamber-grown plants. The bars represent mRNA or enzyme levels measured in either maternal (pericarp) or filial (endosperm and embryo) tissue preparations Standard deviations result from three-fold repetition of enzyme activity measurements. At anthesis, seed tissues contain high amounts of hexose, most probably due to high vacuolar invertase activity of HvSFT6FT1. The enzyme, like vacuolar invertases in maize [94], seems to provide the prominent step in the catabolism of sucrose delivered by the phloem during early seed development. Sucrose cleavage maintains a gradient against the phloem and enables high sucrose influx resulting in high pericarp sink strength. As part of the transport system the hexose transporter HvSTP2 is prominent in the pericarp at this developmental stage, as revealed by its mRNA distribution 2 DAF (Fig. 7D), and should mainly retrieve leaked hexoses back into the cells. Apoplastically produced hexoses are possibly transported, too. The mRNA for a respective apoplastic sucrosecleaving enzyme, HvCWINV2, was detected 5 DAF in the ventral pericarp in median cross sections [24]. However, longitudinal sections of caryopses at the same developmental stage used for in situ localization of HvCWINV2 mRNA show a gradient of expression with high amounts of mRNA immediately below the style, low amounts in the median caryopsis region and non-detectable mRNA amounts above embryo/scutellum (see Fig. 7E). Possibly, the enzyme is involved in the processes of starch mobilization in the style region. This process needs further investigation. Already between 0 and 3 DAF, total cell wall-bound invertase activity in maternal tissues is reduced to one third and stays at nearly 1 µmol/g/min until 10 DAF [24]. Another isoform, HvCWINV3, has been detected during the storage phase but was found by Northern blotting to reside in the embryo/scutellum [10]. The most important invertase isoform with respect to development of the filial grain part is cell wall-bound invertase HvCWINV1, a homologue of maize INCW2. Its mRNA has been localized by in situ hybridization in a larger set of tissue sections to reconstruct

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its 3D expression pattern during early development [95]. The mRNA is typically located at the apoplastic boundary between maternal and filial tissue, i.e. in the cells facing the endospermal cavity on both the maternal and the filial side (see Fig. 6 in [23]). 3-4 DAF, i. e. with the beginning cellularization of the endosperm, high amounts of label were found in that first cell row flanking the nucellar projection (Fig. 9A, 3d modeling of the labeling pattern/ blue region). Additionally, some parts of the syncytial endosperm show strong label (Fig. 9A, green regions). Temporally and spatially associated with HvCWINV1 is the hexose transporter HvSTP1 as revealed by in situ hybridization (Fig. 9D). It certainly helps to provide sink strength to the growing endosperm [23]. Sucrose cleavage at the assimilate entry site allows building up and maintaining filial sink strength. It also provides hexoses for endosperm cell divisions until decreasing activity of cell wall bound invertases causes increasing sucrose concentrations which in turn initiate the SUS pathway of sucrose cleavage and the storage phase. In legumes the sucrose peak at the beginning of the storage phase (see also Fig. 1A for barley) necessary to induce SUS is most probably caused by increased sucrose transporter activity supported by a peak of SPS activity [96]. In barley grains, sucrose is taken up from the endosperm cavity by the sucrose transporter HvSUT1. HvSUT1

Figure 9. Tissue-specific accumulation of levels of HvCWINV1-mRNA, HvSPT1-mRNA and HvCWINV1-mRNA as well as CWINV enzyme activity in the filial part of developing caryopses. A, localization of HvCWINV1-mRNA in a caryopsis 3 DAF, detected by in situ hybridization and integrated in a 3d model representing the median part of a caryopsis of the same developmental stage. B, HvCWINV1 mRNA levels and CWINV enzyme activity. For further explanation see legend to Fig. 8. C, 6 DAF, intensive labeling nearly exclusively found in the endosperm transfer cells; dark field image. D, 3 DAF, HvSTP1-mRNA is found in the first cell rows of the syncytial endosperm, weaker labeling of the integuments and the nucellar projection, dark field image.

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expression increases in endospermal transfer cells during the intermediate stage, and correlative evidence suggests an important role of this transporter in sucrose accumulation and starch biosynthesis in the barley endosperm [17]. Sucrose transporter function is limiting for seed growth as shown by antisense suppression of the rice homologue OsSUT1, which resulted in impaired filling and strongly reduced germination ability of the grains [97]. On the other hand, HvSUT1 over-expression in wheat under barley hordein promoter control increases total C as well as N in ripe grains (W. Weschke et al., unpublished results). Contrary to the apoplastic transport out of the endosperm cavity into the transfer cells, transport within the barley starchy endosperm is symplastic as shown for wheat [17]. A summery on sucrose transport in cereal seeds has been published [98]. Sucrose synthases. In cereal grains mostly three sucrose synthase isoforms have been described (rice: [99]; maize: [100]; barley: [10]). Some hints exist for (at least) one more isoform in rice [66] and barley (our unpublished EST data). However, the putative fourth barley isoform is not or only at low levels expressed in developing grains. The isoforms serve different functions (for instance starch biosynthesis, cellulose biosynthesis, delivery of energy for transfer processes) and/or are active in different cell types (vascular tissue, pericarp, endosperm, embryo). Complex regulation occurs at all levels from transcriptional control to regulation of enzyme activity (see [91] for review). In addition, the isoforms can be regulated differently [101, 102]. Endosperm SUS activity has been correlated with sink-strength [103] and dry matter accumulation [104]. In barley the major isoform active in the starchy endosperm is HvSUS1. During the ongoing storage phase, its mRNA is found across the endosperm (Fig. 10). The high Km of the enzyme (see [105] for the faba bean seed enzyme) for the cleavage reaction demands high sucrose concentrations and guarantees at the same time relatively high levels of uncleaved sucrose. There is circumstantial evidence that HvSUS1 is mainly involved in starch synthesis in both, the pericarp during early development and the starchy endosperm during storage product accumulation (see Fig. 10B, E, H). Besides, the pattern of mRNA accumulation visualized by in situ hybridization points to a role of HvSUS1 in the energetization of transport processes in both, the main vascular bundle of the grain during the pre-storage phase and in endospermal transfer cells during the intermediate phase of grain development (Fig. 10B). On the contrary, no HvSUS2 mRNA expression was found in the vascular bundles of the pericarp (Fig. 10C, F, I). However, its mRNA expression parallels that of HvSUS1 within endosperm transfer cells (Fig. 10F). Both isoforms are predominantly expressed in the starch accumulating endosperm (Fig. 10H, I). However, the intensities of labeling seen by in situ hybridization are quite different as reflected by the type of photographic documentation (bright field for HvSUS1- versus dark field for HvSUS2-mRNA; Fig. 10H, I) as well as – tendentially – by Northern blotting. Furthermore, the level of mRNA expression differs remarkably between the two isoforms during the main storage phase (9-17 DAF, Fig. 11A, B). Hence, whereas HvSUS2 seems to be responsible rather for the initiative phase of starch accumulation, i. e. the transient phase of development, HvSUS1 serves the accumulation of storage compounds (cf. Fig. 11A and C), possibly not only of starch but also of storage proteins. This speculation is supported by analyses of the maize homeolog SUS1 showing specificity of expression for the mainly protein accumulating embryo [106].

