protocols-for-obtaining-zygotic-somatic-embryos-for-studying

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume <em>Medicago truncatula</em>

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

University of Newcastle, Callaghan, New South Wales, Australia

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

Leaf explants of Medicago truncatula were used to investigate the origins of auxin-induced root formation. On the application of auxin there is some callus formation (not the massive amount that occurs in response to auxin plus cytokinin) and roots appear shortly after the first visible callus. Histological examination reveals morphologically distinctive sheets of callus cells that emanate from the veins of the leaf explants and, within this cell type, root primordia are produced as well as some vascular tissue cells. What is suggested is that the vein-derived cells (VDCs) are procambial-like and function as pluripotent stem cells with a propensity to form root meristems or vascular tissues in response to added auxin. The development of root primordia from these pluripotent cells was clearly up-regulated by the use of the sickle (skl) mutant, which is a mutant impaired in ethylene signal transduction while the wild type and the sunn mutant, defective in auxin polar transport, produced similar numbers of roots. The skl mutant in generating many more roots concomitantly formed fewer vascular tissues. The root meristems differentiate similarly to normal roots producing a central cylinder of vascular tissue, which connects with the leaf explant veins. The VDCs appear to be derived from the cells of or near the phloem. The leaf observations suggest that a pool of stem cells exist in vascular tissue that, in combination with auxin and perhaps other factors, drive a diversity of plant development outcomes that is species specific. The way auxin interacts with other hormones is a key factor in determining the stem cell fate. The histological data in this study also assist in the interpretation of the molecular analysis of auxin-induced root formation in cultured leaves of M. truncatula.

Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene.

Three homeologous genes encoding a sucrose (Suc) transporter (SUT) in hexaploid wheat (Triticum aestivum), TaSUT1A, 1B, and 1D, were expressed in germinating seeds, where their function is unknown. All three TaSUT1 proteins were confirmed to be capable of transporting both Suc and maltose by complementation tests with the SUSY7/ura3 yeast (Saccharomyces cerevisiae) mutant strain. The role of Suc transporters in germinating grain was examined by combining in situ hybridization, immunolocalization, fluorescent dye tracer movement, and metabolite assays. TaSUT1 transcript and SUT protein were detected in cells of the aleurone layer, scutellar epidermis, scutellar ground cells, and sieve element-companion cell complexes located in the scutellum, shoot, and root. Ester loading of the membrane-impermeable fluorescent dye carboxyfluorescein into the scutellum epidermal cells of germinating seeds showed that a symplasmic pathway connects the scutellum to the shoot and root via the phloem. However, the scutellar epidermis provides an apoplasmic barrier to solute movement from endosperm tissue. Measurements of sugars in the root, shoot, endosperm, and scutellum suggest that, following degradation of endosperm starch, the resulting hexoses are converted to Suc in the scutellum. Suc was found to be the major sugar present in the endosperm early in germination, whereas maltose and glucose predominate during the later stage. It is proposed that loading the scutellar phloem in germinating wheat seeds can proceed by symplasmic and apoplasmic pathways, the latter facilitated by SUT activity. In addition, SUTs may function to transport Suc into the scutellum from the endosperm early in germination and later transport maltose.

Pathway of sugar transport in germinating wheat seeds.

Transcriptional profiling of embryogenic callus produced from Medicago truncatula mesophyll protoplasts indicated up-regulation of ethylene biosynthesis and ethylene response genes. Using inhibitors of ethylene biosynthesis and perception, it was shown that ethylene was necessary for somatic embryogenesis (SE) in this model legume. We chose several genes involved in ethylene biosynthesis and response for subsequent molecular analyses. One of these genes is a gene encoding a transcription factor that belongs to the AP2/ERF superfamily and ERF subfamily of transcription factors. We demonstrate that this gene, designated M. truncatula SOMATIC EMBRYO RELATED FACTOR1 (MtSERF1), is induced by ethylene and is expressed in embryogenic calli. MtSERF1 is strongly expressed in the globular somatic embryo and there is high expression in a small group of cells in the developing shoot meristem of the heart-stage embryo. RNA interference knockdown of this gene causes strong inhibition of SE. We also provide evidence that MtSERF1 is expressed in zygotic embryos. MtSERF1 appears to be essential for SE and may enable a connection between stress and development.

The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula.

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula.

Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells.

• The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

Xin-Ding Wang

School of Environmental and Life Sciences

University of Newcastle, Callaghan, New South Wales, Australia

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula