Differentiation of legume cotyledons as related to metabolic gradients and assimilate transport into seeds

Legume seed development is closely related to metabolism and nutrient transport. To analyse this relationship, a combination of biochemical, histological and transgenic approaches was used. Sugars within tissue sections have been quantitatively measured by metabolic imaging. During cotyledon differentiation glucose gradients emerge related to a particular cell type, with higher concentrations in non-differentiated premature regions. Sucrose in creases at the beginning of maturation in a layer underneath the outer epidermis expressing a sucrose transporter. Sucrose distribution is initially controlled by uptake activity and the permeability within the parenchyma and, later on, also by differences in growth and starch accumulation. Increased sucrose levels are accompanied by increased levels of sucrose synthase and ADP-Glc pyrophosphorylase mRNAs, but carbon flux into starch is initially still low. Rates increase at a stage when hexose concentrations become low, allowing increased flux through the sucrose synthase pathway. Transfer cell formation represents a regional specification of the cotyledonary epidermis for embryo nutrition characterized by increased transport-active cell surfaces and up-regulated expression of transport-related genes. The E2748 pea seed mutation blocks epidermal differentiation into transfer cells and leads to the loss of epidermal cell identity. Embryos with impaired epidermis cannot tolerate elevated levels of sucrose and respond with disorganized growth. The E2748 gene product is required for transfer cell formation in developing cotyledons with no other function during plant growth. Seed coat permeability provides a hypoxic environment for embryo development. However, at maturity, seed energy supply is not limited indicating fundamental developmental and metabolic adaptations. Results from transgenic seeds show that altered expression of single genes induces complex and unexpected changes. In AGP-antisense seeds the block in starch synthesis leads to pleiotropic effects of water and nitrogen content and induces temporal changes in seed development.     

Seed structure and germination in Cuscuta pedicellata with some notes on C. campestris

Dry seeds of Cuscuta pedicellata have a deeply pitted surface due to invaginated epidermal cell walls. After water uptake these walls bulge outwards and the seed surface becomes papillose. The seed coat consists of an epidermis, two palissade cell layers, and a multiple layer of parenchyma cells. The epidermis contains starch and mucilage, the parenchyma cells are compressed but some contain starch. The endosperm consists of starch–filled cells, but has a peripheral aleuron layer. The endosperm cell walls are gelatinous. The variable structure of the seed coat epidermis is believed to function in wind dispersal and rapid water uptake. Seed dormancy is common in the genus, but does apparently not occur in C. pedicellata.     

Suppression of sucrose synthase gene expression represses cotton fiber cell initiation,elongation, and seed development

Cotton is the most important textile crop as a result of its long cellulose-enriched mature fibers. These single-celled hairs initiate at anthesis from the ovule epidermis. To date, genes proven to be critical for fiber development have not been identified. Here, we examined the role of the sucrose synthase gene (Sus) in cotton fiber and seed by transforming cotton with Sus suppression constructs. We focused our analysis on 0 to 3 days after anthesis (DAA) for early fiber development and 25 DAA, when the fiber and seed are maximal in size. Suppression of Sus activity by 70% or more in the ovule epidermis led to a fiberless phenotype. The fiber initials in those ovules were fewer and shrunken or collapsed. The level of Sus suppression correlated strongly with the degree of inhibition of fiber initiation and elongation, probably as a result of the reduction of hexoses. By 25 DAA, a portion of the seeds in the fruit showed Sus suppression only in the seed coat fibers and transfer cells but not in the endosperm and embryo. These transgenic seeds were identical to wild-type seeds except for much reduced fiber growth. However, the remaining seeds in the fruit showed Sus suppression both in the seed coat and in the endosperm and embryo. These seeds were shrunken with loss of the transfer cells and were <5% of wild-type seed weight. These results demonstrate that Sus plays a rate-limiting role in the initiation and elongation of the single-celled fibers. These analyses also show that suppression of Sus only in the maternal seed tissue represses fiber development without affecting embryo development and seed size. Additional suppression in the endosperm and embryo inhibits their own development, which blocks the formation of adjacent seed coat transfer cells and arrests seed development entirely.     

Sugars as signal molecules in plant seed development.

Higher plants as sessile organisms react very flexible to environmental changes and stresses and use metabolites like glucose, sucrose and nitrate not only as nutrients but also as signals as part of their life strategies. The role of metabolites as signal molecules has attracted considerable interest during recent years. Data reviewed here for developing plant seeds suggest a trigger function of especially sugars also in development in that metabolic regulatory control can override developmental regulation, i.e., the developmental programme only continues normally if a certain metabolic state is sensed at a given time point in a given cell or tissue. Several experimental strategies have provided mainly correlative evidence that certain sugar levels and/or the resulting changes in osmotic values are necessary within defined tissues or cells to maintain a distinct stage of differentiation or to proceed with the developmental programme. In young legume seeds, but certainly also in other tissues, a high hexose (probably mainly glucose) level seems to maintain the capacity of cells to divide whereas - later in seed development - a certain sucrose level is necessary to induce storage-associated cell differentiation. A major determinant of embryo hexose levels in young legume seeds is an apoplastic invertase preferentially expressed in the inner cell layers of the seed coat. The enzyme cleaves the incoming photoassimilate sucrose into glucose and fructose. During development the tissue harbouring the invertase is degraded in a very specific spatial and temporal pattern as part of the developmental programme and is thus creating steep glucose gradients within the cotyledons. These gradients can be measured at nearly cellular resolution and were found to be correlated positively with cell division rate and negatively with cell differentiation and storage activities. A hexose and a sucrose transporter accumulating only in the epidermal cell layer of the cotyledons seem to be essential in creating and maintaining these gradients. To gain further insights into the role of metabolites, especially sugars, as triggers of developmental processes we foremost have to identify receptor molecules already characterised in yeast, and to describe and understand the signal transduction networks involved.     

Developmental changes in the inner epidermis of the bean seed coat

Summary The inner epidermis of the bean seed coat shows remarkable structural changes during seed development. At the globular stage of development, a moderately electron-dense substance begins to accumulate in the outer tangential and radial walls of the cells. The staining and fluorescence characteristics, together with the localization of peroxidase in the wall, suggest that this electron-dense material is a phenolic substance. At the same stage of embryo development, structural specialization can be detected in the cytoplasm of the epidermal cells with an increase in the abundance of organelles, especially the endoplasmic reticulum, mitochondria, and dictyosomes. These structural features are similar to those in the underlying branched parenchyma cells. As the seed rapidly expands during the maturation stage of embryo development, the epidermal cells and the inner layers of the branched parenchyma cells begin to degenerate. Small ruptures can be detected in the epidermis, exposing the branched parenchyma cells. These structural changes are discussed in relation to their possible functions during embryo development.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Seed coat development, anatomy and scanning electron microscopy of Harpagophytum procumbens (Devil's Claw), Pedaliaceae

Seed coat development of Harpagophytum procumbens (Devil's Claw) and the possible role of the mature seed coat in seed dormancy were studied by light microscopy (LM), transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM). Very young ovules of H. procumbens have a single thick integument consisting of densely packed thin-walled parenchyma cells that are uniform in shape and size. During later developmental stages the parenchyma cells differentiate into 4 different zones. Zone 1 is the multi-layered inner epidermis of the single integument that eventually develops into a tough impenetrable covering that tightly encloses the embryo. The inner epidermis is delineated on the inside by a few layers of collapsed remnant endosperm cell wall layers and on the outside by remnant cell wall layers of zone 2, also called the middle layer. Together with the inner epidermis these remnant cell wall layers from collapsed cells may contribute towards seed coat impermeability. Zone 2 underneath the inner epidermis consists of large thin-walled parenchyma cells. Zone 3 is the sub-epidermal layers underneath the outer epidermis referred to as a hypodermis and zone 4 is the single outer seed coat epidermal layer. Both zones 3 and 4 develop unusual secondary wall thickenings. The primary cell walls of the outer epidermis and hypodermis disintegrated during the final stages of seed maturation, leaving only a scaffold of these secondary cell wall thickenings. In the mature seed coat the outer fibrillar seed coat consists of the outer epidermis and hypodermis and separates easily to reveal the dense, smooth inner epidermis of the seed coat. Outer epidermal and hypodermal wall thickenings develop over primary pit fields and arise from the deposition of secondary cell wall material in the form of alternative electron dense and electron lucent layers. ESEM studies showed that the outer epidermal and hypodermal seed coat layers are exceptionally hygroscopic. At 100% relative humidity within the ESEM chamber, drops of water readily condense on the seed surface and react in various ways with the seed coat components, resulting in the swelling and expansion of the wall thickenings. The flexible fibrous outer seed coat epidermis and hypodermis may enhance soil seed contact and retention of water, while the inner seed coat epidermis maintains structural and perhaps chemical seed dormancy due to the possible presence of inhibitors.     

Transporter SlSWEET15 unloads sucrose from phloem and seed coat for fruit and seed development in tomato

Tomato (Solanum lycopersium), an important fruit crop worldwide, requires efficient sugar allocation for fruit development. However, molecular mechanisms for sugar import to fruits remain poorly understood. Expression of sugars will eventually be exported transporters (SWEETs) proteins is closely linked to high fructose/glucose ratios in tomato fruits and may be involved in sugar allocation. Here, we discovered that SlSWEET15 is highly expressed in developing fruits compared to vegetative organs. In situ hybridization and β-glucuronidase fusion analyses revealed SlSWEET15 proteins accumulate in vascular tissues and seed coats, major sites of sucrose unloading in fruits. Localizing SlSWEET15-green fluorescent protein to the plasma membrane supported its putative role in apoplasmic sucrose unloading. The sucrose transport activity of SlSWEET15 was confirmed by complementary growth assays in a yeast (Saccharomyces cerevisiae) mutant. Elimination of SlSWEET15 function by clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated protein gene editing significantly decreased average sizes and weights of fruits, with severe defects in seed filling and embryo development. Altogether, our studies suggest a role of SlSWEET15 in mediating sucrose efflux from the releasing phloem cells to the fruit apoplasm and subsequent import into storage parenchyma cells during fruit development. Furthermore, SlSWEET15-mediated sucrose efflux is likely required for sucrose unloading from the seed coat to the developing embryo.

SlSWEET15, a specific sucrose uniporter in tomato, mediates apoplasmic sucrose unloading from phloem cells and seed coat to support fruit expansion and seed filling.     

Temporary storage compounds and sucrose-starch metabolism in seed coats during pea seed development (Pisum sativum)

Quantitative data for growth, carbohydrate, protein and free amino acid nitrogen content of pea ( Pisum sativum L. cv. Finale) seed coat were obtained during the main stage of seed development. These data allowed us to define the role of the seed coat storage compounds. High amounts of arginine were measured in the seed coat and this amino acid is hypothesized to be synthesized de novo in the seed coat cells. Starch appeared to be stored in a specific parenchyma layer of the seed coat. Starch storage was shown to occur from phloem-unloaded sucrose and high activities of some enzymes of sucrose-starch metabolism (sucrose synthase, EC 2.4.1.13 and ADP glucose pyrophosphorylase, EC 2.7.7.27) were measured. The contribution of seed storage compounds is discussed in terms of buffering embryo nutrition. The sink strength of the young pea seed may be located within the seed coat.     