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Figure 10. Distribution pattern of starch and localization of HvSUS1mRNA and HvSUS2mRNA in median-transverse sections of developing barley caryopses. A, D, G, dark field images of sections stained for starch with iodine-potassium after 2, 5 and 8 DAF, resp.; B, E, H, bright-field images of sections after in situ localization of HvSUS1-mRNA 2, 5, 8 DAF, resp.; C, F, I, dark field images of sections obtained after in situ hybridization of HvSUS2-mRNA 2, 5 and 8 DAF, resp. E and F represent the region boxed in D at higher magnification. Surprisingly, though the mRNA of both isoforms adds 8 DAF to a level at least comparable to that of HvSUS1 mRNA alone between 10 and 12 DAF, the enzyme activity at 8 DAF is only about half of that measured at DAF 12 (see Fig. 11A). The third SUS isoform, HvSUS3, expressed in remarkable amounts during early development, could be preferentially involved in cell wall biosynthesis [10]. Inspite of the described data many questions remain unanswered. We have to work out the temporal-spatial distribution patterns of all isoform mRNAs. We need in situ enzyme histochemistry studies (see [107] for maize) and sucrose imaging data (see [108] for faba bean coyledons), and we would like to detect the phosphorylation state of the enzyme(s) by immuno-histochemistry since the enzyme is activated by phosphorylation [91]. In rice seeds the phosphorylating enzyme is most probably the calcium-dependent protein kinase SPK [109]. These are only a few of the experiments that are necessary to understand SUS function. The same comment principally applies to the other genes/enzymes described above. Such more descriptive approaches have to be complemented by extensive genetic engineering experiments to get a detailed understanding of the role of carbohydrate metabolism during seed development. Nitrogen transporters expressed in developing and germinating barley seeds Plants are highly dependent on nitrogen because it is a constituent of essential cellular components. Cereals take up nitrogen from the soil as NH3, NH4+, nitrate, amino acids and soluble peptides. Ammonium assimilation occurs only in roots whereas nitrate assimilation can occur both in roots and leafs. From roots, nitrate and amino acids are

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Figure 11. Sucrose synthase mRNA and enzyme activity levels compared to starch accumulation in developing caryopses. A, HvSUS1-mRNA (bars) and SUS enzyme activity levels; B, HvSUS2-mRNA levels; C, starch accumulation. HvSUS1- and HvSUS2-mRNA expression was sequentially detected on the same blot. Standard deviation as given by error bars in (A) and (C) was calculated from three independent measurements. transported to mature leaves by the transpiration stream through the xylem. In leafs and other non-root tissues exchange of amino acids occurs between xylem and phloem to cover the demand of roots [110] and supply other N sinks like seeds. In addition to nitrate and amino acid transport, transport of small peptides seems to play an important role in periods of high protein demand such as seed filling and germination where N compounds are redistributed from senescing leaves and chlorophyll-containing tissues surrounding the developing grain and storage organs of the seed (for review, see [111]). Nitrogen uptake from the soil, loading into and exchange between xylem and phloem as well as delivery of N compounds to sink organs like developing and germinating seeds is achieved by a multitude of highly regulated N transporters, which differ in their specificity, capacity and affinity. Here, we concentrate on amino acid permeases (AAPs), peptide- (PTRs) and oligopeptide transporters (OPTs) active in developing and germinating seeds especially of barley.

