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

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

Nitric oxide and hydrogen peroxide involvement during programmed cell death of Sechium edule nucellus

The nucellus is a maternal tissue that feeds the developing embryo and the secondary endosperm. During seed development the cells of the nucellus suffer a degenerative process early after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been characterized as a form of developmentally programmed cell death (PCD). In this work we show that nucellus PCD is accompanied by a considerable production of both nitric oxide and hydrogen peroxide (NO and H₂O₂). Interestingly, each of the two molecules is able to induce the production of the other and to cause cell death when applied to a living nucellus. We show that the induced cell death has features of a PCD, accompanied by profound changes in the morphology of the nuclei and by a massive degradation of nuclear DNA. Moreover, we report that NO and H₂O₂ cause an induction of caspase‐like proteases previously characterized in physiological nucellar PCD.     

Rice caryopsis development Ⅰ: Dynamic changes in different cell layers

Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a systematic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30-day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination(DAP). In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.     

Rice caryopsis development I: Dynamic changes in different cell layers

Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a systematic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30‐day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination (DAP). In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.     

Developmental regulation of a VEIDase caspase-like proteolytic activity in barley caryopsis

Caspases are essential in animal programmed cell death both as initiator and executioner proteases. Plants do not have close caspase homologues, but several instances of caspase-like proteolytic activity have been demonstrated in connection with programmed cell death in plants. It was asked if caspase-like proteases are involved during development of the barley caryopsis. The presence of a caspase-6-like proteolytic activity that preferentially cleaved the sequence VEID was demonstrated. A range of protease inhibitors was tested and only caspase-specific inhibitors showed major inhibitory effects. The profile of VEIDase activity in developing starchy endosperm, embryo, and whole caryopsis was measured and showed a general trend of higher activity in young, rapidly developing tissues. The VEIDase activity was localized in vivo to vesicles, shown to be autophagosomes, in randomly distributed cells of the starchy endosperm. The VEIDase activity detected in barley caryopsis is similar to activities described previously in mammals, spruce, yeast, and thale cress. In mammals, spruce, and yeast, VEIDase activity has been shown to be positively correlated with the occurrence of programmed cell death. Several manifestations of programmed cell death exist in developing barley caryopsis, indicating a connection between VEIDase activity and developmental programmed cell death in barley.     

Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases

The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.     

Storage-protein Deposition in the Developing Rice Caryopsis in Relation to the Transport Tissues

The developing caryopsis of rice (Oryza sativa L.) was examinedhistologically at successive stages of grain-filling in orderto identify the factors which determine the distribution ofstorage protein in the endosperm, and which terminate the depositionof endosperm protein. The storage protein was deposited at theperiphery of the endosperm, and this distribution was apparentlycaused by the radial pattern of cell development in the endosperm,and by the proximity of the peripheral endosperm cells to thenucellar epidermis. The nucellar epidermis directly surroundsthe endosperm and functions as the pathway for amino acid transportto the endosperm. During the later stages of caryopsis developmentthe nucellar epidermis became compressed by being ‘sandwiched’between the expanding endosperm and the rigid hull (the tightlylocked palea and lemma) which encloses the caryopsis. It isproposed that this compression of the nucellar epidermis blocksthe supply of amino acids to the endosperm and thereby terminatesthe deposition of storage protein in the rice grain. Oryza sativa, rice, caryopsis (development), endosperm, grain filling, nucellar epidermis, storage protein     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE CARYOPSIS COAT AND THE ALEURONE CELLS

The results of a light and electron microscopic study of the caryopsis coat and aleurone cells in ungerminated, unimbibed rice (Oryza sativa) caryopses are presented. Surrounding the rice grain is the caryopsis coat composed of the pericarp, seed coat and nucellar layers. The outermost layer, the pericarp, consists of crushed cells and is about 10 μm thick. The seed coat, interior to the pericarp, is one cell thick and has a thick cuticle. Between the seed coat cuticle and endosperm are the remains of the nucellus. The nucellus is about 2.5 μm thick and has a thick cuticle adjacent to the seed coat cuticle. Interior to the caryopsis coat is the aleurone layer of the endosperm. The aleurone completely surrounds the rice grain and is composed of two cell types—aleurone cells that surround the starchy endosperm and modified aleurone cells that surround the germ. The aleurone cells of the starchy endosperm contain many aleurone grains and lipid bodies around a centrally located nucleus. The modified aleurone cells lack aleurone grains, have fewer lipid bodies than the other aleurone cells, and contain filament bundles (fibrils). Plastids of aleurone cells exhibit a unique morphology in which the outer membranes invaginate to form tubules and vesicles within the plastid. Transfer aleurone cells are not observed in the mature rice caryopsis.     

