Contrast observation and investigation of wheat endosperm transfer cells and nucellar projection transfer cells

In cereal seed, there are no symplastic connections between the maternal tissues and the endosperm. In order to facilitate solute transport, both the nucellar projection and its opposite endosperm epithelial cells in wheat caryopsis differentiate into transfer cells. In this paper, we did contrast observation and investigation of wheat endosperm transfer cells (ETC) and nucellar projection transfer cells (NPTC). The experimental results showed that there were some similarities and differences between ETC and NPTC. ETC and NPTC almost developed synchronously. Wall ingrowths of ETC and NPTC formed firstly in the first layer nearest to the endosperm cavity, and formed later in the inner layer further from the endosperm cavity. The mature ETC were mainly three layers and the mature NPTC were mainly four layers. Wall ingrowths of ETC were flange type and wall ingrowths of NPTC were reticulate type. NPTC were not nutrient-storing cells, but the first layer of ETC had aleurone cell features, and the second layer and third layer of ETC accumulated starch granules and protein bodies.     

Development of basal endosperm transfer cells in Sorghum bicolor (L.) Moench and its relationship with caryopsis growth

During sorghum caryopsis development, endosperm epidermal cells near the basal main vascular bundle are specialized by depositing wall ingrowths, differentiating into basal endosperm transfer cells (BETCs). All the BETCs together compose the basal endosperm transfer layer (BETL). BETCs are the first cell type to become histologically differentiated during endosperm development. The initiation and subsequent development of BETCs shows the pattern of temporal and spatial gradient. The developmental process of BETL can be divided into four stages: initiation, differentiation, functional, and apoptosis stage. A placental sac full of nutrient solutions would emerge, enlarge, and eventually disappear between the outmost layer of BETL and nucellar cells during caryopsis development. BETCs have dense cytoplasm rich in mitochondria, lamellar rough endoplasmic reticulum, Golgi bodies, and their secretory vesicles. They show a series of typical characteristics of senescence such as nuclei distortion and subcellular organelle deterioration during their specialization. BETCs probably play an active role in nutrient transfer into the starchy endosperm and embryo. The occurrence, development, and apoptosis of BETCs are in close relation to the caryopsis growth and maturation especially the enrichment of endosperm and the growth of embryo. The timing when BETL is fully developed, composed of three to four layers in radial direction and 70 to 80 rows in tangential direction, consists with the timing when average daily gain of caryopsis dry weight reaches its maximum. It is conceivable that measures that delay the senescence and death of BETCs would help to increase the crop yield.     

The cellular pathway of photosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity

In the developing wheat grain, photosynthate is transferred longitudinally along the crease phloem and then laterally into the endosperm cavity through the crease vascular parenchyma, pigment strand and nucellar projection. In order to clarify this cellular pathway of photosynthate unloading, and hence the controlling mechanism of grain filling, the potential for symplastic and apoplastic transfer was examined through structural and histochemical studies on these tissue types. It was found that cells in the crease region from the phloem to the nucellar projection are interconnected by numerous plasmodesmata and have dense cytoplasm with abundant mitochondria. Histochemical studies confirmed that, at the stage of grain development studied, an apoplastic barrier exists in the cell walls of the pigment strand. This barrier is composed of lignin, phenolics and suberin. The potential capacity for symplastic transfer, determined by measuring plasmodesmatal frequencies and computing potential sucrose fluxes through these plasmodesmata, indicated that there is sufficient plasmodesmatal cross-sectional area to support symplastic unloading of photosynthate at the rate required for normal grain growth. The potential capacity for membrane transport of sucrose to the apoplast was assessed by measuring plasma membrane surface areas of the various cell types and computing potential plasma membrane fluxes of sucrose. These fluxes indicated that the combined plasma membrane surface areas of the sieve element–companion cell (se–cc) complexes, vascular parenchyma and pigment strand are not sufficient to allow sucrose transfer to the apoplast at the observed rates. In contrast, the wall ingrowths of the transfer cells in the nucellar projection amplify the membrane surface area up to 22-fold, supporting the observed rates of sucrose transfer into the endosperm cavity. We conclude that photosynthate moves via the symplast from the se–cc complexes to the nucellar projection transfer cells, from where it is transferred across the plasma membrane into the endosperm cavity. The apoplastic barrier in the pigment strand is considered to restrict solute movement to the symplast and block apoplastic solute exchange between maternal and embryonic tissues. The implications of this cellular pathway in relation to the control of photosynthate transfer in the developing grain are discussed.     