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N transporters expressed in developing seeds. Whereas some biochemical data are available regarding the uptake of amino acids and peptides by the germinating barley embryo (for instance [112, 113]), information about the activity of N transporters supplying the endosperm of developing cereal caryopses is rare. On the contrary, amino acid as well as peptide transporters expressed in the cotyledons of dicot seeds have been cloned and characterized at the molecular as well as functional level ([114, 115] for V. faba; [116] for P. sativum, [117, 118] for A. thaliana). Recently, a larger number of Arabidopsis N transporter sequences became available for molecular and functional characterization [119], and results of such analyses have been published [111, 120, 121, 122]. In an additional paper, amino acid sequences of three completely sequenced eukaryotic genomes - Saccharomyces cerevisiae, Arabidopsis thaliana and Homo sapiens - were compared to identify amino acid transporter superfamilies [123]. We used the IPK barley EST set described before to define a set of 42,073 tentative unigene sequences (TUSs) [124]. Out of these TUSs, 911 ESTs with a predicted function in N transport were selected. The sequences, mostly derived from the 5’ end of the respective cDNAs, were compared to full-length N-transporter sequences available in the net. 20 ESTs for putative amino acid transporters were identified together with 41 ESTs encoding proteins with a putative function in peptide transport and 16 ESTs homologous to transporters showing oligo peptide transport activities. Comparison of these EST sequences to the total of Arabidopsis and rice N-transporter amino acid sequences available leads to the preliminary conclusion that there is no strong correlation between the level of identity and functional similarities (F. Blattner, personal communication). Functional characterizations in yeast mutants and analyses of the tissueand development-specificity of the N transporters selected from the IPK EST set are under way. N transporters expressed in germinating barley seeds. As mentioned before (chapter 2.2), mature barley grains contain a set of storage proteins stored in protein bodies of starchy endosperm, sub-aleuron and especially aleuron, to supply the growing embryo with nutrients during germination. The composition of protein bodies differs between the three compartments of the endosperm, presumably dependent on their function during germination. Whereas the nitrogen reserves of the aleuron are used to produce protein-degrading enzymes like, for instance, carboxypeptidase III and thiol protease [125], proteins accumulated in the starchy endosperm are mobilized by proteolytic cleavage to produce, for instance, transport proteins in the scutellum and to feed the developing embryo. The enzymatic hydrolysis of storage proteins in cereal grain endosperm releases a mixture of small peptides and free amino acids [126, 127]. The products of hydrolysis are taken up into the scutellum, where at least most of the peptides are further hydrolysed into free amino acids [128]. Uptake of these solutes is mediated by carriers localized in the plasma membrane of the scutellar epithelium. By biochemical analyses, two non-specific amino acid uptake systems, one system specific for proline and another one specific for basic amino acids, were identified in barley and wheat [129]. Additionally, evidence was obtained for the presence of two carriers for glutamine in the scutellum of germinating barley [113]. Estimation of peptide transport activity in the scutellar epithelium led to the conclusion that multiple peptide transport mechanisms exist within this tissue [112]. More recently, the barley scutellar peptide transporter HvPTR1 has been cloned and characterized at the molecular level [130, 131].

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HvPTR1 does typically contain 12 putative transmembrane domains. Its mRNA expression peaks 24 h after imbibition and, therefore, coincides with the development of peptide uptake activity by the scutellum. Though the highest abundance of HvPTR1 mRNA was found in the scutellum, low levels of expression were seen in other parts of the developing barley grain, too. Interestingly, this only functionally characterized barley peptide transporter clusters together with the Arabidopsis transporters AtPTR2, 2C and I, the V. faba VfPTR1 and two of our putative peptide transporters provisionally named HvPTR4 and 6, whereas all other putative peptide transporters of the barley EST collection are evolutionary rather distant and more similar to the functional characterized amino acid permeases from V. faba (F. Blattner, personal communication). The physiology and molecular biology of peptide transport in germinating barley seeds has recently been reviewed [132]. Since then no additional studies on other N transporters expressed in germinating or developing cereal grains have been published.

Sugars as signal molecules in seed development Extensive analyses of grain legume seed development revealed changing gradients of glucose and sucrose in the cotyledons. These two sugars play different roles and their concentrations or concentration ratios are correlated with developmental parameters like mitotic index and starch accumulation. Since invertases are important determinants of sugar status especially during the pre-storage phase these enzymes were regarded as control elements of seed development [133, 134]. Despite of the emerging high regulatory complexity of developmental processes in seeds, the decisive role of metabolites, especially sugars, in the process is still well substantiated (for recent reviews see [28, 135]. Studies in barley [23], maize [107, 136] and rice [137] have shown that acid invertases play an equally important role in monocot seed development. Due to the completely different seed structure comparisons with legumes are difficult, and Hirose et al. [137] stressed that even within cereals marked morphological differences imply different mechanisms for assimilate transport and metabolism. However, focusing on (i) maternal and filial tissues and (ii) the major storage organs of cereal and legume seeds, endosperm and cotyledons, respectively, a few important common features can be emphasized. First, tissues undergoing cell division are generally characterized by high hexose contents. This is true for both maternal and filial tissues of all investigated seed types but only in faba bean cotyledons glucose concentration gradients have been visualized and correlated directly to developmental processes as mitotic activity [138]. Generally, the relationship between sugars and the cell cycle via especially cyclin D is documented ([139, 140] but the specific signaling pathway between glucose and cell cycle components especially in seeds is still unknown. However, extensive genetic and biochemical studies revealed a number of genes involved and led to the conclusion that the control mechanism is likely to be combinatorial rather than hierarchical [141]. Second, during the intermediate (transition) phase a switch from a high hexose/sucrose to a high sucrose/hexose ratio is a common characteristic feature of seeds. In parallel, acid invertase activity decreases during that time span whereas expression of a sucrose transporter dramatically increases followed by an increase of SUS at both the transcript and enzyme activity level. By this way sink strength is build up in the filial seed part. Third, the decisive tissue of that switch from