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.     

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.     

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.     

Development and function of caryopsis transport tissues in maize,sorghum and wheat

Cereal caryopsis transport tissues are essential channels via which nutrients are transported into the embryo and endosperm. There are differences and similarities between caryopsis transport tissues of maize, sorghum and wheat. Vascular bundle, endosperm transfer cells, endosperm conducting cells and embryo surrounding region are common in maize, sorghum and wheat. Placentochalaza is special in maize and sorghum, while chalaza and nucellar projection transfer cells are special in wheat. There is an obvious apoplastic cavity between maternal and filial tissues in sorghum and wheat caryopses, but there is no obvious apoplastic cavity in maize caryopsis. Based on the latest research, the development and function of the three cereal caryopsis transport tissues are discussed and investigated in this paper.     

Structure and function of the microtubular cytoskeleton during endosperm development in wheat: an immunofluorescence study

Summary The three-dimensional structure of the microtubular cytoskeleton of developing wheat endosperm was investigated immunocytochemically. Semi-thin sections were prepared from polyethylene glycol embedded ovaries. At the free-nuclear stage the endosperm cytoplasm with regularly distributed nuclei surrounded a large central vacuole and exhibited an extensive network of fluorescent labelled microtubular assemblies radiating from each nucleus. As was found in other coenocytes, this particular and nuclear-dependent cytoskeletal configuration functions in the arrangement of nuclei and in the stabilization of the nuclear positions. At the beginning of cellularization of the endosperm the formation of vacuoles altered the radiating networks. It is likely that the radiating microtubular arrays function in the formation of phragmoplasts, independent of nuclear divisions. The formation of anticlinal cell walls, giving rise to openended cell cylinders, coincides with the occurrence of phragmoplast microtubular arrays which were demonstrated during the period of cell wall elongation. The microtubular system radiating from the nuclei in these cell cylinders anchored the nuclei in stage- and locus-specific positions. During the development of aleurone and inner endosperm cells, cell morphogenesis was related to earlier demonstrated types of microtubular configurations in the cortical cytoplasm. This suggests that a general mechanism is involved.Abbreviations A alveolus - AL aleurone layer - CE central endosperm - CV central vacuole - DAP days after pollination - END endosperm - FITC fluorescein isothiocyanate - GAR-FITC goat anti-rabbit antibodies conjugated with FITC - I integument - IE PC inner epidermis pericarp - II inner integument - N nucleus - NC nucellus cells - NE nucellar epidermis - NUC nucellus - OI outer integument - PBS phosphate buffered saline - PC pericarp - PEG polyethylene glycol - V vacuole     

Seed development in Malpighiaceae species with an emphasis on the relationships between nutritive tissues

Malpighiaceae ovules have a well-developed nucellus; previous observations indicate that during seed development, the endosperm does not proliferate, thus, remaining scarce. This study aimed at identifying the nutritive tissues during seed development in Malpighiaceae, focusing especially on the endosperm. We analysed the seed development of Janusia mediterranea, J. occhionii, Mascagnia cordifolia, and Tetrapterys chamaecerasifolia, which were collected and processed by traditional methods for light microscopy. Ovules are subcampylotropous, crassinucellate and unitegmic in Janusia and bitegmic in M. cordifolia and T. chamaecerasifolia. The nucellus is well developed and protrudes through the micropyle, touching the funicular obturator. During development, a pachychalaza is formed, and the integuments coalesce in bitegmic species. Through a series of nucellar cell divisions, the perisperm is formed. In Janusia species, the endosperm is not produced. In M. cordifolia and T. chamaecerasifolia, the endosperm is nuclear, but it is scarce and ephemeral. The mature seed is exalbuminous, and the perisperm is consumed, and thus, the mature embryo is total. The absence of endosperm in Janusia is newly observed for the family and indicates functional transfer for the abundant perisperm.     

Embryology of Early Abortion Due to Limited Maternal Resources in Pisum sativum L.

