Edward C. Yeung

The Accumulation of Oleosins Determines the Size of Seed Oilbodies in Arabidopsis

We investigated the role of the oilbody proteins in developing and germinating Arabidopsis thaliana seeds. Seed oilbodies are simple organelles comprising a matrix of triacylglycerol surrounded by a phospholipid monolayer embedded and covered with unique proteins called oleosins. Indirect observations have suggested that oleosins maintain oilbodies as small single units preventing their coalescence during seed desiccation. To understand the role of oleosins during seed development or germination, we created lines of Arabidopsis in which a major oleosin is ablated or severely attenuated. This was achieved using RNA interference techniques and through the use of a T-DNA insertional event, which appears to interrupt the major (18 kD) seed oleosin gene of Arabidopsis and results in ablation of expression. Oleosin suppression resulted in an aberrant phenotype of embryo cells that contain unusually large oilbodies that are not normally observed in seeds. Changes in the size of oilbodies caused disruption of storage organelles, altering accumulation of lipids and proteins and causing delay in germination. The aberrant phenotypes were reversed by reintroducing a recombinant oleosin. Based on this direct evidence, we have shown that oleosins are important proteins in seed tissue for controlling oilbody structure and lipid accumulation.

Embryogenesis in Angiosperms: Development of the Suspensor

The zygote in flowering plants usually divides transversely to form a terminal cell, which gives rise to the embryo proper, and a vacuolated basal cell, which often divides rapidly to form a structure known as the suspensor. Angiosperm suspensors vary widely in size and morphology from a single cell to a massive column of several hundred cells (Maheshwari, 1950; Wardlaw, 1955; Lersten, 1983). In most cases, the suspensor functions early in embryogenesis and then degenerates during later stages of development and is not present in the mature seed. Classically, the suspensor was thought to play a passive role in embryo development by holding the embryo proper in a fixed position within the seed (Maheshwari, 1950). It now appears from extensive structural, biochemical, and physiological studies with a variety of angiosperms that the suspensor plays an active role early in development by promoting continued growth of the embryo proper. In addition, growth of the suspensor during early stages of development may be inhibited by the embryo proper (Marsden and Meinke, 1985). Analysis of reproductive development in angiosperms must therefore include a consideration of developmental interactions that occur between the embryo proper and suspensor. Although the suspensor appears to play a critical role in zygotic embryogenesis, it usually fails to develop when somatic embryos are produced in culture. The suspensor should therefore be viewed as a specialized structure that functions primarily to facilitate continued development of the embryo proper within the seed. In this review, we present an overview of the structure and function of the angiosperm suspensor and discuss recent attempts to analyze the development of the suspensor through a combination of descriptive, experimental, and genetic approaches. The recent identification of a large collection of Arabidopsis mutants with abnormal suspensors provides a unique opportunity to examine the underlying genetic factors that influence suspensor development.

Recent advances in conifer somatic embryogenesis: improving somatic embryo quality

Somatic embryogenesis of coniferous species was first reported more than 20 years ago. Since then, there has been an explosion of research aimed at developing and optimizing protocols for efficient regeneration of plantlets. Although routinely used both as a means of propagation, as well as a valuable model system for investigating the structural, physiological, and molecular events occurring during embryo development, in vitro embryogenesis is still problematic for some coniferous species. Major problems include: low number of embryos generated; and low frequency of mature embryos able to convert into viable plantlets. Until recent years, despite the fact that embryogenesis is comprised of a sequence of defined steps which include proliferation of embryogenic tissue, embryo maturation, and germination, attempts at improving the whole procedure have been made almost exclusively during the maturation stage. This strategy was based on the assumption that successful regeneration is related to treatments provided during the development of the embryos. Major optimizations of the maturation medium have involved judicious selections of type and concentration of growth regulators, namely abscisic acid, and adjustments of the osmoticum of the culture medium. Extensive work has been conducted in defining the effects of plasmolysing and non-plasmolysing osmoticum agents during maturation, as well as in improving desiccation techniques required for the completion of the maturation program. In the last 2 years, however, work on spruce has clearly demonstrated that the early events in embryogenesis are crucial for the successful completion of the overall embryogenic program. The use of cell tracking techniques, implemented by physiological and molecular studies, has revealed that manipulations of the culture conditions early in the process can increase both number and quality of embryos produced in culture. Additional manipulations of the germination medium can also enhance germination and conversion frequency of somatic embryos matured in a sub-optimal environment. These new findings, together with the unraveling of molecular mechanisms involved in the control/regulation of embryo development hold considerable promise for clonal propagation in conifers.

Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed

Significance Seeds are complex structures that are comprised of the embryo, endosperm, and seed coat. Despite their importance for food, fiber, and fuel, the cellular processes that characterize different regions of the seed are not known. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets provide unique insights into the cellular processes that occur in understudied seed regions, revealing unexpected overlaps in the functional identities of seed regions and enabling predictions of gene regulatory networks. This dataset is an essential resource for studies of seed biology. Seeds are complex structures that consist of the embryo, endosperm, and seed-coat regions that are of different ontogenetic origins, and each region can be further divided into morphologically distinct subregions. Despite the importance of seeds for food, fiber, and fuel globally, little is known of the cellular processes that characterize each subregion or how these processes are integrated to permit the coordinated development of the seed. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets offer the most comprehensive description of gene activity in seeds with high spatial and temporal resolution, providing unique insights into the function of understudied seed regions. Global comparisons of mRNA populations reveal unexpected overlaps in the functional identities of seed subregions. Analyses of coexpressed gene sets suggest that processes that regulate seed size and filling are coordinated across several subregions. Predictions of gene regulatory networks based on the association of transcription factors with enriched DNA sequence motifs upstream of coexpressed genes identify regulators of seed development. These studies emphasize the utility of these datasets as an essential resource for the study of seed biology.

Got milk? The secret life of laticifers

Laticifers are specialized cells that occur in over 20 plant families in several unrelated angiosperm orders. Although laticifers are likely to be of polyphyletic origin, their occurrence is considered a morphological indicator of relatedness among species. The classification of laticifers is based on developmental patterns and overall morphology. The cytoplasmic latex exuded in response to damage often includes specialized metabolites, such as cardenolides, alkaloids and natural rubber. Laticifers provide an effective location to store defense metabolites, although not all latex-bearing plants accumulate bioactive natural products. Ecophysiological studies have shown that latex and its associated metabolites are vital for the defense of plants against insects. The anatomy, development and physiology of laticifers are discussed with a focus on evolutionary and ecological perspectives.

Production of biologically active hirudin in plant seeds using oleosin partitioning

A plant oleosin was used as a ‘carrier’ for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti-thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin ‘carrier’. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide.

The use of histology in the study of plant tissue culture systems : Some practical comments

SummaryHistological methods have contributed significantly to our understanding of in vitro culture systems. A good histological study based on anatomical and histochemical changes provides insight into cellular processes and provides clues that allow for the proposal of hypotheses for further experimentation. This article serves to draw attention to the use of a histological approach to one’s experimental system. Some of the common mistakes in the handling and processing of explants are discussed. A protocol for the plastic embedding method is detailed.

Maturation of somatic embryos in conifers: Morphogenesis, physiology, biochemistry, and molecular biology

SummaryIn the past 15 years tremendons progress has been made towards the development of systems for the induction and development of somatic embryos of coniferous species. Since the first report in 1985, several species have been induced to produce somatic embryos. This has been rendered possible by the development of rational media and improvement of culture conditions, which have resulted in increased embryo quality and higher conversion frequency. Understanding the physiological and biochemical events occurring during in vivo embryogenesis has been fundamental in the design of new protocols for improving the somatic embryogenic process. Specifically, the inclusions of abscisic acid (ABA) and osmotic agents, such as polyethylene glycol (PEG), have been shown to be necessary for the functional development of somatic embryos. In the past few years, physiological and biochemical investigations have been useful in increasing our knowledge on the mode of action of ABA and PEG during embryo development. In comparison with the flowering plants, our understanding on the molecular mechanisms regulating the embryogenic process in coniferous species is still very limited. The application of new molecular techniques is therefore fundamental towards this end. The emphasis of this review is on recent information dealing with the maturation of conifer somatic embryos.

Shoot Histogenesis in Cotyledon Explants of Radiata Pine

The histological events associated with shoot primordium formation in cultured excised cotyledons from germinated seed of radiata pine (Pinus radiata D. Don) were examined. Cytological changes in the explants were observed by day 1 in culture. Mitotic activity, initially random, became restricted to the epidermal and subepidermal cell layers closest to the medium. This led to the formation of meristematic tissue along the entire length of the cotyledon during the first 3 wk in culture. Within this meristematic zone, meristemoids, shoot primordia, and finally shoots with well-developed apical meristems, needles, and needle primordia were formed.

Comparative Development of Zygotic and Microspore-Derived Embryos in Brassica napus L. CV Topas. I. Histodifferentiation

The structural pattern of tissue and meristem formation during zygotic and microspore embryo development in canola was examined. A defined sequence of cell divisions could be found during zygotic embryogenesis. The tissue pattern was delineated at the globular stage. The shoot apical meristem began to initiate at the late heart stage of development and took on a dome shape by the torpedo stage. The root meristem was derived from hypophyseal derivatives and adjoining cells from the embryo proper. In the microspore embryo, a defined pattern of cell division was not observed during early development. A suspensor was not found in all microspore embryos. Tissue differentiation began with the formation of the protoderm after a globular mass of cells was formed. The organization of the root meristem was not as well defined as in its zygotic counterpart. The shoot meristem was derived from a group of cells located at the terminal end of the embryo. At the cotyledon stage, the shoot meristem organization was similar between the zygotic and microspore embryos. Upon prolonged culture, the tissue pattern began to deteriorate in the microspore embryo. This change corresponded to the lowering of conversion frequency upon transferring the embryos onto germination medium. Large intercellular spaces developed within the shoot pole. The cells of the procambium and the meristems differentiated into parenchyma cells with prominent starch grains. As a result, the procambium and the meristem organization were drastically altered. These observations indicated that the tissues of the microspore embryo were not fully determined