The cellular pathway of photosynthate transfer in the developing wheat grain. I. Delineation of a potential transfer pathway using fluorescent dyes

A potential cellular pathway for photosynthate transfer between the crease phloem and the starchy endosperm of the developing wheat grain has been delineated using fluorescent dyes. Membrane permeable and impermeable dyes have been introduced into the grain through the crease phloem, the endosperm cavity or the dorsal surface of the starchy endosperm. The movement of the symplastic tracer 5-(6)-6-carboxyfluorescein (CF) derived from 5-(6)-6-carboxyfluorescein diacetate (CFDA), from either direction between the crease phloem and the endosperm cavity, indicated that the symplastic pathway was operative from the crease phloem to the nucellar projection. Furthermore, the inward movement of apoplastic tracer trisodium, 3-hydroxy-5,8,10-pyrentrisulphonate (PTS) from the endosperm cavity and that of CF following plasmolysis showed that there was a high resistance to solute transfer within the apoplast of the pigment strand. All dyes entered the modified aleurone and adjacent sub-aleurone bordering the endosperm cavity. Subsequent movement of the symplastic tracers CF and sulphorhodamine G (SRG) into and through the endosperm was rapid. However, the movement of apoplastic tracers PTS and Calcofluor White (CFW) was relatively slow and with tissue plasmolysis, CF was confined to the cytoplasm of the modified aleurone and subaleurone cells. Together, these results demonstrate that there is a high resistance to solute movement within the apoplast of the cells bordering the endosperm cavity. We propose that photosynthate transfer is via the symplast to the nucellar projection where membrane exchange to the endosperm cavity occurs. Uptake from the cavity is by the modified aleurone and small endosperm cells prior to transfer through the symplast to and through the starchy endosperm.     

Structural and histochemical studies on grain-filling in the caryopsis of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The endosperm and embryo that constitute the filial tissues of rice caryopsis are isolated from the maternal tissues by the absence of any symplastic continuity. Nutrients are transported to the endosperm through a single ovular vascular trace present on the ventral side of the ovary. Initially solute enters through the chalaza into the nucellar projection and then into the endosperm. At later stages transport occurs through the nucellar epidermis, centripetally towards the endosperm. The cell walls of the nucellar epidermis are provided with rib-like thickenings. A comparison of grain-filling in C₃ and C₄ cereals suggests that rice has structural features allied to C₃ cereals, such as wheat, but with significant differences.     

The Structural Changes During the Degeneration Process of Antipodal Complex and Its Function to Endosperm Formation in Wheat Caryopsis

During the early developmental stage of wheat caryopsis the antipodal complex (composed of 20 or more cells) located on the chalazal part of embryo sac gradually turns to degeneration and degradation from its outer part to the innermost, undergoing apparent structural changes of protoplasm. The senescent tissue (antipodals) exports its cell contents continually to support the proliferation and enlargement of the adjacent free-nuclear endosperm and accommodate the dual function of both material transport and nurture supply. The lacking of callose deposition on the boundary wails between antipodals and endosperm is much benefit to the solute transport, but not all cell contents in antipodals undergo thorough degradation until exporting, at least, part of the protoplasm only undergoes limited structural disintegration. The disassembled protoplasmic constituents actively migrate through symplast route in the form of macromolecule. This shows another mode of material transport in feeding endosperm. The occurrence of wide cytoplasmic channel in part of boundary wal ls berween antipodals and endosperm shows a special structural transformation of intercellular connection. Therefore, disassembled nuclear materials, cisternae of endoplasmic reticulum and plastids, mitochondria, etc. could migrate from antipodals into the developing endosperm. It is deduced that this mode of material transport may play an important role in supporting rapid proliferation and enlargement of free-nuclear endosperm in the developing caryopsis.     

Structural development of aleurone and its function in common wheat

The wheat aleurone is formed from surface endosperm cells, and its developmental status reflects its biogenesis, structural characteristics, and physiological functions. In this report, wheat caryopses at different development stages were embedded in Spurr’s low-viscosity embedding medium for observation of the development of aleurone cells (ACs) by light microscopy, scanning electron microscopy, and fluorescence microscopy, respectively. According to their structures and physiological characterization, the ACs development process was divided into five stages: endosperm cellulization, spherosome formation, aleurone grain formation, filling material proliferation, and maturation. Furthermore, ACs in different parts of the caryopsis formed differently. ACs near the vascular bundle developed earlier and formed transfer cells, but other ACs formed slowly and did not form transfer cells. ACs on the caryopsis backside were a regular square shape; however, ACs in the caryopsis abdomen were mainly irregular. There were also differences in development between wheat varieties. ACs were rectangular in hard wheat but square in soft wheat. ACs were larger and showed a greater degree of filling in hard compared to soft wheat. The storage materials in ACs were different compared to inner endosperm cells (IECs). The concentrations of minerals such as sodium, magnesium, silicon, phosphorus and potassium were higher in ACs than in IECs. ACs contained many aleurone grains and spherosomes, which store lipids and mineral nutrients, respectively. The cell nucleus did not disappear and the cells were still alive during aleurone maturation. However, IECs were dead and mainly contained amyloplast and protein bodies, which store starch and protein, respectively. Overall, the above results characterized major structural features of aleurone and revealed that the wheat aleurone has mainly four functions.     