Development and functions of seed transfer cells

In secretion or absorption processes, solutes are transported across the plasmalemma between the symplastic and apoplastic compartments. For this purpose, certain plant cells have developed a specialised transfer cell morphology characterised by wall ingrowths, which amplify the associated plasmalemma surface area up to 20-fold. Detailed studies on the function and development of transfer cells in the context of seed filling have been carried out mainly in cereal endosperm, and for the cotyledon and seed coat cells of legumes. The major solutes transferred are amino acids, sucrose and monosaccharides. The contributions of recently identified symporter proteins to solute transfer are reviewed here, as is the role of apoplastic invertases in promoting solute assimilation. Expression of invertase and monosaccharide transporters early in both cereal and legume seed development orchestrates the distribution of free sugars which play an important role in regulating transfer cell function and determining final endosperm or embryo cell number. Transfer cell differentiation is subject to developmental control, and may also be modulated by sugar levels. The most abundant genes specifically expressed in the transfer layer of maize endosperm encode small antipathogenic proteins, pointing to a role for these cells in protecting the developing endosperm against pathogen ingress. The functional characterisation of the corresponding transfer layer-specific promoters has provided a tool for dissecting transfer cell functions. Transfer cells are highly polar in their organisation, the characteristic cell wall ingrowths developing on one face only. The presence of cytoskeletal components bordering wall ingrowths is documented, but their role in establishing transfer cell morphology remains to be established.     

The MADS box genes SEEDSTICK and ARABIDOPSIS Bsister play a maternal role in fertilization and seed development

The haploid generation of flowering plants develops within the sporophytic tissues of the ovule. After fertilization, the maternal seed coat develops in a coordinated manner with formation of the embryo and endosperm. In the arabidopsis bsister (abs) mutant, the endothelium, which is the most inner cell layer of the integuments that surround the haploid embryo sac, does not accumulate proanthocyanidins and the cells have an abnormal morphology. However, fertility is not affected in abs single mutants. SEEDSTICK regulates ovule identity redundantly with SHATTERPROOF 1 (SHP1) and SHP2 while a role in the control of fertility was not reported previously. Here we describe the characterization of the abs stk double mutant. This double mutant develops very few seeds due to both a reduced number of fertilized ovules and seed abortions later during development. Morphological analysis revealed a total absence of endothelium in this double mutant. Additionally, massive starch accumulation was observed in the embryo sac. The phenotype of the abs stk double mutant highlights the importance of the maternal-derived tissues, particularly the endothelium, for the development of the next generation.     

Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein     

A mutational approach to the study of seed development in maize

The maize seed comprises two major compartments, the embryo and the endosperm, both originating from the double fertilization event. The embryogenetic process allows the formation of a well-differentiated embryonic axis, surrounded by a single massive cotyledon, the scutellum. The mature endosperm constitutes the bulk of the seed and comprises specific regions containing reserve proteins, complex carbohydrates, and oils. To gain more insight into molecular events that underlie seed development, three monogenic mutants were characterized, referred to as emp (empty pericarp) on the basis of their extreme endosperm reduction, first recognizable at about 12 d after pollination. Their histological analysis reveals a partial development of the endosperm domains as well as loss of adhesion between pedicel tissues and the basal transfer layer. In the endosperm, programmed cell death (PCD) is delayed. The embryo appears retarded in its growth, but not impaired in its morphogenesis. The mutants can be rescued by culturing immature embryos, even though the seedlings appear retarded in their growth. The analysis of seeds with discordant embryo-endosperm phenotype (mutant embryo, normal endosperm and vice-versa), obtained using B-A translocations, suggests that emp expression in the embryo is necessary, but not sufficient, for proper seed development. In all three mutants the picture emerging is one of a general delay in processes related to growth, as a result of a mutation affecting endosperm development as a primary event.     

砂仁种子的解剖学和组织化学研究

砂仁种子包括假种皮、种皮、外胚乳、内胚乳与胚。假种皮由内表皮、外表皮及其间的６－９层薄壁细胞组成。种皮分为外种皮、中种皮与内种皮。外种皮由１层表皮细胞构成,其壁增厚并略木质化。中种皮包括各含１层细胞的下层皮和半透明细胞层与含３－５层细胞的色素层;下皮层与色素层细胞均含有红综色素,后者的壁呈网状增厚。内种皮由１层内切向壁与径向壁非常增厚的石细胞构成。种皮表面具有许多疣状突起,它们是体积较小的表皮细胞     

The defective seed5 (des5) mutant: effects on barley seed development and HvDek1, HvCr4, and HvSal1 gene regulation

Barley, one of the major small grain crops, is especially important in climatically demanding agricultural areas of the world, with multiple uses within food, feed, and beverage. The barley endosperm is further of special scientific interest due to its three aleurone cell layers, with the potential of bringing forward the molecular understanding of seed development and cell specification from Arabidopsis and maize. Work done in Arabidopsis and maize indicate the presence of conserved seed developmental pathways where Crinkly4 (Cr4), Defective kernel1 (Dek1), and Supernumerary aleurone layer1 (Sal1) are key players. With the use of microscopy, a comprehensive phenotypic characterization of the barley defective seed5 (des5) mutant is presented here. The analysis further extends to molecular quantification of gene expression changes in the des5 mutant by qRT-PCR. Moreover, full-length genomic sequences of the barley orthologues were generated and these were annotated as HvDek1, HvCr4, and HvSal1. The most striking results in this study are the patchy reduction in number of aleurone cells, rudimentary anticlinal aleurone cell walls, and the specific change of HvCr4 expression compared to HvDek1 and HvSal1. The data presented support the involvement of Hvdes5 in establishing aleurone cells. Finally, how these results might affect the current model of aleurone and epidermal cell identity and development is discussed with a speculation regarding a possible role of Des5 in regulating cell division/ secondary cell wall building.     

The importance of starch biosynthesis in the wrinkled seed shape character of peas studied by Mendel

The wrinkled-seed mutant (rr) of pea (Pisum sativum L.) arose through mutation of the gene encoding starch-branching enzyme isoform I (SBE1) by insertion of a transposon-like element into the coding sequence. Two isoforms of starch-branching enzyme have been documented in the developing pea embryo. The second isoform, SBEII, is expressed towards the later stages of embryo development while SBEI is expressed highly in the early stages. Due to mutation of SBEI the total amount of starch and the proportion of amylopectin, a branched starch polymer, are greatly reduced in the wrinkled (rr) line as compared to that in the wild-type, round (RR) line. Consequently, the level of sucrose in the rr line is nearly two fold that of the RR line. Increased sucrose concentration in the developing embryos of this mutant line causes increased uptake of water and thereby increases the cell size and fresh weight. During seed maturation in these mutant seeds a greater loss of water occurs. As a result, the wrinkled seed phenotype develops. Besides this morphological variation, the mutation also causes changes in the amount of lipid and of one storage protein, legumin. This review article discusses the role of the SBEI enzyme in causing such metabolic changes in the developing embryos with the implication that metabolism can play a central role in plant development.     

Embryo sac, endosperm, and seed of Nemophila (Boraginaceae) relative to taxonomy, with a remark on embryogeny in Pholistoma

Studies on embryology and seed morphology are complementary to molecular phylogenetics and of special value at the genus level. This paper discusses the delimitation and evolutionary relationships of genera within the tribe Hydrophylleae of the Boraginaceae. The seven Nemophila species characterized by a conspicuous seed appendage are similar in embryology and seed structure. The ovule is tenuinucellate and unitegmic with a meristematic tapetum. The embryo sac penetrating the nucellar apex is of the Polygonum type, has short-lived antipodal cells, and an embryo sac haustorium. The endosperm is cellular, producing two terminal endosperm haustoria, of which the chalazal has a lateral branch. Embryogeny is of the Chenopodiad type (as in Pholistoma). The seed coat is formed from the small-celled inner epidermis of the integument. The large-celled outer epidermis of the integument disintegrates into scattered cells. Seed pits evolve from irregularly placed inner epidermal cells of the integument. The chalazal part of the ovule produces a cucullus, that functions as an ant-attracting elaiosome. Those species of Nemophila with a conspicuous cucullus form a natural genus. Nemophila is most closely related to Pholistoma. The integumentary seed pits of Nemophila might have evolved from ovular seed pits similar to those in Pholistoma.