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high hexose- to high sucrose conditions is the filial epidermis facing the apoplastic space between maternal and filial tissue on the main assimilate transport route. These epidermal cells (endospermal [barley] or epidermal [pea] transfer cells; see [142] for review) represent an efficient sucrose uptake system responsible for coordinated storage tissue development [16, 23, 28, 143, 144]. Mutations affecting transfer cell function [rgf1 of maize [145] and E2748 of pea [144] severely disturb storage tissue development and function. During the transition phase transfer cells express at the same time hexose- and sucrose transporters, vacuolar and cell wall-bound invertases and at least two isoforms of sucrose synthase in both the studied monocot and dicot systems. This necessitates a complex regulatory network in which changing sugar concentrations certainly play a role, but not firm data are available. Fourth, as described above, seed photosynthesis is an important provider of oxygen allowing oxidative phosphorylation and thus energy provision especially for the high demands during storage product synthesis. The described common features in the development of monocot barley and dicot legume seeds underline that seed development follows general rules in spite of drastic morphological differences.

Acknowledgement Research in our laboratory was supported by Land Sachsen/Anhalt, the German Ministry for Education and Research (BMBF), the German Research Foundation (DFG), and IPK. We wish to thank all members of our group and IPK colleagues of the barley GABI community, especially Lothar Altschmied, Hangning Zhang, Uwe Scholz and Wolfgang Michalek (KWS) for many contributions and continuous discussion.

References 1. Briggs, D.E. 1978, Barley, Chapman & Hall, London. 2. Shewry, P.R. (Ed.), 1992, Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology, CAB International, Wallingford, UK. 3. MacGregor, A.W., Bhatty, R.S. (Eds.) 1993, Barley Chemistry and Technology, Am. Assoc. Cereal Chemists, St.Paul, USA. 4. Slafer, G.A., Molina-Cano, J.L., Savin, R., Araus, J.L. and Romagosa, I. (Eds.) 2002, Barley Science, Food Products Press, New York, London, Oxford. 5. Devos, K.M., Gale, M.D. 2000, Plant Cell, 12, 637. 6. Freeling, M. 2001, Plant Physiology, 125, 1191. 7. Palmer, L.E., Rabinowicz, P.D., O'Shaughnessy, A.L., Balija, V.S., Nascimento, L.U., Dike, S., de la Bastide, M., Martienssen, R.A., McCombie, W.R. 2003, Science, 302, 2115. 8. IGROW 2002, Annual wheat Newsletter 48 (HTML Version). 9. Goldberg, R.B., Barker, S.J., Perez-Grau, L. 1989, Cell, 56: 149-160. 10. Sreenivasulu, N., Altschmied, L., Radchuk, V., Gubatz, S., Wobus, U., Weschke; W. 2004, Plant J. 37: 539-553. 11. Duffus, C.M., Cochrane, M.P. 1993, Barley Chemistry and Technology, A.W. MacGregor and R.S. Bhatty (Eds), Am. Assoc. Cereal Chemists, St.Paul, USA., 31. 12. Olsen, O.-A., Linnestad, C., Nichols, S.E. 1999, Trends Plant Sci., 4, 253. 13. Olsen, O.-A. 2001, Ann. Rev. Plant Phys. Plant Molec. Biol., 52, 233. 14. Engell, K. 1989, Nord J. Bot., 9, 265. 15. Bethke, P.C., Jacobsen, J.V., Russell, L.J. 2000, Seed Technology and its Biological Basis, M. Black, J.D. Bewley (Eds.), Academic Press, Sheffield, 184.

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