Monitoring Phloem Unloading and Post-Phloem Transport by Microperfusion of Attached Wheat Grains

Phloem unloading and post-phloem transport in developing wheat (Triticum aestivum L.) grains were investigated by perfusing the endosperm cavities of attached grains. Relative unloading ratio (RUR) and the rate of sucrose release into the endosperm cavity (SRR) were calculated, respectively, from 14C import and from sucrose washout from the cavity. RUR and SRR continued at or near in vivo rates over a wide range of cavity sap osmolality (90 to approximately 500 milliosmolal) and sucrose concentration (14-430 mM) and for long times (29 h). These are much greater ranges than have been observed for the endosperm cavity in vivo (230-300 milliosmolal, and 40-120 mM, respectively), indicating that neither the cavity sap osmolality nor sucrose concentration are controlling factors for the rate of assimilate import into the cavity. The maintenance of in vivo transport rates over a wide range of conditions strongly implicates the role of transport processes within the maternal tissues of the wheat grain, rather than activities of the embryo or endosperm, in determining the rate of assimilate import into the grain. RUR was decreased by high concentrations of sucrose and sorbitol, but not of mannitol. By plasmolyzing some chalazal cells, sorbitol appeared to block symplastic transport across the crease tissues, but neither sucrose nor mannitol caused plasmolysis in maternal tissues of attached grains. The inhibition of RUR by KCN and carbonyl cyanide m-chlorophenyl (CCCP) and the continued import of sucrose into grains against its concentration gradient suggest that solute movement into the endosperm cavity might occur by active membrane transport. However, the evidence is weak, since KCN and CCCP appeared to act primarily on some aspect of symplastic (i.e. nonmembrane) transport. Also, sucrose could move from the endosperm cavity into the maternal tissues (i.e. opposite to the normal direction of sucrose movement), suggesting that transmembrane movement in the nucellus may be a reversible process. Pressure-driven flow into the grain could account for movement against a concentration gradient.     

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.     

Fertilization and Embryo Development in Wheat x Maize Crosses

The fertilization and embryo development in crosses of hexaploid wheat “Kangxuan 9” X maize “SS 7700” were studied. Of 180 florets fi,ced after pollination 34(18.9%) had embryo and endosperm, 46(25.6%) had only embryo and 12(6.7%) had only endosperm. Percentages of single or double fertilization were higher than that in control (“Chinese Spring” X maize). The hybrid embryos and endosperms obtained were karyotypically unstable and characterized by rapid elimination of the maize chromosomes to produce haploid wheat embryos. The potentials for wheat haploid production and transfer of DNA segments, including transposable elements, from maize to wheat is discussed.     

Sucrose Release into the Endosperm Cavity of Wheat Grains Apparently Occurs by Facilitated Diffusion across the Nucellar Cell Membranes

Nutrients required for the growth of the embryo and endosperm of developing wheat (Triticum aestivum L.) grains are released into the endosperm cavity from the maternal tissues across the nucellar cell plasma membranes. We followed the uptake and efflux of sugars into and out of the nucellus by slicing grains longitudinally through the endosperm cavity to expose the nucellar surface to experimental solutions. Sucrose uptake and efflux are passive processes. Neither was sensitive to metabolic inhibitors, pH, or potassium concentration. p-Chloromercuribenzene sulfonate, however, strongly inhibited both uptake and efflux, although not equally. Except for p-chloromercuribenzene sensitivity, these characteristics of efflux and the insensitivity of Suc movement to turgor pressure are similar to those of sucrose release from maize pedicels, but they contrast with legume seed coats. Although the evidence is incomplete, movement appears to be carrier mediated rather than channel mediated. In vitro rates of sucrose efflux were similar to or somewhat less than in vivo rates, suggesting that transport across the nucellar cell membranes could be a factor in the control of assimilate import into the grain.     

Gene expression patterns in the maize caryopsis: clues to decisions in embryo and endosperm development.