In most flowering plants, many embryos are aborted early intheir development due to limited maternal resources. The kin-conflictinterpretation of plant embryology predicts these abortionsshould be under maternal control. In a study of the abortionprocess in Pisum sativum, we found the first visible indicationof abortion was formation of a weak hypostase. Callose was locallydeposited around the chalazal endosperm haustorium, and ligninalong the outer cell walls of the remnant nucellar tissue. Thenucellus was compressed by proliferating adjacent inner integumentalcells. The endosperm haustorium's cytoplasm was forced backinto the embryo sac cavity. With suppression of haustorial activitythe endosperm nuclei gradually enlarged followed by enlargementof the embryo and suspensor nuclei. Finally, nuclei and cytoplasm throughout the endosperm and embryolost stainability and broke down. Four successive stages wererecognized in seed abortion. In seeds developing to maturity,no hypostase was developed and the haustorium continued to digestboth the remnant nucellus and the proliferated inner integumentalcells. These observations are consistent with the kin-conflicthypothesis. Pisum sativum, garden pea, ovule abortion, histology, hypostase, kin-conflict hypothesis     

ANATOMY OF THE CARYOPSIS OF BRIZA MAXIMA (POACEAE)

Briza maxima (quaking grass) is a cosmopolitan grass common to Europe and North and South America. It grows in disturbed soils and on roadsides. The hemispherical caryopsis is embedded between a leaflike lemma and flattened palea. The embryo is of the festucoid type. The scutellum shows two surrounding ridges at the edge of the scutellum/endosperm boundary, and has lateral lobes. A broad epiblast extends toward the embryo apex and is continuous with the dorsal surface of the coleorhiza. The single-layered aleurone surrounds the starchy endosperm and is discontinuous around the embryo. The caryopsis coat is thin, except at the placental pad where it is thickened by the pigment strand and the nucellar projection.     

The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development

The nucellus, which is the maternal tissue of the wheat grain, degenerates during the early stages of development. We have investigated whether or not this degenerative process may be considered as programmed cell death (PCD). The analysis of DNA of tissues dissected from developing wheat (Triticum aestivum L. cv Chinese Spring) grains at 5-20 days post anthesis (dpa) showed the presence of DNA laddering, which is indicative of internucleosomal fragmentation of nuclear DNA, in maternal tissues but not in the endosperm. The TUNEL assay showed in-situ internucleosomal fragmentation of DNA in nuclei of parenchymal and epidermal cells of the nucellus, as well as in the pericarp, during the early stages of grain development (5 dpa). Furthermore, internucleosomal fragmentation of nuclear DNA was observed in nucellar projection cells in the middle stages of grain development (13-18 dpa), thus showing a process of PCD in these maternal tissues. Electron-transmission microscopy analysis allowed the morphology of PCD to be characterized in this plant tissue. Initially, fragmentation of the cytoplasm was observed, the nuclear envelope appeared dilated and to be forming vacuoles, and the content of heterochromatin increased. A progressive degradation of the cytosolic contents and organelles was observed, and the plasma membrane was disrupted. However, the Golgi apparatus remained intact and apparently functional even in the final stages of cell death.     

Caspase-like proteases regulate aluminum-induced programmed cell death in peanut

Recent evidence has proved that caspase protease activities are detected in both mammals and plants during programmed cell death (PCD). The characteristics and functions of caspase-like proteases play important roles in understanding the mechanisms of PCD in plants. In this work, we report firstly the involvement of caspase-like protease activities and effects in aluminum (Al) stress in two contrasting peanut genotypes. Caspase-like activities in the root tip cells of ‘Zhonghua 2’ (Al-sensitive) and ‘99-1507’ (Al-tolerant) were detected using synthetic caspase substrates during Al-triggered PCD. Caspase-1-, -2-, -3-, -4-, -5-, -6-, -8- and -9-like proteases were found in peanut root tip cells. VDQQDase (caspase-2-like) and WEHD (caspase-5-like) were the first detected in the plants, and almost all of the caspase-like proteases were activated during Al-induced PCD, especially caspase-3-like and caspase-1-like, which was higher in ‘Zhonghua 2’ than in ‘99-1507’. The highest activity levels of caspase-3- and caspase-1-like proteases occurred 8 and 4 h after 100 µM Al treatment, respectively. Compared with 100 µM AlCl₃ treatment alone, specific caspase-3 protease inhibitor Ac-DEVD-CHO inhibited the increase of caspase-3-like protease activity, Al content, Hsr203j expression, cell death and DNA fragmentation, and the decrease in root growth induced by 100 µM AlCl₃ treatment, but it was more obvious in ‘Zhonghua 2’ than in ‘99-1507’. In conclusion, there were different caspase-like proteases in root tips of peanut, and caspase-3-like protease was a crucial executioner in Al-induced PCD. Its effects in the ‘Zhonghua 2’ genotype were higher than in ‘99-1507’. An improved model of the mechanism of Al-induced PCD and Al tolerance differences in different genotypes is proposed.