We will describe gene expression patterns in the maize caryopsis, which provide clues to developmental decisions and questions in the embryo and endosperm. The emphasis will be on the development of the root/shoot axis, which is the main achievement of plant embryogenesis. Data obtained in the vegetative seedling are included as far as they may be relevant to the elaboration of the shoot/root axis. Development of the embryo will be briefly compared to endosperm as both seed compartment exhibit pronounced differences.     

Pseudogamous Apomixis in Maize and Sorghum in Diploid-Tetraploid Crosses

Apomictic seed development is a complex process including formation of unreduced embryo sac, parthenogenetic embryo development from the egg cell, and endosperm formation either autonomously, or due to fertilization of polar nuclei by the sperm (under pseudogamous form of apomixis). In the latter case, an obstacle to the normal endosperm development is disturbance of maternal (m) -to-paternal (p) genomic ratio 2m: 1p that occurs in the cases of pollination of unreduced embryo sac with haploid sperms. Usage of tetraploid pollinators can overcome this problem because in such crosses maternal-to-paternal genomic ratio is 4m: 2p that provides formation of kernels with plump endosperm. Using tetraploid lines as pollen parents we observed formation of plump kernels on the ears and panicles of diploid maize and sorghum accessions. These kernels had hybrid endosperm and diploid maternaltype embryo or hybrid embryo with different ploidy level (2n, 3n, 4n). The frequencies of plump kernels on the ear ranged from 0.2-0.3% to 5.7-6.2% counting from the number of ovaries. Maternal-type plants were found in two maize lines, their frequency varying from 10.7 to 37.5% of the progeny plants. In CMS-lines of sorghum pollinated with tetraploid sorghum accessions, the frequency of plump kernels ranged from 0.6 to 14.0% counting from the number of ovaries; the frequency of maternal-type plants varied from 33.0 up to 96.1%. The hybrid nature of endosperm of the kernels that gave rise to maternal-type plants has been proved by marker gene expression and by SDS-electrophoresis of endosperm proteins. These data testify to variable modes of seed formation under diploid × tetraploid crosses in maize and sorghum both by amphi- and by apomixis. Therefore, usage of tetraploid pollinators might be a promising approach for isolation of apomixis in maize and sorghum accessions.     

Water uptake and splitting of wild oat caryopsis

Imbibition and germination experiments were conducted on the caryopses of wild oats (Avena fatua L.). The embryo envelopes, pericarp and aleurone layer, which completely cover the embryo-endosperm, do not form barriers against water uptake. The initial uptake of water is passive and the water moves across the pericarp with ease as it contains cracks; it is, however, transported across the aleurone layer through its cell walls into the endosperm and embryo of the caryopsis. The starchy endosperm enlarges due to water uptake causing the pericarp to rupture, thus exposing the aleuronelayer-covered seed. The aleurone layer is structurally heterogenous consistings of radially compressed irregular cells and cuboidal or radiallys tretched cells; the latter contains thicker walls. The former type is present along the abaxial side of the embryo and in the crease on the adaxial side of the caryopsis; the latter type covers the endosperm. The physical distention of the endosperm due to water uptake causes the rupture of the pericarp and the aleurone layer, and facilitates the emergence of the radicle and coleorhiza of the embryo during caryopsis germination.     

小麦×高粱的受精率和小麦单倍体的诱导(简报)

远缘杂交无论在植物遗传学理论的建立还是在新品种的培育上都是一项十分有用的技术,特别在小麦上成绩更为突出。近来发现小麦不但和其亲缘属(如山羊草属、偃麦草属,赖草属等)植物可以杂交,而且和亲缘关系较远的植物种属如玉米、珍珠栗、大刍草、摩擦禾等杂交也有很高的成胚率,并且通过杂种胚发育过程中父本染色体的消失,可以获得小麦单倍体。Laurie和Bennett以高粱属的     

Transfer aleurone cells inSetaria lutescens (Gramineae)

Summary Typical aleurone cells occur around the periphery of the caryopsis. These cells are tabular with moderately thick walls and lack cell wall ingrowths. Transfer aleurone cells only occur adjacent to the placental vascular bundle, which supplies the developing embryo and endosperm. These specialized aleurone cells are approximately columnar, with thick walls bearing ingrowths on the outer radial and outer tangential walls. The wall ingrowths of transfer aleurone cells appear similar to those of transfer cells previously described and quite likely also function in short-distance transport of substances.Journal paper No. J-6737 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project No. 1685.     

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize

Background and Aims

Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation.

Methods

Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses.

Key Results

During the period 0–28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo.

Conclusions

The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.